Antigen-antibody interactions – Flashcards
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The study of antibodies and antigens in serum using in vitro immunologic test is known as _____________.
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Serology
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What is the combination of antibody and antigen?
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sensitization
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What is the ability of a test to give a positive result if the entity being tested for is present?
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sensitivity
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How is sensitivity calculated?
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% = (TP/ (TP+FN)) x 100
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What is a test which should be positive but gives negative results
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False negative
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What is the ability of a test to give a negative result if the substance being tested for is not present?
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specificity
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How is specificity calculated?
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% = (TN/(TN+FP)) x 100
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What is a test which should be negative but gives positive results?
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false positive
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What is the ability of a test to detect small amounts of a particular substance
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Analytical sensitivity
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What is the ability to detect only the substance being tested for?
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Analytical specificity
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In analytical specificity, what is not detected in high specificity test?
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Cross-reacting substances.
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What is the strength of the noncovalent bond between paratope of antibody and epitope of antigen
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Affinity
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What is the strength of a bond formed between a complete divalent antibody and its corresponding antigen?
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Avidity
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When are high sensitivity & specificity testing applied?
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In diagnosis and management of patients with infectious diseases, quantitation of chemical constituents in patient blood (tumor markers), Blood compatibility for transfusions, leukocyte differentiation and immune system evaluation.
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How are test with high sensitivity and specificity applied when diagnosing and managing patients with infectious diseases?
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The samples are collected 10 days to 2 weeks apart. Look for a four-fold increase in titer which indicate a current infection.
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What are the types of immunologic reactions?
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Primary, secondary and tertiary.
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What are the types of test sensitivities?
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Nonlattice and Lattice
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Nonlattice is a test sensitivity that is _______________.
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more sensitive
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Lattice is a test sensitivity that is __________________.
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less sensitive
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Serological testing are done to detect either _______________.
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antibodies or antigens
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What is usually performed to detect antibodies and sometimes used to detect antibodies particularly in lag phase of antibody production?
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Serological testing
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Which immunologic reaction is a simple combination of antigen with specific antibody and is the most sensitive type?
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Primary.
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Immunofluorescence, radioimmunoassay, immunoenzymatic tests (all have tags to see if binding occurred) are all _____________________.
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Examples of primary immunologic reactions
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Which immunologic reaction is a antigen-antibody combination based on secondary manifestation?
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Secondary.
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Precipitation, flocculation, agglutination, complement fixation, immunoelectrophoresis are all ___________________.
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Examples of secondary immunologic reactions
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Which immunologic reactions are biologic reactions?
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Tertiary.
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Biologic effects of complement activation (opsonization, phagocytosis, chemotaxis), immune adherence reactions, cellular degradation are all _______________________.
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Examples Tertiary immunologic reactions
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What is the interaction of soluble antigen and antibody in proper proportions resulting in visible precipitate?
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Precipitation
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What substance is an antibody that causes precipitation?
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Precipitin.
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Optimum ratio of soluble antigen to antibody and antibody binding to 2 antigens with its 2 Fab (antigen binding) sites are some _________________.
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principles of precipitation
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In principle of precipitation, what happens to the size of the antigen-antibody complexes?
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They grow to large complexes which settle out due to their size.
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What principle of precipitation deals with precipitation from solution in the form of fleecy or downy masses?
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Flocculation.
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What is the Lattic hypothesis?
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Antibody is divalent, antigen has multiple reactive sites. Antibody may be bound with antigen to form coarse "lattice." Antigen-antibody complexes fall out of solution, equivalence zone and excess of either antigen or antibody may produce false negative results.
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What is an equivalence zone?
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When optimum proportions of antigen and antibody are present.
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What is influenced by quantities of antigen and antibodies present?
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The equivalence zone
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What can cause a false negative according to the Lattice hypothesis?
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Excess of antigen or antibody, the prozone and the postzone.
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What involves antibody excess causing the antibody reacts more strongly diluted than undilute?
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Prozone
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What involves antigen excess causing the inability to form lattice?
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Postzone
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In _________________, Fairly good quantitative estimate of antigen or antibody in an unknown. The _____________ is compared to standard.
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precipitation; Equivalence zone
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Quantities of antigen and antibody, temperature, pH and reaction time and salt concentration are _______________.
