Diagnostic immunology 324 PHT Dr. Sarah I. Bukhari PhD in Clinical Microbiology Department of Pharmaceutics Office: 06 - 3rd floor sbukhari@ksu.edu.sa

Diagnostic immunology Tests based on the interactions of antibodies and antigens These tests determine the presence of antibodies or antigens in a patient. Specificity Determined by the percentage of false positive results it gives Sensitivity Determined by the percentage of positive samples it correctly detects Essential elements of diagnostic tests are sensitivity and specificity. Sensitivity is the probability that the test is reactive if the specimen is a true positive. Specificity is the probability that a positive test will not be reactive if a specimen is a true negative.

Immunologic-based diagnostic tests

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

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

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

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

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

Antibody-based Diagnostic Tests problem that must be overcome in antibody-based diagnostic tests: antibodies cannot be seen directly Antibodies produced in an animal were mixed and limited quantity. Even at magnifications of well over 100,000×, they appear only as fuzzy, ill-defined particles

Nobel prize in 1975 Neils Jerne César Milstein Georges Köehler antibodies were produced by specialized cells (B cells), it was understood that these were a potential source of a single type of antibody. If such a B cell producing a single type of antibody could be isolated and cultivated, it would be able to produce the desired antibody in nearly unlimited quantities and without contamination by other antibodies. Unfortunately, a B cell reproduces only a few times under the usual cell culture conditions. This problem was largely solved with the discovery of a method to isolate and indefinitely cultivate B cells capable of producing a single type of antibody. Neils Jerne César Milstein Georges Köehler

Two identical light chains Antibody Structure Two identical light chains Two identical heavy chains Linked by S-S bonds

Monoclonal Antibodies (Mabs) Pathogens contain many different antigens (and many more epitopes)  Polyclonal antibody response Monoclonal antibody: single type of antibody directed against one specific epitope. Produced by single B cell clone.

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

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

Production of MAbs https://www.youtube.com/watch?v=Gykx5FrQbvU

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.

Production of MAbs Hybridoma Production in the laboratory by fusing a cancerous cell with an antibody-secreting plasma cell The hybridoma cell culture produces large quantities of the plasma cell’s antibodies (Mabs) Scientists have long observed that antibody-producing B cells may become cancerous. In this case, their proliferation is unchecked, and they are called myelomas. These cancerous B cells can be isolated and propagated indefinitely in cell culture. Cancer cells, in this sense, are “immortal.” The breakthrough came in combining an “immortal” cancerous B cell with an antibody-producing normal B cell. When fused, this combination is termed a hybridoma. When a hybridoma is grown in culture, its genetically identical cells continue to produce the type of antibody characteristic of the ancestral B cell. The importance of the technique is that clones of the antibody-secreting cells now can be maintained indefinitely in cell culture and can produce immense quantities of identical antibody molecules.

Monoclonal Antibodies (Mabs) Serological identification (tissue and blood typing) As immunotoxins to treat cancer Measure serum protein and drug levels Identify infectious agents Identify and quantify hormones Treatment of specific diseases But the therapeutic use of these monoclonal antibodies has been limited because they were produced in mouse

Development of New Generations of MAbs

Development of New Generations of MAbs Chimeric mabs (66% human): Genetically modified mice produce Ab with a human constant region. Humanized mabs (90% human): MAbs that are mostly human, except for mouse antigen-binding. Fully human antibodies Bacterial, plant and animal systems under investigation to increase production volumes.

Serological Tests

Precipitation Reactions Interaction of soluble antigens with IgG or IgM antibodies to form large, interlocking molecular aggregates called lattices. When optimal proportions of antigens and antibodies  Lattice formation. Excess of either component decreases lattice formation and subsequent precipitation. Precipitin ring test performed in small tube. Immunodiffusion procedures: precipitation reactions carried out in agar gel medium

Precipitation

Precipitation - Precipitation reactions

Precipitation : Ouchterlony (double diffusion)

Agglutination Reactions Interaction of particulate antigens and antibodies. Antigens may be On a bacterial cell: direct agglutination. Attached to latex spheres: indirect or passive agglutination. E.g.: syphilis test Diseases may be diagnosed by combining the patient’s serum with a known antigen

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 particu Agglutination reactions are used in blood typing, the diagnosis of certain diseases, and the identification of viruses. Figure 18.5

Agglutination - 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.

Neutralization Reactions Harmful effect of a bacterial exotoxin or virus is eliminated by a specific antibody (antitoxins and virus neutralization) Some viruses (mumps, measles, influenza) agglutinate RBCs in vitro. Antibodies against these viruses can be detected by their ability to interfere with viral hemagglutination.

Viral Hemagglutination-Inhibition Tests Hemagglutination involves agglutination of RBCs Some viruses agglutinate RBCs in vitro Antibodies prevent hemagglutination

Fluorescent Antibody Techniques Use antibodies labeled with fluorescent dyes. Direct fluorescent-antibody tests: identify specific microorganisms. Indirect fluorescent-antibody (IFA) tests: demonstrate the presence of antibodies in serum. FACS =Fluorescence-activated cell sorting

Enzyme-Linked Immunosorbent Assay ELISA Enzyme-Linked Immunosorbent Assay

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