SEROLOGY - AGGLUTINATION

CROSSLINKING CELLS BY DIVALENT SPECIFIC ANTIBODIES RESULTS IN VISIBLE CLUMPS OF MANY CELLS BOUND BY SOLUBLE ANTIBODY MOLECULES UNKNOWN BACTERIAL CELLS IDENTIFIED BY KNOWN ANTIBODY WHICH CAUSES AGGLUTINATION TUBES, MICROTITER PLATES, TITER = RECIPROCAL OF HIGHEST DILUTION WHICH AGGLUTINATES HEMAGGLUTINATION CLUMPING OF RBC BY ANTIBODIES WHICH REACT WITH ANTIGENS ON RBC SURFACE ABO BLOOD GROUPS VIRAL HEMAGGLUTINATION INFLUENZA VIRUS HAS HEMAGGLUTININ ON SURFACE, BINDS RBC

Figure 35.12 AGGLUTINATION TESTS

TUBE AGGLUTINATION

Figure 35.12 AGGLUTINATION TESTS

MICROTITER PLATE

Figure 35.11 VIRAL HEMAGGLUTINATION

COMPLEMENT FIXATION (USAGE) MEANS THAT A KNOWN ANTIGEN CAN BE USED TO DETECT ANTIBODY BY COUPLING THE REACTION TO COMPLEMENT FIXATION. THAT IS, COMPLEMENT FIXATION WILL INDICATE THAT AN ANTIGENANTIBODY REACTION OCCURRED AND THAT THAT ANTIBODY WAS PRESENT IN THE UNKNOWN SERUM. TUBE 1 KNOWN ANTIGEN NO ANTIBODY NO REACTION ADD COMPLEMENT NO BINDING ADD RBC AND ANTI-RBC COMPLEMENT BINDS AND LYSES RBC TUBE 2 KNOWN ANTIGEN UNKNOWN SERUM REACTION ADD COMPLEMENT BINDS COMPLEXES ADD RBC AND ANTI-RBC NO COMPLEMENT LEFT TO BIND, NO LYSIS

Figure 35.13 COMPLEMENT FIXATION

ENZYME-LINKED IMMUNOSORBENT ASSAY ELISA

DIRECT (SANDWICH) - DETECTS ANTIGENS KNOWN ANTIBODIES ABSORBED ON PLATE ADD MATERIAL WHICH MIGHT CONTAIN THE ANTIGEN, BINDING OCCURS (OR NOT), WASH AWAY EXCESS MATERIAL ADD KNOWN ANTIBODIES LINKED TO ENZYME, BINDING OCCURS IF THE ANTIGEN WAS PRESENT, WASH EXCESS ADD CHROMOGENIC SUBSTRATE OF THE ENZYME OR CHEMILUMINESCENT SUBSTRATE IF THE ANTIGEN WAS PRESENT, THE SECOND ANTIBODY BOUND TO IT, THE ENZYME LINKED TO THAT ANTIBODY, CATALYZED THE REACTION, PRODUCT OF THE REACTION WAS COLORED OR PRODUCES LIGHT

Figure 35.14 THE ELISA OR EIA TEST

INDIRECT ELISA - DETECTS ANTIBODIES KNOWN ANTIGEN ABSORBED ON TO PLATE ADD TEST ANTISERUM (E.G., HUMAN) IF ANTIBODY IS PRESENT, IT BINDS, WASH ADD ENZYME LINKED ANTI ANTIBODY SERUM, (E.G., MOUSE ANTIHUMAN IMMUNOGLOBULIN), WASH ADD CHROMOGENIC/CHEMILUMINESCENT SUBSTRATE FOR THE ENZYME MEASURE ABSORBANCE OR LIGHT

Figure 35.14 THE ELISA OR EIA TEST

WESTERN BLOT IMMUNOBLOT DETECTS UNKNOWN ANTIGENS WITH KNOWN ANTIBODIES

SEPARATE PROTEINS ACCORDING TO MW BY SDS POLYACRYAMIDE GEL ELECTROPHORESIS SODIUM DODECYL SULFATE NEGATIVELY CHARGED SDS-BOUND PROTEINS MIGRATE TO POSITIVE ELECTRODE SMALLER PROTEINS MIGRATE FASTER THRU GEL TRANSFER SEPARATED PROTEINS TO INERT MEMBRANE PROBE THE MEMBRANE WITH KNOWN ENZYME LINKED ANTIBODIES TO THE ANTIGEN OF INTEREST, WASH AWAY EXCESS PROBE THE MEMBRANE WITH CHROMOGENIC/CHEMILUMINESCENT SUBSTRATE

WESTERN BLOT SDS-POLYACRYLAMIDE GEL

EXTRACT BACTERIAL CELLS OVERPRODUCING PROTEIN OF INTEREST

Figure 35.16 IMMUNOPRECIPITATION