Contents:

Introduction

History & Development

Procedure

Identification & Isolation

Application

Persisting Problems

Improvements

Summary

Conclusion

Introduction

• Hybridoma technology is one kind of Biotechnology.

• It is a hybridization technique which is used to produce antibody producing hybrid cell.

• These hybrid cells are produced by fusing the genetic material of B-lymphocyte with tumour cell.

• The antibodies produced are monoclonal antibodies as they are all of a single specificity.

• Presence of B-lymphocyte genetic material helps in antibody production, whereas capacity to divide indefinitely in the culture due to the presence of tumour cell.

The production of monoclonal antibodies was invented by Cesar Milstein and Georges J. F. Köhler in 1975.They pre-programmed B-lymphocytes to respond to a single type of antigen or antigenic determinant, therefore they produce single type of antibody specific to the specific antigen.When an antigen reacts with B-lymphocyte receptors, lymphocytes divide rapidly and produce a clone of B cells, all these B cells produce antibodies against that specific antigen and this is called as clonalselection. That is B-lymphocytes produce only one type of antibodies which are specific to only one type of antigen or antigenic determinant.But fully differentiated antibody producing B-lymphocyte cells known as plasma cells does not divide when cultured in a laboratory.The term hybridoma was coined by Leonard Herzenberg during his sabbatical in Cesar Milstein's laboratory in 1976/1977.

Step-IMouse is immunized by giving antigen injection against which monoclonal antibodies have to produced along with an adjuvant. This is followed by booster doses of the antigen.

After 72 hrs of immunization the spleen is collected from the mouse.

Then a free cell suspension medium of the spleen is prepared using sterile serum free medium followed by centrifugation.

All these processes are applied for extraction of plasma cells from the spleen.

Step-IIMaintain the plasma cells in the cell culture medium for 16-24 hrs before fusion.

It should be ensured that the cells are in early phase of growth at the time of fusion.

The myeloma cells are selected based on some criteria like these cells themselves should not produce antibodies and also they should contain a genetic markers such as HGPRT(hypoxanthine-guanine phosphoribosyltransferase) .

This genetic marker helps in easy selection of the resulting hybrid cells.

Step-IIIAt the time of fusion both

myeloma & spleen cells are

counted & then mix in the

appropriate ratio.

Depending on the properties of the

tumour cell, the mixing ratio of

spleen to tumour cell may vary

from 5:1 to 2:1

Following the mixing, the cells are

centrifuged into a loose pellet.

The supernatant is removed & the

pellet is mixed with 1 ml of

Polyethylene glycol(PEG) for 3

min.

In doing so the pellet will be broken

up into uniform small clumps.

Step-IV

• Following the fusion, dilute

the cells in serum free

medium slowly to reduce

the osmotic disruption of

the fused cell.

• Then centrifuge the cells &

resuspend in HAT

(Hypoxanthine Aminopetrin

Thymidine) medium.

• Then Hat medium containing

the fused cells are allowed

to incubate in a CO2

incubator for 3-4 days.

Step-V• This mixture of cell population

is then cultured in selective media known as HAT medium along with the drug aminopterin.

• The HGPRT myeloma cells cannot divide in the HAT medium due to the presence of aminopterin.

• The Specific antibody producing B-lymphocytes are unable to divide continuously in the culture medium, therefore eventually they die.

Step-V• Only the hybridoma cells

have got the ability to divide and proliferate on the HAT medium because genome from the B-lymphocyte makes them HGPRT positive and genome from the myeloma cells they can divide indefinitely.

• Thus only the hybridomacells or fused cells are selected using selective media called as HAT medium.

Reclone and cultivate positive clones

ELISA PLATE

Most common screening

techniques are ELISA and RIA)

Low concentration

(1-20 ug/ml)

High concentration

(1-10 mg/ml)

In- Vitro In- Vivo

• The mouse ascites method usually produces very high mAb concentrations that often do not require further concentration procedures that can denature antibody and decrease effectiveness.

• The high concentration of the desired mAb in mouse ascites fluid avoids the effects of contaminants in in -vitro batch-culture fluid when comparable quantities of mAb are used.

• The mouse ascites method avoids the need to teach the antibody producer tissue-culture methods.

• The mouse ascites method involves the continued use of mice requiring daily observation.

• MAb produced by in vivo methods can contain various mouse proteins and other contaminants that might require purification.

• The mouse ascites method can be expensive if immuno-deficient mice in a barrier facility used.

