Development of Enantioselective Polyclonal Antibodies to Detect Styrene Oxide Protein Adducts

Abstract

Styrene has been reported to be pneumotoxic and hepatotoxic in humans and animals. Styrene oxide, a major reactive metabolite of styrene, has been found to form covalent binding with proteins, such as albumin and hemoglobin. Styrene oxide has two optical isomers and it was reported that the (R)-enantiomer was more toxic than the (S)-enantiomer. The purpose of this study was to develop polyclonal antibodies that can stereoselectively recognize proteins modified by styrene oxide enantiomers at cysteine residues. Immunogens were prepared by alkylation of thiolated keyhole limpet hemocyanin (KLH) with styrene oxide enantiomers. Polyclonal antibodies were raised by immunization of rabbits with the chiral immunogens. Titration tests showed all six rabbits generated high titers of antisera that recognize (R)- or (S)-coating antigens accordingly. No cross-reaction was observed toward the carrier protein (BSA). All three rabbits immunized with (R)-immunogen produced antibodies that show enantioselectivity to the corresponding antigen, while only one among the three rabbits immunized with (S)-immunogen generated antibodies with enantioselectivity of the recognition. The enantioselectivity was also observed in competitive ELISA and immunoblot analysis. Additionally, competitive ELISA tests showed that the immunorecognition required the hydroxyl group of the haptens. Immunoblot analysis demonstrated that the immunorecognition depended on the amount of protein adducts blotted and hapten loading in protein adducts. In summary, we successfully developed polyclonal antibodies to stereoselectively detect protein adducts modified by styrene oxide enantiomers.

Development of an Indirect Competitive Enzyme-Linked Immunosorbent Assay for Glycocholic Acid Based on Chicken Single-Chain Variable Fragment Antibodies

“…The purified scFv G11 antibody was subsequently used to evaluate performance by an indirect competitive ELISA. Prior to ELISA, the optimal concentration of coating antigen and dilutions of scFv were determined by a checkerboard assay. , To evaluate the potential interferences that may be encountered, for example, in human urine samples, it was necessary to study the effects of pH and ionic strength on the ELISA. As shown in Figure A, the ELISA was more sensitive under neutral and slightly alkaline conditions than under slightly acidic ones.…”

Conformational adaptability determining antibody recognition to distomer: structure analysis of enantioselective antibody against chiral drug gatifloxacin

“…7,19 However, the prepared enantioselective antibody oen had crossreactivity to distomer, and it indicated that the antibody could still recognize distomer in some extent. [20][21][22] For example, the Sibuprofen antibody still had more than 50% cross-reactivity to the R-enantiomer. 20 Enantioselective antibodies had the ability to recognize the distomer, which would restrict the application of antibodies in the eld of chiral drug separation and detection.…”

Magnetism based electrochemical immunosensor for chiral separation of amlodipine

“…In the past decades, enantioselective immunoassay research has arisen in chiral compounds analysis, since it displays high sensitivity [8], simplicity [9], low cost [10], and high throughput [11]. At the same time, several enantioselective antibodies against small molecules have emerged [9][10][11][12]. However, the stratagem of immunogen design and enantioselective antibody preparation for amlodipine or other dihydropyridines has not been revealed.…”