MethodologyMethodology

used in used in

ImmunologyImmunology

Antigen reacts with antibody to form an aggregate or precipitate

Equivalent concentration of antigen & antibody forms a precipitate

The Precipitin Reaction

Blood TypingBlood Typing

If the Rh antigen is also present, the person is RhIf the Rh antigen is also present, the person is Rh++, if they , if they do not have the Rh antigen, they are Rhdo not have the Rh antigen, they are Rh--

CoombsAnti-globulin testHemolytic anaemia

Detects anti-Rh antibody (IgG)

Direct and Indirect

human

Enzyme Linked Immuno Sorbent Assay

Antigenic site = epitope = Antigenic determinant 1° Antibody can be polyclonal or monoclonalMUST be Specific binding

Enzyme Linked Immuno Sorbent Assay

Antibody used for detection isLabeled with biotinBiotin binds to streptavidin that is linked to an enzyme (HRP)

HRP catalyzes the substrate (NBT/BCIP) to yield a color.

Colorimetric detection

Detection of DIG-labeled nucleotides/antibody is done using anti-DIG antibodies conjugated to alkaline phosphatase.

When a substrate such as CSPD (dioxetane) is added, alkaline phosphatase breaks down the substrate, which emits a photon of light.

Fluorescently-tagged DNA probes or antibodies have the advantage that no enzymatic reaction is necessary for detection. Dye-conjugated nucleotides are incorporated into DNA during labeling eg. PCR labeling.

Antinuclear antibodies ANAAntinuclear antibodies ANAThey are auto-antibodies against nuclear components that can be detected by indirect immunofluorescence on cell nuclei.

The diffuse staining pattern is the commonest, associated with SLEThe speckled pattern is associated with Mixed Connective Tissue Disease The nucleolar pattern is associated with sclerosisAnticentromere staining is associated with a subgroup of sclerosis

The ELISPOT assay

Variation of ELISA

Quantitates the number of cells producing antibodies

Identifies cells producing specific cytokines

Western Blot

for detection of anti HIV antibodies

C + Neg

A positive test shows the presence of p24 AND either gp 120 or gp 160

Immunoprecipitation

For detection of proteins using specific antibodies

• Using an antibody to isolate and purify a protein from a whole cell lysate.

• Normally you will only purify the protein the antibody recognizes.

• Any additional proteins that co-purify are candidates for interacting proteins.

Immunoprecipitation

• affinity purification based on isolation of Ag-Ab complexes

• analyze by gel electrophoresis

• isolation of Ag-Ab complexes

Bacterial proteins that bind IgG (Fc):• protein A (Staphylococcus aureus)• protein G (Streptococcus)

• binds more species and subclasses

Separation of Cells on a Ficoll Hypaque density gradient

Separation of cells using magnetic beads

Mouse Spleen

Magneticcolumn Other cells CD45 Cells

Magnetic beads coated with anti CD45 antibody

Immunofluorescence and Immunohistochemistry

PBMC stained withAnti IL-2 FITC

PBMC stained withAnti IL-2 Peroxidase/DAB

IL-2 producing cellsStained in situ

Flow Cytometry

Light Scatter Gating

90 Degree Scatter0 200 400 600 800 1000

Forw

ard

Sca

tter

0 2

00 4

00 6

00 8

0010

00

Lymphocytes

Monocytes

Neutrophils

Side Scatter Projection

(measures complexity)

(mea

sure

s siz

e)

8

15

20

30

40

50

100

200

1000

Scale

CD45 vs SS

Normal

Loss of CD4+ T cellsHIV ??

Immunophenotype of a patient with CLL

CD19+ and Ig kappa +Indicating B cell monoclonality

Mostly CD45+Indicating leukocytesCD14 -No monocytes

Mostly CD23+ B cells(also excludes mantle cell)

CD5+ and CD20+Indicating CD5+ B lymphocytes

EARLY B-CELL CD19 51.8 % PAN B CD20 49.3 % CD20 INTENSITY 2+PAN T/B CLL CD5 85.5 % CD19+/CD5+ CELLS 41.4 % ACTIVATED B-CELL CD23 44.5 %

SIG KAPPA 97.5 % SIG LAMBDA 0.5 %

CD38 60.0 % CD19+/CD38+ 60.6 %CD79b 20.5 % B SUBSET, FMC-7 9.6 % CALLA CD10 0.6 %CD19+/CD10+ CELLS 0.5 %

T-CELL ANTIGEN CD3 46.2 %

INTERPRETATION B-cells express CD5, CD19, CD20, CD23 and monotypic immunoglobulin KAPPA light chain, c/w B-CLL/SLL.

