multi protein-epitope based sers immunoassay for...

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Research Proposal in response to DBT Call for Proposal in the

area of Tuberculosis

Multi protein-Epitope Based SERS

immunoassay for Mycobacterium

Tuberculosis

Submitted By

Prof Chandrabhas Narayana

Chemistry and Physics of Materials Unit,

Jawaharlal Nehru Centre for Advanced Scientific Research

Bangalore

In collaboration with

1. Dr N Jayasuryan

Microtest Innovation Pvt Ltd, ITPL, Bangalore

2. Dr Mynnedu Prasad

Dept of Microbiology, LRS Institute Of TB and Respiratory Diseases,

New Delhi

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PROFORMA – I

PROFORMA FOR SUBMISSION OF PROJECT PROPOSALS ON RESEARCH

AND

DEVELOPMENT, PROGRAMME SUPPORT

(To be filled by the applicant)

PART I: GENERAL INFORMATION

1. Name of the Institute/University/Organisation submitting the Project Proposal:

Chemistry and Physics of Materials Unit, Jawaharlal Nehru Centre for Advanced

Scientific Research, Jakkur P.O. Bangalore 560064.

2. State: Karnataka . 3. Status of the Institute: Academic, Deemed University

(Please see Annexure-I)

4. Name and designation of the Executive Authority of the Institute/University

forwarding the application:

A. N. Jayachandra,

Senior Administrative Officer

Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur P.O.

Bangalore 560064.

5. Project Title: Multi Protein-Epitope based biosignature and SERS immunoassay

for Mycobacterium Tuberculosis

6. Category of the Project (Please tick) : R&D/ Programme Support

7. Specific Area (Please see Annexure - II): Section 2.8 (Vaccines and Diagnostics)

8. Duration : 3 Years

9. Total Cost (Rs.) 206.68 Lakhs

10. Is the project Single Institutional or Multiple-Institutional (S/M) ? : M

11. If the project is multi-institutional, please furnish the following:

Name of Project Coordinator: Dr Chandrabhas Narayana

Affiliation : Professor

Address : Chemistry and Physics of Materials Unit, Jawaharlal Nehru Centre for

Advanced Scientific Research, Jakkur P.O. Bangalore 560064.

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12. Scope of application indicating anticipated product and processes

The time is ripe to move from single markers for correlates of detection/protection to a

biosignature comprising a well-defined set of robust indicators in TB that can accelerate

rapid screening and early selection of potential drug and vaccine candidates. Sensitive

assays for many possible antigens which could be detected in biological systems are still

found wanting. Project proposes to develop a prototype for high sensitivity quantitative

assay which has implementations in: diagnostics, treatment and drug development. An

ELISA based quantitative assay will be developed for a cocktail of selected highly

specific antigens which represent TB infection. Surface Enhanced Raman Spectroscopy

(SERS) will be used to enhance the sensitivity of ELISA and hence it will improvise the

detection of antigens from mycobacterium from clinical samples. Sensitivity and lower

threshold of detection is very critical for TB diagnosis since the amount of antigenic

load in an infected individual is very miniscule. The multiplex SERS-ELISA developed

will be validated for its utility in measuring these antigens in biological fluids from

patient’s samples. Simple, portable field usable equipment can be developed if prototype

works, which will be very useful in diagnosis of TB.

Anticipated Products and Processes:

1) Cloning, expression and purification of TB antigens for R& D use.

2) Production and purification of Monoclonal Antibodies for the proposed

antigens

3) SERS enhanced highly sensitive ELISA based detection assay for proposed

antigens

4) Validation of SERS-ELISA with patients samples for detection of proposed

TB antigens in biological fluids

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13. Project Summary:

Long-term Objectives of the Proposal:

a) Development of a sensitive Antigen Capture Assay for Mycobacterium

tuberculosis.

b) Enhancement of the sensitivity of the ELISA using Surface Enhanced Raman

Spectroscopy (SERS).

c) Development of field-usable Raman equipment for Point-of-care diagnostics of

Tuberculosis.

Specific Aims:

1. Cloning, expression and purification of TB antigens for R&D use.

2. Production and purification of Monoclonal Antibodies for the proposed antigens.

3. SERS enhanced highly sensitive ELISA based detection assay for proposed

antigens.

4. Validation of SERS-ELISA with patients samples for detection of proposed TB

antigens in biological fluids.

Research Design:

Specific aim 1: Cloning, expression and purification of TB antigens for R& D use:

Selection of Antigens: Antigens of potential diagnostic value will be selected based on

the available information as published literatures as well as genomic information. Some

of them are listed below:

BIOLOGICAL

FLUIDS

ANTIGENS References

CSF * Ag85A (3804c), Ag85B

(1686c)

* 65kD hsp

Wiker and Harboe., 1992

Deng. Set al., 2011

Sputum * Ag85complex

* LAM


Periera et al., 2000

Serum * 38kDa*, 16kDa

* Mtb81 (Rv1837c)

Young et al., 1986

Hendrickson et al., 2000

Urine * MT_1721, MT-1694, LAM Kashino.SS et al 2008,

Napolitano et al., 2008,

Periera et al., 2000

Pleural fluid * 43kDa

Ascitic Fluid * Ag85B (1686c), Ag85C

* 43kDa


Xiu-Yun He et al., 2011

Additional Unknown Antigens coming from Proteomic analysis of PPD (Preliminary

work done)

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The listed antigens were identified in the biological fluid of infected individuals. We

would clone and express about ten of the above mentioned antigens and explore their

potential in a diagnostic ELISA format.

The methodology would involve, Polymerase Chain reaction to amplify the various

genes from the Mycobacterium tuberculosis genome, Cloning of the amplified genes

into bacterial expression vectors, over-expression of the genes using the IPTG inducible

system, purification of the expressed proteins using Ni-NTA and various other column

chromatographic techniques.

Specific Aim 2) Production and purification of Monoclonal Antibodies for the

proposed antigens:

The purified antigens would be used to immunize BALB/c mice. The titers of the

antibody produced would be estimated, following which the mice splenocytes will be

fused with a myeloma fusion partner for the production of hybridoma. The hybridoma

would be screen for the antibody production against the proposed antigens. Monoclonals

identifying different epitopes on the antigen will be fine mapped using a panel of

overlapping peptides for the respective antigens. Monoclonals with higher affinity to

the antigens will be scaled up and antibodies will be ammonium sulphate precipitated

and purified on protein A affinity column procedures. The antibody with extremely good

affinity and recognizing different regions of the antigens will be used for capture and

detection antibodies. The detection antibodies will be conjugated with appropriate

enzymes for the ELISA or would be conjugated to different Raman reporter moieties,

for use in SERS.

Specific Aim 3: SERS enhanced

highly sensitive ELISA based

detection assay for proposed

antigens.

Paramagnetic beads with Fe2O3 core

and a SiO2 shell, which is

functionalized and conjugated to

capture antibodies, would be used to

capture the antigens from the

biological fluids. The beads are then

concentrated using magnetic field and

the remaining solution is decanted.

Detector antibody with Raman

reporter molecules are then added

followed by additional of gold nano

particles. The Raman signal is captured by the instrument. Multiple antigens can be

detected at the same time by using multiple capture antibodies and multiple detector

antibodies, conjugated to unique spectrally non-overlapping Raman moieties. This

multiplex SERS-ELISA thus has enhanced sensitivity and specificity.

Specific Aim 4: Validation of SERS-ELISA with patients samples for detection of

proposed TB antigens in biological fluids.

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The optimized SERS-ELISA protocol would then be used to detect TB antigens

in biological fluids of TB patients. The performance of the assay will be compared with

the commercially available kits. The prototype Raman instrument would be tested in the

clinical set-up and the detection parameters and the procedures would be optimized.

Potential of the research for technological innovation:

The technological innovation is to combine the antigen capture ELISA with Surface

Enhanced Raman Spectroscopy. Paramagnetic beads conjugated to monoclonal capture

antibodies targeting different antigens of Mycobacterium tuberculosis would be used for

capturing TB antigens in biological fluids and the sensitivity of detection would be

enhanced a 1000-fold using Raman reporter moieties and gold nano particles.

Summary of the results:

1) We would have cloned, expressed and purified various antigens of

Mycobacterium tuberculosis.

2) A panel of monoclonal antibodies would be developed towards the proposed

antigens, which can be of use in R&D and for development of diagnostic kits.

3) SERS-ELISA, combining the enhanced sensitivity of SERS and the

dependability of ELISA would augment the sensitivity and specificity of

detection.

4) Point-of-care diagnostic field equipment based on Raman spectroscopy is

envisaged.

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PART II: PARTICULARS OF INVESTIGATORS

(One or more co-investigators are preferred in every project. Inclusion of co-

investigator(s) is mandatory for investigators retiring before completion of the project)

Principal Investigator:

14. Name: Chandrabhas Narayana

Date of Birth: 08-09-1965

Sex (M/F): M

Designation: Professor , Chemistry and Physics of

Materials Unit, JNCASR, Bangalore

Department: Chemistry and Physics of Materials Unit

Institute/University: Jawaharlal Nehru Centre for Advance Scientific

Research,Bangalore

Address: Jakkur P.O., Bangalore INDIA

PIN: 560064

Telephone: +91 (80) 2208 2810

Fax: +91 (80) 2208 2766

Email: [email protected]

Co-Investigator

15. Name: Dr. N. Jayasuryan

Date of Birth : 03-08-1959

Sex(M/F) : M

Designation : Director

Department : Microtest Innovation Pvt Ltd

Address: G-04, Discoverer Block, Whitefield Road, ITPL.

PIN: 560066

Telephone : 9845004989 Fax ..............…............. E-mail: [email protected]

Number of research projects being handled at present: 2

Co-Investigator

16. Name: Dr Vitthal Myneedu Prasad

Date of Birth: 4th

May 1958 Sex (M/F) : M

Designation : Sr Microbiologist and HOD

Department : LRS Institute of Tuberculosis and Respiratory Diseases

Sri Aurobindo Marg, Near Qutub Minar, New Delhi-110030

Telephone : Fax ..............….....

E-mail: [email protected]

[email protected]

mailto:[email protected]

mailto:[email protected]

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PART III : TECHNICAL DETAILS OF PROJECT

(Under the following heads on separate sheets)

16. Introduction (not to exceed 2 pages or 1000 words)

16.1 Origin of the proposal

1. Two-thirds of the world population is infected with Mycobacterium tuberculosis

and the fact that early diagnosis can control further spread of the disease.

2. There is no simple rapid and field applicable point of care diagnosis available for

the disease

3. Available techniques of diagnosis are time consuming, not cost effective as well

as it requires trained individuals to handle it.

4. Immunodiagnostics are the most rapid form of diagnostics available with

sensitivity and specificity.

5. Sensitivity and lower threshold of detection is critical for antigen detection in TB

because very less number of the proliferating organism is present in different

organs and the amount of antigen generated by them is very miniscule.

6. The analysis of Mycobacterium tuberculosis proteome revealed antigens widely

recognized by serum samples from different geographical location, ethnicity and

disease progression.

16.2 (a) Rationale of the study supported by cited literature

1) Even after several decades of research, there is no easy definitive diagnostic

test available at primary health centers for TB diagnosis, excepting the gold

standard of a century old smear examination procedure.

2) A plethora of antigens is available in Mycobacterium tuberculosis genome with

potential to be developed into a diagnostic assay.

3) Mass spectrometric analysis of PPD has shown huge number of antigens which

shows specificity to Mycobacterium tuberculosis, but yet to be analyzed for

their bioavailability to assess their potential for diagnosis.

4) Antigen detection if achieved will have definitive advantage than any other

methodology because uniformly it could be detected in people who have active

tuberculosis.

5) Field usable simple gadgets can be developed for detecting the disease at a

point-of-care diagnostics.

6) Immune response detection in terms of antibody detection has several

problems and this can be surmounted by antigen detection.

7) We already have developed a similar technology for HIV-1 detection (patent

office of Republic of South Africa - Patent Number 2009/03128 in 2009) and

hence we have a strong hold in implementing the same for TB diagnostics.

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(b) Hypothesis

New reagents both purified antigens and antibodies would provide the means to

sensitive and specific assay system for the diagnosis of the disease. A biosignature

comprising cocktail of important biomarkers will make the assay highly specific.

Surface Enhanced Raman Spectroscopy (SERS) technology would enhance the

detection of antigens which are immunoreactive but present in trace amounts. The

enhancement of ELISA sensitivity by SERS is about a 1000-fold. Antigens which are

specific to Mycobacterium tuberculosis and present in body fluids would be preferred

target for specificity in diagnosis. Combination of antigens would enhance the

sensitivity and specificity of the assay system. Our group has successfully applied this

technology for HIV.

