Excellnet serological tests in identification of infectious agents

Serological tests involved in identification of infectious agents Prof M.I.N. Matee

Precipitation Tests Lattice Formation

Precipitation reactions in fluids (p. 142) Precipitation reaction can be seen

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

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

I mmunoelectrophoresis ,[object Object],[object Object],[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 + 

Hemagglutination Used to identify blood group antigens or antibodies to them Variations: Chemically couple haptens to RBC Use other particles - bacteria, antigen-coated latex beads

Agglutination Inhibition Assay for HCG

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]

Immunofluorescence Antibodies can be labeled with fluorescent dye Can localize binding sites on cells Dyes: Fluorescein, rhodamine, phycoerythrin can be conjugated to Fc region of Ab (so antigen binding is unaffected) Absorb at one wavelength and emit at another

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

ELISA- enzyme-linked immunosorbent assay Based on RIA Nearly as sensitive Cheaper and safer Many detection systems have been developed Many variations of the assay have been developed

All ELISAs use an antibody conjugated with an enzyme that turns a colorless substrate into a colored product Direct- detects antigens using a single labeled antibody against that antigen Relatively few applications and permutations

ELI SA Enzyme- L inked I mmunosorbent A ssay (ELISA) © 1998 Gold Standard Multimedia Inc. 1. Antigen HIV Protein 2. Test antibody Human serum 3. Developing Ab Enzyme-Goat anti-Human IgG 4. Substrate Colorless > Blue

E nzyme- L inked I mmunosorbent A ssay (ELISA) HIV Ag Human Serum AP-Goat Anti- Human IgG Colorless > Yellow AP = alkaline phosphatase

Western Blot for HIV Antibodies + - Patient sera

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

Tests for Cell Associated Antigens Lattice formation not required

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 Ag in Patient’s sample Labeled Ab

Tissue Immunofluorescence/Immunohistochemistry

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

Excellnet serological tests in identification of infectious agents

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