Immunology and Serology - 06 Antigen-Antibody Reactions

IMMUNOLOGY AND SEROLOGY • Examples o Herpes virus IgM o Dengue virus o Rabies virus o Scrub and murine typhus • Advantages o Sensitive and specific o Can be used for discrepant analysis Limitations o Expensive (reagents and equipment) o Subjective o Cross reactivity o Non-specific immune-fluorescence Time taken - few minutes to few hours Flow cytometry o Cells in suspension are labeled with fluorescent tag ▪ Direct or indirect fluorescence o Cells analyzed on a flow cytometer ANTIGEN-ANTIBODY REACTIONS OUTLINE • Primary and Secondary Reactions o Primary Serologic Reactions o Secondary Serologic Reactions • Secondary Reactions: Neutralization, Complement Fixation, Flocculation o Neutralization, o Complement Fixation, o Flocculation PRIMARY AND SECONDARY REACTIONS PRIMARY SEROLOGIC REACTIONS • • • • Immunologic/Serologic Assay o Tests that involve the reaction antibody & antigen to detect disease or conditions Immunoassays o Based on immunologic/serologic reactions (ag-ab) o Uses of ligands ▪ Substances that complex to another substance • React ab with ag • React ab to enzymes/hormones ▪ Substance to be measured (analyte) • Analyte - measured Ab bound with ag Affinity o Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site o Affinity is the equilibrium constant that describes the antigen-antibody reaction o Binding of antigen and antibody Avidity o Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies o Avidity is influenced by the valence of the antibody and the valence of the antigen (multivalent ab) o Avidity is more than the sum of the individual affinities o Speed of antigen-antibody reaction • • • RADIO-IMMUNOASSAY (RIA) • • • • • Assays utilizes labels/labelling substance • IMMUNOFLOURESCENCE ASSAY (IFA) • • • Aka Fluorescence Immunoassay (FIA) Label: fluorochrome/fluorescent dye Principle o Use fluorescein isothiocyanate (FITs) labeledimmunoglobulin to detect antigens or antibodies according to test systems ▪ Emits green color o Requires a fluorescent microscope • Label: radioactive isotopes o I-125 - commonly used Principle o Radioactively labelled-antibody (or antigen) competes with the patient’s unlabeled antibody (or antigen) for binding sites on a known amount of antigen (or antibody) o Reduction in radioactivity of the antigen-patient antibody complex compared with control test is used to quantify the amount of patient antibody/antibody bound Example o HBsAg o Thyroid function test Advantages o Highly sensitive o Can be used for detection of small quantities o Quantification possible Limitations o Expensive o Requires isotopes o Can be hazardous due to radioactivity Time taken - 1 day ENZYME LINKED IMMUNO-SORBENT ASSAY (ELISA) • • • • Label: enzymes o Alkaline phosphatase, horseradish phosphatase Principle o Use of enzyme-labeled immunoglobulin to detect antigens or antibodies o Signals are developed by the action of hydrolyzing enzyme on chromogenic substrate o Optical density measured by micro-plate reader (based on spectrophotometry) o Can be interpreted visually or through spectrometry Examples o Hepatitis A (Anti-HAV-IgM, anti-HAV Iggy) Types of ELISA (Ag Abs tests) o Competitive o Non-competitive • • • Advantages o Automated, inexpensive o Objective o Small quantities required o Class specific antibodies measurable Limitations o Expensive initial investment o Variable sensitivity/specificity of variable tests o Cross contamination Time taken - 1day CHEMILUMINESCENT IMMUNOENZYMATIC ASSAY • • • Label: Organic label o Luminol - commonly used Process for the quantitative and qualitative determination of antigens, antibodies, and their complexes by means of a chemiluminescing labeling substance activated or excited to chemiluminescence’s by an analytical reagent By means of a serological reaction, initially and antigen/antibody complex is formed which is treated with a chemiluminescing conjugate containing chemiluminescing triphenylmethane dyes and the chemiluminescence of the chemiluminescing complex formed is measured. SECONDARY SEROLOGIC REACTIONS PRECIPITATION REACTIONS • • • Competitive ELISA o Antigen or antibody are labeled with enzyme and allowed