(c) Key questions.

1. Can we combine the robustness of ELISA and sensitivity of SERS technology

and to improve sensitivity of detection and build a diagnostic assay for TB?

2. By including novel antigen(s) which has a possibility to be detected in

biological fluids, can we develop a newer diagnostic kit?

3. Can detection of multiple antigens and synergistic accumulation of signals

from multiple Raman reporters enhance the specificity and sensitivity of

detection?

4. Can we detect in biological specimens like sputum and serum, circulating

antigens which will give a definitive diagnosis for smear negative, culture

positive cases?

5. Can we develop field-usable portable Raman equipment for point-of-care

diagnosis of tuberculosis infection?

16.5 Current status of research and development in the subject (both

international and national status)

Although molecular diagnostics are simple and rapid in approach its usability remains

inaccessible to remote areas and middle income group. Analysis of the genome of M.

tuberculosis and M.bovis (vaccine strain) has revealed certain regions of difference

(RD). Quanti-FERON-TB Gold (Cellestis, Carnegie, Australia) (Chegou et al., 2009)

and T-Spot TB test (Oxford Immunotec, Oxford, United Kingdom), which is based on

RD1 antigens ESAT-6 and CFP-10, have been evaluated in clinical studies and showed

better performances than that of TST (Meier et al., 2005). A card based antigen MPT-64

detection from culture supernatant is available for routine diagnosis from BD bioscience

and Standard Diagnostics, INC.

Novel diagnostic tests have emerged as promising alternative to the previously available

tests. These include, IGRA (Interferon gamma release assay) like QuantiFERON-TB

Gold assay and T-SPOT®.TB test. IGRAs are in vitro blood tests that are based on

interferon-gamma (IFN- γ) release after stimulation by TB specific antigens (e.g. ESAT-

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6 and CFP-10) (Diel et al., 2009). However, IGRA assay meets gold standard for active

state of TB but not for LTBI (Walzl et al.,2011). IGRA yields discordant results

depending upon age, BCG vaccination and migration (Machado et al., 2009). Xpert

MTB/RIF is a highly sensitive, rapid nucleic acid amplification test that identifies

targeted nucleic acid sequence in TB genome by real time PCR and can detect

rifampicin resistant TB. But Xpert MTB/RIF is expensive and cannot detect LTBI.

Over the last few years, there has been a great deal of effort in various laboratories to

develop SERS techniques for use in biomedical diagnostics, pathogen detection, gene

identification, gene mapping, and DNA sequencing. There have been attempts by

various groups to develop techniques to identify and detect the reactions of individual

molecules in living cells with improved spatial and temporal resolution. Raman imaging

is emerging as a rapid and nondestructive analytical tool that yields highly compound-

specific information for chemical analysis and has great potential for high-throughput

analysis and direct imaging. Recent reports have shown an increasing trend in applying

Raman spectroscopy in cellular and tissue imaging studies (Peltier et al., 2002; Van de

Poll et al., 2002; Timlin et al., 2000; Schaeberle et al., 1999). The development of SERS

techniques for biological analysis and medical diagnostics has been investigated some of

the laboratories with varying amounts of success (Dinh et al., 1988; 1998; 2002; Stokes

et al., 2000; Allain et al., 2002; Culha et al., 2003; ). In addition, SERS studies on living

cells using gold nanoparticles (Kneipp et al., 2002) and coherent anti-Stokes Raman

scattering (CARS) for cellular imaging have been reported (Zumbusch et al., 1999). In

the national scenario, SERS has been used to demonstrate the potency of the substrates

developed either as thin films or as nanoparticles. A vast literature exists on the

synthesis of nanoparticles for SERS applications, but seldom any of these techniques

have been used and demonstrated for applications of bio-diagnostics or Raman imaging.

We strongly believe that we are one of the only a few groups in India working on bio-

diagnostic applications of SERS in biological systems.

16.6 The relevance and expected outcome of the proposed study

Point of care diagnostics is not available for TB diagnosis. With nearly two-

thirds of the Indian population being affected, there is a strong need for rapid diagnosis.

The outcome of the proposed study is a field-usable Raman instrument which combines

ELISA and Surface enhanced Raman Spectroscopy, thus enhancing the sensitivity,

specificity and rapidity of diagnosis. Multiple antigens can be captured simultaneously

on paramagnetic beads with capture antibodies, and they can be differentially recognized

using non-overlapping, spectrally distinct Raman reporter, which enhance the signal of

ELISA by a 1000-fold. To our knowledge, we would be the first people in India to use

this technology for TB detection and this has a possibility of developing new IPR and

rapid diagnosis is the need of the hour.

16.7 Preliminary work done so far

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1. Identification of proteins present in PPD from Mycobacterium tuberculosis as a

collaborative project by Microtest Innovations Pvt Ltd

We carried out a proteomic profiling of Connaught Tuberculin (CT68), prepared from a

human strain of M. tuberculosis cultured using a protein-free medium, using high

resolution Fourier transform mass spectrometry (LTQ-Orbitrap Velos). Mass

spectrometry-derived data was searched using - Sequest algorithm embedded in the

Proteome Discoverer 1.3.0.339 software (Thermo Scientific, Bremen, Germany) against

a protein database of M. tuberculosis from NCBI RefSeq. Search of 5,205 MS/MS

spectra were resulted in 1,146 peptide-spectrum matches, which corresponded to 695

unique peptides. Representative MS/MS spectra are provided in Figure 1. Based on

these 695 unique peptides, we identified 265 proteins of M. tuberculosis in Connaught

Tuberculin (CT68).

Figure 1: Representative MS/MS spectra for proteomic profiling of Connaught

Tuberculin (CT68), prepared from a human strain of M. tuberculosis.(Manuscript

under preparation)

2. Development of monoclonals and p24 capture assay at Microtest innovations pvt

ltd.

Microtest Innovation is exploring diagnostic possibilities & its implication in

understanding more about the disease process in our country. Main area of expertise is

immuno diagnostics, genetic diagnostics, recombinant protein expression, purification,

refolding & molecular biology. The expertise MTI has in immuno biology & molecular

biology are being capitalized in development of newer assays. MTI has excellent

expertise in the entire spectrum of antibody generation & characterization starting from

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high precission monoclonal, polyclonal &

recombinant antibodies. MTI with collaborating

team has excellent expertise in the area of

virology, one of them being HIV which also

includes mapping B cell epitope of some of the

important antigens of HIV to develop

appropriate Immuno diagnostics in this area.

While infectious disease being the priority, MTI

team is critically looking in other areas of

medicine like cardiovascular disease,

endocrinology & cancer. MTI is developing

different research assays with acute sensitivity

and specificity. Contract research is another area

of operation. Microtest is also building a good

Bioinformatic and structural biology team to

cater to all our needs in the development

process.

Immuno Diagnostics- Production, purification and characterization of Monoclonal

Antibodies:

Monoclonal antibodies have been generated to p24 (from HIV) and have been purified

and characterized. The epitope recognizing these antibodies have been determined. This

includes

Generation and characterization of functional monoclonals

Gene cloning and expression

Scaling up and purification of monoclonals

Characterization in terms of mapping of epitopes

Identifying different classes of antibodies

Detecting affinity constant of monoclonals

Generating labeled antibodies

Identifying functional antibodies

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Development of Functional Monoclonal Antibody for Taq Polymerase:

Anti Taq Polymerase antibody was developed which binds to an antigen and has an

effect that can be demonstrated in laboratory tests.

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3. Surface-enhanced Raman spectroscopy (SERS)

We have now developed magnetic core gold or silver shell nanoparticle which support

the adsorption or chemically linking the antibodies on them. The reagents can then be

immobilised easily within an ependoff by placing the tube in magnetic field. The key to

this technique is that the nanoparticles are paramagnetic as well as they are not bigger

than a few hundred nanometers. Since we can decant the supernatant after immobilizing

the reagents, we remove the washing cycle all together as well as we do the whole

reaction in solution. This reduces not only the time but also increases the sensitivity of

the detection.

In order to validate their novel approach to SERS immunoassay, we have tested the

method on the well-known HIV kit, where we used the Raman tag as the OPD, which

upon oxidation produces an azo compound, which has a very strong Raman signature.

Using this we have been able to increase the sensitivity of the ELISA by 2 orders of

magnitude as shown in the Figure 1.

0.0

0.5

1.0

1.5

2.0

10 1 0.1 0.01 0.001 0.0001 No Ag

Antigen concentration (ng/ml)

Ab

s (

49

5 n

m)

0.0

0.5

1.0

1.5

2.0

10 1 0.1 0.01 0.001 0.0001 No Ag


Ab

s (

49

5 n

m)

500 1000 1500

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

0.0001 ng/mL0.001 ng/mL

0.01 ng/mL

0.1 ng/mL

1 ng/mL

10 ng/mL

control

Inte

nsi

ty (

a.u

.)

Raman shift (cm-1)

• NIH kit sensitivity is 1 ng/ml in ELISA (Rs. 4,000)

• Assay sensitivity is enhanced by 100-fold by SERS ) 0.01 ng/ml

0.0

1.0

2.0

3.0

4.0

10 1 0.1 0.01 0.001 0.0001 No Ag


SERS

Absorbance

ELISA only

ELISA-SERS

Comparison of ELISA vs ELISA-SERS using

low-sensitivity HIV-1 p24 kit

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We were able to determine the presence of this marker in solution at a concentration as

low as 1-10 picograms per millilitre. This is about 100 to 1000 times more sensitive than

ELISA and the assay time took less than 1 h, including the optical detection steps.

Their proof of principle not only bodes well for developing the approach for other

biomarkers but could also have clinical application for assaying for TB.

17. Specific objectives (should be written in bulleted form, a short paragraph indicating

the methods to be followed for achieving the objective and verifiable indicators of

progress should follow each specific objective)

Objective 1

Cloning, Expression and Purification of proposed antigens

Method(s) to be followed:

The genes related to these antigens will be PCR amplified, cloned and expressed using

IPTG induction protocol. The recombinant proteins will be purified by a combination of

standard methods of column chromatography and salt precipitation etc.

Variable indicators of progress:

1. Recombinant expressed TB antigens and expression vectors.

2. Highly purified antigens

Objective 2

Development of highly specific Monoclonal antibodies for the proposed antigens.


1. Bioinformatics analysis for epitope sequences for generation of highly specific

monoclonal antibodies.

2. Custom synthesis of peptides.

3. Immunization in Balb/C mice for antibody production.

4. Generation and Screening of Hybridomas for the proposed antigens.

5. Purification and validation of monoclonal antibodies.


Purified and characterized monoclonal antibodies that recognize the recombinant and

native proteins from Mycobacterium tuberculosis

Objective 3

Development of Antigen Capture ELISA


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Monoclonal antibodies to the proposed antigens would be generated, following which

the higher affinity antibodies would be used as Capture and/or detector antibodies.

Using these antigen capture assay/ Sandwich ELISA would be developed.


1. Two different monoclonal antibodies for each antigen targeting different epitopes.

2. Optimization of Sandwich ELISA.

Objective 4:

Increasing the sensitivity of the assay using SERS


Paramagnetic beads with Fe3O4 core, coated with SiO2, which would be functionalized

and linked with the monoclonal Capture antibodies. The detector antibodies would be

conjugated with different Raman reporter moieties. Addition of gold nanoparticles will

enhance the signal from Raman reporters which can be detected and analyzed using the

equipment.


1. Preparation of paramagnetic beads.

2. Conjugation of antibodies to beads.

3. Conjugation of Raman reporter moieties to antibodies.

4. Optimization of detection of complex.

Objective 5

Clinical Validation


Culture positive smear negative and smear positive samples would be categorized and

the patients various biological fluids like sputum, serum, urine etc would be tested using

the optimized SERS-ELISA and the results compared and validated using established

methodologies.


1. Analysis of sensitivity and sensitivity of our assay with existing methodologies.

2. A field usable prototype Raman equipment.

17

18. WORK PLAN

Specific aim 1: Cloning, expression and purification of TB antigens for R& D use:

a) Selection of Antigens: Antigens of potential diagnostic value will be selected

based on the available information as published literatures as well as genomic

information. The already identified secreted antigens which are of diagnostic

importance is listed as a table below

BIOLOGICAL FLUIDS ANTIGENS

CSF

* Ag85A (3804c), Ag85B (1686c)

* 65kD hsp

* 43kDa antigen

Sputum * Ag85complex

* LAM

Serum * 38kDa*, 16kDa

* Mtb81 (Rv1837c)

Urine * MT_1721, MT-1694, LAM

Pleural fluid * 43kDa

Ascitic Fluid * Ag85B (1686c), Ag85C

* 43kDa

The listed antigens were identified in the biological fluid of infected individuals. We

would clone and express about ten of the above mentioned antigens and explore their

potential.

b) Cloning and expression of the selected antigens:

Polymerase Chain Reaction (PCR):

PCR amplification of the genes encoding the proteins using gene specific 5’ and 3’

primers. PCR will be carried out by standard methods of amplification.

