摘要:

Mycotoxins are fungal secondary metabolites that sometimes contaminate agrifoods such as corn, wheat and peanuts prior to harvest and during storage. Because of their capacity to affect human and animal health, two key areas of mycotoxin research have been analysis and toxicity assessment. Research initiated in the late 1970s proposed the application of immunochemical assay to the analysis of aflatoxin B1(AFB1) and other mycotoxins. The most common assay, ELISA, involves the competition between a free mycotoxin in a sample extract with an enzyme‐labeled mycotoxin for an antibody binding site. This and other assays have been made available commercially for the rapid assay of mycotoxins in food. Important points to consider relative to development and application of mycotoxin im‐munoassays are: method of antibody production, adaptability to food analysis and multi‐analyte methods. In recent years, there has been increasing evidence that mycotoxins alter the intricate balance of the immune system. In experimental animals, vomitoxin and potentially other trichothecenes impair normal immune function and induce an autoimmune disease, IgA nephropathy. Trichothecene‐mediated immune suppression is readily explainable by its potent capacity to inhibit protein synthesis. Its ability to stimulate certain immune functions such as IgA hyper‐production IgA might be explained by superinduction of T‐cell cytokines.

ISSN:0954-0105    

DOI:10.1080/09540109409354833

出版商:Taylor & Francis Group    

年代:1994

数据来源: Taylor

摘要:

New Zealand pastoral agriculture relies heavily on the intensive grazing of pasture. To protect both the animal and the consumer, this feedstuff must be of high quality; the detection of mycotoxin contamination is thus essential. The analysis of mycotoxin contamination of herbage feed requires robust assays which can reliably cope with this complex matrix. Conventional analysis of herbage by high‐pressure liquid chromatography, gas chromatography/mass spectrometry is at best tortuous, relying on extensive extract cleanup and purification prior to analysis to avoid matrix interferences. No routine methods exist as yet for the analysis of the trichothecenes in pasture samples. Recent advances in analytical procedures have seen the rapid development of immunoassays, such as enzyme‐linked immunosorbent assays (ELISAs), which have the advantage of simplicity, speed and sensitivity—but these too frequently suffer matrix interferences when applied to herbage samples. We describe here a simple extraction and solvent partition clean‐up procedure which minimizes the interferences caused by the grass matrix on the analysis of herbage samples for 3‐acetyldeoxynivalenol (3‐AcDON) by an indirect competitive ELISA.

ISSN:0954-0105    

DOI:10.1080/09540109409354834

出版商:Taylor & Francis Group    

年代:1994

数据来源: Taylor

摘要:

The detection of illegal growth promoters in cattle urine by conventional immunoassays, such as radioimmunoassay (RIA) and enzyme‐linked immunosorbent assay (ELISA) has been extensively described. However, improved speed and simplicity, with the development of a fast and simple on‐site test as the ultimate goal, remains an interesting immunochem‐ical challenge. This paper describes two approaches to this problem. To avoid time‐consuming sample preparations, attempts were made to omit the extraction and hydrolysis step. Secondly, instead of the classical microtiter plate as the solid phase, magnetizable beads, which provided a solid phase that was dispersed throughout the sample, were used. In this design, the diffusion limitations of the free immunoreagents to the solid‐phase‐bound immunoreagent (which slow down the kinetics of the antibody‐antigen reaction) were circumvented. This principle was applied to the detection of the β‐agonist clenbuterol and the anabolic steroid nortestosterone in cattle urine. The assay of unextracted urine for the presence of nortestosterone failed due to matrix effects. For clenbuterol, a magnetic particle‐based immunoassay on unextracted urine samples was developed. The test, which can be performed within 90 min using pre‐coated beads, compared favourably to microtitre plate ELISA (with a4h incubation) and to a commercial RIA. The results on test samples were confirmed by gas chromatography/mass spectrometry.

