Purification of hapten conjugates

For EIA, it is important to establish the degree of substitution, the specific and the total activity of the enzyme recovered, as well as the extent of recovery of the immune reactivity. The number of amino groups substituted with haptens in carrier molecules is conveniently established by the determination of the number of free amino groups before and after substitution (Habeeb, 1966 Table 12.9). Another method is provided by the use of radioactive haptens (Abraham et al., 1971). 

Determination of amino groups in proteins by the color development due to the reaction of 2,4,6-lrinitrobenzenesulfonic acid (TNBS) with e-amino groups 

This determination is performed under more gentle conditions than the ninhydrin method and is linear up to 1 mg/mi. SDS interferes, particularly when the pH of the sample is below pH 8.5 in the first step. 

Add 1 ml 4% NaHCOjand 1 ml 0.1% TNBS. respectively, to 1 ml of protein solution (0.6-1.0 mg/ml). The blank contains 1 ml water instead of protein solution. 

Read at 335 nm (molar extinction coefficient of TNBS modified amino group of different proteins are all close to 10 M cm ). 

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