Solid-phase immobilized epitope

Fig. 7. Principle of the solid-phase immobilized epitope immunoassay (SPIE-IA).

Sandwich assays employ two antibodies, and are useful for ligands possessing two or more distinct epitopes. The primary, or unlabeled antibody, is immobilized onto a solid support, and must be present in excess quantities relative to the total [Ag] present in standards and unknowns. Following incubation with antigen, to allow quantitative capture Ag, and a rinse step, the labeled antibody is added in excess, and a second incubation proceeds. The labeled antibody reacts only with the antigen that has been retained on the solid phase by the primary antibody. After a second rinse, the quantity of immobilized Ab Ag Ab sandwich is then determined, and is equal to the quantity of antigen in the sample. 

Plastic is by far the most popular solid phase, since it makes the procedures extremely simple. However, plastics may also have some important limitatons (i) they are immunoreactant-consumptive, i.e. often require 10 times more reactants than particulate solid phases or membranes (ii) the avidity of immobilized antibodies for large antigens decreases by 1-2 orders of magnitude (Zwolinski, G. Jo-sephson, L. cited by Parsons, 1981), probably due to the wide spacing of epitopes or paratopes (iii) the rate of antibody-antigen interactions is slower than in solution or with particulate solid phases (hours instead of minutes), due to the necessity of the free immuno-reactant to diffuse to the solid phase (association kinetics is largely dictated by diffusion rate Section 8.4) and, (iv) few suitable antibod-... 

Direct non-competitive solid-phase enzyme immunoassays This approach is little used for quantitative investigations, but often in qualitative tests, e.g. for the detection of antigens on protein blots (Chapter 16) or in EIH (Chapter 17). For each epitope a corresponding labeled antibody is needed. The immobilized antigen (] = Ag) reacts directly with the conjugate (Ab E) ... 

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