SEROLOGY AGGLUTINATION

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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

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Figure 35.12 AGGLUTINATION TESTS
TUBE AGGLUTINATION
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Figure 35.12 AGGLUTINATION TESTS
MICROTITER PLATE
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Figure 35.11 VIRAL HEMAGGLUTINATION
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• 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 ANTIGEN-ANTIBODY REACTION OCCURRED AND THAT
  THAT ANTIBODY WAS PRESENT IN THE UNKNOWN SERUM.
• TUBE 1 TUBE 2
• KNOWN ANTIGEN KNOWN ANTIGEN
• NO ANTIBODY UNKNOWN SERUM
• NO REACTION REACTION
• ADD COMPLEMENT ADD COMPLEMENT
• NO BINDING BINDS COMPLEXES
• ADD RBC AND ANTI-RBC ADD RBC AND
  ANTI-RBC
• COMPLEMENT BINDS NO COMPLEMENT
• AND LYSES RBC LEFT TO BIND, NO LYSIS

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Figure 35.13 COMPLEMENT FIXATION
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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

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Figure 35.14 THE ELISA OR EIA TEST
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• 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 ANTI-HUMAN IMMUNOGLOBULIN), WASH
• ADD CHROMOGENIC/CHEMILUMINESCENT SUBSTRATE FOR
  THE ENZYME
• MEASURE ABSORBANCE OR LIGHT

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Figure 35.14 THE ELISA OR EIA TEST
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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/CHEMILUMINESCE
  NT SUBSTRATE

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WESTERN BLOT SDS-POLYACRYLAMIDE GEL
EXTRACT BACTERIAL CELLS OVERPRODUCING PROTEIN OF
INTEREST
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Figure 35.16 IMMUNOPRECIPITATION