Cloning in expression vectors for recombinant expression:

The PCR fragments will be gel purified using the PerfectprepR gene clean up kit from

Eppendorf per the manufacturer’s instructions and taken for digestion. The PCR

fragments will be digested with appropriate enzymes selected for cloning into pET21B

for E.coli expression. The transformation and selection of clones from all the expression

systems can be done by using the standard molecular biology protocols.

Sequencing of the clones:

All the genes cloned in pET21B for E.coli expression will be completely sequenced by

dideoxy chain termination method (outsourced to MWG Biotech) before proceeding

with expression and purification.

Protein expression and purification:

The confirmed clones will be checked for expression by IPTG induction protocol which

can be further scaled up after expression optimization at different temperature, time

scale and IPTG concentrations. The purification of the recombinant protein from the

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expression systems would be by a combination of standard methods of column

chromatography and salt precipitation etc. and would have to be optimized individually

for each of the proteins. The genes are cloned into pET21b (+) backbone and the genes

would have a C-terminal 6X-Hisdine Tag. The proteins expressed can purified by the

standard Ni-NTA technology.

Specific Aim 2) Production and purification of Monoclonal Antibodies for the

proposed antigens:

a) Immunization

A group of young BALB/c mice, six to eight weeks old will be primed subcutaneously

with 50 µg of protein/mouse emulsified in CFA (Complete Freund’s adjuvant). This will

be followed by a few boosters of the antigen in IFA (Incomplete Freund’s Adjuvant).

Antigen specific antibody titers will be evaluated in ELISA. Mice generating highest

immune response will be used for fusion and hybridoma generation. Three days prior to

fusion, selected mice will be given a final booster with soluble antigen without adjuvant

through the tail veil.

b) Fusion and hybridoma selection

One week before fusion the myeloma cell line P3X63/SP20 will be expanded in

complete DMEM medium to obtain 10 x 106 cells on the day of fusion. One day before

fusion, cells will be split into fresh medium. Spleens from the selected mice will be

harvested aseptically, a homogenous single cell suspension of splenocytes will be

prepared and the cells washed with complete DMEM medium without serum. Cell

number will be determined and both the fusion partners will be mixed at equal

proportion. Cell fusion will be performed at 37ºC using 50% PEG according to standard

protocols. Following fusion, cells will be suspended in complete DMEM medium with

20% FCS at a cell density of 2.5 x 106 cells/ml. 100 l of cell suspension will be

dispensed per wells in a number of 96-well plates and the plates will be incubated for 24

hours. On day-2, 100 µl of complete medium supplemented with HAT will be added to

each well. During the subsequent week, every day, half the medium from each well will

be replaced with fresh medium. HAT selection will be imposed for 14 days and on day

15; cells will be fed with HT medium. The subsequent day cells will be fed with

complete medium without HT. During the following days, all the wells with cell

proliferation will be marked and the spent medium will be rapidly screened for antibody

in ELISA and positive wells will be expanded. Screening will be performed using

different recombinant proteins or peptides. Several clones of hybridoma will be

systematically analyzed and stored (frozen) in aliquots.

c) Screening of the hybridoma:

Selected hybridoma will be grown in culture medium at high cell density to generate

adequate concentration of monoclonal antibodies. Antibodies will be purified by

ammonium sulfate precipitation, dialyzed and protein concentration determined.

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The culture supernatant antibodies will be ammonium sulphate precipitated and purified

on protein A affinity column. This antibody will be further characterized for its class and

sub-class and will be utilized for developing quantitative assay for western blot analysis

and for neutralization and for diagnostic development depending upon the need. We will

use these assays to quantify infected samples. The protein concentration of the purified

antibodies will be evaluated and monoclonal antibodies will be stored in smaller aliquots

at -20ºC.

d) Fine mapping of the monoclonal antibodies:

Epitopes of the antigen identified by the monoclonal antibodies will be characterized

using the synthetic peptides. A panel of overlapping peptide will be designed,

synthesized and will be used to fine map the monoclonals. Antibodies recognizing

different epitopes will be used as antibody pairs in the capture assay.

e) Develop quantitative assays using these reagents:

The antibody with extremely good affinity and recognizing different regions of the

antigens will be used for capture and detection antibodies. The detection antibodies will

be conjugated with appropriate enzymes for the ELISA and they would be conjugated to

different RAMAN reporter moieties, for use in SERS. Standard would be made using

recombinant proteins and these assays will be validated for antigen detection in

biological fluids.

Specific Aim 3: SERS enhanced highly sensitive ELISA based detection assay for

proposed antigens.

a) Enhanced detection of OPD derivative:

Conversion of o-phenylenediamine to azoaniline by peroxidase is a common

reaction of routine ELISA. Azoaniline is a highly reactive Raman reporter. We

tested if the formation of azoaniline could help enhance the sensitivity of the

HIV-1 p24 ELISA if

SERS of this compound,

instead of the color, is

determined. Using two

different commercial p24

kits of different

sensitivity, we

demonstrated that by the

application of silver

nanoparticles and SERS

detection, the detection

sensitivity of both of the kits could be enhanced by two orders of magnitude as

compared to colorimetry (Figure-1). This result has direct implication on the

novel Raman reporter dyes we would like to develop through the present project.

b) Preparation of paramagnetic beads:

20

We have prepared several Fe2O3 based

and silica and PEG-capped

paramagnetic beads of uniform size

(Figure-2:). These beads will be

instrumental in extracting TB antigens from

the biological fluids. One such preparation

is shown. These paramagnetic beads can be

functionalized on the outer silica, which

will allow conjugation of the monoclonal

capture antibody.

c) Optimization of SERS-ELISA:

The paramagnetic beads coupled to capture antibody would react with the

biological fluid containing the TB antigens and the antigens would be get bound

to the beads. The beads, along with the sample are incubated for a small period

of time and the tubes are then exposed to magnetic field. Unbound antigens and

samples are eliminated by just decanting the sample tube. The bound beads are

then disbursed in the releavant solution and the second antibody, specific to a

different epitope of the antigen are added. The detector antibody used in this

reaction is already conjugated to a Raman reporter moiety. After the incubation

period, the sample tube is exposed to magnetic field and the unbound samples

are eliminated. Gold nanoparticles are then added to the reaction and the signal

that comes from the Raman reporter are then recorded in the instrument. Signal

is obtained only when all the three components are present together, namely, the

Capture antibody, the antigen and the detector antibody. The basic principle is

represented in a schematic below:

21

The strengths of this approach are:

1) There are no special washing steps required.

2) The time duration for the assay is about an hour only.

3) Combination of beads conjugated to different monoclonals can be used to

capture multiple antigens, which gives the strength of multiplexing.

4) Different Raman reporters can be conjugated to different detector antibodies,

thereby, enhancing the multiplexing and help in distinguishing the antigen

present.

5) The assay has a very high signal to noise ratio.

6) The assay is a 1000-fold more sensitive than the conventional ELISA.

Specific Aim 4: Validation of SERS-ELISA with patients samples for detection of

proposed TB antigens in biological fluids.

Firstly, ELISA would be optimized for the different monoclonal pairs and their

corresponding purified recombinant antigens. The ideal capture and detection antibody

pair would be identified using a matrix analysis and affinity based titrations. The affinity

of the antibody antigen reactions would be evaluated using competitive ELISA, in the

presence of chaotropic agents. Once the antigen-capture assay is optimized, the

sensitivity of the ELISA will be enhanced using SERS technology. The optimized

SERS-ELISA protocol would then be used to detect TB antigens in biological fluids of

TB patients. The performance of the assay will be compared with the commercially

available kits. The prototype Raman instrument would be tested in the clinical set-up

and the detection parameters and the procedures would be optimized.

Alternate strategies (if the proposed experimental design or method does not work

what is the alternate strategy):

We have previously optimized the capture and detection of HIV-1 single

stranded DNA detection using this SERS technology. ( A high sensitivity assay for

molecular typing of biological sample, probes and a kit therof - Inventors: Ranga

Udaykumar, Chandrabhas Narayana and N. Jayasuryan - patent granted by patent

office of Republic of South Africa - Patent Number 2009/03128 in 2009.)

We would employ the similar strategy for detection of M. tuberculosis genetic material

in the sample using the same technology.

22

The methodology followed is outlined below:

a) Capture and detection of HIV single-stranded DNA to solid substrate: The

objective of this experiment is to ensure the specificity of the capture and detector

probes for the viral nucleic acid. Single-stranded DNA of the viruses representing

the viral LTR was generated in the laboratory using asymmetric PCR. Three

capture probes (CP-1, specific probe, should capture viral DNA; CP-2 and CP-3

mutant probes, should not capture the viral DNA) were chemically cross-linked to

glass surface. Different amount of viral DNA were captured and d etected with a

detector probe conjugated to Rhodamine-6-G. Subtype-specific Raman signals

were detected only with CP-1 but not with other two probes confirming the assay

specificity. Sensitivity of the assay is approximately 104 molecules with a 1

second acquisition time (Figure-3). Sensitivity of the assay could be enhanced by

scanning the entire spot, using high power lasers, increasing acquisition time and

other molecular manipulations to improve the detection probe.

b) Capture and detection of HIV

RNA to solid substrate: Full-length

viral RNA, isolated from 500 μl of

culture supernatants, was directly

captured and detected in this

experiment using a strategy similar to

‘a’ above. Viral RNA was specif

ically detected by CP-1 but not CP-2

23

at two different template concentrations (Figure-4). This result ascertained that

our novel strategy can detect the viral RNA without amplification.

Similar approach can be used for the

detection of M. Tb DNA from the

biological samples. Considering the

genome of M. Tb is so vast in

comparison with HIV-1 genome, it will

be easier to design multiple specific

windows in the genome of M. Tb, which

are highly suitable for the capture and

hence the detection.

Connectivity of the participating institutions and investigators (in case of

collaborating / multi- institutional projects only)

Microtest Innovation

Pvt Ltd.

• Cloning, expression and purification of Antigens

• Generation of monoclonal antibodies and component Development

• Setting up quantitative ELISA

JNCASR

• Subject matter expert on SERS. Enhancing the sensitivity of ELISA kit by SERS

(Clinical Group at

LRS)

• Providing Clinical Samples for Assay Development

• Assay Validation

JNCASR will

Supervise and

coordinate the project

24

References:

1. Allain,L.R., T. Vo-Dinh. (2002) Anal. Chim. Acta 469, 149 410.

2. Chegou,N.N. Gillian F Black, Martin Kidd, Paul D van Helden and Gerhard

Walzl.(2009), BMC Pulmonary Medicine 9:21.

3. Culha, M., D. L. Stokes, L. R. Allain, T. Vo-Dinh. (2003) Anal. Chem. 75, 6196

410.

4. Danielle R. Napolitano, Nira Pollock, Suely S. Kashino, Virmondes Rodrigues,

Jr.,3 and Antonio Campos-Neto. (2008) Clinical and Vaccine Immunology, Apr.,

p. 638–643.

5. Diel, R., et al., Chest, (2009). 135(4): p. 1010-8.

6. Dinh, T.V., L. R. Allain, D. L. Stokes. (2002) J. Raman Spectrosc. 33, 511 410.

7. Dinh, T.V. (1998) Trends Anal. Chem. 17, 557 410.

8. Dinh,T.V., A. Alak, R. L. Moody. (1988) Spectrochim. Acta B 415, 605.

9. Hendrickson, R.C., John F. Douglass, Lisa D. Reynolds, Patricia D. Mcneill,

Darrick Carter, Steven G. Reed, AND Raymond L. Houghton. (2000) Journal of

Clinical Microbiology, June, p. 2354–2361.

10. Kashino.SS et al (2008) Clin Exp Immunol. Jul;153(1):56-62.

11. Kneipp,K., A. S. Haka, H. Kneipp, K. Badizadegan, N. Yoshizawa, C. Boone,

K. E. Shafer- Peltier, J. T. Motz, R. R. Dasari, M. S. Feld. (2002) Appl.

Spectrosc. 56, 150 410.

12. Machado, A., Jr., et al.,. (2009) Int J Tuberc Lung Dis,. 13(4): p. 446-53.

13. Meier T, Eulenbruch HP, Wrighton-Smith P, Enders G, Regnath T (2005). Eur.

J. Clin. Microbiol. Infect. Dis. 24 (8): 529–36.

14. Pereira arias-bouda, Lan N. Nguyen, Ly M. Ho, Sjoukje Kuijper, Henk M.

Jansen and Arend H. J. Kolk. (2000) Journal of clinical microbiology, June, p.

