Non-competitive assays with antibodies immobilized on the solid phase

Non-competitive assays with antibodies immobilized on the solid phase Immobilization of antibodies or their F(ab )2 on 

Crook and Payne (1980) compared the direct (Section 14.2.1.1), indirect (Section 14.2.1.2) and the double-antibody sandwich methods (variant A) for their ability to detect and discriminate between several granulosis viruses. The indirect method was the most sensitive (1 ng/ml), the direct method was the least sensitive (15 ng/ml) and method A had an intermediate detectability (10 ng/ml). 

There are, however, some specific characteristics and limitations for these variants. When anti-Ig antibodies are used, they should not be reactive with the immobilized antibody, i.e., for the more sensitive indirect methods (variants B, E and G) primary antisera from two different species should be available. This is not always practical or feasible and various ways have been developed to circumvent this problem (De Jong, 1983 Section 14.2). In variants C and D, the solid phase is coated with Fab or F(ab )2 and the complete antibody is applied in the third layer, together with enzyme-conjugated anti-lg antibodies (C) or enzyme-labeled SpA (which is Fc specific D) (Barbara and Clark, 1982 Koenig and Paul, 1982). Although this variant requires strictly Fc-specific antibodies, most anti-IgG antibodies, produced against complete IgG, are rather Fc-specific. If this is not the case, the anti-IgG serum could always be absorbed with an immunosorbent prepared with Fab or F(ab )2. In variants F and G biotinylated antibody and biotinylated enzyme are used 

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