8 ag ab reactions07

Ag-Ab reactions Tests for Ag-Ab reactions

Nature of Ag/Ab Reactions ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Source: Li, Y., Li, H., Smith-Gill, S. J., Mariuzza, R. A., Biochemistry 39, 6296, 2000 http://www.med.sc.edu:85/chime2/lyso-abfr.htm

Affinity = ∑ attractive and repulsive forces Affinity ,[object Object],Ab Ag High Affinity Ab Ag Low Affinity

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

Avidity ,[object Object],Y K eq = 10 4 Affinity Y 10 6 Avidity Y Y Y Y Y 10 10 Avidity

Specificity ,[object Object],[object Object]

Cross Reactivity ,[object Object],[object Object],Anti-A Ab Ag A Anti-A Ab Ag B Shared epitope Anti-A Ab Ag C Similar epitope Cross reactions

Factors Affecting Measurement of Ag/Ab Reactions ,[object Object],[object Object],[object Object],[object Object],Ab excess Ag excess Equivalence – Lattice formation

Tests Based on Ag/Ab Reactions ,[object Object],[object Object]

Agglutination Tests Lattice Formation

Agglutination/Hemagglutination ,[object Object],[object Object],Y Y Y +  ,[object Object],[object Object]

Agglutination/Hemagglutination ,[object Object],[object Object],[object Object],1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512 1/1024 Pos. Neg. Titer 64 8 512 <2 32 128 32 4 Patient 1 2 3 4 5 6 7 8

Agglutination/Hemagglutination ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512

Passive Agglutination/Hemagglutination ,[object Object],[object Object],[object Object],Y Y Y + 

Coombs (Antiglobulin)Tests ,[object Object],[object Object],[object Object],+  Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Patient’s RBCs Coombs Reagent (Antiglobulin)

Coombs (Antiglobulin)Tests ,[object Object],[object Object],Y Y Y Y Y Patient’s Serum Target RBCs +  Step 1 +  Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Coombs Reagent (Antiglobulin) Step 2

Coombs (Antiglobulin)Tests ,[object Object],[object Object],[object Object]

Agglutination/Hemagglutination Inhibition ,[object Object],Y Y Y +  Prior to Test Y +  Y Y + Test Patient’s sample

Agglutination/Hemagglutination Inhibition ,[object Object],[object Object],[object Object],[object Object],[object Object]

Precipitation Tests Lattice Formation

Radial Immunodiffusion (Mancini) ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Ag Concentration Diameter 2 Ag Ag Ag Ag Ab in gel

I mmunoelectrophoresis ,[object Object],[object Object],[object Object],[object Object],[object Object],Ag - + Ag Ab Ag Ab

I mmunoelectrophoresis ,[object Object],[object Object],[object Object],[object Object]

Countercurrent electrophoresis ,[object Object],[object Object],[object Object],[object Object],[object Object],Ag Ab - +

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

Competitive RIA/ELISA for Ag ,[object Object],[object Object],[object Object],Y Y +  Prior to Test Labeled Ag Y Y +  Test + Patient’s sample Labeled Ag +

Competitive RIA/ELISA for Ag ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Y Y +  Test + Patient’s sample Labeled Ag + Solid Phase Solid Phase

Solid Phase Non-Competitive RIA/ELISA ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Solid Phase Y Ag Immobilized Y Ab in Patient’s sample Labeled Anti-Ig

Solid Phase Non-Competitive RIA/ELISA ,[object Object],[object Object],[object Object],[object Object],[object Object],[object Object],Solid Phase Y Ag Immobilized Y Ag in Patient’s sample Labeled Ab

Tests for Cell Associated Antigens Lattice formation not required

Immunofluorescence ,[object Object],[object Object],Ag Y Fluorochrome Labeled Ab Tissue Section

Immunofluorescence ,[object Object],[object Object],[object Object],[object Object],Ag Y Y Fluorochrome Labeled Anti-Ig Tissue Section Unlabeled Ab

Immunofluorescence ,[object Object],[object Object],[object Object],[object Object],Flow Tip Laser FL Detector Light Scatter Detector

Immunofluorescence ,[object Object],[object Object],Green Fluorescence Intensity Number of Cells Unstained cells FITC-labeled cells One Parameter Histogram Red Fluorescence Intensity Green Fluorescence Intensity Two Parameter Histogram

Assays Based on Complement Lattice formation not required

Complement Fixation ,[object Object],[object Object],[object Object],[object Object],Ag Ag ,[object Object],Y Y Patient’s serum Y Y Y Y Y Y Y Y Ag No Ag

8 ag ab reactions07

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8 ag ab reactions07