David L. Brandon

ELISA Analysis of Soybean Trypsin Inhibitors in Processed Foods

Soybean proteins are widely used in human foods in a variety of forms, including infant formulas, flour, protein concentrates, protein isolates, soy sauces, textured soy fibers, and tofu. The presence of inhibitors of digestive enzymes in soy proteins impairs the nutritional quality and possibly the safety of soybeans and other legumes. Processing, based on the use of heat or fractionation of protein isolates, does not completely inactivate or remove these inhibitors, so that residual amounts of inhibitors are consumed by animals and humans. New monoclonal antibody-based immunoassays can measure low levels of the soybean Kunitz trypsin inhibitor (KTI) and the Bowman-Birk trypsin and chymotrypsin inhibitor (BBI) and the Bowman-Birk foods. The enzyme-linked immunosorbent assay (ELISA) was used to measure the inhibitor content of soy concentrates, isolates, and flours, both heated and unheated; a commercial soy infant formula; KTI and BBI with rearranged disulfide bonds; browning products derived from heat-treatment of KTI with glucose and starch; and KTI exposed to high pH. The results indicate that even low inhibitor isolates contain significant amounts of specific inhibitors. Thus, infants on soy formula consume about 10 mg of KTI plus BBI per day. The immunoassays complement the established enzymatic assays of trypsin and chymotrypsin inhibitors, and have advantages in (a) measuring low levels of inhibitors in processed foods; and (b) differentiating between the Kunitz and Bowman-Birk inhibitors. The significance of our findings for food safety are discussed.

Effects of purification on the bioavailability of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. They are primarily produced by the gram-positive, anaerobic spore-forming bacterium, Clostridium botulinum. In bacterial cultures, secreted BoNTs are associated with non-toxic accessory proteins forming large complexes. Neurotoxin-associated proteins have been shown to play an important role in the oral toxicity of BoNTs by protecting them from degradation and digestion by gastric acid and enzymes. Most toxicity studies using BoNTs have been performed using highly purified toxin. In this study, the toxicities of purified and crude BoNT/A toxin preparations were compared. Protein components secreted into culture supernatants along with BoNT/A were identified by mass spectrometry and the contribution of extra proteins found in the soluble crude toxin extracts to the toxicity of BoNTs was determined in mouse models of oral and parenteral botulinum intoxication. Analysis of crude toxin composition permitted assessment of the impact of accessory proteins on the oral bioavailability of BoNT/A toxin in food matrices.

Internalisation of the Bowman-Birk Protease Inhibitor by Intestinal Epithelial Cells

Protease inhibitors have been shown to be effective suppressors of carcinogenesis in vitro and in vivo. For example, the soybean-derived Bowman-Birk inhibitor (BBI) suppresses dimethylhydrazine-induced colon carcinogenesis in mice. Relatively little is known about the effects of protease inhibitors on intestinal epithelial cells. In the present study, we have investigated the interaction of the anticarcinogenic BBI with intestinal epithelial cells. At the concentrations examined, BBI was non-toxic and had no effect on the doubling time, saturation density or rate of DNA synthesis by these cells. This compound was taken up by these cells in a time dependent manner and was present in the cells for 12 h following a 2 h incubation with BBI. Subcellular fractionation experiments demonstrated that the bulk of the internalised inhibitor was present in the cytosol. Analysis of BBI from treated cells on a chymotrypsin affinity column revealed that active inhibitor was present in the cells. Our results indicate that the BBI is internalised by colonic epithelial cells which would allow BBI to inhibit critical intracellular proteases and thus suppress malignant transformation.

Development of monoclonal antibodies specific for Ricinus agglutinins

Abstract Ricin is a highly toxic, dichain ribosome-inactivating protein present in the seeds of Ricinus communis (castor), grown principally as a source of high quality industrial lubricant and as an ornamental. Because of its presence in industrial byproducts and its documented use for intentional poisoning, there is a need for analytical methodology to quantify ricin in both castor extracts and food matrices. We developed a panel of monoclonal antibodies to ricin, with most having strong cross-reactivity with RCA-1, a homologous but less toxic castor agglutinin. Some of the IgM-producing hybridomas appeared to produce a second, IgG isotype and were further analysed by fluorescence-activated cell sorting. The antibodies were effective in various ELISA formats, many with IC50s in the range of 0.1–10 ng/mL and minimal matrix effects in skim milk. Assay specificity can be adjusted for analytical needs by varying the combination of antibodies in a sandwich ELISA format.