2278–228.

15. Schaeberle, M.D., H. R. Morris, J. F. Turner, P. J. Treado. (1999) Anal. Chem.

71, 175A 410.

16. Shafer-Peltier., K.E., A. S. Haka, M. Fitzmaurice, J. Crowe, J. Myles, R. R.

Dasari, M. S. Feld: (2002) J. Raman Spectrosc. 33, 552 410.

17. Stokes,D.L T. Vo-Dinh: (2000) Sens. Actuat. B Chem. 69, 28 410.

18. Timlin,J.A., A. Garden, M. D. Morris, R. M. Rajachar, D. H. Kohn. (2000)

Anal. Chem. 72, 2229 410.

25

19. Van de Poll, S.W.E., T. C. Bakker Schut, A. Van der Laarse, G. J. Pupples.

(2002) J. Raman Spectrosc. 33, 544 410.

20. Walzl, G., et al., 2011. Nat Rev Immunol. 11(5): p. 343-54.

21. Wiker, H.G and Harboe M. (1992). Microbiological reviews, Dec. 1992, p. 648-

661.

22. Young,D., L. Kent, A. Rees, J. Lamb, AND J. Ivanyi. (1986) Infection and

Immunity, oct., p. 177-183.

23. Zumbusch,A., G. R. Holtom, X. S. Xie. (1999) Phys. Rev. Lett. 82, 4142 410.

26

19. Timelines: (Please provide quantifiable outputs)

Period of study Achievable targets

6 Months

1. Procurement of chemicals, consumables, equipments.

2. Identification of epitopes and Peptide synthesis and purification.

3. Cloning and expression of Antigens.

4. Development of SERS molecules with high SERS cross section

12 Months

1.Cloning and Expression of Antigens

2. Purification of antigens.

3. Immunization and evaluating the antibody titers.

4. Development of magnetic and noble metal nanoparticles for various

laser excitations, optimization, characterization.

18 Months

1. Hybridoma Screening for Monoclonals.

2. Purification of Monoclonal antibodies.

3. Development of both solution-based and substrate based assays for

SERS detection.

4. Development of SERS technique to detect the antigen-antibody

complex (biosystems) in very low concentrations.

24 Months

1. Development of ELISA using combinatorial approach including all

the antigens.

2. Validation with patient samples in the clinical set-up.

30 Months

1. Development of Raman system for optimum detection with

combination of lasers and microscope objectives.

2. Applying SERS to enhance the sensitivity to standardized ELISA

protocol.

3. Standardization of Protocol and Validation of ELISA kit as per ICH

guidelines.

36 Months

1. Screening clinical samples and Validation.

2. Development of Raman system for optimum detection with

combination of lasers and microscope objectives.

3. Development of prototype of the assay with the Raman system

developed for diagnostics.

20. Name and address of 5 experts in the field Sr.No. Name Designation Address

27

PART IV: BUDGET PARTICULARS

Jawaharlal Nehru Center For Advanced Scientific Research

Budget (In Rupees)

A. Non-Recurring (e.g. equipments, accessories, etc.)

S.

No.

Item Year 1 Year 2 Year 3 Total

1.

Laser source 488 nm Rs. 4

lakhs

-NA- -NA-

2. Laser source 785 nm Rs. 8

lakhs

-NA- -NA-

3. Ultra long working distance

Microscope Objectives

(20x and 2 No. and 50x and 2

No.)

Rs. 8

lakhs

-NA- -NA-

4. Raman Edge Filter sets Rs. 5

lakhs

-NA -NA-

5. Microscope stage Cryostat

(From Room temperature to 4 K

and 1 No.)

Rs. 25

lakhs

-NA- -NA-

Sub-Total (A): 50 Lakhs

B. Recurring

B.1 Manpower (See guidelines at Annexure-III)

S.

No.

Position

No.

Consolidated

Emolument

Year 1 Year 2 Year 3 Total

1. JRF Rs. 16000 + Rs

250 Medical (for

Year 1 and

2)+30%HRA

7,48,800 7,48,800 8,42,400 23,40,000

Sub-Total (B.1) =23.4 Lakhs

B.2 Consumables

S.

No.

Item

Quantity Year 1 Year 2 Year 3 Total

10.00 10.00 10.00 30.00

Sub-Total (B.2) =30.00 Lakhs

Other items Consolidated

Emolument


B.3 Travel

1.00 1.00 1.00 3.00

28

B.4 Contingency

1.00 1.00 1.00 3.00

B.5 Overhead

(If applicable)

3.96 3.96 3.96 11.88

Sub-total of B

(B.1+B.2+B.3+B.4+B.5)

71.28

Lakhs

Grand Total (A + B) 121.28

Justification: High end equipments are required for successful implementation of

the project. High grade analytical reagents are required for the synthesis of

paramagnetic particles and Gold Nanoparticles. A minimum of 1 JRF is required

to carry out the work.

29

PART V: EXISTING FACILITIES

(Jawaharlal Nehru Centre for Advanced Scientific Research)

Resources and additional information

1. Laboratory:

a. Manpower

Five Ph.D students, One Research Associate and 2 project assistants

b. Equipments

We have a well-equipped central instrumentation facility at the

university.

Custom built Raman spectrometer

Atomic Force Microscope

Tramission Electron Microscope

Field emission Scanning Electron Microscope

Confocal Microscope

Mass spectrometer

Flow cytometer

ELISA reader

Laminar Hoods

C02 incubators

2. Other resources such as clinical material, animal house facility, glass house.

Experimental garden, pilot plant facility etc.

We have a well equipped animal house in the campus.

30


Microtest Innovations, Pvt Ltd.

Budget (In Rupees)


S.

No.


-NA-

Sub-Total (A)

B. Recurring


S.

No.

Position

No.

Consolidated

Emolument


1

JRF

Rs. 16000 + Rs

250 Medical (for

Year 1 and

2)+30%HRA

7,48,800 7,48,800 8,42,400 23,40,000

Sub-Total (B.1) =23, 40,000/-

1. Gene synthesis, Cloning and Expression 2. Scaling up, purification and

characterization of recombinant proteins

3. Peptide synthesis and purification

4. An adequate number of overlapping peptides for multiepitope analysis

4. Immunoassays for peptides and proteins

All these would require a minimum of 1 JRF and hence this manpower is justified.

B.2 Consumables

S.

No.

Item


1.

Chemicals

15.00 10.00 10.00 35.00

Sub-Total (B.2) =35.00

Justification: 1.Molecular biology reagents

2. Immunology reagents

3. Recombinant technology

The budget for consumables proposed in project is minimum. High quality reagents are

extremely critical for this purpose.


Emolument


B.3 Travel

1.00 1.00 1.00 3.00

B.4 Contingency

1.00 1.00 1.00 3.00

31

B.5 Overhead

(If applicable)

-NA

Sub-total of B

(B.1+B.2+B.3+B.4+B.5)

64.4

Lakhs


Lakhs

Note : Please give justification for each head and sub-head separately mentioned in the

above table.

Financial Year : April - March

In case of multi-institutional project, the budget estimate to be given separately for each institution.


1. Laboratory:

Microtest innovation is a well-equipped laboratory located in the International

Technology Park, the technology hub of government of Karnataka. The company is

undertaking research and development programs in several frontier areas of national

significance, requiring specialization in biotechnology such as immunology, hybridoma

technology, recombinant protein production, vaccine development and protein folding.

2. Manpower

Three PhD, One MD, Four M.Sc Graduates in Biotechnology

Equipments

Sl No Equipment

1 MilliQ water

2 Autoclaves

3 Microfuges

4 PCR machine

5 -20ºC deep freezer

6 Refrigerators

7 Incubator

8 Electrophoresis unit Agarose

9 Semi-preparative and Analytical HPLC

10 SDS PAGE unit

11 Ultra filtration systems

12 Incubator shaker

13 Sonicator

14 ELISA plate reader

15 Laminar Air flow Hood

16 CO2 Incubators

17 Amicon protein concentrator

18 Table top centrifuge

19 Weighing balance

20 Magnetic Stirrer

21 pH meter

22 CO2 incubator

23 Heating Block

24 Hot air oven

25 Western blotting apparatus

32


(LRS Institute of Tuberculosis and Respiratory Diseases)

Budget (In Rupees)


S.

No.


-NA-

Sub-Total (A)

B. Recurring


S.

No.

Position

No.

Consolidated

Emolument


-NA-

Sub-Total (B.1) =

B.2 Consumables

S.

No.

Item


1.

Chemicals

5.00 5.00 5.00 15.00

Sub-Total (B.2) =15.00


Emolument


B.3 Travel

1.00 1.00 1.00 3.00

B.4 Contingency

1.00 1.00 1.00 3.00

B.5 Overhead

(If applicable)

-NA-

Sub-total of B

(B.1+B.2+B.3+B.4+B.5)

21.00

Lakhs


Lakhs

Justification:

Antigens and peptides needs to tested against the serum collected from the patients.

Assays need to be validated at each and every step of the project. Hence the budget is

justified.

33


LRS Institute of TB and Respiratory Diseases is an Autonomous Institute under the

Ministry of Health & Family Welfare, Govt. of India.

The department of Microbiology regularly carries out the following procedures:

i) Specimen Collection

ii) Sputum Smear Microscopy

iii) Culture (Mycobacteria)

iv) Identification Tests for Mycobacteria

v) Sensitivity testing for M. Tuberculosis

vi) BACTEC Tests

The Bacteriology and Mycology

i) Culture & Sensitivity testing of pyogenic and fungii from specimen from ICU

post operative, and other chest diseases patients.

Serology

This section carries out investigations for HIV, HBsAg and Widal test. IHA for

Aspergillosis.

The department is equipped with latest laboratory equipments like fluorescent

microscope, Binocular microscope and BACTEC 460 TB system, ELISA reader etc.

Shortly it will be acquiring gas liquid chromatography system for anaerobic bacteria and

Mycobacteria and PCR system for diagnosis of Mycobacterial diseases. A new lab.

extension was developed in which state of art bio safety laminar flow cabinets (class-I

cabinet) were installed for specifically carrying out tests for tuberculosis and related

mycobacterial diseases.

It has been identified for expansion for conducting research under RNTCP. The

department is identified as National Reference Laboratory (NRL) and will take-up the

responsibilities of External Quality Assurance and Drug Resistance Survillance

activities under RNTCP in designated states. It is recently certified by CDC, Atlanta,

USA, for its quality in Drug Sensitivity Testing for M. tuberculosis. It is also proposed

to establish the bio-safety Lab-III for Molecular biology based tests for Mycobacteria

and for Epidemiological research.

The department is conducting training programmes for WHO fellows, actively

involved in the training of paramedical personnel (Lab. Technician, Sr.TB Lab.

Supervisors, Treatment organisers, Sr.Treatment supervisors ) who visit from other

states of our country, in implementation of strategies under Revised National

Tuberculosis Control Programme (RNTCP).

34

PART VI: DECLARATION/CERTIFICATION

It is certified that

a) the research work proposed in the scheme/project does not in any way duplicate the

work already done or being carried out elsewhere on the subject.

b) the same project proposal has not been submitted to any other agency for financial

support.

c) the emoluments for the manpower proposed are those admissible to persons of

corresponding status employed in the institute/university or as per the Ministry of

Science & Technology guidelines (Annexure-III)

d) necessary provision for the scheme/project will be made in the

Institute/University/State budget in anticipation of the sanction of the

scheme/project.

e) if the project involves the utilisation of genetically engineered organisms, we agree

to submit an application through our Institutional Biosafety Committee. We also

declare that while conducting experiments, the Biosafety Guidelines of the

Department of Biotechnology would be followed in toto.

f) if the project involves field trials/experiments/exchange of specimens, etc. we will

ensure that ethical clearances would be taken from concerned ethical

Committees/Competent authorities and the same would be conveyed to the

Department of Biotechnology before implementing the project.

g) it is agreed that any research outcome or intellectual property right(s) on the

invention(s) arising out of the project shall be taken in accordance with the

instructions issued with the approval of the Ministry of Finance, Department of

Expenditure, as contained in Annexure-V.

h) we agree to accept the terms and conditions as enclosed in Annexure-IV. The same

is signed and enclosed.

35

i) the institute/university agrees that the equipment, other basic facilities and such other

administrative facilities as per terms and conditions of the grant will be extended to

investigator(s) throughout the duration of the project.

j) the Institute assumes to undertake the financial and other management

responsibilities of the project.

