Amanda S. Hill

Altering the fine specificity of an anti-Legionella single chain antibody by a single amino acid insertion.

Antibody engineering provides the potential to clone and manipulate antibody genes to produce fragments with altered specificity. We have produced an anti- Legionella single chain fragment with broader specificity towards Legionella serotypes than the parent monoclonal antibody. Using this relationship between the parent monoclonal and the recombinant antibody derived from it as a model, we attempted to identify those residues responsible for this change in fine specificity. Sequence analysis of this recombinant antibody revealed the deletion of a conserved residue, Asp101, in the CDR-H3 region. Using site-directed mutagenesis, we have created a mutant form of this single chain fragment with an aspartic acid insertion mutation at position 101 of the antibody heavy chain. This mutant scFv demonstrates improved specificity compared to the wild-type recombinant antibody, indicating an important role for Asp101.

An improved immunoassay for chlorpyrifos‐methyl (Reldan) in grain

An improved immunoassay for chlorpyrifos‐methyl (Reldan) in grain has been developed, based on an immobilized polyclonal antibody. This assay had greater sensitivity (limit of detection of 0.02 ppm in grain, or 0.05–0.1 ppb in buffer), was less susceptible to interference from methanol (used to extract residue from the grain) and was of greater precision than the earlier monoclonal antibody assay (Skerritt et al., 1992b). The polyclonal antibody exhibited greater cross‐reaction with chlorpyrifos‐ethyl (not used as a grain protectant), but less with fenchlorphos and bromophos (used occasionally as grain protectants), and employed a more stable peroxidase conjugate than the monoclonal antibody assay. Good correlations were obtained between chlorpyrifos‐methyl residue levels in wheat grain determined by the improved immunoassay and by gas chromatogra‐phy. The properties of the polyclonal antibody should also allow its use in a rapid field assay.

Determination of Gluten in Foods Using a Monoclonal Antibody‐based Competition Enzyme Immunoassay

A sensitive competition enzyme‐immunoassay for quantification of gluten in foods was developed, using horseradish peroxidase‐labelled monoclonal antibodies. Selected antibodies specific for wheat omega‐gliadin components were used, and these antibodies bound proteins from the related cereals, rye and barley, which are also toxic to individuals with gluten‐intolerance (coeliac disease). Binding of these antibodies was not inhibited by heating of gluten during cooking or baking and the assay did not detect cereals not toxic in coeliac disease, such as maize or rice. Gluten could be quantified at higher levels in meat products or in cereal products such as flours or baked goods. Results were not affected by wheat variety. Quantitiative results could be obtained using simple extraction techniques and solvents (40% or 70% ethanol). Detection of gluten was quantitative in a wide range of foods, except for certain products containing gluten proteins that had been subjected to severe heat, enzymic or chemical tre...

Monoclonal antibody based two‐site enzyme immunoassays for wheat gluten proteins. 1. Kinetic characteristics and comparison with other Elisa formats

Solid‐phase two‐site enzyme immunoassays were developed using a number of monoclonal antibodies with differing specificities for wheat grain gluten proteins. While polystyrene microwells performed well in the assay, very high levels of non‐specific binding occured with PVC (Polyvinylchloride) microwells in the absence of ‘capture’ (solid‐phase bound) antibody. This was due to binding of complexes of antigen and labelled antibody, and could only be reduced by the use of rather stringent blocking conditions. Assays involving either the simultaneous or sequential addition of antigen and labelled antibody were developed, the latter providing superior results. A number of successful two‐site assays were constructed using single monoclonal antibodies, suggesting that repeating epitopes, known to occur in gluten proteins, were commonly recognized. Pairs of monoclonal antibodies with dissimilar antigenic specificites did not function in the two‐site assay. A comparison of monoclonal antibody based two‐site and an...

Homologies between grain storage proteins of different cereal species. 2. Effects of assay format and grain extractant on antibody cross-reactivity

A variety of monoclonal antibodies with specificities for different wheat grain storage proteins has been shown to exhibit a number of differing cross-reaction patterns with grain storage proteins from related cereal species ( Skerritt and Lew, J. Cereal Sci. 11 (1990) 103-121 ). This antibody library provides an opportunity to measure the effects of a range of immunoassay methodological variables on the cross-reactivity of these antibodies. Variation of the extractant used for grain proteins from different cereals altered both the amount and composition of protein extracted and thus the cross-reaction of the antibodies. Antibody cross-reactivity was greater when nitrocellulose membrane solid phases compared with polystyrene microwells were used for indirect enzyme linked immunosorbent assay (ELISA). Quite differing cross-reactivity results were obtained when the same grain protein extracts were used in indirect, antigen-competition and sandwich-type assay formats. In the sandwich assay format, alteration of either the solid phase-bound or labelled antibody altered apparent antibody specificity. These results indicate that it is important when reporting immunological homology data, to define cross-reaction of antibodies with respect to the assay format used. Antibody specificity (or cross-reactivity) can be manipulated by variation of sample extractant, solid phase and the format of the assay.