to compete with unlabeled ones 9in patient serum) for binding to the same target o Hydrolysis signal form AB-Ag complex (enzymelabeled) is measured o Antigen or antibody in serum is then calculates o No need to remove the excess/unbound Ag or Ab from the reaction plate or tubes Non-competitive ELISA o Commonly performed o Must remove excess/unbound Ag or Ab before every step of reactions o Materials ▪ Diluent ▪ Wash solution - separates bound & unbound ab/ag ▪ Antibody conjugate - attaches labeled enzyme ▪ Substrate A & B - color developer ▪ Stop solution - stops the rxn o Direct ELISA ▪ Detect antigen o Indirect ELISA ▪ Detects antibody o Sandwich ELISA ▪ Commonly performed ▪ Uses 2 ab to “sandwich” antigen or vice versa o Ab Capture ELISA ▪ Similar to sandwich ELISA but in 1st step, anti-Ig (M or G) is coated on the plate ▪ Then, antibodies in patient serum are allowed to capture in the next step • • • • Precipitation o Involves combining soluble antigen with soluble antibody to produce insoluble complexes that are visible in the form of precipitate Principle o Soluble antigen + antibody (in proper proportions) → visible precipitation o Lattice formation (antigen binds with Fab sites of 2 antibodies) Precipitation Reactions o Precipitin (soluble antibody) combines with precipitinogen (soluble antigen) in 2 steps: ▪ Rapid invisible formation of antigen-antibody aggregates (sensitization) ▪ Slowly forming visible complex lattice of interlocking aggregates (lattice phenomenon/hypothesis) • Cross-linking of ag and ab Precipitation curve o Prozone – antibody excess o Postzone – antigen excess o Zone of equivalence – optimum antigen and antibody concentration Gel Immunodiffusion - most commonly used technique o Examples ▪ Double Diffusion (Ouchterlony) ▪ Single diffusion (radial immunodiffusion) ▪ Immunoelectrophoresis ▪ Immunofixation o Oudin Method ▪ Single diffusion, single dimension ▪ Only antigen diffuses, antibody is already present in the agarose gel o Radial Immunodiffusion (Mancini) ▪ Single diffusion in double dimension ▪ In radial immunodiffusion, antibody is incorporated in the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. ▪ As antigen diffuses into gel, it reacts with antibody and when the equivalence point is reached, a ring of precipitation (precipitin ring) is formed o Oakley and Fulthrope ▪ Double diffusion, single dimension ▪ Both antigen & antibody diffuse ▪ Produces single precipitin line Double Immunodiffusion by Ouchterlony ▪ Used more often ▪ Double diffusion, double dimension ▪ Presence of precipitin lines ▪ Antigen and antibody are allowed to diffuse towards each other in an agar medium, e.g. from separate wells cut in an agar plate or in Petri dish. ▪ When antigen and antibody meet in optimal proportions they produce a thin line of precipitate. ▪ Position of the precipitate line depends on concentrations of both antigen and antibody and on their diffusion coefficient ▪ This reaction is used for diagnosing various bacterial, viral, fungal, and autoimmune disease, for recognizing toxin production by Corynebacterium diptheria ▪ Diffusion patterns • Fusion of lines at their junction to form an arc o Serologic identity/presence of common epitope • Crossed lines o Demonstrates 2 separate reactions o Compared antigens shared no common epitopes • Fusion of 2 lines with spur o Partial identity o o • Ring test ▪ Aka Fluid precipitation ▪ Layering of antigen solution over a column of antiserum in a narrow tube ▪ Precipitate at the junction (cloudiness/turbidity between ag-ab) ▪ E.g. Ascoli’s inter thermoprecipitin test, grouping of Streptococci by Lancefield Test Immunoelectrophoresis (IEP) Qualitative o A serum sample is electrophoresed through an agar medium o A trough is cut in the agar and filled with Ab o A precipitin arc is then formed o Because Ag diffuses radially and Ab from a trough diffuses, the reactants meet in optimal proportions for precipitation o Counter Immunoelectrophoresis ▪ Reactions occur between migrating Ag’s and Ab’s during electrophoresis (Ag and Ab migrate towards each other by electrophoresis) ▪ Used only when Ag and Ab have opposite charges ▪ Pairs of wells are punched in agarose