Quantification of antibody immobilization using peroxidase enzyme–substrate reaction

Abstract

Due to the multitude of antigens and antibodies that are currently being studied by immunoscientists and researchers, numerous and diverse antibody immobilization techniques were developed. Newer and improved methods of detection and immobilization are constantly being developed. As a result, especially when no prior research exists, scientists and researchers are faced with the challenge of choosing the best immobilization technique. Assessment of antibody immobilization based on the final antibody–antigen reaction may not always be accurate, as certain unknown processes or reactions may account for a part (or whole) of the transducer response. The present paper describes an alternative method for scientific quantification of antibody attachment on the biosensing surface or transducer using the peroxidase enzyme–substrate (TMB) colorimetric reaction. The proposed method can be used to pre-screen various immobilization techniques or pre-coatings, or can be used as a secondary quantification method. Four different pre-coatings on piezoelectric crystals and three affinity membranes were tested to evaluate the degree of antibody attachment. Results indicate that polystyrene-coated crystals had an average immobilization of 10.55% and the Immobilon membrane had 18.22% immobilization. Although the precision of the proposed technique varied significantly between different transducer surfaces (CV=1.7–21.7%), results clearly demonstrate that polystyrene coating on crystal is approximately 450% superior to glutaraldehyde coating. Similarly, the Immobilon membrane had 720% higher antibody immobilization than Ultrabind membrane. The sensitivity of the overall technique is so high that each test requires only 3 μl of diluted, labeled antibody solution (1 μg/ml).