Approach for ochratoxin A fast screening in spices using clean-up tandem immunoassay columns with confirmation by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).

An approach for ochratoxin A (OTA) fast cost-effective screening based on clean-up tandem immunoassay columns was developed and optimized for OTA detection with a cut-off level of 10 microg kg(-1) in spices. Two procedures were tested and applied for OTA detection. Column with bottom detection immunolayer was optimized for OTA determination in Capsicum ssp. spices. A modified clean-up tandem immunoassay procedure with top detection immunolayer was successfully applied for all tested spices. Its main advantages were decreasing of the number of analysis steps and quantity of antibody and also minimizing of matrix effects. The total duration of the extraction and analysis was about 40 min for six samples. Chilli, red pepper, pili-pili, cayenne, paprika, nutmeg, ginger, white pepper and black pepper samples were analyzed for OTA contamination by the proposed clean-up tandem immunoassay procedures. Clean-up tandem immunoassay results were confirmed by HPLC-MS/MS with immunoaffinity column clean-up. Among 17 tested Capsicum ssp. spices, 6 samples (35%) contained OTA in a concentration exceeding the 10 microg kg(-1) limit discussed by the European Commission. All tested nutmeg (n=8), ginger (n=5), white pepper (n=7) and black pepper (n=6) samples did not contain OTA above this action level.

Novel developments in rapid mycotoxin detection.

Rapid antibody-based mycotoxin screening techniques are designed to be used outside a laboratory environment, at the place of sampling. Results are expected immediately, so that commodities can be further processed without delay. Because they are used for mycotoxin analysis, very low levels (ppb and ppt range) should be detected. A further requirement is that the obtained results are accurate with a false negative rate of <5% at the level of interest.At first, plastic microtiter plates were used as solid phase materials for immobilizing antibodies (enzyme-linked immunosorbent assays). However, to increase speed and user-friendliness, plastics were replaced by microporous membranes. As an example a flow-through enzyme immunoassay for the detection of fumonisins in cornflakes with a cut-off value of 275 µg/kg is described. No false negative results were observed and the false positive rate was 18%. However, enzyme labels, used to enable visual evaluation of results, did not seem to be completely satisfactory in terms of stability and repeatability of the generated signal. Therefore microparticle labels such as colloidal gold particles are used more and more,e.g. in a lateral flow dipstick immunoassay. When applied to the detection of aflatoxin B1 in pig feed a cut-off value of 5 µg/kg could be reached with no false negative results and a false positive rate of only 10%. Sample pretreatment for screening techniques should be rapid and simple. Preferably a simple solvent extraction is used, followed by a filtration and dilution step. However, for strongly coloured or complex food matrices, this did not seem to work. The combination of clean-up and detection in one single test device is a new approach. When using this clean-up tandem assay column for the detection of ochratoxin A in roasted coffee, a cut-off value of 6 µg/kg was reached. No false positive results were obtained, however, the false negative rate was 8%.

Development of a sensitive ELISA for the determination of fumonisin B(1) in cereals.

Monoclonal fumonisin B(1) antibodies with high titer were raised by using FB(1)-glutaraldehyde-keyhole limpet hemocyanin immunogen prepared by a short cross-linker reagent (glutaraldehyde). Mean cross-reactivities of the selected monoclonal antibody for FB(1), FB(2), and FB(3) were 100, 91.8, and 209%, respectively; no reactivity was found with hydrolyzed fumonisin. A direct, competitive enzyme-linked immunosorbent assay for the quantitative determination of FB(1) in cereals has been developed with this antibody. Fifty percent acetonitrile-based solvent with some additives was used for extraction of cereals, and the diluted extracts were used without cleanup in the test. The mean within-assay and interassay coefficients of variation for the standard curve were <10%. The measuring range of this test is 10-500 ng/g, with a detection limit of 7.6 ng/g FB(1). The toxin recovery from cereals infected with 50-200 ng/g of FB(1) varied between 61 and 84%. According to the comparable results of naturally infected maize samples, this test proved to be suitable for the rapid screening of food and feed samples for the presence of FBs.

Analysis of serum and seminal plasma after feeding ochratoxin A with breeding boars.