plates in which Ag is Ag is placed in one well of each pair and Ab in the other ▪ Following electrophoresis, precipitin lines will be visible between the wells of a pair of wells of matching specificity ▪ Qualitative ▪ Its major advantage is its speed. o Rocket Immunoelectrophoresis ▪ Aka spike immunoelectrophoresis ▪ Antigen is electrophoresed into gel containing antibody ▪ The distance from starting well to front of rocket shaped arc is related to antigen concentration AGGLUTINATION REACTIONS • • • • Agglutination o soluble antibody (agglutinin) forms a lattice with an insoluble (particulate or cellular) surface antigen Types of particles that participate in such reactions o Erythrocytes o Bacterial cells o Inert carriers such as latex particles Steps In Agglutination o Primary Phenomenon (Sensitization) ▪ First reaction involving Ag-Ab combination ▪ Single antigenic determinant on the surface particle ▪ Initial reaction - rapid and reversible ▪ Cross link formation → visible aggregates (stabilization) o Secondary Phenomenon (Lattice Formation) ▪ The Fab portion of the Ig molecule attaches to antigens on 2 adjacent cells-visible results in agglutination ▪ If both antigen and antibody are soluble, reaction will become visible over time (ie, precipitation) Direct Agglutination o Principle ▪ Combination of an insoluble particulate antigen with its soluble antibody • Forms antigen=antibody complex • Particles clump/agglutinate ▪ Used for antigen detection o Examples ▪ Bacterial agglutination test for sero-typing and sero-grouping (eg. Vibrio cholera, Salmonella spp.) o Slide Agglutination Test o Tube Agglutination TestS • • • • • Reverse Passive Agglutination Tests o Antibody rather than antigen is attached to a carrier particle o For the detection of microbial antigens such as ▪ Group A and B streptococcus ▪ Staphylococcus aureus ▪ Neisseria meningitides ▪ Heamophilus influenza ▪ Rotavirus ▪ Cryptococcus neoformans ▪ Mycoplasma pneumoniae ▪ Candida albicans Viral Hemagglutination o Some viruses and microbes contain proteins which bind to erythrocytes (RBCs) causing them to clump to together o Examples ▪ NDV ▪ Adenovirus III ▪ AIV ▪ IBV ▪ Mycoplasma Coomb’s Test o An agglutination test o The Coomb’s Test is actually two separate tests: the direct and indirect Coomb’s test. Both aim to identify autoimmune hemolysis of RBCs o Applications ▪ Detection of anti-Rh ab ▪ Autoimmune hemolytic anemia Agglutination Inhibition tests o Pregnancy Test ▪ Classic example of agglutination inhibition ▪ Human chronic gonadotropin (hCG) • Appears in serum & urine early in pregnancy o Weil-Felix Test ▪ Heterophile agglutination test ▪ The agglutination reactions based on antigens common to both organisms, determine the presence and type of Rickettsial infection ▪ Because Rickettsia are both fastidious and hazardous, few laboratories undertake their isolation and diagnostic identification ▪ Weil-Felix test based on cross-reactive antigens on OX-19 & OX-2 strains of Proteus vulgaris ▪ Latex Agglutination o Antibody molecules can be bound to each latex beads o It will increase the potential number of exposed antigen-binding sites o When an antigen is present in test specimen, it may bind to the latex bead thus forming visible cross-linked aggregates o Latex particles can be coated with antigen (pregnancy testing, rubella antibody testing) SECONDARY REACTIONS: NEUTRALIZATION, COMPLEMENT FIXATION, FLOCCULATION • NEUTRALIZATION • • • Neutralizing antibody plus complement, neutralizes infectivity of microorganisms Antibody (antitoxin) can neutralize microbial toxin Neutralization reactions o Generally, positive control samples used in inhibition or neutralization tests show no reaction and negative control samples show a reaction (opposite of results in direct agglutination testing) o Example of inhibition: Hemagglutination inhibition test for rubella o Example of neutralization: anti-streptolysin O Test (ASO) COMPLEMENT FIXATION (CF) • • • • • • • • Viral Neutralization o Viruses introduced into appropriate cell cultures will invade and kill the cells, a phenomenon called cytopathic effect o The ability of a virus to kill culture cells is neutralized