Rapid antibody-based field test to distinguish between Helicoverpa armigera (Lepidoptera: Noctuidae) and Helicoverpa punctigera (Lepidoptera: Noctuidae).

Abstract Helicoverpa armigera (Hübner) and Helicoverpa punctigera (Wallengren) are the two most important insect pests of cotton production in Australia and require application of insecticides to control them. H. armigera has developed resistance to several insecticides but H. punctigera has not. Cost-effective management of insecticide resistance requires that growers be able to determine the proportion of H. armigera eggs or young larvae present on their crop before applying insecticides. This is impossible visually. We generated two monoclonal antibodies that reacted with the insect protein “lipophorin” and were capable of discriminating individuals of the two species at all life-stages. The antibodies were incorporated into a rapid test kit that was tested under field conditions over two growing seasons. Results obtained with the kit agreed closely with those obtained by rearing larvae through to second instar.

Monoclonal antibody based two‐site enzyme immunoassays for wheat gluten proteins. 2. Specificity analysis

The specificity characteristics of monoclonal antibody based two‐site enzyme immunoassays for gluten proteins were analyzed using unlabelled antibody‐labelled antibody competition assays with whole gliadin, and by two‐site assays using various gluten protein fractions. Specificities for gliadin and glutenins were often predictable from direct ELISA or immunoblotting results, although some additional antigenic specificities were noted that would not have been predicted from direct ELISA. Monoclonal antibody based two‐site assays could be used to quantify related prolamins from other cereal grains. Applications of these immunoassays are discussed.

Sample Matrix Interference in Immunoassays for Organochlorine Residues in Plant-derived Foods and Some Strategies for Their Removal

Immunoassays for two groups of organochlorine insecticides, cyclodienes (endosulfan and heptachlor) and DDT were applied to the analysis of a diverse range of plant-derived foods. Water-miscible solvent extracts of high-moisture, low-fat foods such as cauliflower, cabbage, green and red blue grapes and tomato caused little or no interference with the assays, enabling methanol or acetonitrile extracts of the foods to be analysed directly by immunoassay, after dilution in assay buffer. Reasonable recoveries of spikes of these pesticides were obtained by direct analysis of extracts of spiked commodities, with reliable detection down to 0.025 mg kg−1 heptachlor or endosulfan and 0.1 mg kg−1 DDT in the commodities. Acetonitrile extracts of milk could also be analysed directly for DDT. In contrast, extracts of low moisture, non-fatty (rice) and fatty (cottonseed) food commodities interfered appreciably with the assays, reducing assay colour and detection sensitivity. Some simple cleanup methods were developed to remove interference and enable detection of spiked organochlorines in these foods. Extracts of coloured foods, such as tea, coffee and spinach caused similarly major interference in the assays, and a number of simple clean-up methods were ineffective in removing interference. However, use of an immunoaffinity chromatography method for cyclodienes enabled quantitative recoveries to be obtained in extracts of several of these foods when analysed by either ELISA or gas chromatography. Direct analysis was suited for screening purposes but immunoaffinity chromatography results were more quantitative. These results indicate that ELISAs can be applied under developing country conditions to a range of diverse foods, but that cleanup strategies need to be tailored to different types of foods.

Rapid field tests for the organophosphorus pesticides, fenitrothion and pirimiphos-methyl—Reliable estimates of residues in stored grain

Abstract In order for grain handlers and traders to reliably estimate residues of grain protectants in the field, antibody-based tests were developed for the organophosphorus pesticides, fenitrothion and pirimiphos-methyl. To complement the rapid analysis, rapid but efficient extraction techniques were developed. In these tests, a pesticide-containing methanol extract of the grain sample and an enzyme-labelled component are added dropwise to precoated tubes containing buffer. After a brief incubation, the tubes are rinsed out in tap water and a substrate/chromogen for the enzyme is added. The colour developed is stabilized by acidification and the test result read either by eye or in a portable field photometer. Significant levels of the particular pesticide result in a pale colour compared to a dark green pesticide-free control. No calculations were required to provide a quantitative estimate of residue as this could be read directly from a graph of colour yield (absorbance) vs logarithm of pesticide concentration, using standard solutions of pesticide. For fenitrothion, the test had a limit of detection of 4 ng/ml (0.1 ppm in grain) and gave quantitative estimates in the range 0.5–15 ppm (in the grain), while the pirimiphos-methyl test had a limit of detection of 1 ng/ml (0.03 ppm in grain) and gave quantitative estimates over the range 0.1–15 ppm. Thus both tests can be used to segregate “pesticide-free” grain, with residues below 0.1 ppm. Data obtained using the field test correlated well with those obtained using laboratory methods, including both gas-liquid chromatography and immunoassay using microwell plates. The field immunoassay reagents were formatted into a small prototype test kit, and the components stabilised for field use.

Detecting Gluten and Related Prolamins in Food

The universal approach to the management of coeliac disease is the exclusion of gluten from wheat, rye and barley from the diet.