• In vivo methods can cause significant pain or distress in mice.

In vitro methods reduce the use of mice at the antibody-production stage but can use mice as a source of feeder cells when antibody generation is under way).

In vitro methods are usually the methods of choice for large-scale production by the pharmaceutical industry because of the ease of culture for production, compared with use of animals, and because of economic considerations.

In vitro methods avoid the need to submit animal protocols to IACUCs.

In vitro methods avoid or decrease the need for laboratory personnel experienced in animal handling.

In vitro methods using semipermeable-membrane-based systems produce mAb in concentrations often as high as those found in ascitic fluid and are free of mouse ascitic fluid contaminants.

It should be noted that each of the items below pertains to only a fraction (3–5%) of hybridomas, but they indicate some of the difficulties associated with in vitro methods.Some hybridomas do not grow well in culture or are lost in culture.The loss of proper glycosylation of the antibody (in contrast with in vivo production) might make the antibody product unsuitable for in vivo experiments because of increased immunogenicity, reduced binding affinity, changes in biologic functions, or accelerated clearance in vivo.In general, batch-culture supernatants contain less mAb(typically 0.002-0.01) per milliliter of medium than the mouse ascites method. Note that semipermeable-membrane-based systems have been developed that can produce concentrations of mAbcomparable with concentrations observed in mouse ascites fluid.

Dose determination of a medicineTo detect allergies & viral disease to detect certain type of cancer; to monitor the

presence or appearance of malignant cells after surgical or radio-therapeutic treatments.

For purification of complex biological mixture.Envisaged for the labelling & precise

identification of some specialized cell such as neurone for gaining better knowledge of the way of association & operation.

For purification of structural cell membrane protein.

Mab has a great role in serotherapy

Used to produce ImmunotoxinUsed in the preparation of very specific vaccines,

particularly against certain virus strain or against some parasites.

Neutrilize the action of lymphocytes responsible for the rejection of grafts & destroy the auto-antibodies produced in auto-immune disease.

Mab could considerably increase the effectiveness of the medicinal substances on the target cells, while avoiding the serious side-effects of cancer therapies.

The production of monoclonal antibodies remains

inefficient and labor intensive

The process of immortalizing a B cell is so inefficient that

only 1 in 20,000, or sometimes 1 in 200,000, B cells forms

a viable hybridoma.

Generation of useful monoclonal an- tibodies to weak

immunogens are very difficult as If the antigen is poorly

immunogenic, available in only small amounts, or impure,

the numbers of B cells making antibody to that antigen will

be low and only a few positive hybridomas will be

identified.

The efficiency of fusion decreases greatly if one waits until

the end of the immune response. when B cells are no

longer proliferating rapidly and synchronously, even though

this is when the highest affinity antibodies are being

made.

IMPROVEMENTS IN THE HYBRIDOMA TECHNOLOGY

Enrichment of the cells making the antibody(B- Cells) of interest using in vitro immunization would reduce the amount of screening and make it possible to start with larger populations of cells and immortalize the more number of B cells.

In one version of this technique, immune cells are incubated with antigen-biotin conjugates and then mixed with biotin-coated myeloma cells. Avidin is added to bring the B cells and the fusion partner(Tumour Cell) together and they are then fused by electrofusion

When antibody-forming cells are grown in culture, most of the B cells die, but those that are stimulated by antigen will proliferate and over- grow than the rest of the population.

Harvest Ab

Monoclonal antibodies

Myeloma cells

Grow indefinitely in

cell culture but don't

secrete the desired

antibody

FUSE Hybridoma cells

Secrete antibody but

don't grow in tissue

culture

Grow indefinitely in

cell culture AND

secrete antibody

Conclusion

Many European & North-American firms are interested in the applications of Mab. In california, for-instance, certain medicine companies are preparing diagnostic kits designed for the screening of certain lethal diseases. It is anticipated that the future support of some aspects of this hybridomas based monoclonal antibody technology will be tailored to the needs of aquaculture industry of each developing country of the world.

• http://www.medarex.com/Development/UltiMAb.htm • http://wwwext.amgen.com/media/media_pr_detail.jsp?y

ear=2006&releaseID=837754• http://www.regeneron.com/velocimmune.html• http://www.ukbusinesspark.co.uk/cay92125.ht• http://www.biotecharticles.com/Others-

Article/Hybridoma-Technology-A-Biotechnology-Technique-378.html

Links:

Book:Fish Biotechnology by Ranga & Shammi (Pg No. 230-237)