摘要:

The performance of a monoclonal antibody (MAb)-based indirect competitive ELISA (ICELISA) for fumonisin detection in corn was evaluated using 150 post-harvest corn samples from the April 1995 and March-April 1996 crop, in the State of Paraná, Brazil. Compared to high performance liquid chromatography (HPLC), the correlation coefficient (r) of the results from the two methods was 0.94. The IC-ELISA detected total fumonisin levels of 1.1 to 1.5-fold higher than HPLC in 85 samples (56.6%). The concentration range of 1.0 to 5.0 μg g−1was observed in 26% of samples analysed by ELISA and in 29% by HPLC. In addition, four samples (2.7%) showed an ELISA/HPLC ratio of 0.7 to 0.99 and seven samples (4.7%) a 1.0 ratio. In 29 (13.3%) samples, IC-ELISA detected 1.6 to 2.0-fold higher values than HPLC. These data showed high correlation (r= 0.91) of IC-ELISA and HPLC for fumonisin concentration in corn contaminated with ≤ 10 μg g−1, with a detection limit of 93 ng g−1. Matrix interference was minimized by diluting the sample before the ELISA assay. MAb-based IC-ELISA may be effectively applied for fumonisin screening in corn, with advantages of simplicity, ease of sample preparation and sensitivity.

ISSN:0954-0105    

DOI:10.1080/09540100099580

出版商:Taylor & Francis Group    

年代:2000

数据来源: Taylor

摘要:

Polyclonal antibodies (PAbs) against eight different sulphonamides were raised in rabbits. The aromatic amino group, common to all sulphonamides, was used for linking the different sulphonamides to the carrier proteins (bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH)) and enzyme (horseradish peroxidase (HRP)), using different coupling procedures. The competitive direct ELISAs (cdELISAs) developed with these antisera and HRP-conjugates showed high sensitivity (0.2- 8.0 ng ml−1at 50% inhibition) and high specificity. The performances of these antibodies were compared with PAbs raised in mice against two sulphonamide derivatives (N1-[4-(carboxymethyl)-2-thiazolyl]sulphanilamide (TS) andN1-[4-methyl-5-\[2-(4-carboxyethyl-1-hydroxyphenyl)]-azo-2-pyridyl]sulphanilamide (PS)) linked to proteins (BSA and KLH) in such a way that the common aromatic amino group was distal to the protein. In competitive indirect ELISAs (ciELISAs), these PAbs recognized several structurally different sulphonamides. The PAbs from mice immunized with TS-BSA reacted with sulphonamides containing thiazolyl, thiadiazolyl, pyridazinyl and isoxazolyl groups. The PAbs from mice immunized with PS-KLH reacted with sulphonamides containing pyrimidinyl, pyridazinyl, quinoxalinyl and pyridinyl groups. The spleen cells of the mice were fused with myeloma cells to obtain monoclonal antibodies (MAbs) producing hybridomas. So far, with only one of the mice (immunized with TS-BSA), this resulted in four different MAbs which recognized several sulphonamides. By use of the best MAbs (27G3A9B10 and 4E10B12B6E12) and an optimized ciELISA protocol, eight structurally different sulphonamides showed 50% inhibition at concentrations less than 100 ng ml−1or 5 ng/well. However, other relevant sulphonamides (such as sulphadimidine, sulphatroxazole and sulphachloropyrazine) were detected at a high level only.

ISSN:0954-0105    

DOI:10.1080/09540100099599

出版商:Taylor & Francis Group    

年代:2000

数据来源: Taylor

摘要:

The one step strip test described is a competitive immunoassay in which the detector reagent consists of colloidal gold particles coated with affinity purified monoclonal anti(dihydro)streptomycin antibodies. The capture reagent in the assay is a streptomycin- bovine serum albumin conjugate which is immobilised on the lateral flow membrane of the test device. In the test procedure, three drops of raw milk are brought into the sample well of the test device and allowed to migrate over the membrane. The more analyte present in the sample, the more effectively it will compete with the streptomycin immobilised on the membrane for binding to the limited amount of antibodies of the detector reagent. A sufficient amount of (dihydro)streptomycin in the sample will thus prevent the binding of the detector reagent to the streptomycin immobilised on the membrane, resulting in disappearance of the test line in the read out zone. Using raw milk samples, spiked with either streptomycin or dihydrostreptomycin, complete disappearance of the test line was obtained with 160 ng ml−1and 190 ng ml−1, respectively. This demonstrates that the test is applicable for screening raw milk samples for the presence of (dihydro)streptomycin residues at MRL level (EU: 0.2 mg (dihydro)streptomycin kg−1milk). The major advantages of the one step strip test are that results can be obtained within 10 min and that all reagents are included in the test device.

ISSN:0954-0105    

DOI:10.1080/09540100099607

出版商:Taylor & Francis Group    

年代:2000

数据来源: Taylor

摘要:

Coeliac disease (CD) is a gastrointestinal afliction triggered by the ingestion of prolamins from wheat, barley, rye and possibly oats. The only treatment for CD is a strict diet, free of the toxic components. Immunochemical methods are usually applied to certify foods aimed at coeliac patients. The characterization of a panel of four anti-prolamin monoclonal antibodies (MAbs) to be used to certify gluten-free products is described here. To this aim, purified gliadin, secalin and hordein fractions were obtained by preparative electrophoresis at acid pH. This procedure provides purified fractions not exposed to denaturing conditions. The specificity of the MAbs was tested by ELISA against purified fractions and ethanol extracts of wheat, barley, rye, oats, rice, maize, buckwheat, sorghum and soy. The four MAbs recognized only coeliac-toxic cereals. Each MAb reacted strongly with gliadins and showed differential reactivity against the different prolamin purified fractions. Some MAbs showed a broad pattern of recognition whereas others presented a more restricted one. The reactivity observed corresponded to structural homologies among gliadin, secalin and hordein fractions. It is remarkable that some fractions obtained by electrophoresis in the presence of sodium dodecylsulphate were not recognized by some MAbs, whereas the same components obtained by preparative A-PAGE showed high reactivity. This reinforces the suitability of the purification method employed in this study to isolate prolamin fractions. Using these purified prolamins, characterization of anti-prolamin MAb reactivity was achieved.

