Gary M Brett

Design and development of immunoassays for detection of proteins

Abstract Immunoassay methodology is currently the method of choice for the quantitative and semi-quantitative detection of many types of proteins in complex mixtures. A combination of sensitivity, specificity and cost-effectiveness in terms of analytical performance are allied to a diverse array of assay formats suitable for laboratory and field use. In the present article, the problems of setting up immunoassays for novel food proteins, and the questions that need to be answered if a successful outcome is to be achieved, are discussed. Speculation on future developments in immunochemistry leads to the conclusion that antibody technology will play an important role in detection of novel proteins from genetically modified organisms.

The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin

The effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through the determination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and a synthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act as crosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodium polygalacturonate demonstrated that a conformational change was induced as a result of their complexation. In addition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin network in water.

Use of the indirect competitive ELISA for the detection of Brazil nut in food products

Food related allergic reactions following inadvertent ingestion are increasingly common, with nuts, including Brazil nut, placed firmly in the top 10 food groups whose presence within a product should be declared. The presence of hidden allergens as a result of adulteration or contamination of ingredients presents a problem for both the food industry and the consumer. A sensitive and specific immunoassay for Brazil nut is described with a limit of detection of 1 ppm. Based upon the detection of the abundant 2S protein the assay is suitable for detection of raw and roasted Brazil nut in a range of food matrices.

Production of monoclonal antibodies to gluten proteins and their use in developing tests for gluten quality

There is a need for rapid, simple tests to give added assurance in gluten quality evaluation which address the needs of plant breeders and the milling and baking industry. As part of fundamental research into gluten protein structure‐function relationships, a library of monoclonal antibodies (MAbs) has been developed to various gluten protein fractions, including both high and low molecular weight subunits of glutenin. The difficulties presented by working with insoluble, heterogeneous gluten proteins in raising MAbs of the requisite specificity are discussed. Several of the MAbs have been used to develop rapid immunoassays which are capable of analyzing a single sample in 10 min, and these have been applied to the quantification of total glutenin proteins, and high and low molecular weight subunits of glutenin in flour samples, illustrating the potential of immunotechnology for monitoring flour quality.

Development of an ELISA specific for Listeria monocytogenes using a polyclonal antibody raised against a cell extract containing internalin B

We have developed a new enzyme-linked immunosorbent assay (ELISA) that is specific to the foodborne pathogenic micro-organism Listeria monocytogenes. It is based on an antibody raised against an L. monocytogenes cell preparation optimized for extraction of internalin B. Only in a sandwich ELISA format was the protein A-purified antibody specific to L. monocytogenes. In a competitive ELISA format, the antibody recognizes other Listeria species. The sandwich ELISA shows no recognition of L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, or L. grayii. It has a minimum detectable level for L. monocytogenes of log10 6.37 cfu ml−1 in pure culture, is reproducible, and is unaffected by the presence of high numbers (approximately log10 8.0 cfu ml−1) of the other Listeria species. Possible reasons for the format-dependent specificity are discussed. When the ELISA was applied to milk samples inoculated with L. monocytogenes reference material (5 cfu ml−1), there was a strong response to the enrichment cultures. The new assay may prove useful in detection of L. monocytogenes in enrichment cultures of food samples.

Temperature-dependent binding of monoclonal antibodies to C hordein

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by enzyme-linked immunosorbent assay (ELISA). The K(d) of IFRN 0614 for the consensus peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and beta-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.