Ab2 fluorescently labeled; specific for Ab1 (b) Ab2 attaches to Ab1-visible fluorescence Ab1 in serum Positive No Ab in Known Ag Ab2 cannot serum attach-no fluorescence Negative (a)
Observe figure and make note of the several steps in the
indirect ELISA test. What four essential events are necessary to
develop a positive reaction (besides having antibody A)? Hint: What
would happen without rinses?
Indirect ELISA is an immunoassay in which the antigen is bound by the primary antibody which is then detected by a labeled secondary antibody.
Indirect ELISA is used to capture antibodies in the serum sample. The indicator antibody is complexed to an enzyme that causes a change in the color if the serum sample is positive for the infecting agent. The starting reactant is a known antigen that is adsorbed to a hard surface and unknown test serum is added to it.
After rinsing, an enzyme-Ag reagent that can react with an unknown test antibody is placed in the well. The substrate is added and change in color is observed. Change in color indicates that the suspected antibody was present in the serum sample. The final positive reaction is obtained by means by the antigen-antibody reaction.
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