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Factors affecting precipitation
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What is the pH in precipitation and what happens if it is too high or too low?
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Between 6.0-7.5. If not within this range it causes dissociation of immune complex.
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In high salt concentrations, what happens to the immune complexes?
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Increases its solubility and cause dissociation of the complexes.
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In low salt concentrations (<0.15M), what happens to the degree of precipitation?
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There is a striking decrease in amount of precipitate.
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A ring test, immunodiffusion, flocculation, electroimmunodiffusion are all ________________.
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Uses of precipitation reactions.
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What technique involves patient serum overlaid with soluble antigen (in gel) in test tube or capillary tube and the antibody diffusing upward forming a fine line of precipitate during overnight incubation?
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Ring test
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This is considered the simplest form of precipitation reaction
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Ring test
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What test involves the forming of precipitate by soluble antigens interacting with antibody which are fine particles visible by particles that are forced to remain in a confined space?
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Flocculation
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RPR and VDRL are ________________.
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Examples of flocculation.
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Which test includes radial single diffusion and Double diffusion?
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Immunodiffusion
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What technique in immunodiffusion involves antiserum incorporated in gel, antigen (patient sample) added to well cut in gel and at optimal proportions of both a circle of precipitation forms with the precipitin diameter proportional to concentration of Ag with a single Ag giving single precipitation circle and multiple Ags giving multiple circles?
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Radial Immunodiffusion (RID)
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What is another name for radial immunodiffusion and what is it used for?
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Radial single diffusion. It quantitates immunoglobulins, complement and other serum proteins.
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What technique involves Gel has circular wells cut in it, antibody (reagent) and antigen (patient serum) are added to wells, incubate both antigen and antibody diffusing and when well size and shape, distance between wells, temperature and incubation time are optimal, lines of visible precipitate form at the point of equivalence?
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Double diffusion
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What is another name for diffusion precipitation in gel and what is it used for?
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Ouchterlony. It identify antibodies associated with autoimmune disorders.
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What are some examples of the autoimmune disorders that the ouchterlony detects?
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Rheumatoid arthritis, SLE, systemic sclerosis and Sjogren's syndome.
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What are the different types of electroimmunodiffusion?
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Rocket electrophoresis and countercurrent electrophoresis.
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What technique of electroimmunodiffusion involves antibody incorporated into gel, antigen added to wells using voltage applied to increase rate of diffusion with precipitation pattern resembling a shooting rocket and antigen concentration proportional to the height of rocket?
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Rocket electrophoresis
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What is another name for rocket electrophoresis and what are the techniques combined in this one technique?
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Laurell. Combines single diffusion precipitation with electrophoresis.
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Which electroimmunodiffusion technique involves Ag & Ab placed in well opposite from one another, the pH chosen so that Ab travels to cathode and Ag migrates to anode (pH 8.6) and Ag & Ab driven toward each other with precipitin line forms where reactants meet in optimal proportions?
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Countercurrent electrophoresis
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What are the techniques combined in countercurrent electrophoresis and when is it used?
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Double diffusion precipitation and electrophoresis. Utilized when rapid results are necessary as in diagnosis of bacterial meningitis.
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What semiquantitative technique involves Ag migrating through gel under electrical current, the current is discontinued, the trough in agar is filled with Ab, incubated, the Ag diffusing radially and Ab diffusing in a plane with reactants meeting in optimal proportions for precipitation to form an arc and being closest to the trough at point where antigen is in concentration?
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Immunoelectrophoresis (IEP)
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What is the use of immunoelectrophoresis?
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To differentiate antigens in a mixture, identify monoclonal proteins (k and l free light chains), immunoglobulin classes and urine proteins.
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Why is IEP considered a semiquantitative method?
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The size of the arc indicates amount of antibody present. The shape and position of the arc may indicate monoclonality of protein.
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Agglutination is similar to precipitation reactions. What is the difference between agglutination and precipitation?
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Antibody combines with particulate antigen rather than soluble antigens and results in visible clumping.
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What are some similarities between agglutination and precipitation in reference to formation and equivalence zone?
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It has lattice formation and is subject to prozone and postzone.
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What can cause a prozone in agglutination?
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antibody excess, presence of IgG blocking antibodies and weak avidity.
