Synthesis and characterization of a functional benzoylecgonine - bovine serum albumin conjugate for quantification of a humanized monoclonal anti-cocaine antibody

Abstract

We have developed a humanized monoclonal anti-cocaine antibody (h2E2), as a potential solution for cocaine use disorder. A key milestone was the development of an assay to quantify this monoclonal antibody (mAb) in animal and human blood. Thus, we synthesized a novel benzoylecgonine-1,4-diaminobutane-BSA (BE-diab BSA) conjugate as an antigen for quantifying h2E2 using ELISA. We report here the method of synthesis of this conjugate, BE-diab BSA, and assessment of its binding to the h2E2 mAb using an ELISA and a fluorescence quenching assay. Compared with four commercial BE-BSA conjugates, BE-diab BSA demonstrated markedly stronger mAb binding in ELISA-three of the commercial conjugates showed less than 10 % relative binding. Fluorescence quenching assays confirmed this binding superiority, with the commercial conjugates showing minimal mAb interaction, while BE-diab BSA induced robust intrinsic fluorescence quenching. SDS-PAGE analyses identified structural differences consistent with binding results between our functional conjugate and the commercial preparations. This functional and reproducible in-house conjugation has been integrated into a GLP-validated ELISA, which is now in use for pharmacokinetic analyses and for qualifying antibody release lots for clinical deployment.

Keywords: Anti-cocaine antibody; Antigen-synthesis; BE-diab BSA; Cocaine; ELISA; Humanized; Monoclonal.

Graphical abstract

Fig. 1

Synthetic strategy used for the synthesis of BE-diab BSA conjugate. BSA is reacted with the spacer 1,4 diamino butane in the presence of EDC in acidic conditions. The resulting BSA-spacer linked via an amide linkage is then reacted with BE resulting in the BE-BSA conjugate, wherein the carboxyl group of BE is in an amide linkage with the amino group of the spacer (see text for complete details).

Fig. 2

Fluorescence quenching of h2E2 mAb by BE-BSA conjugates. Panel A: Fluorescence emission at 330 nm (excitation at 280 nm for tyrosine and tryptophan or 295 nm for tryptophan only) shows h2E2-induced quenching for functional in-house conjugates (analysts 2 and 3) but not analyst 1, who used a different, non-functional synthetic method. Panel B: Comparison of analyst 1 and 2 conjugates with four commercial BE-BSA preparations: Fitzgerald Cat# 801037 (Fitz#1), Fitzgerald Cat# 801B29 (Fitz#2), My Biosource (MBS3033688), and BiosPacific (BSP V52000501). All commercial conjugates showed minimal quenching, with BSP demonstrating partial activity (∼30 %).

Fig. 3

ELISA evaluation of h2E2 mAb binding to BE-BSA conjugates All figures are a plot of absorbance vs log of the concentration of the antibody h2E2 tested against 96 well plates coated using 2 μg/mL of BE-diab BSA synthesized in our laboratory by 3 different analysts (2, 3 and 4), using the synthetic protocol described in this study. Panel A: each product was tested on 3 separate days in assays using triplicates each day (n = 9). All antigens were tested against a range of h2E2 mAb concentrations (0–0.4 μg/mL). Panel B: Binding comparison between in-house antigen (Ag5) and commercial conjugates—Fitz#1 (Ag1), Fitz#2 (Ag2), MBS (Ag3), BSP (Ag4). All antigens were tested under identical conditions against h2E2 at a concentration range of 0–0.4 μg/mL. Commercial antigens were tested once (n = 3); for the in-house antigens a mean of absorbance obtained at each concentration of the antibody (h2E2) using analyst 3 (n = 3) or analyst 4 (n = 3) conjugates assayed on two separate days. Panel C: Antigens tested were the in-house synthesis (Ag5), Fitzgerald antigen 801037 (Ag1, FITZ#1), Fitzgerald antigen 801b29 (Ag2, FITZ#2), My Biosource antigen (Ag3, MBS) and BiosPacific antigen (Ag4, BPS). All commercial antigens were tested at a concentration of 2 μg/mL against vendor supplied antibody at a concentration range of 0–0.4 μg/mL. The in-house synthesized antigen (Ag5, mean absorbances of analyst 3 and 4, n = 3 for each) was tested against h2E2 (concentration 0–0.4 μg/ml).

Fig. 4

SDS-PAGE analyses of various BE-BSA conjugates. Panel A: Reducing and non-reducing SDS-PAGE (7 % gel) of three in-house conjugates (3 μg/well). Functional conjugates from Analysts 2 and 3 show distinct migration compared to non-functional conjugate from Analyst 1, made using an alternative method. Panel B: Comparative SDS-PAGE of in-house conjugates (Analyst 1 and 2) and commercial BE-BSA products (Fitz#1, Fitz#2, MBS, BSP), all analyzed under both reducing and non-reducing conditions at 3 μg/well. Analyst 1 synthesis (a previous, undescribed conjugation synthetic strategy that generated a non-functional conjugate) differs from analysts 2 and 3, who synthesized antigen using the methods described in this work. Differences in band migration correlated with functional assay results.