SEROLOGY

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SEROLOGY USE OF ANTIGEN-ANTIBODY REACTIONS IN
LAB

• KNOWN ANTIGENS USE CAN DETECT ANTIBODIES (CAN
  SHOW THEY ARE PRESENT, IF THEY ARE PRESENT)
• PURIFIED HIV ANTIGENS DETECT THE PRESENCE OF
  ANTI-HIV ANTIBODIES IN INDIVIDUAL
• HIV PERSON (EXCEPT NEWBORNS WHO RECEIVE
  ANTI-HIV ANTIBODIES FROM MOM, NATURAL
  PROCESS)
• KNOWN ANTIBODIES (ANTISERA) USE CAN DETECT AND
  IDENTIFY SPECIFIC ANTIGENS WHICH CAN REACT WITH
  THOSE ANTIBODIES, IF PRESENT)
• PURIFIED (KNOWN) ANTISERA DETERMINE THE
  ANTIGENS PRESENT ON INDIVIDUALS RBC (ABO
  BLOOD GROUP)

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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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ENZYME-LINKED IMMUNOSORBENT ASSAY ELISA

• DIRECT (SANDWICH) - DETECTS A CERTAIN
  ANTIGEN (IF PRESENT)
• KNOWN ANTIBODIES TO THAT ANTIGEN ABSORBED ON
  PLATE
• ADD MATERIAL WHICH MIGHT CONTAIN THE ANTIGEN,
  BINDING OCCURS (OR NOT), WASH AWAY EXCESS
  MATERIAL
• ADD KNOWN ANTIBODIES TO THAT ANTIGEN 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,
  CATALYZES THE REACTION, PRODUCT OF THE REACTION
  IS COLORED OR PRODUCES LIGHT INDICATING
  PRESENSE OF THE ANTIGEN

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A. KNOWN ANTIBODIES TO THAT ANTIGEN ABSORBED ON
PLATE
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B. ADD MATERIAL WHICH MIGHT CONTAIN THE ANTIGEN,
BINDING OCCURS (OR NOT), WASH AWAY EXCESS
MATERIAL
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C. ADD KNOWN ANTIBODIES TO THAT ANTIGEN LINKED TO
ENZYME BINDING OCCURS IF THE ANTIGEN WAS
PRESENT, WASH EXCESS
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D. ADD CHROMOGENIC SUBSTRATE OF THE ENZYME
OR CHEMILUMINESCENT SUBSTRATE
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Figure 35.14 THE ELISA OR EIA TEST
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• INDIRECT ELISA - DETECTS SPECIFIC ANTIBODIES
  IF PRESENT
• KNOWN ANTIGEN ABSORBED ON TO PLATE
• ADD TEST ANTISERUM (E.G., HUMAN) IF ANTIBODIES
  WHICH CAN
• REACT WITH THAT ANTIGEN ARE PRESENT, THEY BIND.
• 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 INDICATING THAT THE
  SPECIFIC ANTIBODIES WERE PRESENT

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A. KNOWN ANTIGEN ABSORBED ON TO PLATE
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B. ADD TEST ANTISERUM (E.G., HUMAN) IF
ANTIBODIES WHICH CAN REACT WITH THAT ANTIGEN ARE
PRESENT, THEY BIND
WASH
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C. ADD ENZYME LINKED ANTI-ANTIBODY SERUM, (E.G.,
MOUSE ANTI-HUMAN IMMUNOGLOBULIN) WASH
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D. ADD CHROMOGENIC/CHEMILUMINESCENT
SUBSTRATE FOR THE ENZYME
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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/ CHEMILUMINESCENT
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