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What qualitative/semiquantitative technique involves Ab attaching to particulate or insoluble Ag (RBC, latex particle, bacteria, yeast), random collisions of sensitized Ags forming bridges or lattice between antigens linking the together resulting in visible clumps?
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Agglutination
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Why is agglutination considered a qualitative/semiquantitative technique?
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The strenght of antibody is reciprocal of highest dilution that gives a positive reaction.
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What factors influence agglutination reactions?
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Quantities of antigen and antibody, temperature, motion, neutral pH, and antibody class.
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Which antibody is a good agglutinin and why?
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IgM due to its large size allowing binding up to 5 antigens.
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Which antibody is a poor agglutinin and why?
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IgG. It often require special conditions in order to produce agglutination.
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What are the types of agglutination?
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Direct, passive, and reverse passive.
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Which agglutination reaction has the antigen as a natural constituent of the particle?
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Direct.
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When is direct agglutination reaction used?
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Hemagglutination, RBCs in blood banking and testing for cold agglutinins.
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Which agglutination reaction consist of the antigen being firmly attached to insoluble particles?
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Passive
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What are the subtypes of passive agglutination?
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Passive hemagglutination, reverse passive hemagglutination and passive latex agglutination.
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Which passive agglutination reaction consist of the antigen coating the RBC and treating the RBC with tannic acid to enhance attachment (coating)?
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Passive hemagglutination.
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Which passive agglutination reaction consist of the antibody attaching to the RBC?
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Reverse passive hemagglutination.
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Which passive agglutination reaction involves the latex being used as an insoluble particle?
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Passive latex agglutination.
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What are the insoluble particles involved in both reverse and passive hemagglutination?
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Erythrocytes.
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Which agglutination reaction consist of the antibody attaching to a carrier and is a test for antigen?
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Reverse passive.
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What are the steps in agglutination inhibition?
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The patient's serum (antibody) is incubated with known antigen. The indicator cells (antibody attached to RBC or latex-coated particle) are then added.
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What does it mean to have no agglutination in agglutination inhibition?
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Positive test. The antigen and antibody in original system were specific and no antigen is left to react with antibody on indicator cells.
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What does it mean to have agglutination in agglutination inhibition?
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Negative test. The antigen and antibody were not specific leaving antigen free to react with antibody on indicator cells.
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What is the use of agglutination inhibition?
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To detect HCG in serum or urine, rubella, or soluble A, B and H substances in body fluids.
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What technique involves an antigen and antibody combining in the presence of complement, the complement is "fixed" by antigen-antibody complexes?
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Complement fixation test.
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What stage of complement fixation involves heating patient serum to 56 degrees, Ag (known) & Ab (unknown) are mixed, a specific amount of C' (from guinea pig which is more active as a source of C' than most other species) is added, and if specific Ag or Ab is present for the known Ag or Ab, they will combine and fix C?
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The first stage.
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What stage of complement fixation involves indicator system consisting of sensitized RBCs (coated with rabbit antibodies to SRBCs - called hemolysins) being added?
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The second stage
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What are the test results if C' was fixed in 1st stage, C' is not available to attach to sensitized RBCs and they remain unhemolyzed?
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Positive test (no hemolysis)
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What are the test results if Ag-Ab reaction did not take place in 1st stage, C' is available to hemolyze sensitized RBCs of indicator?
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Negative test (hemolysis)
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Why must C' be carefully standardized?
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Excessive amounts may cause hemolysis even though Ag-Ab reaction occurred in 1st stage.
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Why are specimens not stored for long periods of time?
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Ab titers decrease and sera may become anti-complementary
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What must happen to antigen in complement fixation?
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It must be quantitated.
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How are result expressed in complement?
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The highest dilution of serum that gives positive results
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Why is it necessary for elaborate controls?
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To validate results
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What are the uses of complement fixation?
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In diagnosis of syphilis and some parasitic diseases.
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Complement fixation is only applicable to what antibodies and what is the consequence of this?
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IgM. It will have limited usefulness.
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What are the sources of error in complement fixation tests?
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False positive and false negative.
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Inactive complement, improper dilution of patient serum, anti-complementary components in patient serum and anti-complementary antigen are all_____________________.
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Causes of false positives in C' fixation test causing no or decreased lysis.