36

PART VII: PROFORMA FOR BIOGRAPHICAL SKETCH OF INVESTIGATORS

Appendix J

BIOGRAPHICAL SKETCH

To be submitted separately for the Project Coordinator and each Key Investigator

Name : Dr Chandrabhas Narayana

Designation :Professor

Name of the Department/Institute/University :

Chemistry and Physics of Materials Unit

Jawaharlal Nehru Centre for Advanced Scientific Research,

Jakkur P.O., Bangalore 560064. India.

Date of Birth : 08-09-1965 Sex (M/F) : M

Education (Graduation onwards)

Sl

No. Institution

Degree

Awarded Year Field of Study

1.

University of Mysore

B.Sc B.Ed

1982-1986

Physics,

Chemistry,

Mathematics,

Psycology,

Sociology,

Science

Methodology

2. University of Mysore

M.Sc and

B.Ed

1986-1988 Physics and

Science

Methodology

3. Indian Institute of

Science, Bangalore

Ph.D 1989-1995 High Pressure

Materials

Research

4 Cornell university,

Ithaca, NY, USA

Post Doc 1995-1998 Metalization of

Hydrogen

Professional Career (Starting with the most recent employment)

Sl

No. Institution Position From (Date) To (Date)

1.

Jawaharlal Nehru Centre

for Advanced Scientific

Research Bangalore

Professor

May 11 Till Date

37

2.



Research, Bangalore

Dean,

Fellowships

and Extension

Programmes

Oct09 Oct11

3.



Research, Bangalore

Associate

Professor

May05 May11

4.



Research, Bangalore

Assistant

Professor

Sept98 May05

Honors/ Awards

MRSI Medal in 2007: This award was given by Materials Research Society of India for

“outstanding contributions in Material Science and Engineering”.

Sir C.V. Raman Young Scientist Award in 2008: This award was given by the Karnataka

Government for outstanding contributions in the area of Material Science and Physical

Sciences.

CNR Rao Oration Award in 2009: This award was given by Jawaharlal Nehru Centre for

Advanced Scientific Research, Bangalore.

Publications (Numbers Only) 75

1. Books: 1 2. Research Papers, Reports : 3.General

articles :

4. Patents : 6 ( 3 Indian, 3 International) 5. Others (Please specify):

Publications:

1. Gayatri Kumari and Chandrabhas Narayana, New Nano Architecture for SERS Applications, Journal

of Physical Chemistry Letters, 3, 1130 - 1135 (2012). , Read abstract.

2. Soumik Siddhanta and Chandrabhas Narayana, Surface Enhanced Raman Spectroscopy of Proteins:

Implications in Drug Designing, Nanomaterials and Nanotechnology, 2, 1 - 13 (2012). , Read abstract.

3. Narendra Kurra, Venkat Srinu Bhadram, Chandrabhas Narayana and G.U. Kulkarni, Field effect

transistors based on graphitized patterns of carbon contamination, ACS Applied Materials and

Interfaces, 4, 1030 - 1036 (2012). , Read abstract.

4. Diptikanta Swain, Venkat Srinu Bhadram, Papia Chowdhury and Chandrabhas Narayana, Raman and

X-ray Investigations of Ferroelectric Phase Transitions in NH4HSO4, Journal of Physical Chemistry A,

116, 223 - 230 (2012).

5. Harikrishnan S. Nair, Diptikanta Swain, Hariharan N., Shilpa Adiga, Chandrabhas Narayana and Suja

Elzabeth, Griffiths phase-like behavior and spin-phonon coupling in double perovskite

38

Tb2NiMnO6,Journal of Applied Physics, 110, 123919 - 123925 (2011). , Read abstract.

6. Supti Das, Venkata Srinu Bhadram, Chandrabhas Narayana and Aninda J. Bhattacharyya, Brillouin

Scattering Investigation of Solvation Dynamics in Succinonitrile-Lithium Salt Plastic Crystalline

Electrolytes, Journal of Physical Chemistry B, 115, 12356 - 12361 (2011). , Read abstract.

7. P. Mandal, Venkat Srinu Bhadram, Y. Sundarayya, Chandrabhas Narayana, A. Sundaresan and C.N.R.

Rao, Spin-Reorientation, Ferroelectricity, and Magnetodielectric Effect in YFe(1-x)Mn(x)O(3)(0.1 <= x

<= 0.40), Physical Review Letters, 107, 137202 - 137205 (2011). , Read abstract.

8. G. V. PAVAN KUMAR, N. RANGARAJAN, B. SONIA, P. DEEPIKA, NASHIOUR ROHMAN and

CHANDRABHAS NARAYANA, Metal-coated magnetic nanoparticles for surface enhanced Raman

scattering studies.,Bulletin of Materials Science, 34, 207 - 216 (2011). , Read abstract.

9. Partha P. Kundu, G. V. Pavan Kumar, K. Mantelingu, Tapas K. Kundu and Chandrabhas

Narayana, Raman and surface enhanced Raman spectroscopic studies of specific, small molecule activator

of histone acetyltransferase p300, Journal of Molecular Structure, 999, 10 - 15 (2011). , Read abstract.

10. Gopal Krushna Pradhan, Chandrabhas Narayana, Mala Narasappaya Rao, Samrath Lal Chaplot,,

Olivier Pages and et al, The Phonon Percolation Scheme for Alloys: Extension to the Entire Lattice

Dynamics and Pressure Dependence, JAPANESE JOURNAL OF APPLIED PHYSICS, 50, 05FE02

(2011). , Read abstract.

11. Gopal K. Pradhan, Anil Kumar, S.K. Deb, Umesh V. Waghmare and Chandrabhas Narayana, Elastic

and structural instability of cubic Sn3N4 and C3N4 under pressure, Physical Review B, 82, 144112 -

144115 (2010). , Read abstract.

12. Awadesh K. Mallik, S. R. Binu, L. N. Satapathy, Chandrabhas Narayana, Md. Motin Seikh, S. A.

Shivashankar and S. K. Biswas, Effect of substrate roughness on growth of diamond by hot filament

CVD,Bulletin of Materials Science, 33, 251 - 255 (2010). , Read abstract.

13. S. Murugavel, C. Vaid, Venkat Srinu Bhadram and Chandrabhas Narayana, Ion Transport

Mechanism in Glasses: Non-Arrhenius Conductivity and Non-Universal Features, Journal of Physical

Chemistry B, 114, 13381 - 13385 (2010). pdf , Read abstract.

14. M. Viswanathan, P.S. Anil Kumar, Venkata Srinu Bhadram, Chandrabhas Narayana, A.K. Bera and

S.M. Yusuf, Influence of lattice distortion on the Curie temperature and spin-phonon coupling in

LaMn0.5Ca0.5O3, Journal of Physics: Condense Matter, 22, 346006 - 346013 (2010). pdf ,

Readabstract.

15. Diptikanta Swain, Venkata Srinu Bhadram, Venakataprasad Bhat, Gopal K. Pradhan, Chandrabhas

Narayana and C.N.R. Rao, Superionic phase transition in KHSO4: A temperature dependent Raman

investigation, Journal of Physical Chemistry A, 114, 10040 - 10044 (2010). pdf , Read abstract.

16. Sudip Mohapatra, Soumik Siddhanta, D. Ravindar Kumar, Chandrabhas Narayana and Tapas K.

Maji, A facile bottom up approach for fabricating multifunctional silver nanorods, European Journal of

http://desktop.jncasr.ac.in/uploaded/cbhas/pdfnew/jpcb2010v114p13381.pdf

http://desktop.jncasr.ac.in/uploaded/cbhas/pdfnew/jpcm2010v22p346006.pdf

http://desktop.jncasr.ac.in/uploaded/cbhas/pdfnew/jpca2010v114p10040.pdf

39

Inorganic Chemistry, 31 , 4969 - 4974 (2010). , Read abstract.

17. T Bhuvana, Anurag Kumar, Aditya Sood, Roger H. Gerzeski, Jianjun Hu, Venkata Srinu

Bhadram,Chandrabhas Narayana and Timothy S. Fisher, Contiguous Petal-like Carbon Nanosheet

Outgrowths from Graphite Fibers by Plasma CVD, ACS Applied Materials and Interfaces, 2, 644 - 648

(2010).

18. Gopal K. Pradhan, Chandrabhas Narayana, O. Pagès, A. Breidi, J. Souhabi, A. V. Postnikov, S. K.

Deb, F. Firszt, W. Paszkowicz, A. Shukla and F. El Haj Hassan, Pressure-induced phonon freezing in the

Zn1−xBexSe alloy: A study via the percolation model, Physical Review B, 81, 115207 - 115212 (2010).

19. Gopal K. Pradhan, Diptikant Swain, T.N. Guru Row and Chandrabhas Narayana, High-Temperature

Phase Transition Studies in a Novel Fast Ion Conductor, Na2Cd(SO4)2, Probed by Raman

Spectroscopy,Journal of Physical Chemistry A, 113, 1505 - 1507 (2009).

20. R.V.K. Mangalam, Chandrabhas Narayana and A. Sundaresan, Pressure-dependent phase transition

in the ordered BaBi0.7Nb0.3O3 perovskite, High Pressure Research, 29, 272 - 277 (2009).

21. Vishnu Shankar, Saroj L. Samal, Gopal K. Pradhan, Chandrabhas Narayana and Ashok K.

Ganguli, Nanocrystalline NaNbO3 and NaTaO3: Rietveld studies, Raman spectroscopy and dielectric

properties, Solid State Sciences, 11, 562 - 569 (2009).

22. Chandrabhas Narayana, R. G. Greene and Arthur L. Ruoff, Studies on silane to 70 GPa, Journal of

Physics: Conference Series, 121, 042019 - 042023 (2008). , Read abstract.

23. G. V. Pavan Kumar, R. Selvi, H. Kishore, T. K. Kundu and Chandrabhas Narayana, Surface

enhanced Raman spectroscopic studies of coactivator-associated arginine methyltransferase, Journal of

Physical Chemistry B, 112, 6703 - 6707 (2008).

24. R. V. K. Mangalam, Gopal K. Pradhan, Chandrabhas Narayana and A. Sundaresan, Spin state

transition in ferromagnet Sr0.9Ce0.1CoO2.85, Solid State Communications, 146, 110 - 114 (2008).

25. G. V. Pavan Kumar and Chandrabhas Narayana, Adapting a Fluorescence Microscope to Perform

Surface Enhanced Raman Spectroscopy, Current Science, 93, 778 - 781 (2007).

26. C. SubRamaniam, T. S. Sreeprasad, T. Pradeep, G. V. Pavan Kumar, Chandrabhas Narayana, T.

Yajima, Y. Sugawara, H. Tanaka, T. Ogawa and J. Chakrabarti, Metal-semiconductor transition induced

visible fluorescence from single walled carbon nanotube-noble metal nanoparticle composites, Physical

Review Letters, 99, 167404 - 167407 (2007).

27. K. Mantelingu, B. A. Ashok Reddy, V. Swaminathan, A. Hari Kishore, N. B. Siddappa, G.

Nagashankar, G. V. Pavan Kumar, N. Natesh, S. Roy, P. P. Sadhale, Udaykumar Ranga, Chandrabhas

Narayana and Tapas K. Kundu, Nonspecific to specific HATs off: Alters global gene expression and

repress HIV multiplication ,Chemistry and Biology, 14, 645 - 657 (2007).

28. K. Mantelingu, A. Hari Kishore, K. BalasubRamanyam, G.V. Pavan Kumar, M. Altaf, S. Nanjunda

Swamy, Ruthrotha Selvi, Chandrima Das, Chandrabhas Narayana, K.S. Rangappa and Tapas K.

40

Kundu, Activation of p300 histone acetyltransferase by small molecules altering enzyme structure: probed

by surfaced enhanced Raman spectroscopy, Journal of Physical Chemistry B, 111, 4527 - 4534 (2007).

29. G. Kavitha and Chandrabhas Narayana, Pressure-induced structural transitions in n-Pentane: A

Raman study, Journal of Physical Chemistry B, 111, 7003 - 7008 (2007).

30. T. Bhuvana, G. V. Pavan Kumar, G. U. Kulkarni and Chandrabhas Narayana, Carbon assisted

electroless gold for surface enhanced Raman scattering studies, Journal of Physical Chemisry C, 111,

6700 - 6705 (2007).

31. T. Bhuvana, G. V. Pavan Kumar, Chandrabhas Narayana and G. U. Kulkarni, Nanogranular Au films

deposited on carbon covered Si substrates for enhanced optical reflectivity and Raman

scattering,Nanotechnology, 18, 145702 - 145708 (2007).

