Practical Applications of Immunology

Serological Tests Agglutination: Particulate antigens Hemagglutination: Agglutination of RBCs Precipitation: Soluble antigens Fluorescent-antibody technique: Antibodies linked to fluorescent dye Complement fixation: RBCs are indicator Neutralization: Inactivates toxin or virus ELISA: Peroxidase enzyme is the indicator

Nature of Ag/Ab Reactions Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000 http://www.med.sc.edu:85/chime2/lyso-abfr.htm Lock and Key Concept Non-covalent Bonds Hydrogen bonds Electrostatic bonds Van der Waal forces Hydrophobic bonds Multiple Bonds Reversible

Avidity The overall strength of binding between an Ag with many determinants and multivalent Abs Y Keq = 104 Affinity Y 106 Avidity Y 1010 Avidity

Cross Reactivity Cross reactions The ability of an individual Ab combining site to react with more than one antigenic determinant. The ability of a population of Ab molecules to react with more than one Ag Anti-A Ab Ag C Similar epitope Cross reactions Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope

Factors Affecting Measurement of Ag/Ab Reactions Ab excess Ag excess Affinity Avidity Equivalence – Lattice formation Ag:Ab ratio Physical form of Ag

Tests Based on Ag/Ab Reactions All tests based on Ag/Ab reactions will have to depend on lattice formation or they will have to utilize ways to detect small immune complexes All tests based on Ag/Ab reactions can be used to detect either Ag or Ab

. Clone: a strain of cells descended form single cell. Monoclonal antibody Definition: . Mono: one . Clone: a strain of cells descended form single cell. . Antibody: a molecule of animal origin that has immunological activity only against the antigen to which it was made.

Monoclonal antibodies MAbs produced from a single clone of B cells Monoclonal antibodies all have identical antigen-binding sites. Thus they all bind to the same epitope with the same affinity Mostly produced by fusing a B cell secreting the desired antibody with a myeloma cell capable of growing indefinitely in tissue culture

HISTORY

ANTIBODIES Polyclonal antibodies Monoclonal Antibodies Produced by: Many B cell clones A single B cell clone Bind to: Multiple epitopes of all A single epitope of a single antigens used in the antigen immunization Antibody class: A mixture of different All of a single Ab class Ab classes (isotypes) Ag-binding sites: A mixture of Abs with All Abs have the same antigen different antigen-binding binding site sites Potential for cross-reactivity: High Low

Hybridoma 1975 Kohler and Milstein Fusions generated by Sendai virus or PEG

Antibodies Polyclonal Monoclonal recognize multiple antigenic sites Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media Antibodies that are collected from sera of exposed animal recognize multiple antigenic sites of injected biochemical. recognize ONE antigenic site of injected biochemical

PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY 1) Immunize animal (mouse or rabbit) 2) Isolate spleen cells (containing antibody-producing B cells) 3) Fuse spleen cells with myeloma cells (e.g. using PEG - polyethylene glycol) 4) Allow unfused B cells to die 5) Add HAT culture to kill unfused myeloma cells 6) Clone remaining cells (place 1 cell per well and allow each cell to grow into a clone of cells) 7) Screen supernatant of each clone for presence of the desired antibody (ELISA) 8) Grow the chosen clone of cells in tissue culture indefinitely. 9) Harvest antibody from the culture supernatant.

Ab titre reached in Serum PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 1: - Immunization Of Mice & Selection Of Mouse Donor For Generation Of Hybridoma cells ANTIGEN ( Intact cell/ Whole cell membrane/ micro-organisms ) + ADJUVANT (emulsification) Ab titre reached in Serum Spleen removed (source of cells)

PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 2: - Screening Of Mice For Antibody Production After several weeks of immunization Serum Antibody Titre Determined (Technique: - ELISA / Flow cytometery) Titre High Titre too low 2 weeks BOOST (Pure antigen) BOOST (Pure antigen)

Step 3: - Preparation of Myeloma Cells Immortal Tumor Of Lymphocytes PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 3: - Preparation of Myeloma Cells + 8 - Azaguanine Myeloma Cells Immortal Tumor Of Lymphocytes Myeloma Cells HGPRT- High Viability & Rapid Growth hypothanthin-aminopetrin Medium HGPRT = Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)

PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY Step 4: - Fusion of Myeloma Cells with Immune Spleen Cells & Selection of Hybridoma Cells PEG FUSION MYELOMA CELLS SPLEEN CELLS Feeder Cells Growth Medium Plating of Cells in HAT selective Medium Scanning of Viable Hybridomas HYBRIDOMA CELLS ELISA PLATE HAT Medium

PRODUCTION OF MONOCLONAL ANTIBODY HYBRIDOMA TECHNOLOGY

Disadvantages Need high experience qualification High risk of contamination Some mcABs are very liable so activity may be lost on freezing and thawing or long term storage The frequent need to purify a mcAB from monoclonal culture medium Advantages -useful for the production of a specific antibodies to impure or mixed immunogenes.

