Antibody Interactions withRicinus communisAgglutinins Studied by Biolayer Interferometry

Abstract

Two related agglutinins are present in the seeds of Ricinus communis (castor): ricin, a dichain ribosome-inactivating protein and Ricinus communis agglutinin-1, a much less toxic tetrameric hemagglutinin. The immunochemical analysis of these agglutinins is of special interest because ricin toxicity has resulted in both accidental and intentional poisonings, while it has also provided a potential cancer chemotherapeutic in the form of an immunoconjugate. We previously characterized a panel of monoclonal antibodies (mAbs) for the analysis of potential contamination with ricin in several food matrices. In this study, an optical sensing technique, biolayer interferometry (BLI), was used to study the binding of two mAbs to the agglutinins. MAbs were immobilized on sensors with amine-reactive, Fc-binding, and streptavidin-coated tips to study the interactions with the agglutinins and with ricin A-and B-chains in solution. The kinetically determined equilibrium dissociation constants generally agreed with the relative binding observed in ELISA, although binding was less predictable for the isolated ricin chains. BLI analysis of kinetic constants for mAb 1797 was not affected by nonfat milk (0.5% by volume). BLI provides a useful method to characterize the binding of antibodies, with the potential for immunodiagnostic applications in food matrices.

Immunosorbent analysis of toxin contamination in milk and ground beef using IgY-based ELISA

“…Although the study of thermal stability indicated that IgY was more stable than mouse polyclonal Ab at 65°, mouse mAbs used in many of our studies shared the high thermal stability of IgY (e.g. mAbs 1443 and 1797; Brandon, Adams, et al, 2014). IgY could be especially useful when a large quantity of PBS pAb is required, but it remains to be seen whether other features of IgY, such as its altered hinge region, afford advantages in sensor methodology.…”

“…These assays were conducted at neutral pH at ambient temperature (about 20°). However, we also used the mAbs in an optical sensor system and found that mAb 1642 was too acidlabile to permit regeneration using 10 s pulses with 10 mM glycine-HCl, pH 1.7 (Brandon, Adams, Yang, & Korn, 2014). We hypothesize that the same structural features than confer acid lability also render mAb 1642 heat-labile.…”

Milk Matrix Effects on Antibody Binding Analyzed by Enzyme-Linked Immunosorbent Assay and Biolayer Interferometry

“…Asialofetuin was obtained from Sigma (St. Louis, MO). The principal buffers and milk samples used in ELISAs and BLI were phosphate-buffered saline (PBS), PBS–0.05% (v/v) Tween-20 (PBST), and PBST with 10 mg/mL bovine serum albumin (BSA), and 0.01% (w/v) thimerosal (BPT). , For binding in the BLI system, both PBS and kinetics buffer (KB, PBST with 1 mg/mL BSA) were used at pH 7.4. Biotinylated mAbs were detected with horseradish peroxidase (HRP)–streptavidin (SA) conjugate (Invitrogen, Eugene, OR) at 0.3–1 μg/mL.…”

“…The immunochemistry of ricin has been extensively studied, particularly to optimize therapeutic immunotoxins containing ricin A chain and to define potentially protective and therapeutic antibodies . Exploitation of ricin antibodies and the amplification afforded by the polymerase chain reaction (PCR) has contributed new tools for food defense. , In an earlier study, we used BLI to study the interaction of ricin and RCA-1 with antibodies . In this report, we describe BLI and ELISA experiments to quantitate milk matrix effects and mitigate their impact on the analysis of a carbohydrate-binding protein, such as ricin.…”

“…20,21 In an earlier study, we used BLI to study the interaction of ricin and RCA-1 with antibodies. 22 In this report, we describe BLI and ELISA experiments to quantitate milk matrix effects and mitigate their impact on the analysis of a carbohydrate-binding protein, such as ricin.…”