The use of avidin-biotin interaction in immunoenzymatic techniques.

TLDR

Avidin’s four active sites enable multivalent binding, forming the basis for the avidin–biotin interaction used in immunoenzymatic assays. Biotin is covalently attached to antibodies, antigens, and enzymes, and the resulting biotin–avidin complexes are employed in two quantitative enzyme‑immunoassay and enzyme‑immunohistochemical staining procedures—BRAB, which uses sequential incubation with biotin‑labeled antibody, avidin, biotin‑labeled enzyme, and measurement, and LAB, which uses biotin‑labeled antibody followed by enzyme‑labeled avidin for detection. Optimal conditions for enzyme‑immunoassay and enzyme‑immunohistochemical staining with the BRAB and LAB techniques were determined.

Abstract

Biotin was covalently attached to antibodies, antigens and enzymes, and the effects of this labeling on the antigen and antibody binding capacity and on enzymatic activity were tested. Based on avidin-biotin interaction, the labeled proteins were used in quantitative enzyme-immunoassay and enzyme-immunohistochemical staining procedures. Two procedures were developed. In the first procedure, named the Bridged Avidin-Biotin (BRAB) technique four steps were used sequentially in order to quantify or detect an immobilized antigen: 1) incubation with biotin-labeled antibody; 2) incubation with avidin; 3) incubation with biotin-labeled enzyme; 4) measurement or histochemical staining of the enzyme. The technique is based on the observation that avidin possesses four active sites. In the second procedure, named the Labeled Avidin-Biotin (LAB) technique, biotin-labeled antibody and enzyme-labeled avidin are used sequentially. Enzyme-associated antigen is then quantified or revealed immunohistochemically. The optimal conditions for enzyme-immunoassay and enzyme-immunohistochemical staining using BRAB and LAB procedures were established.