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Fragile sheep red blood cells, failure to heat inactivate serum, failure to add antigen or antibody and patient serum over diluted are all __________________.
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Causes of false negatives in C' fixation tests.
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When is neutralization of toxins useful?
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When neither precipitation or agglutination is successful in demonstrating Ag-Ab reaction.
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What reaction involves testing for neutralizing antibodies to toxins due to bacterial infection producing toxins more damaging to the host than the bacteria?
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Neutralization of toxins
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What technique involves patient serum incubated with Streptolysin O reagent (toxin), incubate, RBCs are added and if patient has neutralizing antibodies, RBCs will not hemolyze and is an example of neutralization of toxins test?
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Antistreptolysin O test
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In the antistreptolysin O test, if the patient has neutralizing antibodies what will happen?
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The RBCs will no hemolyze.
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What semiquantitative technique involves specific antibody being labeled with fluorescent dye and then layered on a tissue section containing suspected antigen?
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Fluorescent antibody reactions (immunofluorescence)
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What fluoresenct dye(s) are used in immunofluorescence?
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fluorescein or rhodamine.
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What are the techniques used in immunofluorescence?
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Direct and Indirect.
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Which immunofluorescence technique is used for detection of antigen in unknown tissue and identifies unknown antigen by using known fluorescein-labeled antibody with the unknown antigen being exposed to labels antibody, washed, and examined with fluorescent microscope?
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Direct or single layer.
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Which immunofluorescence technique is used for detecting an antibody in unknown sera by using Ag in tissue (reagent) on a slide, adding patient serum (containing antibody) then adding labeled AHG and then examined for fluorescence which indicate reaction between antigen, the unknown antibody, and labeled antiglobulin?
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Indirect or double-layer.
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Which quantitative immunofluorescence technique is based on change in polarization of fluorescent light emitted from a labeled molecule when it is bound to an Ab, incident light directed at sample is polarized with a filter so that light waves are oriented in one plane?
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Fluorescent polarization immunoassay (FPIA)
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Which molecules would cause an FPIA interpretation consisting of polarization rotating slowly, emitting a high degree of polarized light?
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Large molecules such as immune complexes with fluorescent labels.
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Which molecules would cause an FPIA interpretation consisting of polarization rotating rapidly, emitting light in many directions resulting in depolarization?
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Small molecules such as free fluorescent labeled haptens.
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What quantitative technique involves fluorescent-labeled Ag competing with unlabeled Ag in patient sample for limited amount of Ab with the amount of fluorescence being inversely proportional to concentration of Ag in patient sample?
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Competitive FPIA
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What happens if the antigen concentration in patient sample is high in competitive FPIA?
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There will be less bound fluorescence-labeled antigen and less polarization
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What is the use of competitive FPIA and what is required?
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Small molecules which rotate freely. Sophisticated instrumentation is needed.
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What are the small molecules that rotate freely in competitive FPIA?
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Therapeutic drugs and hormones.
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What quantitative technique Employs radioactive isotopes as tags such as 125I, 131I, 58Co, high sensitivity of detectors for small amounts of radioactivity and high specificity for antibody, allow quantitation of very small amounts of biologic substances, accurate to ng/mL or pg/mL, useful for measuring hormones, vitamins and drugs and need a ligand which is any substances that will complex to another substance to be measured in this technique?
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Immunoassay
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What reagents are required in immunoassay?
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Antibody specific for the antigen (patient serum) being measured, labeled antigen, standard preparation of antigen and system to separate bound antigen from free antigen.
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What are the different techniques in immunoassay?
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Radioimmunoassay (RIA) and Enzyme immunoassay (EIA).
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Which Immunoassay is antibody specific and have sensitivity of detectors for small amounts of radioactivity?
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Radioimmunoassay
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What are some disadvantages to RIA?
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Exposure to radiation, costly and inconvenient waste disposal, short shelf-life of labeled reagents, labor intensive, costly procedures and bothersome bureaucratic procedures (licensing and inspections).
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Which immunoassay technique is similar to RIA but is labeled with stable enzyme instead, depends on competition between enzyme-labeled and unlabeled antigen or hapten for binding to limited amount of antibody which commonly use horseradish peroxidase and alkaline phosphatase?
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Enzyme Immunoassay (EIA)
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In EIA, what effects choice of enzymes?