Measuring protein and drug levels in serum Typing tissue and blood Uses Measuring protein and drug levels in serum Typing tissue and blood Identifying infectious agents Identifying clusters of differentiation for the classification and follow-up therapy of leukemias and lymphomas Identifying tumor metastasis Identifying and quantifying hormones Immunoaffinity Purification

Detect Ag from patient sample . Detect Ab from patient sample Serological Tests Direct Indirect Detect Ag from patient sample . Detect Ab from patient sample

Precipitation : ( Continued ) Precipitation curve :

Precipitation Reactions Involve soluble antigens with antibodies

Precipitation : ( Continued ) - Precipitation reactions

Precipitation : ( Continued ) Ouchterlony (double diffusion)

Agglutination Reactions Involve particulate antigens and antibodies Antigens may be: On a cell (direct agglutination) Attached to latex spheres (indirect or passive agglutination)

Antibody Titer Is the concentration of antibodies against a particular antigen Figure 18.5

Agglutination : ( continued ) - Agglutination reactions involve whole cell antigens, while precipitation reactions involve soluble antigens. - Cellular/molecular view of agglutination and Precipitation reactions that produce visible Ag-Ab complexes.

Hemagglutination Hemagglutination involves agglutination of RBCs. Viral hemagglutination inhibition tests for antibodies by the antibodies' ability to prevent viruses from agglutinating RBCs. Figure 18.7

Neutralization Reactions Eliminate the harmful effect of a virus or exotoxin

Agglutination Tests Lattice Formation 32

Agglutination/Hemagglutination Definition - tests that have as their endpoint the agglutination of a particulate antigen Agglutinin/hemagglutinin Y +  Qualitative agglutination test Ag or Ab

Agglutination/Hemagglutination Quantitative agglutination test Titer Prozone 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 4 Patient 1 2 3 5 6 7

Agglutination/Hemagglutination Definition Qualitative test Quantitative test 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 Applications Blood typing Bacterial infections Fourfold rise in titer Practical considerations Easy Semi-quantitative

Passive Agglutination/Hemagglutination Definition - agglutination test done with a soluble antigen coated onto a particle Y +  Applications Measurement of antibodies to soluble antigens

Coombs (Antiglobulin)Tests Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes +  Y Patient’s RBCs Coombs Reagent (Antiglobulin)

Coombs (Antiglobulin)Tests Indirect Coombs Test Detects anti-erythrocyte antibodies in serum anti-rhesus factor (Rh) antibodies Y Patient’s Serum Target RBCs +  Step 1 +  Y Coombs Reagent (Antiglobulin) Step 2

Y Complement Fixation Methodology Ag No Ag Ag mixed with test serum (Preheated) to be assayed for Ab Standard amount of complement is added Erythrocytes coated with Abs is added Amount of erythrocyte lysis is determined Ag No Ag Ag Y Patient’s serum Ag Y

Complement Fixation

Complement Fixation

Complement fixation test

Fluorescent Antibody Techniques (Direct) Figure 18.10a

Fluorescent Antibody Techniques (Indirect)

Enzyme-Linked Immunosorbent Assay (Direct ELISA)

Enzyme-Linked Immunosorbent Assay (Indirect ELISA)

ELISA Enzyme-Linked Immunosorbent Assay

Test antigen Test antiserum

Visualization of reaction

ELISA to test MAb production itself The dark blue spot represents a positive clone. The light blue spots are positive controls, the next two wells to the right are negative controls +ve clone -ve control +ve control

The enzyme choice based on three parameters: 1- the enzyme should have high turnover rate of substrate to product. 2- the product should be detected with max sensitivity. 3- the enzyme activity should be minimally susceptible to interference from factors may be present in the sample. The commonly used Enzymes: Alkaline phosphatase. 2) β-Galactosidase. 3)Horseredish peroxidase.

The developed color is read by eye as qualitative assays The Enzyme The developed color is read by eye as qualitative assays To perform quantitative assays take the sample for serial dilutions ,the highest dilution to give +ve result is taken for potency measurement by spectrophotometer.

Microtitre Plate It is a disposable plate with 96 wells. Ether Antigen or antibody can be adsorbed to the wall of each well. Subsequent washing and reagent addition become very easy using the microtitre system specially in using of multihead pipettes. Because of its simplicity ,ELISA has become the method of choice for screening mcAB production cultures.

Serological Tests

Tests for Cell Associated Antigens Lattice formation not required

Immunofluorescence Direct Y Ab to tissue Ag is labeled with fluorochrome Ag Y Fluorochrome Labeled Ab Tissue Section

Qualitative to Semi-Quantitative Immunofluorescence Indirect Ab to tissue Ag is unlabeled Fluorochrome-labeled anti-Ig is used to detect binding of the first Ab. Ag Y Fluorochrome Labeled Anti-Ig Tissue Section Unlabeled Ab Qualitative to Semi-Quantitative

Immunofluorescence Flow Cytometry Cells in suspension are labeld with fluorescent tag Direct or Indirect Fluorescence Cells analyzed on a flow cytometer Flow Tip Laser FL Detector Light Scatter

Green Fluorescence Intensity Two Parameter Histogram Immunofluorescence Flow Cytometry cont. Data displayed Red Fluorescence Intensity Green Fluorescence Intensity Two Parameter Histogram One Parameter Histogram Unstained cells Number of Cells FITC-labeled cells Green Fluorescence Intensity