FLOW CYTOMETRY - Lymph Node, FNA

41.1 %

0 200 400 600 800 1000SSC-Height

10 0 10 1 10 2 10 3 10 4CD19 FITC

10.4 %

44.1 %

10 0 10 1 10 2 10 3 10 4KAPPA FITC

10 0 10 1 10 2 10 3 10 4LAMBDA PE

CD

19 P

ER

CP

CD

19 P

ER

CP

CD

5 PE

F SC

- Hei

g ht

expression of kappa & lambda in CD19+/CD5+ lymphocytes

41.4%

Fluorescence Activated Cell Sorting

Some Applications of Flow Cytometry

Surface antigens.• Lymphocyte sub-sets.• Immunophenotyping : peripheral blood & bone marrow. • Leukemia diagnosis• Stem cell counting. (CD34).• Cytokine receptors & intracellular flow cytometry

DNA Analysis (Ploidy)

Apoptosis

Cell Cycle and DNA analysis

DNA Ploidy

Detection of Apoptosis using Annexin V

Normal cellApoptotic cell

Detection of Apoptosis using Annexin V

Detection of Apoptosis by

TUNEL

Functional assays for lymphocytes

Measurement of Cell Proliferation

Measurement of Cytotoxicity

Hemacytometer: Use 10XUse 10Xobjectiveobjective

1 2

3 4

5

Count the number of cells in squares 1Count the number of cells in squares 1--4; determine average # cells/square4; determine average # cells/squareAverage # cells/square X 10Average # cells/square X 1044 = # cells/ml= # cells/ml

Parameter Methods

Phagocytosis Migration and chemotaxis, detection of respiratory burst

Lymphocyte subsets

Flow cytometry, immunohistochemistry

Lymphocyte activity

Lymphocyte proliferation test, Cytotoxicity assay, mixed lymphocyte reaction

Cytokines Bioassay, ELISA, PCR

Immunoglobulin levels

Radial immunodiffusion RID, ELISA

Complement Hemolytic activity, ELISA

CRP, lysozyme and other humoral factors

ELISA, turbidimetry

•Immunoassays (e.g., ELISA)

•Protein-protein interaction assays (e.g., interaction mapping)

•Enzyme assays (e.g., kinase, phosphatase, and protease assays)

•Receptor-ligand assays

•Protein-nucleic acid assays

Liquid Bead arrays

The Luminex machine

Luminex Multiplexed Assay Principle

UCUCSSFF

5.6 micron polystyrene beads internally dyed with red and infrared fluorophores.

100 bead sets available, each with different dye ratios and each with a unique spectral signature determined by its red/infrared ratio.

Capture reagents (e.g., antibodies, oligos, peptides, receptors) chemically coupled to beads.

Small size/surface area and 3-dimensional exposure of the beads allows for nearly solution-phase kinetics.

The red laser identifies which assay is carried on the bead, while the green laser quantifies the analyte.

Principles of the Luminex Technology

The Microsphere is a 5.6mM polystyrene bead with two fluorescent dyes incorporated into it in different ratios.

The Microsphere is a 5.6mM polystyrene bead with two fluorescent dyes incorporated into it in different ratios.

A primary antibody specific for the analyte isconjugated to the bead surface by an amine coupling reaction

A primary antibody specific for the analyte isconjugated to the bead surface by an amine coupling reaction

The primary antibody binds to the specific analyte – no crossreactivity with other analytes occurs.

The primary antibody binds to the specific analyte – no crossreactivity with other analytes occurs.

A biotinylated, analyte specific reporter antibody is added to the assay after another wash step.

A biotinylated, analyte specific reporter antibody is added to the assay after another wash step.

After another wash step Streptavidin PE is added to the assay. The biotinylated reporter antibody binds to one of the four available sites.