32. G. V. Pavan Kumar, S. Sruthi, B. Vibha, B. A. Ashok Reddy, T. K. Kundu and Chandrabhas

Narayana, Hot spots in Ag core-Au shell nanoparticles potent for surface enhanced Raman scattering

studies of biomolecules, Journal of Physical Chemistry C, 111, 4388 - 4392 (2007).

33. Neena Susan John, G. U. Kulkarni, Ayan Datta, S. K. Pati, F. Komori, G. Kavitha, Chandrabhas

Narayana and M. K. Sanyal, Magnetic Interactions in Nickel Alkanethiolates, Journal of Physical

Chemistry C (Letters), 111, 1868 - 1870 (2007).

34. Harshad B. Ghodke, Kasinath Vignesh, G. V. Pavan Kumar, Ramya Krishnan, Chandrabhas

Narayanaand Yamuna Krishnan, The I-tetraplex building block: Rational Design and Controlled

Fabrication of robust 1D DNA Scaffolds via non-Watson Crick self assembly, Angewandte Chemie

(International Edition), 46, 1 - 5 (2007).

35. G. Kavitha and Chandrabhas Narayana, Raman spectroscopic investigations of pressure-induced

phase transitions in n-hexane, Journal of Physical Chemistry B, 111, 14130 - 14135 (2007).

36. Mohammed Arif, G. V. Pavan Kumar, Chandrabhas Narayana and T. K. Kundu, Autoacetylation

Induced Specific Structural Changes in Histone Acetyltransferase Domain of p300: Probed by Surface

Enhanced Raman Spectroscopy, Journal of Physical Chemistry B (Letters), 111, 11877 -

11879 (2007).

37. G. V. Pavan Kumar, B. A. Ashok Reddy, Md. Arif, T. K. Kundu and Chandrabhas Narayana, Surface

Enhanced Raman Scattering Studies of Human Transcriptional Coactivator p300., Journal of Physical

Chemistry B, 110, 16787 - 16792 (2006).

38. Md. Motin Seikh, Chandrabhas Narayana, A. K. Sood, P. Murugavel, M.W. Kim, P. A. Metcalf, J. M.

Honig and C. N. R. Rao, A Brillouin study of the temperature-dependence of the acoustic modes across

the insulator-metal transitions in V2O3 and Cr-doped V2O3, Solid State Communications, 139, 466 -

471 (2006).

39. G. Kavitha and Chandrabhas Narayana, Raman Scattering Studies on n-Heptane under High

Pressure,Journal of Physical Chemistry B, 110, 8777 - 8781 (2006).

41

40. Md. Motin Seikh, Chandrabhas Narayana, A. K. Cheetham and C. N. R. Rao, A comparative study of

the electro- and hole-doped compositions of single crystalline Nd1-xCaxMnO3 (x = 0.6 and 0.4), Solid

State Science, 7, 1486 - 1491 (2005).

41. Tokeer Ahmad, G. Kavitha, Chandrabhas Narayana and Ashok K. Ganguli, Nanostructured barium

titanate prepared through a modified reverse micellar route: Structural distortion and dielectric, Journal of

Materials Research, 20, 1415 - 1421 (2005).

42. Md. Motin Seikh, Chandrabhas Narayana, P. A. Metcalf, J. M. Honig and A. K. Sood, Brillouin

scattering studies in Fe3O4 across the Verwey transition, Physical Review B, 71, 174106 - 174110

(2005).

43. Md. Motin Seikh, A. K. Sood and Chandrabhas Narayana, Electronic and vibrational Raman

spectroscopy of Nd0.5Sr0.5MnO3 through the phase transitions, PRamana, 64, 119 - 128 (2005).

44. L. Sudheendra, Md. Motin Seikh, A. R. Raju, Chandrabhas Narayana and C. N. R. Rao, Dielectric

properties of rare earth cobaltates, LnCoO3 (Ln = La, Pr, Nd), across the spin-spin

transition,Ferroelectrics, 306, 227 - 234 (2004).

45. S. Mandal, G. Kavitha, Chandrabhas Narayana and S. Natarajan, Solvothermal synthesis of an open-

framework zinc chlrophosphate [C8N4H26]Zn3Cl(HPO4)3(PO4)], with a layer structure, Journal of

Solid State Chemistry, 177, 2198 - 2204 (2004).

46. Md. Motin Seikh, L. Sudheendra, Chandrabhas Narayana and C. N. R. Rao, A Raman study of the

temperature-induced low-to-intermediate-spin state transition in LaCoO3, Journal of Molecular

Structure, 706, 121 - 126 (2004).

47. Md. Motin Seikh, Chandrabhas Narayana, L. Sudheendra, A. K. Sood and C. N. R. Rao, A brillouin

scattering study of La0.77Ca0.23MnO3; across the metal-insulator transition, Journal of Physics:

Condense Matter, 16, 4381 - 4390 (2004).

48. Pallavi Teredesai, D. V. S. Muthu, N. Chandrabhas, S. Meenakshi, V. Vijayakumar, P. Modak, R. S.

Rao, B. K. Godwal, S. K. Sikka and A. K. Sood, High pressure phase transitions in metallic LaB6: Raman

and x-ray diffraction studies, Solid State Communications, 129, 791 - 796 (2004).

49. G. Kavitha, S. R. C. Vivek, A. Govindaraj and Chandrabhas Narayana, A low-cost Raman

spectrometer design used to study Raman scattering from a single-walled carbon nanotube, Proceedings

Indian Academy of Sciences (Chemical Science), 115, 689 - 694 (2003).

50. Md. Motin Seikh, Chandrabhas Narayana, Sachin Parashar and A. K. Sood, Temperature-dependent

Brillouin scattering studies of surface acoustic modes in Nd0.5Sr0.5MnO3, Solid State

Communications, 127, 209 - 214 (2003).

51. Chandrabhas Narayana, Brillouin scattering studies on charge-ordered manganite, Journal of Indian

Institute of Science, 82, 093 - 103 (2002).

52. L. Sudheendra, Md. Motin Seikh, A. R. Raju and Chandrabhas Narayana, An Infrared spectroscopic

42

study of the low-spin to intermediate-spin state (1A1 - 3T1) transition in rare earth cobaltates, LnCoO3

(Ln = La, Pr and Nd), Chemical Physics Letters, 340, 275 - 281 (2001).

53. P. Murugavel, N. Chandrabhas, A. R. Raju, A. K. Sood and C. N. R. Rao, Magnetic excitations in

charge-ordered Nd0.5Ca0.5MnO3 : A Brillouin scattering study, Europhysics Letters, 52, 461 - 467

(2000).

54. P. Murugavel, Chandrabhas Narayana, A. Govindaraj, A. K. Sood and C. N. R. Rao, Brillouin

scattering from C70 and C60 Films: Comparative study of elastic Properties, Chemical Physics

Letters, 331, 149 - 153 (2000).

55. P. Murugavel, Chandrabhas Narayana, A. K. Sood and C. N. R. Rao, A Brillouin scattering study of

the quasi-one-dimensional blue bronze, K0.3MoO3, Journal of Physics: Condense Matter, 12, (2000).

56. Jon Orloff, Chandrabhas Narayana and A. L. Ruoff, Use of focused ion beams for making tiny

sample holes in gaskets for diamond anvil cells, Reviews of Scientific Instruments, 71, 216 - 219

(2000).

57. A. L. Ruoff, T. Li, A. C. Ho, M-F. Pai, H. Luo, R. G. Greene, Chandrabhas Narayana, J. C. Molstad,

S. S. Trail, F. J. DiSalvo and P.E. van Camp, Jr., Sevenfold coordinated MgSe: Experimental internal

atom position determination to 146 GPa, diffraction studies to 202 GPa, and theoretical studies to 500

Gpa, Physical Review Letters, 81, 2723 - 2726 (1998).

58. Chandrabhas Narayana, Huan Luo, Jon Orloff and A. L. Ruoff, Solid hydrogen at 342 GPa: no

evidence for an alkali metal, Nature, 393, 46 - 49 (1998).

59. Chandrabhas Narayana, V. J. Nesamony and A. L. Ruoff, Phase transformation of BeS and equation

of state studies to 96 Gpa, Physical Review B, 56, 14338 - 14343 (1997).

60. A. K. Sood, N. Chandrabhas, D. V. S. Muthu and A. JayaRaman, Phonon Interference in BaTiO3 :

High Pressure Raman Study, Physical Review B, 51, 8892 - 8895 (1995).

61. N. Chandrabhas and A. K. Sood, Raman Study of Pressure-induced Phase Transitions in

RbIO4,Physical Review B, 51, 8795 - 8800 (1995).

62. N. Chandrabhas, A. K. Sood, D.V.S Muthu, C. S. Sundar, A. Bharathi, Y. Hariharan and C. N. R.

Rao, Pressure-induced Amorphization in solid C70 : Raman and Photoluminescence Study, Physical

Review Letters, 73, 3411 - 3414 (1994).

63. N. Chandrabhas, A. K. Sood, D. SundaRaman, S. Raju, V. S. Raghunathan, G. V. N. Rao, V. S.

Sastry, T. S. Radhakrishnan, Y. Hariharan, A. Bharathi and C. S. Sunder, Studies on Structure and

Vibrational Properties of Carbon Tubules, PRamana - Journal of Physics, 42, 375 - 385 (1994).

64. A. K. Sood, N. Chandrabhas, D. V. S. Muthu, Y. Hariharan, A. Bharathi and C. S. Sunder, Pressure-

induced Band gap Reduction, Orientational Phase Transition and Re- versible Amorphization in C70

43

Crystals: Photoluminescence and Raman Study, Philosophical Magazine B, 70, 347 - 358 (1994).

65. M. N. Shashikala, N. Chandrabhas, K. Jayaram, A. JayaRaman and A. K. Sood, High Pressure

Raman Spectroscopic Study of LiCsSO4: Pressure-Induced Phase Transitions and

Amorphization, Journal of Physics and Chemistry of Solids, 55, 107 - 112 (1994).

66. D. V. S. Muthu, N. Chandrabhas, A. K. Sood, K. Venkatesan, P. Venugopalan and A. JayaRaman, A

High Pressure Raman Study of K and T Forms of Octachloro Cyclic Phosphazene Tetramer

P4N4Cl8,Journal of Raman Spectroscopy, 23, 611 - 614 (1993).

67. N. Chandrabhas, K. Jayaram, D. V. S. Muthu, A. K. Sood, R. Seshadri and C. N. R.

Rao, Orientational Phase Transitions in C70: A Raman Spectroscopic Investigation, Physical Review B

(Rapid Communication), 47, 10963 - 10966 (1993).

68. M. N. Shashikala, N. Chandrabhas, K. Jayaram, A. JayaRaman and A. K. Sood, Pressure-Induced

Phase Transitions in LiRbSO4: A Raman Spectroscopic Study, Journal of Raman Spectroscopy, 24,

129 - 132 (1993).

69. N. Chandrabhas, M. N. Shashikala, D.V. S. Muthu, A. K. Sood and C. N. R. Rao, Pressure-Induced

Orientational Ordering in C60 Crystals as revealed by Raman Spectroscopy, Chemical Physics

Letters, 197, 319 - 323 (1992).

70. A. K. Sood, N. Chandrabhas, D. V. S. Muthu, A. JayaRaman, N. Kumar, H. R. Krishnamurthy, T.

Pradeep and C. N. R. Rao, Pressure-Induced Shift of the Photoluminescence Band in Single Crystals of

Buck-minster Fullerene C60 and its Implications for Superconductivity in Doped Samples, Solid State

Communications, 81, 89 - 92 (1992).

71. N. Chandrabhas, D. V. S. Muthu, A. K. Sood, H. L. Bhat and A. JayaRaman, Raman Study of

Pressure Induced Structural Transitions in CsIO4 to 12 GPa, Journal of Physics and Chemistry of

Solids, 53, 959 - 965 (1992).

72. K. S. Harshavardhan, M. N. Vijayaraghavan, N. Chandrabhas and A. K.Sood, Raman investigations

of diamond films prepared by combustion flames, Journal of Applied Physics, 68, 3303 - 3306 (1990).

73. B. N. Meera, A. K. Sood, N. Chandrabhas and J. Ramakrishna, Raman study of lead borate

glasses,Journal of Non-Crystalline Solids, 126, 224 - 230 (1990).

74. C. N. R. Rao, R. Nagarajan, R. Vijaya Raghavan, N. Y. Vasanthacharya, G. V. Kulkarni, G. Ranga

Rao, A. M. Umarji, P. Somasundaram, G. N. Subbanna, A. R. Raju, A. K. Sood and N.