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Stability, cost, generation of suitable chromophore or fluorochrome that is easily quantified.
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What are the advantages of EIA?
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Easily automated, stable, does not possess hazards of radioactivity and tissue preparations do not fade as in fluorescence procedures.
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What quantitative technique involves antibody having the same avidity for labeled and unlabeled antigen, the unlabeled antigen (patient serum) is added to a limited amount of specific antibody, a known concentration of radio-labeled antigen is added, the labeled and unlabeled compete for binding on antibody, at equilibrium the bound ag-ab complexes are separated from unbound and the radioactivity is counted?
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Competitive RIA
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How is a competitive RIA interpreted?
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Ratio of antibody-bound, labeled Ag decreases as concentration of unlabeled Ag (patient serum) increases. Readings compared to standard curve. The higher the concentration of Ag in patient, the lower the radioactive counts.
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What are some examples of competitive RIA?
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The radioallergosorbent test (RAST) and the radioimmunosorbent test (RIST).
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What does the RAST measure?
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This specialized RIA procedure measure the amount of serum IgE against specific inhaled or ingested antigens.
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What quantitative technique involves the antibody having different avidities for labeled and unlabeled antigen (patient serum with the unlabeled Ag (patient serum) is mixed with excess Ab and incubated to achieve equilibrium, the labeled Ag is added & allowed to reach equilibrium and after separation the bound radioactive counts are determined and compared to standards?
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Sequential RIA
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What quantitative technique involves the Ab attaching to solid phase support, the unknown Ag (patient) is allowed to react with Ab-coated beads, a wash removes free Ag, radiolabeled Ab is added that reacts with bound Ag at different site (sandwich technique), a wash removes excess unbound labeled Ab and the counts are read with concentration of Ag (unknown) being directly proportional to counts ?
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Immunoradiometric assay (IRMA)
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Immunoradiometric Assay (IRMA) is also known as ________________.
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"Sandwich" RIA.
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IRMA is used for large molecules and not small molecules. Why is this so?
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The antigen must be able to bind the two antibodies simultaneously.
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What quantitative technique involves Ab adsorbed on solid phase surface, patient serum(antigen) added, Ag (if present) binds to fixed Ab, wash, add excess of enzyme-labeled Ab which binds to all open binding sites of Ag, wash to remove excess labeled Ag, add enzyme substrate, incubate and measure absorbance of color produced with Ag concentration being proportional to absorbance?
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Enzyme-linked Immunosorbent Assay (ELISA)
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What is Enzyme-linked immunosorbent assay (ELISA)?
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HETEROGENEOUS immunoassay system necessary to separate bound labeled antigen-antibody complex from free label.
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ELISA can also quantify antibodies. How is this done?
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The antigen is bound to solid phase and sample with antibody is added. After washing, labeled antibody (from a different species) directed against the first antibody is added. A substrate is added and absorbance is measured and the concentration of antibody read from standard curve.
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ELISA is used to indentify viruses and antibodies against viruses, bateria and parasites. what is its primary use and official method for?
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It measures large molecules requiring two antibodies to bind to the antigen. It is sanctioned for screening of HIV/AIDS.
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What quantitative technique involves patient serum added to enzyme-drug complex (reagent), anti-drug Ab is added, incubate, add enzyme substrate, incubate and read absorbance of color formation?
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Enzyme-multiplied immunoassay (EMIT)
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What is Enzyme-multiplied immunoassay (EMIT) and what is its use?
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A homogeneous system that does not need separation of bound from free hapten. It is best suited for the analysis of small molecules such as drugs, hormones and thyroxine and requires that antibody when count to antigen be in sufficient close proximity to enzyme to inactivate it or the antibody may bind at a site too far from the enzyme to hinder its activity for large antigens.
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In EMIT, why is separation unnecessary?
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Enzymatic activity is severely reduced when it binds to an antibody.
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In EMIT, the drug in the patient's serum attaches to the anti drug antibody leaving active sites on enzyme available for binding with substrate which causes color formation. What is this an indication of?
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A positive test.
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In EMIT, the anti-drug antibody attaches to the drug on the enzyme-drug complex blocking the active sites on the enzyme leaving no available binding site on enzyme for substrate. No color is formed. What is this an indication of?
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A negative test.