ISSN:0954-0105    

DOI:10.1080/09540109409354835

出版商:Taylor & Francis Group    

年代:1994

数据来源: Taylor

摘要:

Several programme offices of the United States Envirnmental Protection Agency (USEPA) have been investigating the use of immunoassay methods for environmental applications for several years, but with limited success. Most of the problems concerned method ‘rugged‐ness’ (i.e. they worked well in clean, spiked matrices, but not well on actual samples). The situation has changed significantly since January 1992, when the Office of Solid Waste received its first rugged immunoassay method (for pentachlorophenol) that worked on field samples. In the past 30 months, several more have followed. This paper addresses several major topics including: an overview of the USEPA's major regulatory programmes; how analytical methods are used in regulatory programmes and a brief overview of the regulatory approval process; general guidelines for the development of screening methods; specific validation criteria for immunoassay methods; the current status of the USEPA immunoassay method development programme; current and potential environmental applications for immunoassay technology; and barriers to implementation of immunoassay methods and the steps being taken to overcome them. The overall future of the technology for environmental monitoring and analysis looks very bright. It offers a cost‐effective way to generate reliable information on which sound environmental decisions can be based.

ISSN:0954-0105    

DOI:10.1080/09540109409354836

出版商:Taylor & Francis Group    

年代:1994

数据来源: Taylor

摘要:

An enzyme‐linked immunosorbent assay (ELISA) for 2,4‐dichlorophenoxy acetic acid (2,4‐D) was developed using polyclonal antibodies raised in sheep, immunized with a derivative of 2,4‐D (2,4‐dichlorophenoxy acetyl‐2‐succinamyl hydrazine) conjugated to keyhole limpet haemocyanin. The mean percentage recovery of 2,4‐D from fortified water samples was 104% and ranged from 96 to 120% for fortified orange juice. The sensitivity of the assay without concentration of sample or sample clean‐up was 0.2 ng ml−1for water and 50 ng ml−1for orange juice. With dichloromethane extraction of 2,4‐D from orange juice, 1 ng ml−1could be detected. 2,4,5‐T was the only herbicide investigated which cross‐reacted significantly with the 2,4‐D antisera. The ELISA also detected 2,4,5‐T in water, being sensitive to 0–66 ng ml−1. The ELISA is suitable for direct screening of water and fruit juice samples for 2,4‐D contamination.

ISSN:0954-0105    

DOI:10.1080/09540109409354837

出版商:Taylor & Francis Group    

年代:1994

数据来源: Taylor

摘要:

The synthetic pyrethroid permethrin has widespread use in agriculture and, as a result, is found in a variety of matrices. Chemical methods based on gas chromatography/mass spectrometry operated in the negative chemical ionization mode (GC‐NCI/MS) have been developed. Sample extraction techniques such as ultrasonication and steam distillation have also been examined. Significant improvements have been made, compared with traditional methods, in both sample extraction time and detection level for a range of matrices. Enzyme‐linked immunosorbent assay (ELISA) methods have also been developed for permethrin. This study addresses the comparison of ELISA and chemical assays for permethrin in a variety of both environmental and laboratory‐spiked samples. The sensitivities, specificities and potential applications of these methods are discussed.

ISSN:0954-0105    

DOI:10.1080/09540109409354838

出版商:Taylor & Francis Group    

年代:1994

数据来源: Taylor

摘要:

During the past 4 years, several laboratories have developed new methods of cloning antibody combining site genes from hybridomas or B‐lymphocytes, and functionally expressing them in bacteria, yeast, mammalian cells or plants. At least three research groups have also constructed ‘antibody display’ libraries with vastly diverse sequence permutations of combining site genes in bacteriophage. These semi‐synthetic combinatorial libraries present an antibody repertoire orders of magnitude greater than that accessed by conventional hybridoma technology. They may yield antibodies not obtainable through conventional immunization. We have cloned the immunoglobulin genes from a hybridoma specific for the phenylurea herbicide, diuron, into a phage display vector. The cloned antibody fragments (Fabs) were as sensitive as the parent monoclonal antibody for detecting free diuron in competition enzyme immunoassays. We also derived diuron hapten‐specific clones from synthetic combinatorial phage libraries, but only a few weakly recognized free diuron. The relative merits of deriving Fabs from synthetic phage display libraries, and the steps in engineering new specificities and other properties into recombinant antibodies, are discussed.

