IMMUNOLOGIC TESTS IN THE DIAGNOSIS OF DISEASE DR. MARTIN MIGUEL I.

AMOR
Antigen-Antibody Reactions antigen-antibody reactions are highly-specific o an antigen will react only with antibodies elicited by itself or by a closely related antigen o basis of serologic reactions results are expressed as titers o highest dilution of the specimen that gives a positive reaction in the test o an antibody titer of 1/64 contains more antibodies (higher titer) than a serum with a titer of 1/4 Major Uses of Serologic Tests DIAGNOSIS OF INFECTIOUS DISEASES o when the organism cannot be cultured o when the organism is too dangerous to culture o when culture techniques are not readily available o when the organism takes too long to grow DIAGNOSIS OF AUTOIMMUNE DISEASES o antibodies against various normal body components DETERMINATION OF BLOOD TYPE AND HLA TYPE o known antibodies are used to determine blood types (ABO, Rh) and class I or class II HLA proteins Detection of Antigens Detection of Antigens DIRECT IMMUNOFLUORESCENCE RADIOIMMUNOASSAY ELISA/EIA FOR ANTIGEN (SANDWICH) LATEX AGGLUTINATION FOR ANTIGEN Immunofluorescence fluorescent dyes (fluorescein and rhodamine) are covalently attached to antibodies visualized under UV light in a fluorescence microscope labeled antibody can be used to identify antigens on the surface of bacteria, in cells in histologic section, or in other specimens Types of Immunofluorescence DIRECT IMMUNFLUORESCENCE o labeled antibody interacts directly with unknown antigen INDIRECT IMMUNOFLUORESCENCE o PRIMARY ANTIBODY known antigen is attached to slide, patient's serum is added and preparation is washed if serum contains antibody, it will remain fixed to slide o SECONDARY ANTIBODY addition of a fluorescent dye labeled antibody to human IgG and examination by UV microscopy more sensitive and more flexible than direct immunofluorescence Enzyme-Linked Immunosorbent Assay (ELISA) also called enzyme immunoassay (EIA) can be used for the quantitation of either antigens or antibodies in patient specimens nearly as sensitive as RIA yet requires no special equipment or radioactive labels ELISA Procedure covalently linking an enzyme to a known antigen or antibody reacting the enzyme-linked material with the patient's specimen assaying for enzyme activity by adding the substrate of the enzyme

Radioimmunoassay used for quantitation of antigens that can be radioactively labeled procedure: o competition for specific antibody between labeled and the unlabeled concentration of material o separation of complexes that form between the antigen and antibody o measurement of amount of radioactivity EXAMPLES: o hormone assays, drug assays, RAST for IgE

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IMMUNOLOGIC TESTS IN THE DIAGNOSIS OF DISEASE DR. MARTIN MIGUEL I. AMOR

Sandwich ELISA for Antigens

Latex Agglutination an inert particle (latex beads) is coated with an antigen or antibody antigen-antibody reaction leads to clumping (agglutination) simple procedure which can be done in a small cup or tube or with a drop on a slide EXAMPLE: ABO blood group determination

Detection of Antibodies Detection of Antibodies LATEX AGGLUTINATION COMPLEMENT FIXATION EIA/ELISA (INDIRECT) HEMAGGLUTINATION NEUTRALIZATION IMMUNODIFFUSION INDIRECT IMMUNOFLUORESCENCE Latex Agglutination for Antibody

Complement Fixation known antigen and unknown antibody are mixed measured amount of complement is added if the antigen and antibody match, they will combine and use up (fix) the complement an indicator system (sensitized RBCs) is added to check for complement-mediated RBC lysis

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IMMUNOLOGIC TESTS IN THE DIAGNOSIS OF DISEASE DR. MARTIN MIGUEL I. AMOR

Indirect ELISA for Antibodies Zones of Precipitation ZONE OF EQUIVALENCE o optimal proportions of antigen and antibody combine o maximal amount of precipitates forms ZONE OF ANTIBODY EXCESS o too much antibody for efficient lattice formation, and precipitation is less than maximal ZONE OF ANTIGEN EXCESS o all antibody has combined, but precipitation is reduced because many antigenantibody complexes are too small to precipitate

Hemagglutination ACTIVE HEMAGGLUTINATION o viruses clump red blood cells from one species or another o inhibited by antibody specifically directed against the virus (hemagglutination inhibition) PASSIVE HEMAGGLUTINATION o RBCs also can absorb many antigens o when mixed with matching antibodies, RBCs will clump together because they are passive carriers of the antigens

Neutralization use the ability of antibodies to: o block effect of toxins o block infectivity of viruses can be used in cell culture (inhibition of cytopathic effect and plaque-reduction assays) or in host animals (mouse protection tests)

Methods of Precipitation PRECIPITATION IN SOLUTION o can be made quantitative o antigen or antibody can be measured in terms of micrograms of nitrogen present o used primarily in research PRECIPITATION IN AGAR o done as either single or double diffusion o can also be done in the presence of an electric field Immunodiffusion also called radial immunodiffusion procedure: o antibody is incorporated into agar o antigen is placed into a well o as the antigen diffuses with time, precipitation rings form depending on antigen concentration the greater the amount of antigen in the well, the farther the ring will be from the well Double Diffusion also known as Ouchterlony method procedure: o antigen and antibody are placed in different wells in agar and allowed to diffuse and form concentration gradients o where optimal proportions occur (zones of equivalence), lines of precipitate form indicates whether antigens are identical, related but not identical, or not related MEDICAL MICROBIOLOGY HANDOUT - PAGE 3 of 4

Precipitation antigen is placed in solution antibody cross-links antigen molecules in variable proportions formation of aggregates (precipitates)

IMMUNOLOGIC TESTS IN THE DIAGNOSIS OF DISEASE DR. MARTIN MIGUEL I. AMOR

Precipitation in Agar with an Electric Field IMMUNOELECTROPHORESIS o procedure: serum sample is placed in a well in agar on a glass slide current is passed through the agar proteins move according to their charge and size a trough is cut into the agar and filled with antibody as the antigen and antibody diffuse toward each other, they form a series of arcs of precipitate o permits the serum proteins to be characterized in terms of their presence, absence, or unusual pattern COUNTER-IMMUNOELECTROPHORESIS o during passage of electric current through agar: o antigen moves toward cathode (positive terminal) o antibody moves toward anode (negative terminal) o meeting of the antigen and antibody is greatly accelerated (visible in 30 to 60 minutes) o EXAMPLE: detection of bacterial and fungal polysaccharide antigens in cerebrospinal fluid Indirect Immunofluorescence

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