The aim of the experiment was to investigate whether or not ochratoxin A (OA) can be detected in seminal plasma after feeding the toxin in five and 10 times of the human tolerable daily intake with breeding boars and how toxin profiles of serum and seminal plasma correspond to each other. In addition to that, the effect of the toxin challenge on motility and longevity of boar semen was also evaluated. OA from samples was analyzed by microplate ELISA. Percentage of progressive motility of spermatozoa was determined initially and after 24, 48, 96, 120 and 144 h of storage. OA appeared in serum and seminal plasma shortly after toxin application had started. Significant reduction of initial motility and impaired longevity was observed after toxin withdrawal. These findings suggest that OA might have the potential to affect sperm production and semen quality of boars, but further research is required to elucidate whether OA exerts direct effect on germinal epithelium or disturbs sperm cell maturation only.

Glycoprotein B of Aujeszky's disease virus: topographical epitope mapping and epitope-specific antibody response.

A panel of 26 monoclonal antibodies (mAbs) against glycoprotein B (gB) of Aujeszky's disease (pseudorabies) virus (ADV), a glycoprotein complex consisting of three glycoproteins, gBa, gBb, and gBc, was produced by two research groups and was used for the topographical epitope mapping of gB. An epitope map was constructed in which the identified epitopes of gB were situated in 14 topologically distinct antigenic domains; ten antigenic domains represented by 22 mAbs were localized on gBc, while four antigenic domains represented by four mAbs resided on gBb of the gB complex. All the epitopes located on gBc appeared to be conformation-dependent, whereas all the epitopes on gBb were conformation-independent. The identified epitopes of gB were conserved among laboratory, vaccine and field ADV strains. Conformation-dependent epitopes were shown to contribute largely to the overall antibody response to gB in naturally infected swine and immunized mice. Moreover, it was found that most of the infected animals responded relatively weakly to the identified conformation-independent epitopes of gB, while a group of immunodominant epitopes that induced a strong antibody response was represented exclusively by conformation-dependent epitopes from different antigenic domains. The results clearly demonstrated that conformation-dependent epitopes of gBc play a crucial role in inducing the humoral immune response to gB of ADV during the natural infection of swine and immunization of mice. The application of mAbs of our panel as research and diagnostic tools is discussed.

Ochratoxin A content of human sera determined by a sensitive ELISA.

A sensitive, monoclonal antibody-based ELISA test was developed and used for quantitative determination of ochratoxin A (OA) in human sera. The measuring range of this test (without sample dilution) was 0.2-2.0 ng/mL, and the detection limit was 0.2 ng/mL. The OA concentrations of 355 sera samples varied from < 0.2 to 10 ng/mL OA, but 75% of the samples contained 0.2-1.0 ng/mL. This amount reflects a tolerable daily intake (TDI) value of toxin. However, in some cases (6.8%), more than 1.0 ng/mL OA was measured, which is probably a result of elevated intake of OA, which may even exceed the "virtually safe dose". Our data indicate that, like in many other countries, OA is present in food or feed products available in Hungary, and in order to save the health of consumers, their regular control is desirable.

A new monoclonal antibody detecting ochratoxin A at the picogram level.

A monoclonal antibody against ochratoxin A was produced after immunization of Balb/c mice with ochratoxin A-BSA. This antibody was of the IgG1 heavy chain subclass with a kappa type light chain. The 50% inhibition value was 0.45 ng ml-1 in a direct competitive ELISA and the detection limit was 42 pg ml-1. This antibody is very specific, cross-reacting only with ochratoxin B (9.3%).

Latent immunization to produce high-affinity monoclonal antibodies to progesterone.

A simple immunization method to obtain high-affinity monoclonal antibodies to progesterone is described in this article. The method is based on the theory of affinity maturation. A long interval between antigen priming and booster ("latent immunization") permits an undisturbed completion of affinity maturation, resulting in the accumulation of memory B lymphocytes with high-affinity Ig receptors, and consequently, in a higher rate of hybridoma clones producing high-affinity antibody after cell fusion. Antibodies obtained after hyperimmunization and latent immunization are compared in a homologous, direct, competitive ELISA. The average numbers of high-affinity antibodies per fusion were 1.3 and 5.7 in the hyperimmunized and latent immunized groups, respectively. There was no significant difference in the specificities between the two immunization groups.

Monoclonal antibody-based enzyme-linked immunosorbent assay of Fusarium T-2 and zearalenone toxins in cereals.

