摘要:

A convenient new bacteriophage display vector, pSD3, has been constructed and used to generate rabbit monoclonal anti-pesticide antibody fragments. Following amplification of immunoglobulin light chain, and heavy chain variable region gene libraries, restriction enzymes Sfi I and PflM I are used to assemble scFv libraries in pSD3. This allows the number of stages involving the polymerase chain reaction and restriction enzyme digestion to be minimized to optimize maintenance of the original diversity of the variable region genes in the libraries. The vector also incorporates an amber codon, a 6xHis tag and a c-myc epitope to facilitate soluble single-chain Fv production detection and purification. Using the pSD3 system two anti-atrazine single-chain Fvs were isolated from a library derived from the spleen cells of a rabbit immunized with bovine serum albumin-atrazine conjugate. Characterization of single-chain Fvs by competition and equilibrium ELISA indicated good specificity and affinity to atrazine.

ISSN:0954-0105    

DOI:10.1080/09540109999870

出版商:Taylor & Francis Group    

年代:1999

数据来源: Taylor

摘要:

A rapid immunoassay using an optical biosensor (BIAcoreTM) for determining the presence of sulphadiazine (SDZ) residues in pig bile was developed. SDZ was immobilised onto the surface of a dextran-coated silicon chip and a solution containing SDZ antibody, sample and buffer was injected over the chip surface. The level of antibody binding to the chip was determined after 20 s and the surface of the chip was then regenerated over a 1-min period prior to another sample injection taking place. Standard curves were constructed to allow quantification of SDZ presence in sample. Concentrations ranging from 0 to 10.64 mu g ml-1 SDZ were detected in bile samples taken from experimentally fed pigs and randomly selected pigs taken from a local slaughterhouse. These results were compared to the concentrations of SDZ detected by high-performance liquid chromatography in associated tissues. When concentrations in bile exceeded 0.6 mu g ml-1 SDZ, the corresponding edible tissue was above the maximum residue level (MRL), i.e. 0.1 mu g g-1 in 13 out of 14 cases. When the bile concentration was less than 0.6 mu ml-1 the associated tissue concentrations never exceeded the MRL. This experiment has indicated that biosensor analysis of bile is a highly effective method for detecting violative SDZ residues in meat.

ISSN:0954-0105    

DOI:10.1080/09540109999889

出版商:Taylor & Francis Group    

年代:1999

数据来源: Taylor

摘要:

Using a partially purified norsolorinic acid reductase (NSR) preparation as the immunogen, monoclonal antibodies (mAbs) against NSR, an enzyme responsible for the conversion of norsolorinic acid to averantin in the early stage of aflatoxin biosynthesis, were produced. An ELISA, using partially purified NSR as coating antigen and a second antibody-peroxidase conjugate as indicator, was established for measurement of the antibody titre and enzyme level. A total of 12 hybridoma cell lines that produced antibodies against various proteins were obtained. HPLC-postcolumn ELISA, immunoblot analysis and enzyme inhibition study revealed that a mAb elicited in cell line 10D2 specifically bound with the 43 kDa NSR. Immunoblot and ELISA analyses of extracts from various fungal cultures showed that the 10D2 mAb was highly specific to the NSR; only culture extracts with positive NSR activities contained the 43 kDa band and had a positive reaction with the antibody in the ELISA. The mAb produced by 10D2 cell line was capable of partially neutralizing the NSR activity and has also been used for detection of expression of the gene encoding the enzyme.

ISSN:0954-0105    

DOI:10.1080/09540109999898

出版商:Taylor & Francis Group    

年代:1999

数据来源: Taylor

摘要:

Chicken antibodies have several advantages over mammalian antibodies due to the phylogenetical differences between birds and mammals, resulting in an increased sensitivity and a decreased background in many immunological assays. So far, there has been a very limited use of avian antibodies. This is probably partly due to tradition but also because there are very few optimized methods for the labelling of chicken antibodies. We report upon the suitable conditions for peroxidase-labelling of chicken antibodies with a modified periodate method.

ISSN:0954-0105    

DOI:10.1080/09540109999906

出版商:Taylor & Francis Group    

年代:1999

数据来源: Taylor

摘要:

Soy proteins are used in products of pork butchery for their binding properties and/or their protein content. French legislation limits the addition of binding proteins to 2% in some meat products. Current detection methods are unable to quantify vegetable proteins in these products: the results differ according to the manufacturing process. We have developed a method to quantify these proteins by using immunohistochemical techniques associated with image analysis. This process is based on measuring the areas occupied by labeled soy proteins in sections mounted on slides, and not on estimating amounts of proteins in solution.

