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