when the virus is first mixed with antibodies against it o Absence of cytopathic effect indicates the presence of antibodies against the virus o This test can also be used to test toxins o Advantages - sensitive and specific enough to identify whether an individual has been exposed to a particular virus or viral strain Toxin Antitoxin Neutralization o In neutralization reactions, the harmful effects of a bacterial exotoxin or virus are eliminated by a specific antibody o An antitoxin is an antibody produced in response to a bacterial exotoxin or a toxoid that neutralizes exotoxin o In a virus neutralization test, the presence of antibodies against a virus can be detected by the antibodies’ ability to prevent cytopathic effect sof viruses in cell cultures Occurs after the binding of antigen and antibody, uptake of complement can be used as an indicator of the presence of either specific antigen or antibody. 2 systems involved: o Test system/bacteriolytic system o Indicator system/hemolytic system Antibody and antigen allowed to combine in presence of complement If complement is fixed by specific antigen-antibody reaction, it will be unable to combine with indicator system Precautions o Serum must be heat-activated o Stored serum become anti-complementary o Extensive QC/standardization required o Only use for IgM antibodies Methodology o Ag mixed with test serum to be assayed for Ab o Standard amount of complement is added o RBCs coated with Abs is added o Amount of RBC lysis is determined REVIEW • FLOCCULATION • • Tube flocculation test o Khan test for syphilis o Standardization of toxins and toxoids o Serial dilutions of toxins and toxoids added to fixed quantity of antitoxin o Amount of toxin/toxoid that flocculates with fixed quantity of antitoxin is defined as 1 Lf dose Agglutination of colloidal particles Slide Flocculation Test o Antigen & antiserum placed on slide & mixed by shaking o Appearance of floccules • Primary Reactions o Reaction is not observable o Use labeling substances o Immunoassays ▪ Based on preprocedural requirement on ligand • Separation technique, separate bound from unbound • Requires washing • Use of solid base (eg. ELISA) • Heterogenous - require solid base & washing • Homogenous - doesn’t require solid base & washing ▪ Based on presence/absence of labels • Use of labeling substance ( to facilitate reaction) • Non-label based assay o Usually, labelled assays ▪ Heterogenous ▪ Homogenous o FIA Secondary Reactions o Has observable reaction o Employ lattice formation/hypothesis o • Involves two stages ▪ Sensitization ▪ Lattice formation o Precipitation ▪ Fluid precipitation - original, named by Ascoli (ring-precipitin test) ▪ Gel precipitation/immunodiffusion • Occur in petri dish by semi-solid agarose gel • Oudin - single diffusion, single dimension • Mancini - single diffusion, double dimension o Radial immunodiffusion - Fahey & Mancini o Mancini - measures diffusion within 24h o Fahey - measures diffusion within 18h • Oakley & Fulthorpe - double diffusion, single dimension • Ouchterlony - double diffusion, double dimension ▪ Immunoelectrophoresis - combination of diffusion and electrophoresis • Countercurrent - (+) precipitin line/s • Rocket - (+) sharp precipitin arc o Agglutination ▪ Direct ▪ Indirect • Passive • Reverse passive ▪ Viral hemagglutination ▪ Antiglobulin test ▪ Agglutination inhibition o Hemolysis o Neutralization ▪ Antibody reduces harmful effect of antigen (pathogens: S. pyogenes) ▪ Infectivity of an antigen is reduced ▪ Toxic - (+) = no hemolysis ▪ Viral - (+) = no hemolysis o Flocculation ▪ In syphilis ▪ Combination of precipitation and agglutination ▪ (+) - presence of floccules ▪ Floccule - discreet flaky substance o Complement Fixation ▪ Two stages • Ag is reacted with antibody o If ab is present in serum, complement will fix when there is ab-ag rxn causing complement activation & destruction o If ab is absent, complement is unfixed • Addition of ag-ab complex (indicator) o To fix complement to observe lysis ▪ (+) = no hemolysis ▪ (-) = hemolysis Tertiary Reaction o Reactions are not observable but observed on affected tissue or cells o Phagocytosis o Opsonization o Chemotaxis o