Enzyme conjugation with reduced antibodies

Figure 20.4 Reduction of the disulfide bonds within the hinge region of an IgG molecule produces half-anti-body molecules containing thiol groups. Reaction of these reduced antibodies with a maleimide-activated enzyme creates a conjugate through thioether bond formation.

A final consideration is to provide adequate desalting of the reduced antibody molecule from excess reducing agent. If even a small amount of a thiol-containing reductant remains, subsequent conjugation with a maleimide-activated enzyme will be inhibited. 

Yazynina et al. (1999) introduced novel reagents into the inhibition step with the goal of decreasing the long analysis times typically required for ELISA measurements. A polyanion-protein A conjugate was introduced into the atrazine antibody-enzyme conjugate solution. Antibody bound by protein A was in turn bound to the walls of a microplate coated with a polycation. This innovation reduced overall time of analysis to 40 min and achieved a detection limit of 0.03 ppb of atrazine. 

Homogeneous enzyme immunoassays have also been developed for serum T4 determination. These procedures are rapid and simple to use and have also been applied to several major automated instruments.For example, the enzyme-multiplied immunoassay technique (EMIT) for T4 measurement uses glucose-6-phosphate dehydrogenase covalently hnked to T4 as the enzyme label.Binding of T4 specific antibody to this label reduces enzyme activity, perhaps as a result of steric or allosteric inhibition As the concentration of unlabeled T4 increases, less enzyme-labeled hormone is bound by the antibody. As a result, the catalytic activity of the unbound enzyme conjugate increases in direct proportion to the amount of T4 in the specimen. The indicator reaction involves oxidation of glucose-6-phosphate with simultaneous reduction of nicotinamide-adenine dinu-... 

The immune complex transfer assay has allowed the development of very sensitive EIAs for the detection of antibodies. In one example, the assay utilizes a 2,4-dinitrophenyl (DNP)-biotin-conjugated antigen. This is incubated with the sample antibody to be measured, and, after the immune reaction, the immune complexes are trapped onto a primary solid phase coupled with anti-DNP antibody. After washing away unbound materials, immune complexes are eluted with DNP-lysine and transferred to a secondary streptavidin-coated solid phase. Next, an enzyme-labeled anti-immunoglobulin antibody is added, and, after washing, enzyme activity is assayed. In this case, the enzyme activity correlates with the amount of antibody in the sample. The sensitivity depends on the amount of nonspecific binding of the enzyme-labeled antibody on the second solid phase. Thus, several modified assay systems have been developed to reduce background. 

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