AgAb reactions Tests for AgAb reactions Nature of
Ag-Ab reactions Tests for Ag-Ab reactions
Nature of Ag/Ab Reactions • Lock and Key Concept http: //www. med. sc. edu: 85/chime 2/lyso-abfr. htm • Non-covalent Bonds – Hydrogen bonds – Electrostatic bonds – Van der Waal forces – Hydrophobic bonds • Multiple Bonds • Reversible Source: Li, Y. , Li, H. , Smith-Gill, S. J. , Mariuzza, R. A. , Biochemistry 39, 6296, 2000
Affinity • Strength of the reaction between a single antigenic determinant and a single Ab combining site High Affinity Low Affinity Ab Ab Ag Ag Affinity = ∑ attractive and repulsive forces
Calculation of Affinity Ag + Ab Ag-Ab Applying the Law of Mass Action: Keq = [Ag-Ab] [Ag] x [Ab]
Avidity • The overall strength of binding between an Ag with many determinants and multivalent Abs Keq = 104 Affinity 106 Avidity 1010 Avidity
Specificity • The ability of an individual antibody combining site to react with only one antigenic determinant. • The ability of a population of antibody molecules to react with only one antigen.
Cross Reactivity • 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 Cross reactions Anti-A Ab Ag A Ag B Ag C Shared epitope Similar epitope
Factors Affecting Measurement of Ag/Ab Reactions • Affinity • Avidity Ab excess Ag excess • Ag: Ab ratio • Physical form of Ag Equivalence – Lattice formation
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
Agglutination Tests Lattice Formation
Agglutination/Hemagglutination • Definition - tests that have as their endpoint the agglutination of a particulate antigen – Agglutinin/hemagglutinin • Qualitative agglutination test – Ag or Ab + ↔
Agglutination/Hemagglutination • Quantitative agglutination test Neg. Pos. 1/1024 1/512 1/256 1/128 1/64 1/32 1/16 1/8 1/4 Patient 1/2 – Titer – Prozone Titer 1 2 3 64 8 512 4 5 <2 32 6 7 8 128 32 4
– Bacterial infections –Fourfold rise in titer • Practical considerations – Easy – Semi-quantitative 1/512 1/256 1/128 1/64 1/32 1/16 1/8 1/4 • Definition • Qualitative test • Quantitative test • Applications – Blood typing 1/2 Agglutination/Hemagglutination
Passive Agglutination/Hemagglutination • Definition - agglutination test done with a soluble antigen coated onto a particle + ↔ • Applications – Measurement of antibodies to soluble antigens
Coombs (Antiglobulin)Tests • Incomplete Ab • Direct Coombs Test – Detects antibodies on erythrocytes + Patient’s RBCs ↔ Coombs Reagent (Antiglobulin)
Coombs (Antiglobulin)Tests • Indirect Coombs Test – Detects anti-erythrocyte antibodies in serum Step 1 + Patient’s Serum ↔ Target RBCs Step 2 + ↔ Coombs Reagent (Antiglobulin)
Coombs (Antiglobulin)Tests • Applications – Detection of anti-Rh Ab – Autoimmune hemolytic anemia
Agglutination/Hemagglutination Inhibition • Definition - test based on the inhibition of agglutination due to competition with a soluble Ag Prior to Test + ↔ Test + + ↔ Patient’s sample
Agglutination/Hemagglutination Inhibition • Definition • Applications – Measurement of soluble Ag • Practical considerations – Same as agglutination test
Precipitation Tests Lattice Formation
Radial Immunodiffusion (Mancini) • Method Ab in gel – Ag in a well Ag Ag Ag – Diameter of ring is proportional to the concentration • Quantitative Diameter 2 • Interpretation – Ig levels Ag Concentration Ag
Immunoelectrophoresis • Method – Ags are separated by electrophoresis – Ab is placed in trough cut in the agar + - Ag Ag Ab • Interpretation – Precipitin arc represent individual antigens
Immunoelectrophoresis • Method • Interpretation • Qualitative – Relative concentration
Countercurrent electrophoresis • Method – Ag and Ab migrate toward each other by electrophoresis – Used only when Ag and Ab have opposite charges - + Ag • Qualitative –Rapid Ab
Radioimmuoassays (RIA) Enzyme-Linked Immunosorbent Assays (ELISA) Lattice formation not required
Competitive RIA/ELISA for Ag • Method – Determine amount of Ab needed to bind to a known amount of labeled Ag – Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor Prior to Test + ↔ Labeled Ag Test + Labeled Ag + ↔ Patient’s sample +
Competitive RIA/ELISA for Ag • Method cont. – Determine amount of labeled Ag bound to Ab • ↓ NH 4 SO 4 • ↓ anti-Ig • Immobilize the Ab Test + Solid Labeled Ag Phase + + ↔ Patient’s sample Solid Phase – Concentration determined from a standard curve using known amounts of unlabeled Ag • Quantitative – Most sensitive test
Solid Phase Non-Competitive RIA/ELISA • Ab detection – Immobilize Ag – Incubate with sample – Add labeled anti-Ig – Amount of labeled Ab bound is proportional to amount of Ab in the sample • Quantitative Labeled Anti-Ig Ab in Patient’s sample Immobilized Ag Solid Phase
Solid Phase Non-Competitive RIA/ELISA • Ag detection – Immobilize Ab – Incubate with sample – Add labeled antibody – Amount of labeled Ab bound is proportional to the amount of Ag in the sample • Quantitative Labeled Ab Ag in Patient’s sample Ag Immobilized Solid Phase
Tests for Cell Associated Antigens Lattice formation not required
Immunofluorescence • Direct – Ab to tissue Ag is labeled with fluorochrome Fluorochrome Labeled Ab Ag Tissue Section
Immunofluorescence • Indirect – Ab to tissue Ag is unlabeled – Fluorochrome-labeled anti. Ig is used to detect binding of the first Ab. • Qualitative to Semi. Quantitative Unlabeled Ab Fluorochrome Labeled Anti-Ig Ag Tissue Section
Immunofluorescence • Flow Cytometry – Cells in suspension are labeld with fluorescent tag • Direct or Indirect Fluorescence – Cells analyzed on a flow cytometer Flow Tip FL Detector Light Scatter Detector Laser
Immunofluorescence • Flow Cytometry cont. – Data displayed Two Parameter Histogram Number of Cells Unstained cells FITC-labeled cells Green Fluorescence Intensity One Parameter Histogram Red Fluorescence Intensity
Assays Based on Complement Lattice formation not required
Complement Fixation • Methodology – Ag mixed with test serum to be assayed for Ab – Standard amount of complement is added – Erythrocytes coated with Abs is added – Amount of erythrocyte lysis is determined No Ag Ag Patient’s serum
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