Andrew A. Frohlich

Use of chicken egg‐yolk antibodies against K88+ fimbrial antigen for quantitative analysis of enterotoxigenic Escherichia coli (ETEC) K88+ by a sandwich ELISA

An enzyme-linked immunosorbent sandwich assay (ELISA) has been developed to detect and quantify both the K88+ fimbrial antigen and the concentration of bacterial cells in fecal and other samples using anti-fimbrial chicken egg-yolk and anti-fimbrial rabbit serum antibodies. The assay has a sensitivity of 40 ng ml−1 for K88+ fimbrial antigen and 107 CFU ml−1 for intact cells and cell homogenate. The inter- and intra-assay coefficients of variation (CV) for intact cells, homogenates, and fimbrial antigens were similar, suggesting that reproducible results can be obtained with any of the three preparations. In all cases the CV increased with decreasing concentration of the antigen, especially when very low concentrations of antigen were used. Fimbrial antigens had lower CV (values ranged from 2 to 37%, depending on its concentration) than standards prepared from either whole cells or homogenized cells (values ranged from 1 to 67%). The correlation between the actual number of cells as counted and the number as estimated from the ELISA using the amount of fimbrial antigen was r = 0.98, P < 0.001. The anti-K88+ polyclonal antibodies from chicken serum and egg yolk had little or no cross-reactivity with K99, 987P ETEC. The correlation (r) between the fecal score for diarrhea and the number of E coli in the fecal swab was high, r = 0.80 (n = 64, P < 0.0001). The assay is sensitive and specific and provides a good estimate of the amount of fimbrial protein or the number of K88+ ETEC in the sample. © 1999 Society of Chemical Industry

Enzymatic and Immunological Approaches for the Quantitation and Confirmation of Ochratoxin A in Swine Kidneys

Several techniques for the quantitation and confirmation of ochratoxin A (OA) in swine kidneys were examined. Naturally and artificially contaminated swine kidneys were analyzed for OA by conventional high-performance liquid chromatography (HPLC) analysis. Samples were additionally tested by enzyme-linked immunosorbent assay (ELISA) or treated with carboxypeptidase A followed by HPLC analysis (enzymatic method). Correlations (r values) between the conventional HPLC procedure and the ELISA, using artificially contaminated samples, were 0.88 and 0.81 (P < 0.05) respectively, while the corresponding values between the conventional HPLC procedure and the enzymatic method were 0.89 and 0.98 (P < 0.05). The ELISA gave a more direct estimation of OA contamination than the enzymatic procedure. The enzymatic method also had a reproducible tendency to underestimate or overestimate the amounts of OA in kidney. This was found to be dependent on the source of contamination, as artificially and naturally contaminated kidney samples resulted in linear regression analysis slopes of 0.38 and 2.8, whereas the slopes for the ELISA method were 1.13 and 0.92, respectively. The results with the naturally contaminated kidneys suggest that other naturally occurring forms of OA also occurred in swine kidney. Regardless of this effect, the enzymatic method accurately confirms the presence of OA and related compounds in kidney. The techniques are simple and will complement conventional HPLC analysis for the detection, quantitation, and confirmation of OA in swine kidneys.

The development of a quantitative ELISA for aflatoxin B1 in ground peanuts using antibodies isolated from the yolk of a laying hen

Antibodies directed against Aflatoxin B1 (AFB1) were produced in laying hens, isolated from egg yolk and applied in an enzyme-linked immunosorbent assay (ELISA) for AFB1 in ground peanuts. The ELISA sensitivity was improved by reduction of the amount of the coating antigen absorbed onto the microplate well surfaces. The main aflatoxins B1, B2, G1, G2, M1, aflatoxicol, sterigmatocystin were found to cross-react in the competitive ELISA, 100, 25, 23, 4, 0, 0, 0%, respectively. Aflatoxin B1 could be reproducibly detected and quantified in spiked ground peanuts at levels greater than 5 ppb using a hexane and methanol solvent based peanut extraction protocol.

Evaluation of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) excreta on ochratoxin production in stored wheat

Abstract The influence of excreta from the red flour beetle, Tribolium castaneum (Herbst), on the growth of Aspergillus alutaceus (formerly A. ochraceus Wilhelm NRRL 3174) and ochratoxin A (OA) production in sterile wheat was investigated. When sterile excreta (3–6%) was added to wheat, OA production by A. alutaceus during the first 2 weeks of growth was significantly ( P > 0.05) reduced. A similar trend was observed with fungal growth. The addition of equivalent levels of uric acid (76.8 or 153.5 mg/g wheat) to sterile wheat resulted in no significant ( P > 0.05) effect on OA production although fungal growth appeared slightly enhanced. In non-sterile wheat, the addition of sterile excreta (5%) significantly ( P > 0.05) reduced OA production compared to the control. The presence of excreta in a grain bulk may favor fungal competition thereby inhibiting OA production.

Comparative studies on the specificity and sensitivity of rabbit and laying‐hen antisera to ochratoxin a

Polyclonal antisera specific for ochratoxin A (OA) were developed in laying‐hens and rabbits immunized with OA conjugated to bovine serum albumin. The rabbits and layinghens were immunized and tested under parallel conditions. OA‐specific antiserum activity was monitored using an indirect, competitive enzyme‐linked immunosorbent assay (cELISA). Rabbits and hens produced OA‐specific antisera within the first 51 days of the immunization schedule. The rabbits consistently produced OA antisera in 10‐fold higher amounts than the laying‐hens. Rabbit and laying‐hen antisera were found to be equally specific at most times in the immunization schedule for ochratoxins B and C, but not α. Antisera from both sources could be used to quantify OA in spiked wheat. The rabbit antisera could reproducibly detect OA at concentrations of 3 μg kg‐1 or more, whereas antisera from hens could only detect 50 μg kg‐1 or more. Similar results were obtained when OA was quantified in spiked wheat with both antisera when assayed at th...

Isolation, purification and identification of intracellular protein targets of ochratoxin A: mitochondrial aldehyde dehydrogenase class 2 is a high-affinity ochratoxin A-binding protein

An affinity chromatography technique was utilised to isolate and purify the soluble intracellular protein targets of ochratoxin A (OA). OA was covalently immobilised on a beaded agarose matrix. Several proteins (approximately nine) in the soluble fraction of crude protein extracts of different bovine tissues were specifically bound to the immobilised OA. The binding of these proteins to the modified agarose could not be dissociated with mild buffer (buffer plus 0.5 M NaCl and 1% Tween 20), but the proteins were competitively eluted with free 25 mM OA in mild buffer. Only one major protein (55 kDa) remained tightly bound to the immobilised OA when the column was incubated with bovine liver extract and then washed extensively with higher concentrations of salt and detergent (2 M buffer plus NaCl and 5% Tween 20). The bound protein was eluted competitively with 25 mM OA and further purified using SDS-PAGE. The N-terminal sequence of 24 residues of the high-affinity OA-binding protein was shown to be AATQAVPTPNQQPEVLYNQIFINN. This sequence is identical to the 24 residues of the N-terminal sequence of bovine mitochondrial aldehyde dehydrogenase class 2 (ALDH2, EC 1.2.1.3). The molecular weight and tissue distribution of ALDH2 are the same as those of the high-affinity protein isolated in this study. Immobilised OA can serve as an excellent affinity matrix for the identification of target proteins of OA and for the purification of ALDH2 and possibly other proteins. © 1999 Society of Chemical Industry