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J Immunol Methods. 1985 Dec 27;85(2):409-19.

Methods for reducing non-specific antibody binding in enzyme-linked immunosorbent assays.


Enzyme-linked immunosorbent assays were used to study (i) binding of rabbit antibodies (raised against litter mate liver plasma membrane fraction) to the immunizing membrane fraction, and (ii) binding of human antibodies to liver membrane fractions and to liver-specific lipoprotein (a liver membrane-derived antigen complex). When assays were conducted using the non-ionic detergent Tween 20 as blocking agent, high non-specific binding was encountered. With the low titre rabbit antisera high binding of non-immune test antibody and of second antibody (anti-rabbit IgG) to the immunogen, and also directly to the solid phase, was found. This was abolished by replacement of Tween 20 in the antibody diluent buffers by a non-reactive protein, casein proving to be a more effective blocking agent than either bovine serum albumin or gelatin. With human sera, high binding of human IgG to the solid phase was noted. This too was blocked by casein, but only when the anti-microbial agent Thimerosal was included in the casein buffer, and when Tween 20 in the wash buffer was replaced by casein-Thimerosal so that the solid phase was exposed to casein before incubation with the test serum. The casein buffers described may prove of general value in solid-phase assays where high non-specific binding is encountered.

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