Dot-blot analysis of the degree of covalent modification of proteins and antibodies at amino groups

The present study describes a rapid and sensitive dot-blot assay approach for determining the degree of covalent modification of amino groups in proteins. N-hydroxy-succinimide ester of acetic acid was used for irreversible, covalent modification of proteins whose reactive primary amino groups were reversibly blocked (or protected) with 2,3-dimethylmaleic anhydride prior to processing. Immobilon(TM) AV affinity membrane was utilized for differential covalent attachment of the proteins to the activated ester on the membrane matrix, primarily through their protected ε-amino group of lysins. The efficacy of the method was demonstrated for a murine monoclonal antibody and for two human plasma proteins. The degree of covalent modification of proteins at their amino groups as estimated by the proposed method is compared with that obtained by using the conventional trinitrobenzenesulfonic acid (TNBS) method. Several advantages of the present method over the TNBS method are emphasized. The new method, which requires only nanograms of protein, is shown to be more sensitive than the TNBS method where the limit of detection is in the milligram range. The proposed assay is very specific and facile, and the advantage of small sample size requirement (1 μl) provides sequential detection of multiple samples facilitating much higher precision in data obtained than that of the TNBS assay.