Direct, competitive enzyme-linked immunosorbent assays (ELISAs) with monoclonal antibodies have been developed for quantitative determination of trichothecene T-2 toxin (T-2), and zearalenone (F-2) from different cereals: Among the several extraction solvents tried, 89% acetonitrile with additives was chosen. The extracts were then used without cleanup in the ELISA. With appropriate dilution of the samples (1:25 or 1:50), the matrix effects caused by lipid and/or protein content of the samples can be diminished to the extent that the assay is no longer impaired. The mean recoveries from cereals infected with 100 to 2,000 ng of T-2 and 50 to 500 ng of F-2 per g were 85 and 91%, respectively. The measuring range of the T-2 test is 100 to 2,000 ng/g, and that of the F-2 test is 25 to 400 ng/g. The mean within-assay and interassay coefficients of variation of standard curves are both less than 10%. According to recovery results with artificially infected cereals, our tests proved to be suitable for rapid screening of food and feed samples for the presence of T-2 and F-2 toxins.

In vitro neutralization of T-2 toxin toxicity by a monoclonal antibody.

A T-2 toxin specific monoclonal antibody, IgG1 K, with a low level of ELISA cross-reactivity to Acetyl T-2, HT-2, and iso T-2 toxins has been produced. The ability of this monoclonal antibody to neutralize the cytotoxicity of T-2 toxin in PHA stimulated cultures of human lymphocytes was determined by the MTT method. The complete neutralization of the toxic effect of 0.02 microM T-2 toxin was obtained with 0.03 microM of MoAb, whereas the 50% neutralizing dose (ND50) was observed at 0.009 microM of MoAb. Partial neutralization was observed with Acetyl T-2 toxin (ND50 = 0.038 microM) and HT-2 (ND50 = 0.94 microM). These results could represent a rational for clinical use of T-2 toxin specific monoclonal antibody in prophylaxis and therapy of T-2 toxemia.

Clinical and pathological effects of the dermonecrotic toxin of Bordetella bronchiseptica and Pasteurella multocida in specific-pathogen-free piglets.

The role of dermonecrotic toxin (DNT) of Bordetella bronchiseptica and Pasteurella multocida, purified by repeated chromatography in Sephacryl S-200 gel, in the pathogenesis of atrophic rhinitis (AR) of swine was studied bacteriologically, clinically and pathologically. Two-week-old specific pathogen-free (SPF) piglets were parenterally treated with 30 micrograms of DNT 3 times at 2-day interval and 7-week-old piglets were treated with 15 micrograms of DNT twice a week for 5 weeks. In 2- to 3-week-old piglets, both B. bronchiseptica DNT and P. multocida DNT produced nasal turbinate lesions with similar severity, characterized by damage of the cilia, epithelial metaplasia, intensive proliferation of osteoblasts, regressive changes, and diffuse osteocytic osteolysis. In 7- to 12-week-old piglets, treatment with B. bronchiseptica DNT failed to produce progressive changes in the nasal turbinates. Histopathological examination revealed osteogenic processes and osteoid synthesis besides the proliferation of osteoblasts and mild osteocytic osteolysis. Moreover, severe gross pathological lesions developed in the stomach, liver, kidneys, and lymphoid organs. The piglets' appetite and body weight gain gradually decreased during the DNT treatment and in the last week when the toxic signs appeared. Treatment of 7- to 12-week-old piglets with P. multocida DNT resulted in progressive AR. Histopathologically, diffuse osteocytic osteolysis was observed in the nasal turbinates. Neither clinical signs nor pathological lesions of the visceral organs developed in these piglets. The authors emphasize that the DNT of B. bronchiseptica basically differs from that of P. multocida in biological properties, though there are certain similarities between the DNTs.

Enzyme-linked immunosorbent assay in the serodiagnosis of syphilis.

Enzyme-linked immunosorbent assay using ultrasonic lysate of Treponema pallidum and Treponema reiteri as antigens was used for the detection of antisyphilitic antibodies in various stages of syphilis. The conjugate was goat antiserum to human IgG labelled with horseradish peroxidase. A comparison with results of the T. pallidum immobilization test, Rapid Plasma Reagin test, Kolmer complement fixation reaction using cardiolipin and Reiter protein antigens showed that ELISA was more sensitive but less specific with T. pallidum antigen, whereas less sensitive but more specific with T. reiteri antigen. Absorption of group specific treponemal antibodies was needed to make the method reliable.

Characterization of enhancing factor (EF) prepared from spleen of mice immunized with tetanus toxoid.

In cultures of spleen cells from tetanus toxoid-primed mice a soluble, nonspecific material appeared, which enhanced antibody response in vivo. The active material was purified by gel filtration on Sephadex-G-150 and by affinity chromatography on Con-A lectin. According to the immunological and physiochemical investigation the active material does not contain carbohydrate and its molecular weight is in the 100 000 dalton range.