Immunology (elective) MLIL-101 Prepared by: Dr. Mohamed S. Abdel-Latif

Ag-Ab reactions Tests for Ag-Ab reactions

Learning Outcome: At this time you should know the following: 1. To describe the nature of Ag-Ab reactions 2. To compare and contrast antibody affinity and avidity. 3. To delineate the basis for antibody specificity and cross reactivity. 4. To discuss the principles of commonly used tests for antigen/antibody Reactions.

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,

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

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

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

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 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 Affinity Avidity Ag:Ab ratio Physical form of Ag Ab excess Ag excess 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 –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

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

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 Patient’s Serum Target RBCs +  Step 1 +  Coombs Reagent (Antiglobulin) Step 2

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 Applications –Measurement of soluble Ag Practical considerations –Same as agglutination test Definition

Precipitation Tests Lattice Formation

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

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

I mmunoelectrophoresis 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 Qualitative –Rapid Ag 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 +  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

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

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

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

Tests for Cell Associated Antigens Lattice formation not required

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

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

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

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

Assays Based on Complement Lattice formation not required

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

Assignment: As a part of the semester activity, Group of students are selected every week to prepare a short seminar about his/her point of interest in one of the lecture topics. That to be discussed and evaluated during the next lecture.