Success and Failure in Antibody Recognition by Surface-Type Sensors: Essential Prerequisites

Abstract

In order to determinate small molecular compounds (so-called haptens) in biological media, especially when the concentrations of compounds are at trace concentrations, it is necessary to produce antibodies with high affinity and high selectivity. Since the hapten is not able to stimulate the animals to produce specific antibodies directly, it should be bound to protein carrier. The manner of chemical binding of the hapten to a protein determines the character of the antibody specificity to small molecules which are analyzed. One of the highly sensitive methods in the small molecule determination at low concentration is competitive SPR-based immunoassay. To use the method effectively, a definite sensor surface sensitive to the specific antibody is needed to achieve the lowest value in the limit of detection (LOD) in immunoassay. The most evident class of small molecules to be determined in biological media at the lowest concentration is the steroid hormones, particularly estrogens. We have developed the sensor surface by binding the target molecules (estradiol, E2) directly to the gold surface through the specific linker to provide the closest distance to the surface along with biocompatibility to achieve maximal response in antibody–antigen interaction. As an antibody we have used a commercial monoclonal antibody raised to the 6-position in E2 with BSA (E26*BSA_CC). We failed to observe the specific binding of the antibody to the sensor surface. Then, we suggested that the main factor hindering this interaction is the wrong choice regarding the hapten–carrier conjugated with carrier protein. In order to confirm the assumption, we took the serum obtained from animal immunization by the antigen where BSA- is attached to the 3- or 17-position in E2 (E23*BSA_CC or E217*BSA_CC) instead of 6-position. Only the polyclonal antibody obtained with E23*BSA_CC resulted as expected in its successful binding to the sensitive sensor surface identification of low molecular weight analytes.

Keywords

• low-molecular-weight analytes
• 17β-Estradiol
• antibody recognition