ISSN:0954-0105    

DOI:10.1080/09540109409354839

出版商:Taylor & Francis Group    

年代:1994

数据来源: Taylor

摘要:

Samples of bovine milk, from cows vaccinated and non‐vaccinated against a wide range of pathogenic bacteria includingSalmonella enteritidis,previously heated to different temperatures between 37°C and 75°C, were examined for specificity and the ability of immunoglobulin G (IgG) to bind to antigen (lipopolysaccharide fraction fromS. enteritidis)in a particle concentration fluorescence immunoassay (PCFIA). Antigen binding activity falsely increased with heating temperature. This trend was not observed when using enzyme‐linked immunosorbent assays (ELISAs). In order to identify the factor(s) responsible for the increased fluorescence values when using PCFIA, purified bovine and chicken IgG molecules were heat treated and the fluorescence values recorded. The fluorescence of the purified immunoglobulins also increased with heating temperature. Tryptophan (trp) is present at the active binding site of IgG molecules. Quenching of trp residues with 0.6 M‐acrylamide demonstrated that the increase in fluorescence observed with increased heating temperature was associated with the presence of trp residues in the immuno globulin molecule (antigen binding site). When the Stern‐Volmer equation was applied to the fluorescence data, the results indicated that none of the proteins studied gave an evident non‐linear plot. The bimolecular quenching constant, kqincreased from 21°C to 75°C, indicating increased exposure of trp residues to solvent. A method is proposed to use acrylamide as a trp fluorescence quencher in studies involving the determination of biological activity of previously heat‐treated immunoglobulins by PCFIA. The measured fluorescence is then only associated with the concentration of fluorescein isothiocyanate‐labeled antibody bound to the antigen. The results are then similar to trends observed using ELISA.

ISSN:0954-0105    

DOI:10.1080/09540109409354840

出版商:Taylor & Francis Group    

年代:1994

数据来源: Taylor

摘要:

Polyclonal antibodies were raised in male New Zealand White rabbits against a 0.5% (v/v) Lubrol PX protein extract of crude chicken red bone marrow. Following serum protein removal by ammonium sulphate precipitation, the serum was affinity‐purified against muscle protein from a selection of different species. The antiserum, when assayed by enzyme‐linked immunosorbent assay, was shown not to recognize any proteins from specimens other than chicken hand‐deboned (HDM) and mechanically separated (MSM) meats, with the reaction towards MSM chicken being significantly stronger than that against HDM chicken. When the same samples were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred to nitrocellulose by Western blotting and immunochemically stained with the affinity‐purified antiserum, they showed specific staining of protein bands within chicken HDM and MSM, with no reaction towards any proteins of any other species. A band common to both chicken HDM and MSM at about 45 kDa , and a band specific for MSM at 70 kDa were the major immunoreactive proteins.

ISSN:0954-0105    

DOI:10.1080/09540109409354841

出版商:Taylor & Francis Group    

年代:1994

数据来源: Taylor

摘要:

Immunoassays have been developed to facilitate authenticity testing of fish and seafood products. Three specific authenticity issues were addressed: substitution of canned sardine with other species, adulteration of canned tuna with bonito and adulteration of reformed crustacean meat with white fish. Antisera were raised to water‐soluble protein extracts of canned sardine and canned tuna and, where appropriate, their specificities were adjusted using simple blocking and/or immunoadsorption methods. The antisera were used to construct several immunoassays, one of which allowed discrimination between canned sardine and non‐sardine meat, and could be used to detect substitution of whole sardines with non‐sardine species. A second set of assays allowed discrimination between fish and crustacean meat, and should allow the detection of fish in crustacean products. However, with a third set of immunoassays, only limited discrimination between bonito and tuna could be achieved and these assays require further development.

ISSN:0954-0105    

DOI:10.1080/09540109409354842

出版商:Taylor & Francis Group    

年代:1994

数据来源: Taylor