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Ag-Ab reactions Tests for Ag-Ab reactions. Nature of Ag/Ab Reactions Lock and Key Concept Non-covalent Bonds – Hydrogen bonds – Electrostatic bonds –

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Presentation on theme: "Ag-Ab reactions Tests for Ag-Ab reactions. Nature of Ag/Ab Reactions Lock and Key Concept Non-covalent Bonds – Hydrogen bonds – Electrostatic bonds –"— Presentation transcript:

1 Ag-Ab reactions Tests for Ag-Ab reactions

2 Nature of Ag/Ab Reactions Lock and Key Concept Non-covalent Bonds – Hydrogen bonds – Electrostatic bonds – Van der Waal forces – Hydrophobic bonds Reversible Multiple Bonds Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000

3 Affinity = ∑ attractive and repulsive forces Ab Ag High Affinity Ab Ag Low Affinity Affinity Strength of the reaction between a single antigenic determinant and a single Ab combining site

4 Calculation of Affinity Ag + Ab Ag-Ab K eq = [Ag-Ab] [Ag] x [Ab] Applying the Law of Mass Action:

5 Avidity The overall strength of binding between an Ag with many determinants and multivalent Abs K eq = 10 4 Affinity 10 6 Avidity 10

6 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.

7 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 Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope Anti-A Ab Ag C Similar epitope Cross reactions

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

9 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

10 Agglutination Tests Lattice Formation

11 Agglutination/Hemagglutination Definition - tests that have as their endpoint the agglutination of a particulate antigen –Agglutinin/hemagglutinin + ↔ Qualitative agglutination test –Ag or Ab

12 Agglutination/Hemagglutination Quantitative agglutination test –Titer –Prozone 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer < Patient

13 Agglutination/Hemagglutination Definition Qualitative test Quantitative test Applications – Blood typing – Bacterial infections –Fourfold rise in titer Practical considerations – Easy – Semi-quantitative 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512

14 Passive Agglutination/Hemagglutination Definition - agglutination test done with a soluble antigen coated onto a particle + ↔ Applications –Measurement of antibodies to soluble antigens

15 Coombs (Antiglobulin)Tests Incomplete Ab Direct Coombs Test – Detects antibodies on erythrocytes + ↔ Patient’s RBCs Coombs Reagent (Antiglobulin)

16 Coombs (Antiglobulin)Tests Indirect Coombs Test –Detects anti-erythrocyte antibodies in serum Patient’s Serum Target RBCs + ↔ Step 1 + ↔ Coombs Reagent (Antiglobulin) Step 2

17 Coombs (Antiglobulin)Tests Applications –Detection of anti-Rh Ab –Autoimmune hemolytic anemia

18 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

19 Agglutination/Hemagglutination Inhibition Applications –Measurement of soluble Ag Practical considerations –Same as agglutination test Definition

20 Precipitation Tests Lattice Formation

21 Radial Immunodiffusion (Mancini) Interpretation –Diameter of ring is proportional to the concentration Quantitative –Ig levels Method – Ab in gel – Ag in a well Ag Concentration Diameter 2 Ag Ab in gel

22 I mmunoelectrophoresis Method –Ags are separated by electrophoresis Interpretation – Precipitin arc represent individual antigens Ag - + Ab Ag Ab –Ab is placed in trough cut in the agar

23 I mmunoelectrophoresis Method Interpretation Qualitative –Relative concentration

24 Countercurrent electrophoresis Method –Ag and Ab migrate toward each other by electrophoresis –Used only when Ag and Ab have opposite charges Qualitative –Rapid Ag Ab - +

25 Radioimmuoassays (RIA) Enzyme-Linked Immunosorbent Assays (ELISA) Lattice formation not required

26 Competitive RIA/ELISA for Ag Method –Determine amount of Ab needed to bind to a known amount of labeled Ag + ↔ Prior to Test Labeled Ag + ↔ Test + Patient’s sample Labeled Ag + –Use predetermined amounts of labeled Ag and Ab and add a sample containing unlabeled Ag as a competitor

27 Competitive RIA/ELISA for Ag Method cont. –Determine amount of labeled Ag bound to Ab ↓ NH 4 SO 4 ↓ anti-Ig Immobilize the Ab Quantitative – Most sensitive test + ↔ Test + Patient’s sample Labeled Ag + –Concentration determined from a standard curve using known amounts of unlabeled Ag Solid Phase Solid Phase

28 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 Solid Phase Ag Immobilized Ab in Patient’s sample Labeled Anti-Ig

29 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 Solid Phase Ag Immobilized Ag in Patient’s sample Labeled Ab

30 Tests for Cell Associated Antigens Lattice formation not required

31 Immunofluorescence Direct – Ab to tissue Ag is labeled with fluorochrome Ag Fluorochrome Labeled Ab Tissue Section

32 Immunofluorescence Indirect –Ab to tissue Ag is unlabeled –Fluorochrome-labeled anti- Ig is used to detect binding of the first Ab. Ag Fluorochrome Labeled Anti-Ig Tissue Section Unlabeled Ab Qualitative to Semi- Quantitative

33 Immunofluorescence Flow Cytometry – Cells in suspension are labeld with fluorescent tag Direct or Indirect Fluorescence – Cells analyzed on a flow cytometer Flow Tip Laser FL Detector Light Scatter Detector

34 Immunofluorescence Flow Cytometry cont. – Data displayed Green Fluorescence Intensity Number of Cells Unstained cells FITC-labeled cells One Parameter Histogram Red Fluorescence Intensity Green Fluorescence Intensity Two Parameter Histogram

35 Assays Based on Complement Lattice formation not required

36 Complement Fixation –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 Ag Patient’s serum Ag No Ag Ag Methodology


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