Manual of basic techniques for a health laboratory - libdoc.who.int

11. Immunological and serological techniques 331Enzyme immunoassayIn an enzyme immunoassay, an antigen or antibody is conjugated with a labelledenzyme and a colour change is produced by the enzyme reacting with its substrate.This change can be detected visually or with a spectrophotometer. The bindingevent can be either competitive or non-competitive. Competitive binding dependson competition between a labelled (known amount) and an unlabelled (unknownamount) antigen for the same antibody (when measuring antigen) or between alabelled (known amount) and an unlabelled (unknown amount) antibody for thesame antigen (when measuring antibody) (Fig. 11.3). The amount of binding ofthe labelled antigen (or antibody) is related to the amount of unlabelled antigen (orantibody) present.In non-competitive binding (sandwich technique), the antigen or antibodyis adsorbed (or immobilized) to a solid phase, which may be an insoluble particle(bead) or the sides of a test-tube or the bottom of a microtitre plate. The test samplecontaining the corresponding antibody or antigen is then added. A labelledantibody or antigen (conjugate) is added last to form the top layer of the sandwich(Fig. 11.4). When testing for an antibody, the conjugate would contain anti-immunoglobulinand when testing for an antigen, the conjugate would containan antibody specific for that antigen. The amount of binding of the conjugate isdirectly related to the amount of antigen or antibody in the test sample and onlythe bound portion is measured in this assay.An example of this technique is the enzyme-linked immunosorbent assay (ELISA).The enzymes used in ELISAs include horseradish peroxidase, alkaline phosphatase,lysozyme and b-galactosidase.The enzyme immunoassays are replacing many radioimmunoassays because of theiradvantages over the latter, which include longer reagent shelf-life, cheaper and simplerequipment, no restrictive regulations for reagent disposal, and saferreagents.Incubatethen washEIncubatethen washEEEE+ EnzymesubstrateESolid-phaseadsorbedantigenAntibody-containingserumEnzyme-labelledanti-immunoglobulinFig. 11.3 Principle of competitive enzyme immunoassaysColourIncubatethenwashEEIncubatethenwashEE+ EnzymesubstrateEEESolid-phaseadsorbedantigenAntibody-containingserumEnzyme-labelledanti-immunoglobulinFig. 11.4 Principle of non-competitive enzyme immunoassaysColour