Labeling Techniques

 

Testing of antigen-antibody binding

 

Many Types of Labels used

lRadioactive Labels (RIA)

lEnzymes

lImmunofluorescent

 

RIA

lAntigen is added to microplate containing antibody

lExcess washed away

lLabeled antibody is added with excess being washed away

lNeed gamma counter to read

lPro- Can detect very small amts.

lCon- Radioactive waste

 

Enzyme

lUses enzyme labeled antibody or antigen

lEnzyme is conjugated to a substrate that will produce a color change

lFast and economical, can easily be automated

 

Immunofluorescent

lDirect or indirect

lDirect uses fluorescent labeled antibodies to look for antigens in tissue (ex: chalmydia)

lNeed to use fluorescent microscope to read

lIndirect uses known antigen as a substrate fixed on a slide

lPatient serum (ab) is added, then fluorescent AHG is added

 

Misc Techniques

lOlder procedure, uses the fixation of complement as an endpoint (Complement Fixation)

lPatient serum containing antibody is added to antigen. Complement is then added.

lSheep RBCs are the indicator of hemolysis

lPositive test no hemolysis

lNegative test hemolysis

lCumbersome and many controls needed

 

 

Polymerase Chain Reaction

lDouble stranded DNA is separated

lEach single strand serves as a template and synthesizes new complementary strand

lProcess is repeated 30 times

lLow amt. of DNA is amplified

lRadioactive labeled DNA that is complementary to the amplified strand then is used 

 

Western Blot

lUsed to confirm HIV antibodies

lAntigenic components are separated by electrophoresis

lPatient sample containing antibody is then added

lAntibody specificity is then characterized