Chandrabhas, Superconducting cuprates of the series Bi2Ca1-xLnxSr2Cu2O8+d (Ln = rare earth or

Y), Superconductors - Science and Technology, 3, 242 - 248 (1990).

75. R. Vijayaraghavan, A. K. Ganguli, N. Y. Vasanthacharya, M. K. Rajumon, G. U. Kulkarni, G. Sarkar,

D. D. Sarma, A. K. Sood, N. Chandrabhas and C. N. R. Rao, Investigations of novel cuprates of the

TlCa(1-x)LnxSr2Cu2O7-d (Ln = rare earth) series showing electron-or hole superconductivity depending

on the composition, Superconductors - Science and Technology, 2, 195 - 201 (1989).

44

Professional Experience relevant to the Project

Dr. Chandrabhas Narayana is an expert in ultra High Pressure Research (Mbar Pressures)

and Raman spectroscopy (RS). His laboratory in JNCASR is the only laboratory in India

practicing Brillouin Spectroscopy and he has helped in setting up this facility. The work

carried out during and after his Ph.D. has been recognized by both Indian and foreign

scientists alike. During the past 5 years, Dr. Chandrabhas Narayana has used this knowledge

of RS to work in an interdisciplinary area of Physics, Chemistry and Biology and has

developed a new field of research in JNCASR which is fast gaining popularity world over.

He uses an important technique, Surface Enhanced Raman Spectroscopy (SERS), to look at

two aspects in biological systems: (i) small molecule protein interactions and (ii) DNA/RNA

detection of pathogens without polymerase chain reaction (PCR). The success can be seen

from his paper on Raman of p300 which appeared in Journal of Physical Chemistry B

(2006), 110, 16787-16792 has already been sighted more than 22 times in such a short time.

He has demonstrated the effectiveness of SERS in predicting the interaction of the potential

drug molecule derived from anacardic acid (cashewnut shell) with p300 histone

actyltranferase (HAT) region (Journal of Physical Chemistry B 111, 4527-4534 (2007)).

This was subsequently proven to be correct from x-ray studies of synthesized HAT domain

and drug molecule interactions (Nature 451, 846 (2008)). Recently, he has shown that SERS

can be used to study secondary and tertiary structure changes due to cationic substitution in

proteins, like C-protein. This work is under review now. This would be a new tool in the

hands of Biologists to study the drug-protein interactions and protein structural changes.

Another major achievement of his has been the demonstration of (in the case of HIV) the

viral RNA detection without PCR amplification using SERS. PCR is a well known

technique and a very useful one, but requires sophisticated laboratory (recent incident of the

pandemic SWINE FLU is one such example). It is a challenge for the molecular biologists

to find non PCR based techniques. Dr. Chandrabhas Narayana along with Dr. Udaykumar

Ranga (a highly trained virologist at JNCASR) came up with an assay (recently entered the

national phase: International Patent Number: WO2008062479-A2; WO2008062479-A3;

National Patent Number IN200602161-I4) which can detect about a few thousand copies of

viral RNA without PCR amplification. The implication of this is a massive cost reduction, a

micro array kind of system, normal clinical laboratories can carry out this test, possible

mobile testing, use in various other viral pathogen like Dengue, Chickenguniya, detection,

use in seed industries and agriculture like the propagation of markers into future generation

etc. In order to develop a cost effective diagnostics, Dr. Chandrabhas Narayana has been

working with interested companies to miniaturize Raman spectrometer (National Patent

Numbers: IN200800787-I4; US Patent: US20090244534-A1). He has recently synthesized

improved Raman reporters as well as nanoparticle to detect a few copies of viral RNA. He

has been working on the cutting edge research which would soon set a new trend in medical

diagnostics.

(b) Summary the most significant recent work:

Dr. Chandrabhas Narayana’s research has shown the potential of Surface Enhanced Raman

Spectroscopy to understand the drug-protein interactions and in diagnostics, namely,

detection of viral or bacterial DNA/RNA without polymerase chain reaction. To make the

diagnostics reach clinical laboratories, he has developed Raman instrumentation.

(c) Names of the industries in which the technology (ies) has (have) been used:

The products have not yet been commercialized, but discussions are on with various

industries and possibilities of startup companies are also being explored. One of the

multinational companies, TISMO Technologies Solutions (P) Ltd. (Bangalore branch) is

already working on the prototype of miniaturized Raman Spectrometer based on the patent

of Dr. Chandrabhas NaNarayana (Indian patent IN200800787-I4 and US Patent

US20090244534-A1).

45

Ongoing Research Projects

Sl

No. Title of Project Source of Funds Amount

Duration

(from – to -- )

1 Multiplexed immune and

DNA-based diagnosis of

tuberculosis

Swedish Research

Links programme

Rs.

18,00,000/=

(Indian side

funding)

December

2010 –

December

2013

Completed Research Projects (State only major projects of last 3 year)

Sl

No. Title of Project Source of Funds Amount

Duration

(from – to -- )

1 HIV-1 viral RNA

diagnostics

DST Nano Science

and Nano

Technology (India

Rs.

40,00,000

2007

2 Lattice dynamical and

structural study of Be

based II-VI

semiconductor alloys at

ambient and high

pressures''

Indo-French Centre

for Promotion of

Advanced Research

Rs.

10,00,000/

2004

3 Application of surface-

enhanced Raman

spectroscopy to study the

role of Tat protein in

HIV-1 pathogenesis''

Inter-departmental

Collaborative

Project (Jawaharlal

Nehru Centre for

Advanced

Scientific Research

10,00,000/ 2005

It is certified that the above particulars submitted are true and correct.

Place: Bangalore Signature :

Date : 30.04.2012

46

Name : Jayasuryan Narayana

POSITION TITLE: Director

Date of Birth: 3rd

August, 1959

Address : Director

Microtest Innovations Pvt. Ltd.,

International Tech Park,

White Field Road,

Bangalore – 560066,

India.

Phone: +91 80 41262037

Mob : +91 9845004989

E-mail: [email protected]

[email protected]

Education (Post-Graduation onwards & Professional Career)

Education : University of Bombay 1992 Ph.D.

Sl No. Institution

Place

Degree

Awarded

Year Field of Study

1 University of Bombay Ph.D. 1992 Biochemistry

A. Position and Honors

Position and Employment (Starting with the most recent employment)

Sl No. Institution

Place

Position From (Date) To (date)

1 Microtest Innovations Pvt

Ltd Director Present

Director 2002 Present

2 International Center for

Genetic Engineering and

Biotechnology, New Delhi,

India

Post Doc

1992 1995

Honors/Awards

1987-1988: Awarded Fellow of National Science Foundation (USA) to work

under Dr. Rosalyn. S.Yalow, Nobel Laureate in Medicine,

Soloman. A. Berson Research Laboratory, Veterans

Administration Hospital, New York.

1987-1990: Senior Research Fellow, Indian Council of Medical Research,

India.

1991-1995: Post Doctoral Research, at the International Center for Genetic

Engineering and Biotechnology under UNIDO.

1996-2001: Consultant and Advisor in various biotechnology companies and

research laboratories for development of various biotechnology

programs, All Indian Institute of Medical Sciences, New Delhi

2002- Present: Director, Microtest Innovations Pvt.Ltd, International Tech

Park, Bangalore.

47

Professional Experience and Training relevant to the Project

After trained with Dr. Rosanlyn Yalow, who laid the fundamental basis for

modern immunodiagnostics (for which she was awarded noble prize in medicine in

1977), my doctoral and post–doctoral, research was in the area of basic and applied

aspects of infectious diseases. I have worked in the area of Tuberculosis, Viral Hepatitis

and HIV and have extensive experience in diagnostic development (both Molecular and

Immuno diagnostic). I have also been involved with developing assay systems for

conducting large population-base studies in a cost-effective manner by developing

indigenous technology. Currently, as the Director of Microtest Innovation Pvt Ltd. (a

Biotechnology R&D Company), my focus is on developing novel indigenous diagnostic

tests. Accordingly, Microtest Innovations as a company is dedicated to R&D in the area

of biology and medicine with special emphasis on disease diagnostics. In the past seven

years of its existence, the team in Microtest Innovations has been inventing and

innovating appropriate and affordable diagnostics for existing and emerging health

problem through and by collaborative indigenous scientific and technological effort. Our

motto is to lay a strong foundation in basic research to build a product line in novel

diagnostics and diagnostic components.

B. Publications (Numbers only)

Books : Research Papers, Reports : .General articles :

Patents : .Others (Please specify):

Selected peer-reviewed publications (Ten best publications in chronological order)

1. Gupta BP, Jayasuryan N, Jaleel SJ. Direct detection of hepatitis B virus from

dried blood spots by polymerase chain reaction amplification. Coo Microbiol, 30 (8),

19136, 1992.

2. Gupta A, Mal TK, Jayasuryan N, Chauhan. VS assignment of disulphide bonds

in the X-protein (HBx) of hepatitis B virus. Biochemi Biophys Res Commun, 212

(3), 919-24, 1995.

3. Kumar V, Jayasuryan N, Kumar R. A truncated mutant (residues 58-140) of the

hepatitis B virus X protein retains transactivation function. Proc Natl Acad Sci USA,

93 (11), 5647-52, 1996.

4. Kumar V, Jayasuryan N, Reddi H, Sabal D, Panda SK. A monoclonal antibody

against the X protein of hepatitis B VffuS : fine mapping of its epitope and

application in a quantitative ELISA of the X protein in sera of hepatitis B patients.

Hybridoma, 17 (12), 157-64, 1998.

5. Kochupillai N, Jayasuryan N, Godbole MM, Pandav CS. Benefit and cost of

application of Radioimmuno assay in tuberculosis and iodine deficiency disorders:

two major problems of developing countries. Symposia of the giovanni lorenzini

foundation, Volume 18.1 Cost / benefit and predictive value of radio immuno assay.

Elsevier Publication.

6. Udaykumar Ranga, Raj Shankarappa, Nagadenahalli B. Siddappa, Lakshmi

Ramakrishna, Ramalingam Nagendran, Marthandan Mahalingam, Anita Mahadevan,

48

Narayana Jayasuryan, Parthasarathy Satishchandra, Susarla K. Shankar, and

Vinayaka R. Prasad J, Tat Protein of Human Immunodeficiency Virus Type 1

Subtype C Strains Is a Defective Chemokine. J.Virol. 2004. 78: 2586-2590.

7. Nagadenahalli B. Siddappa,Prashanta K.Dash, Anita Mahadevan, Narayana

Jayasuryan, Bethany Hoffman, Randy Keefe, Kadappa S. Satish, Bhuthiah Satish,

Kuttan Sreekanthan, Ramdas Chatterjee, Kandala Venu, Parthasarathy

Satishchandra,Vasanthapuram Ravi,Susala K. Shankar, Raj Shankarappa, and Uday

Kumar Ranga, Identification of Subtype C Human Immunodeficiency Virus Type 1

by Subtype-Specific Polymerase Chain Reaction and Its Use in the characterization

of Viruses Circulating in the Southern Parts of India. Journal of Clinical

Microbiology, June 2004, p. 2742–2751.

8. Nagadenahali Byrareddy Siddappa , Prashanta Kumar Dasha, Anita Mahadevan,

Anita Desaib,Narayana Jayasuryan, Vasanthapuram Ravi b, Parthasarathy

Satishchandrad, Susarla K. Shankar and Udaykumar Ranga, Identification of unique

B/C recombinant strains of HIV-1 in the Southern state of Karnataka, India. AIDS

2005, Vol 19 No.13, 1426 – 1429

9. Nagadenahalli Byrareddy Siddappa, Mohanram VenkatRamanan, Prasanna

Venkatesh, Mohanbabu Vijayamma Janki3, Narayana Jayasuryan, Anita Desai,

Vasanthapuram Ravi and Udaykumar Ranga, Transactivation and signaling

functions of Tat are not correlated: biological and immunological characterization of

HIV-1 subtype-C Tat protein, 18 August 2006 Retrovirology.

10. Nagadenahalli Byrareddy Siddappa, Venkatesh Prasanna Kashi , Mohanram

VenkatRamanan,Anangi Balasiddaiah,Narayana Jayasuryan,Anita

Mahadevan,Anita Desai,Kadappa S Satish Susarla K,Shankar,Vasanthapuram Ravi

and Udaykumar Ranga. Gene Expression Analysis from Human Immunodeficiency

Virus Type 1 Subtype C Promoter and Construction of Bicistronic Reporter Vectors,

AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 23, Number 10,

2007.

C. Research Support

Ongoing Research Projects

Sl

No.