After another wash step Streptavidin PE is added to the assay. The biotinylated reporter antibody binds to one of the four available sites.

In assays with high analyte numbers excess biotinylated reporter antibodies bind to the Strep-PE in a non-specific manner.

In assays with high analyte numbers excess biotinylated reporter antibodies bind to the Strep-PE in a non-specific manner.

Free excess streptavidin-PE binds to the non-specifically bound biotinylated reporter antibody leading to a signal amplification.

Free excess streptavidin-PE binds to the non-specifically bound biotinylated reporter antibody leading to a signal amplification.

The phycoerythrin is excited by the reporter laser and emits a fluorescence which is quantified by the Luminex reader.

The phycoerythrin is excited by the reporter laser and emits a fluorescence which is quantified by the Luminex reader.

The immune-complex/ microsphere is then excited by the ****** laser. The bead specific emmission is quantified by the luminex and the bead identified.

The immune-complex/ microsphere is then excited by the ****** laser. The bead specific emmission is quantified by the luminex and the bead identified.

Beadlyte Immunoassay format for the Luminex Machine.

TSPE Reaction•TSPE primers are chimeric oligos with a virus-specific sequence and Tag sequence complimentary to anti-Tag sequence bound to microbead

•TSPE primers are extended by Taq polymerase and incorporate biotin reporter molecule

•Biotinylated amplicons are detected with a fluorescent SA-Phycoerythrin conjugate and signals are read using the Luminex instrument

Tag Anti-Tag>

BiotinBiotin

Bead Hybridization

Cytokines

UCUCSSFF

Aβ 40 Aβ 42 BDNF DR-5 EGF ENA-78 Eotaxin Fatty Acid Binding Protein FGF-basic G-CSF GCP-2 GM-CSF GRO αGRO-KC HGF I-TAC ICAM-1 IFN-αIFN-γ

IL-1αIL-1βIL-1ra IL-1ra/IL-1F3 IL-2 IL-3 IL-4 IL-5 IL-6 IL-7 IL-8 IL-9IL-10 IL-11IL-12 p40/p70 IL-13 IL-15 IL-16 IL-17 IL-18 IP-10

JE/MCP-1 KC KC/GROa LIF Lymphotacin M-CSF MCP-1 MCP-1(MCAF) MCP-2 MCP-3 MCP-5 MDC MIG MIP-1αMIP-1βMIP-1γMIP-2 MIP-3 βOSM PDGF-BB RANTES

Rb (pT821) Rb (total) Rb pSpT249/252 Tau (pS214) Tau (pS396) Tau (total) Tissue Factor TNF-αTNF-βTNF-RI TNF-RII TNF-SF5 VCAM-1 VEGF

Immune Cell Function Test

• Measures the Net State of Immune Function –based on lymphocyte proliferation assay.

•Used to MonitorTherapy Response

Immune Function is Independent of CD4 Count

LuminometerATPATP ATPATP

ATPATP

Cell lysis to release ATP

ATP detection reagents

Measure light intensity

LymphocyteStimulation w/PHA

WashIncubate

15-18 hrs.

Magnetic separationof CD4 cells.

J. Britz et. al.,“In Vitro CMI: Rapid Assay for Measuring Cell-Mediated Immunity”, CRC Press, 2002

Immuknow

ImmuKnow™: Stratification of Immune Response

Kowalski, R. et. al., Clinical Transplantation:17:77-88, 2003

FDA Claim: ImmuKnow™ is used for the detection of cell-mediated immunity in an immunosuppressed population.

Strong

Low

Moderate

Healthy Adults Stable Transplant Recipients

I mm

un

e R

esp o

ns e

(A

T P n

g /m

L)

0

200

400

600

800

415

258

(n=155) (n=127)

P < 0.0001

Immune Function Correlates With Clinical Outcomes

Healthy StableRejection Infection

Imm

un

e R

espo

nse

(A

TP n

g/m

L)

0

200

400

600

800

1000

415

249

488

111

Strong

Low

Moderate

(n=155) (n=39) (n=504) (n=66)

P < 0.001

P < 0.001

Meta-Analysis: NIDDK, UCLA, JHU, UPMC, NZTI, LifeLink, UTMB, UAB, UMD, INOVA

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