Immunosorbent analysis of ricin contamination in milk using colorimetric, chemiluminescent and electrochemiluminescent detection

Analytical methodology to detect active ricin in food matrices is important because of the potential use of foodborne ricin as a terrorist weapon. Monoclonal antibodies (mAbs) that bind ricin were used as both capture and detection ligands in sandwich enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescent (ECL) immunosorbent assays. ELISA employed two types of substrate for colorimetric or chemiluminescent detection. Although both fat content and protein content of samples influenced the recovery of ricin, the lower limit of detection (LOD) in ELISA and ECL systems permitted detection of 0.1 ng/mL for milk samples containing 0–4% fat. The assay systems detect pure ricin- or crude ricin-containing castor extract, but do not significantly respond to isolated ricin chains, heat-denatured ricin or the related agglutinin, Ricinus communis agglutinin 1 (RCA-1). Using the standard 96-well-plate formats, the assays detect less than 0.01% of an adult human lethal dose in a typical serving of milk.

Detection of Ricin Contamination in Liquid Egg by Electrochemiluminescence Immunosorbent Assay

UNLABELLED A monoclonal antibody-based electrochemical luminescence method was developed for detecting and quantifying ricin in liquid egg, with a limit of detection of 0.2 ng/mL. Because this highly toxic protein, present in the seeds of Ricinus communis (castor), has been used for intentional poisoning in the past, it is important to have sensitive and reliable analytical methodology to detect ricin in food matrices such as liquid egg. The detection of this quantity of pure or crude ricin spiked into commercial samples of liquid egg provides approximately 50000-fold greater sensitivity than required to detect a toxic dose of ricin (>1 mg) in a 100 g sample. PRACTICAL APPLICATION Because ricin has been used for intentional poisoning, there is a need for analytical methodology to detect ricin in food matrices to assure a safe food supply. Using monoclonal antibodies to ricin developed in our laboratory, we explored an assay readout system known as electrochemiluminescence. This technique afforded sensitive and specific analysis of ricin intentionally added to liquid egg and could potentially be used to monitor egg-based vaccine production.

Antibody Interactions with Ricinus communis Agglutinins Studied by Biolayer Interferometry

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.

Electrochemiluminescence immunosorbent assay of ricin in ground beef: biotinylated capture antibodies and matrix effects

Abstract Ricin is a highly toxic protein present in the seeds of castor (Ricinus communis), grown principally as a source of high quality industrial lubricant. Because of the past use of ricin for intentional poisoning, there is a need for analytical methodology to detect ricin in food matrices. Ground beef and other fatty, solid matrices present challenges for extraction and detection of protein constituents. This study focused on the use of streptavidin-coated assay plates, with biotinylated monoclonal antibodies (mAbs) immobilised as capture reagents. It explored matrix effects on immunosorbent analyses of ricin in enzyme-linked and electrochemiluminescent detection systems. A variety of mAb pairs enabled assays with predetermined specificity for ricin vs. the related protein, Ricinus communis agglutinin-1 (RCA-1). Extraction of samples at low dilution (1:5) and inclusion of 100 mM galactose in the extraction medium produced excellent quantification of ricin in the 1–20 ng/g range in ground beef.

Monoclonal Antibody to Fenbendazole: Utility in Residue Studies

A monoclonal antibody-based ELISA was developed for fenbendazole, a widely used benzimidazole anthelmintic, with approved uses in cattle and other food animals. The antibody was elicited using as hapten 2-succinamido-5(6)-phenylthiobenzimidazole, which was conjugated with bovine serum albumin to produce an immunogen and with horseradish peroxidase to prepare a labeled ligand. The ELISA was performed on aqueous extracts of bovine liver or diluted milk samples. In bovine liver, the limit of detection was 200 ppb; in the milk matrix, the limit of detection was 3 ppb. The ELISA method is a simple approach to screen food samples for residues of fenbendazole.

Competitive ELISA of Thiabendazole Residues in Produce Using Indirectly Immobilized Monoclonal Antibodies

An enzyme‐linked immunosorbent assay (ELISA), developed originally for analysis of thiabendazole (2‐[4‐thiazolyl] 1H‐benzimidazole, TBZ) residues in bovine liver, was used to analyze residues of this post‐harvest fungicide in the peel of apple, potato, orange, grapefruit and banana. Residues were extracted by soaking peels overnight in 80% methanol and filtering the decanted supernatants. A 20‐fold dilution eliminated significant matrix effects. The ELISA has a limit of detection of 0.1 ppm in peel samples, corresponding to 10–40 ppb in the whole fruit or tuber. The ELISA employs a monoclonal antibody layer, immobilized indirectly via a rabbit anti‐mouse IgG secondary antibody, and horseradish peroxidase‐conjugated 5‐succinamido‐TBZ. Assay wells coated in this way could be dried and stored for at least 6 months without detectable changes in assay parameters. The method exceeds the required sensitivity for regulatory monitoring of TBZ residues in the US, and offers greater speed and specificity than previo...