ISSN:0954-0105    

DOI:10.1080/09540109999915

出版商:Taylor & Francis Group    

年代:1999

数据来源: Taylor

摘要:

A number of monoclonal antibodies were produced against Escherichia coli and two of them were chosen for further study. Antibody 2D1 reacted with all 34 E. coli strains assessed but not with a variety of other bacterial strains, including 45 Salmonella. Antibody 4E1 was more broadly reactive and bound to all E. coli and Salmonella strains tested as well as some species of Enterobacteriaceae. The isotype for 2D1 was found to be IgG2a and for 4E1, IgG2b, both with kappa light chains. The 2D1 antibody was further characterised, and shown to identify a heat modifiable outer membrane protein of E. coli. The effect of temperature, detergent and 2-mercaptoethanol was assessed on exposing the 2D1 epitope using SDS-PAGE and immunoblotting. 4E1 binding was investigated with rough mutants and the antibody was found to identify an inner core, heptose-associated, structure.

ISSN:0954-0105    

DOI:10.1080/09540109999924

出版商:Taylor & Francis Group    

年代:1999

数据来源: Taylor

摘要:

Forty lambs were immunised against a cocktail of five 'lactic acid' spoilage bacteria typical of those found on vacuum packaged chilled meat. The animals were slaughtered and their hind legs removed within 20 min. Half of the legs (one from each carcass, were inoculated with viable Lactobacillus delbrueckii cells (one of the organisms used in the vaccine formulation), vacuum packaged and stored at - 1.5 C. Forty non-immunised controls were similarly processed and packaged. The vaccine elicited an immune response as determined by ELISA assay. Over a 16-week storage period, meat derived from immunised animals generally returned lower numbers of bacteria than meat derived from non-immunised animals. Sensory analysis determined that non-immunised product stored for longer than 12 weeks had a higher level of undesirable odours and flavours than immunised product. These findings support the view that immunising lambs against spoilage organisms has the potential to extend the storage life of packaged chilled meat.

ISSN:0954-0105    

DOI:10.1080/09540109999933

出版商:Taylor & Francis Group    

年代:1999

数据来源: Taylor

摘要:

The acidic polypeptide chain of soybean glycinin G1 was identified as one of the several IgE-binding proteins by immuno-blotting with pooled sera from soy-allergic individuals. The region of IgE-binding within glycinin G1 was narrowed by in situ digestion with endoproteinase GluC followed by immuno-blotting of peptide products. This procedure resulted in a single IgE-binding fragment of approximately 15 kDa corresponding to residues 192 to 306 of the acidic chain of glycinin G1.

ISSN:0954-0105    

DOI:10.1080/09540109999942

出版商:Taylor & Francis Group    

年代:1999

数据来源: Taylor

摘要:

Lactic acid bacteria (399 meso- and thermophilic strains or mixed cultures) were screened for their ability to reduce the antigenicity of bovine alpha -lactalbumin ( alpha -la) and beta -lactoglobulin ( beta -lg). Residual antigenicity of these proteins in whey from the fermented milks was determined by indirect ELISA and competitive ELISA for alpha -la and beta -lg, using rabbit polyclonal antibodies. The antigenicity was related to allergenicity, as determined by skintesting with selected samples of whey from the fermented milks. The antigenicity of whey proteins was found to be reduced by over 99% as compared to raw milk after the lactic acid fermentation of sterilized cow's milk. The allergenicity of alpha -la and beta -lg was not, however, eliminated, being only slightly attenuated.

ISSN:0954-0105    

DOI:10.1080/09540109999951

出版商:Taylor & Francis Group    

年代:1999

数据来源: Taylor

摘要:

Fifty-eight sera from humans allergic to cow's milk proteins were analysed for the specificity of their IgE response to the whole casein fraction of milk from different ruminant and nonruminant species (e.g. cow, sheep, goat, rabbit and rat). IgE-specific responses were determined by an enzyme allergosorbent test using the purified casein fractions as immobilized antigen and an anti-human IgE monoclonal antibody labelled with acetylcholinesterase. Co-and/or cross-sensitizations to caseins of the different ruminant species occurred extensively, though IgE responses to ovine and caprine casein appeared to be lower than that obtained with bovine casein. Cross-reactivity is suggested by the significant reactivity of rat and rabbit casein toward human IgE. In terms of specificity and intensity, the IgE response to caseins demonstrates a great variability. Structural homologies in caseins of such different species, that can share common epitopes for the IgE of some patients, suggest that prevention of cow's milk allergy cannot be achieved by using milk from other species as substitutes.

ISSN:0954-0105    

DOI:10.1080/09540109999960

出版商:Taylor & Francis Group    

年代:1999

数据来源: Taylor