ISSN:0954-0105    

DOI:10.1080/09540100099616

出版商:Taylor & Francis Group    

年代:2000

数据来源: Taylor

摘要:

Gliadin and glutenin contents are known to affect gluten functional properties. ELISA tests, which are rapid and easy to perform, can provide very convenient methods for industrial quality control. The purpose of this study was to develop a competitive ELISA for quantification of gliadins in wheat flour and compare the results with those obtained by nitrogen and reverse-phase high-performance liquid chromatography (RP-HPLC) determination of gliadin content after sequential extraction. Asαβ-gliadin and total gliadin contents are highly correlated, polyclonal antibodies directed against the C-terminal peptide ofαβ-gliadins were used in a sequential competitive ELISA to quantify total gliadins. In 21 flour samples, the results with ELISA were closely correlated with those obtained by nitrogen and RP-HPLC determinations (r> 0.82,P< 0.001), indicating that the ELISA test based on detection ofαβ-gliadins allowed fairly accurate quantification of total gliadins. Immunoblotting analysis after acid-PAGE showed that someβ-gliadin components were poorly detected or undetected by the antiserum, which could account for differences between the immunochemical and biochemical values observed for a few cultivars.

ISSN:0954-0105    

DOI:10.1080/09540100099625

出版商:Taylor & Francis Group    

年代:2000

数据来源: Taylor

摘要:

This paper presents the characterisation of polyclonal antibodies directed against two different N-terminal sequences ofω-gliadins. Both antisera recognised specifically the corresponding omega-gliadin fractions but showed different reactivities againstω-gliadins extracted from bread and durum wheats. Antibodies directed against the 'SRLL' type (ω5 type) recognised severalω-gliadin components in bread and durum wheat extracts whereas antibodies directed against the 'AREL' type (ω2 type) reacted specifically with anω-gliadin component from bread wheat. The narrow specificity of this antiserum made it potentially interesting for the detection of bread wheat additions in durum wheat pasta. Moreover, the reactivity of this antiserum was not modified by an increase in drying temperature of pasta.

ISSN:0954-0105    

DOI:10.1080/09540100099634

出版商:Taylor & Francis Group    

年代:2000

数据来源: Taylor

摘要:

This study is a critical comparison of reported methods of purification of IgY from hen egg yolk. Five methods of lipid removal were compared, followed by a comparison of three methods of immunoglobulin precipitation. Each of these methods was tested three times. Lowry assays were used to measure the protein content of the various purified fractions, and densitometric analysis of SDS-polyacrylamide gels was used to quantify the proportion of IgY. Peak IgY yields of 15.6 and 15.1 mg of IgY per ml of egg yolk, with greater than 60% purity, were obtained after lipid removal using dextran sulphate and calcium chloride or phosphotungstic acid and magnesium chloride, respectively. Further precipitation of IgY from these fractions using 12% PEG, the most effective method of immunoglobulin precipitation, recovered pure IgY preparations, with mean yields of 8.80 and 8.62 mg per ml of egg yolk. Alternatively, a simpler and more cost effective method of lipid removal by freezing and thawing of egg yolk at neutral pH recovered 13.1 mg of IgY per ml of egg yolk at a purity of 71.1%. Subsequent Ig precipitation also recovered a pure IgY preparation with a mean yield of 7.49 mg per ml of egg yolk.

ISSN:0954-0105    

DOI:10.1080/09540100099643

出版商:Taylor & Francis Group    

年代:2000

数据来源: Taylor

摘要:

Agglutinin and toxin was isolated and purified fromAbrus precatoriusseeds using conventional biochemical techniques such as Sepharose 4B affinity chromatography, Sephadex G-100 gel filtration and analytical HPLC chromatography. The yield of purified agglutinin was 272 mg per 50 g of seeds which was composed of 8.0% agglutinin and 6.1% toxin. SDS gel electrophoresis of purified protein under reducing and non-reducing conditions of native and denatured protein revealed four bands with molecular weights ranging from 31 to 66 kDa by protein and glycoprotein staining procedures. The adjuvant property of abrus agglutinin was evaluated using rat as an animal model, in comparison with Freund's complete adjuvant. Ovalbumin was used as a test antigen and was injected along with Freund's complete adjuvant (Group I experimental animals) and along with denatured abrus protein (Group II experimental animals). The humoral response was compared between two groups of animals (Group I and II) for total (by sandwich ELISA) and specific response (by antibody capture assay). Group II animals which received denatured abrus protein along with test antigen showed a significantly higher IgG response of 23.0 ± 2.37 mg ml−1(p< 0.001) when compared to Group I animals (14.0 ± 2.50 mg ml−1). However, there was no statistically significant difference between Group I and Group II, with respect to specific IgG response to test antigen. Thus, the present investigation suggests that the denatured abrus agglutinin can be used as an effective alternative protein adjuvant.

ISSN:0954-0105    

DOI:10.1080/09540100099652

出版商:Taylor & Francis Group    

年代:2000

数据来源: Taylor