Novel cross-linked enzyme–antibody conjugates for Western blot and ELISA

Covalent cross-linking of enzymes to antibodies to produce immunoconjugates for Western blot analysis and ELISAs was achieved using in vacuo cross-linking methodology [Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Covalent cross-linking of proteins without chemical reagents. Protein Sci. 11, 1558.]. The advantageous feature of this methodology for producing enzyme–antibody conjugates is that the cross-linking is accomplished without the use of chemical modifying or activating reagents. This reduces the potential activity loss due to chemical modification and allows easy recovery of any free antibody or native enzyme. In vacuo cross-linking of horseradish peroxidase (HRP) to anti-rabbit immunoglobulin G (IgG) produced an enzyme-linked antibody with an improved sensitivity for antigen detection compared to a commercial conjugate prepared by conventional chemical cross-linking methods. A soluble multi-enzyme-based immunoconjugate was prepared by the in vacuo cross-linking of HRP to a high molecular weight polyglutamic acid polymer followed by the in vacuo cross-linking of a limiting amount of antibody to yield an antibody–(HRP)n–polyglutamate complex. This complex had a detection signal 100-fold greater than that of the 1:1 enzyme–antibody conjugates prepared by chemical cross-linking.