Title of Project Funding Agency Amount Date of

sanction and

Duration

1 Mechanisms of Chikungunya virus

disease: mouse model, virulent and

fitness determinants and the

development of novel therapeutics

Department of

Biotechnology,

Govt. of India

3 years

May 2010

Completed Research Projects (State only major projects of last 3 years)

Sl

No.

Title of Project Funding Agency Amount Date of

completion

1 Development of a Cost-

effective Viral –Load

Pharmaceuticals Research

and development Support

Jan 2005 to

Jan 2008

49

Assay and its Commercial

Application in

Fund Programme

(PRDSF),Department of

Science and Technology

2 Functional analysis of the

NF-kB polymorphism in

the long terminal repeat of

HIV-1 subtype-C viruses

Department of

Biotechnology, Govt. of

India

Mar 2005 to

Feb 2008

Place: Bngalore Signature of Investigator

Date: 30-04-2012

50

Curriculum Vitae

Dr. Myneedu Vithal Prasad.

D-3, Staff Quarters,

LRS Institute of TB & RD

New Delhi-110030

26529974(H)

26517829 Ext. 204 (R) 207(O)

9871102482

[email protected]

[email protected]

Educational Qualifications:

Undergraduate Studies

S.S.C.(1973) :Government Model Higher Secondary School for Boys, Kurnool, A.P. India.

Intermediate (1975): Government Junior College, Kurnool, A.P. India.

Professional Qualifications:

MBBS (1981): Kurnool Medical College, A.P. India.

MD (1988): All India Institute of Medical Sciences, New Delhi, India.

Internship/Residency Experience:

Internship (compulsory): 1 year (07.22.1981 to 07.21.1982), Government general Hospital,

Kurnool, A.P. India.

Senior House Physician: 04.01.1983 to 06.27.1983, Department of Skin and STD,

Government General Hospital, Kurnool, A.P. India.

Junior Resident: 1 year (07.04.1983 to 07.03.1984, Departments of Dermatology & STD

and Medicine, Dr. RML Hospital, New Delhi, India

Junior Resident: Post Graduate Residency Program and Senior Research Fellow 07.04.1984

to 06.30.1988, Department of Microbiology, AIIMS, New Delhi, India.

Special Training programs attended:

1. August 15- 17, 2005, SAARC Regional Training for Laboratory Trainers on

Imparting Training on Quality Assurance in Sputum Microscopy In Tuberculosis.

SAARC TB Center and Government of Sri Lanka, Colombo, Sri Lanka.

2. December 1st 2002 to February 28

th 2003 – Fogarty Fellowship AIDS and

Tuberculosis International Training at New York University School of Medicine,

New York and National TB Center, New Ark, NJ, USA.

3. July 25th

and 26th

2002 – ISO 9000 for Hospital and Health Care Services, National

Institute for Standardization and Quality Management, Bureau of Indian Standards,

New Delhi, India.

4. September 7th

to October 12th

1996 – Modern Mycobacteriology, Centers for

Disease Control and Prevention, Atlanta, GA. USA and NY State Department of

Health Laboratories.

5. September 11th

& 12th

1996 – Radiation Safety in the Laboratory, Centers for

Disease Control and Prevention, Atlanta, GA. USA.

6. September 10th

1996 – Bio Safety in the laboratory (9396), Centers for disease

Control and prevention, Atlanta, GA.USA.

51

7. July 19th

to 30th

.1993 – Revised Strategy for Controlling Tuberculosis, National TB

Institute, Bangalore, India.

8. December (4weeks) 1992 – Training in Mycobacteriology, Supra National

Reference Laboratory, Tuberculosis Research center, Chennai, India.

Professional Experience

Senior Microbiologist(SAG) 02. 02. 2008 to date ,Department of Microbiology,

LRS Institute of TB and respiratory Diseases, New Delhi, India

Senior Microbiologist (Specialist Grade I, Senior Scale):02.12.1998 to 1.2 2008,

Department of Microbiology, LRS Institute of TB and Respiratory Diseases,

New Delhi-30, India.

Bacteriologist/Microbiologist (Specialist II Senior Scale): 02.12.1994 to

02.11.1998, Department of Microbiology, LRS Institute of TB and Respiratory

Diseases, New Delhi-30, India.

Bacteriologist (Specialist Grade II): 02.12.1990 to 02.11.1994, Department of

Microbiology, LRS Institute of TB and Respiratory Diseases, New Delhi-30,

India.

Senior Demonstrator: 08.03.1989 to 02.11.1990, Department of Microbiology,

UCMS and GTB hospital, GTB Enclave, Delhi-92, India.

Senior Research Fellow: 12.11.1988 to 08.02.1989, Department of

Microbiology, Institute of Pathology (ICMR) New Delhi, India.

Job Profile of the present post:

Post: Senior Microbiologist (Senior Administrative Grade) of Microbiology and

National Reference Laboratory , LRS Institute Of TB and Respiratory Diseases, /

Head, Department Institute: 520 bed Tertiary care and Post Graduate teaching facility for TB and Respiratory

Diseases.

Department Team: Microbiologists – 8, Senior Residents 2,Technical officer- 1, Senior

Laboratory Technicians (contractual) – 5, Medical laboratory Technicians -7, Laboratory

assistants -2, Laboratory attendants – 5.

Functions:

Plan and organize all the functions of the Level IV Mycobacteriology Laboratory and the

Department of Microbiology and Dept of Molecular Medicine and Biotechnology

1. To assist Revised National TB Control Program (RNTCP) as a National Reference

Laboratory (NRL) for External Quality assurance in Smear Microscopy and Drug

Resistance Surveillance.

2. Conduct training Programs for TB Laboratory Technicians, Senior TB laboratory

Supervisors and other Para Medical personnel under RNTCP.

3. To participate in the teaching activities of post graduate course in the institute.

4. To participate in the operational research in the institute.

52

5. To participate in the External quality control program of the Supra National

Reference laboratory of the region.

6. To plan and monitor infection control activities in the institute.

Membership of Professional Bodies:

1. Permanent Registration with Andhra Medical council, Hyderabad, A.P., India.

2. Registered with Delhi Medical council, New Delhi, India.

3. Life Member – Indian Association of Medical Microbiologists – Delhi Chapter.

4. Life Member – Hospital Infection Society of India.

5. Life Member - Geriatrics Society of India.

6. Life Member – Bio materials and Artificial Organs of India.

7. Member – National Laboratory Committee, Central TB Division, Ministry of

Health and family Welfare, Government of India.

8. Member – Expert Group, DOTS – PLUS Committee, Central TB Division, Ministry

of Health and Family Welfare, Government of India.

9. Member - Doctoral Committee, Department of Microbiology, AIIMS, New Delhi,

India.

10. Reviewer- Indian Journal Of Tuberculosis

11. Reviewer- The Indian journal Of Chest and Allied Diseases

Research Experience:

MD Thesis: “Detection of circulating serum antibodies by CIEP in the serological

diagnosis of Typhoid Fever”.

Guide – M.Sc. (Biosciences) Thesis – In vitro effect of Nitrogen Laser on the

standard strain of M.tuberculosis (H37 RV), Jamia millia Islamia University, New

Delhi, India.

Guide - M.Sc. (Microbiology) Thesis – Profile of Bacteria and Fungi in children

with respiratory tract infections at LRS institute, New Delhi, India. Thesis

submitted to Bundelkhand University, Jhansi, India. (2001).

Co-guide – M.Biotech Thesis entitled ‘Study of M.bovis infections in cultured

strains of human strains of mycobacterium’. Thesis Submitted to AIIMS, New

Delhi, India. (2001)

Co Investigator – Department of Biotechnology funded project entitled “Efficacy

and safety of Immune modulator (Mycobacterium w) as an adjunct therapy in

category II pulmonary TB. (Double Blind, randomized, placebo controlled, multi

centric clinical trial). (2005).

Administrative Experience:

1.Head, Department Of Microbiology:

The department grew from a simple smear microscopy center (level I Lab) to NRL (Level

IV Lab) under RNTCP.

Dept is recognized for conducting Doctoral Programme (PhD) of IGNOU, New Delhi,

India

Dept is recognized for conducting post doctoral Training in Mycobacteriology

2. Chairman, Hospital Infection control committee LRS Institute since 2000:

Works under taken:

Planned and implemented infection control strategies in the institute.

53

The department got upgraded by installing Bio Safety Level III Facilities.

Developed facilities for safe disposal of infectious materials.

Developed plans for separate wards for MDR-TB patients.

3. Chairman, Estate committee of the institute since 2002.

4. Public Grievances officer, since 2004.To 2006

5. Appellate Authority, Under Right to Information Act 2005. Till 2006.

Achievements:

Started laboratory services in the institute after taking over as In-charge of laboratory.

Over the period the lab grew and Microbiology, Pathology, Biochemistry and Mol.

Medicine divisions were established as full-fledged Departments.

Department of Microbiology is recognized as a National reference Laboratory for TB under

RNTCP programme of India. It is also recognized and approved by IGNOU for doctoral

programme in Mycobacteriology and Microbiology. It is externally quality tested by WHO

Supra National reference lab of Institute of Tropical Medicine Antwerp Belgium. It is

accreditated for testing for TB and DST for all anti TB drugs by conventional as well as by

MGIT method.

It is one of the few labs which have got state of the art BSL III lab facilities in the country.

Research Papers Published

1. Rajnish Gupta, M.sircar, A.Jaiswal, V.K.Arora, K.Gupta, P.Visalakshi and

V.P.Myneedu. A thyroid Tubercular abscess and bilateral symmetrical hilar

lymphadenopathy; a rare association. Ind. j Chest Dis. Allied Sci. 2004: 46: 121-

126

2. Manju Sharma, M.Alfamilamo, H.K.Prasad, V.P.Myneedu, N.Nand.

Characterization by single strand conformation polymorphism of mutations in the

rpoB gene of Rifampicin resistant M.tuberculosis strains from Vancouver, Mexico

city and New Delhi. J Asso Phy of Ind., 2000, 48: 568 -572.

3. Sunil Bhatt,Subrato Mukherjee, V.P.Myneedu,R.C.Jain, R.Sarin, Unsupervised

intermittent short course chemotherapy with intensive health education .Ind J Tub.,

1998, 45: 145 – 151.

4. R. Rajalingam, N.K. Mehra, R.C. Jain, V.P.Myneedu, J.N.Pande. Polymerase chain

reaction based sequence specific oligonucleotide hybridization analysis of HLS

class II antigens in Pulmonary Tuberculosis relevance to chemotherapy and disease

severity. J inf. Diseases 1996, 173: 669 – 676.

5. M.M.Puri, Myneedu.V.P. Jain.R.C., Nitrogen and Helium-neon Laser therapy in the

treatment of Drug resistant Pulmonary tuberculosis cases. Laser Therapy,1995,

7:123

6. Rupak Singla, V.P.Myneedu, M.M.Puri, A.Jaiswal, R.C.Jain,. A study of

Ethionamide, cycloserine, Isoniazide, sodium PAS, and Kanamycin in the re

treatment of drug failure pulmonary TB cases. Ind J. Tub, June 1995, 42: 23 – 26.

54

7. Ruth Stavrum Vithal Prasad Myneedu, Virendra K. Arora, Niyaz Ahmed, Harleen

M.S. Grewal In –Depth Molecular Characterization of Mycobacterium tuberculosis

from New Delhi – Predominance of Drug Resistance Isolates of the ‘Modern’ (Tb

D12) Type PLoS One.2009; 4(2):e4540. Epub 2009 Feb 23.

8. Ajay Wanchu., Yuxin Dong., Sunil Sethi, V.P. Myneedu, Arthur Nadas, Zhentong

Liu, John Belisle, Suman Laal A. Biomaekers for Clinical and Incipient

Tuberculosis: Performance in a TB-Endemic Country. PLoS One.2008 Apr 30; 3

(4):e2071.

9 H.Syre, V.P Myneedu, V.K. Arora, and H.M.S. Grewal Direct Detection of

Mycobacterial Species in Pulmonary Specimens by Two Rapid Amplifiaction Test,

the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test and the Geno

Type Mycobacteria Direct Test. J.Clin.Microbiol.2009 Nov.,

47(11):36359.Epub2009Sep 30.

10 VisalakshiP, Meharwal S, Myneedu V.P, Behera D (2009) Evaluation of direct

method of drug susceptibility testing of Mycobacterium tuberculosis to rifampicin

and isoniazid by nitrate reductase assay in a national reference laboratory

Diagonistic Microbiology and Infectious Disease 66:148-152

11 .MyneeduV.P ,Visalakshi, Varma.A.K., Behera.D, Bhalla.M, Prevalence of XDR

TB cases – A retrospective study from a tertiary care TB hospital (Accepted for

publication In Ind.J.Tub)

multi protein-epitope based sers immunoassay for...

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