A rapid, sensitive, user-friendly method for detecting storage mites

Abstract

The study reported here forms the first part of work to develop a rapid method for detecting storage mite pest infestation in cereals and their derived products. This was achieved by examining the suitability of an existing monoclonal antibody for detection of mites by immunoassay. The specificity of the antibody was investigated to 13 species of mite and four species of insect pest. Its sensitivity was examined by studying the reaction to different numbers of mites.

The results demonstrated that, in the absence of grain, the monoclonal antibody could:

- detect and accurately quantify unknown numbers of the cosmopolitan food mite, Lepidoglyphus destructor, ranging between 0-100 adult mites;

- detect both dead and live adult mites of L. destructor and its faeces;

- detect the house mite, Glycyphagus domesticus with similar sensitivity to L. destructor.

In the presence of grain the sensitivity of the assay decreased. This was probably because the existing antibody had not been raised for this purpose. However, further investigation using an additional, polyclonal, antibody gave satisfactory results in accurately detecting a range of 0 to 100 mites seeded in 5g of both wheat and barley. This demonstration of an immunoassay for the quantification of L. destructor mites in stored grain is a significant advance on previous immunoassays which have focused on detecting mites in dust.

The work reported here has proven the potential of the immunoassay method for the rapid and sensitive detection of storage mites. It is clear that the existing monoclonal antibody is insufficient by itself for detecting mites in cereal grain and, although we have shown how this can be overcome by using the additional polyclonal antibody, this would still have limitations. The supply of polyclonal antibody is not guaranteed and the assay will only detect the Glycyphagid mites L. destructor and G. domesticus.

It is therefore proposed that future work should raise an antibody against a common storage mite protein rather than crude extracts of mites, with the additional precaution that the selection pressure includes lack of reactivity to cereal grains. This would allow detection of a wider range of mite pests even in the presence of grain. Previous experience in related studies suggests that this approach would be highly successful and the best way to exploit the encouraging results obtained in the present study.

Summary

Storage mites threaten the quality of UK grain, oilseeds and their derivatives. They damage stored grain and cause occupational health problems to farmers and workers in the grain and milling industry. Storage mites have also been found in processed cereals destined for human consumption and cited as the cause of potentially life-threatening anaphylaxis after ingestion of foods with heavy infestations. Pesticide resistance is now widespread in all three major mite species found infesting grain in farm and central grain stores, Acarus siro (L.), Lepidoglyphus destructor (Schrank) and Tyrophagus longior.

Grain quality is difficult to control and prove to potential purchasers if it cannot be monitored. Several methods have been suggested to determine the presence of mites but all have disadvantages being for example time consuming or unreliable. The purpose of the present study was to investigate the use of an existing antibody as the basis for an immunoassay method of detection which would overcome these disadvantages.

The existing antibody, monoclonal 42B6, was found to be very sensitive and able to detect single mites for both Glycyphagid species L. destructor and Glycyphagus domesticus.

The antibody responded best to these two species and to the less common Aleuroglyphus ovatus. Protein analysis by Western Blot showed there was a common band at 39 KDa in all three of these species, which would explain their reactivity with this antibody. Response was marginal with Tyrophagus longior, Tyrophagus putrescentiae, Acarus siro, Acarus farris and Blomia tropicalis. There was virtually no reactivity with Tyrophagus palmarum, Glycyphagus ornatus, Caloglyphus berlesei or the house dust mite, Dermatophagoides pteronyssinus.Neither was there any significant reactivity with any of four storage insect pest species tested, Ahasverus advena, Oryzaephilus surinamensis, Sitophilus granarius and Cryptolestes ferrugineus.

Of the different life stages of L. destructor, adults had the greatest response while responses to tritonymphs and nymphs were marginal and there was no response with eggs. Live adult mites of this species, dead adult mites and mite faeces all gave strong responses. Responses to spent food and to a mixture of faeces and food were marginal, while there was little response to food alone. Detecting dead mites and mite faeces could be of an advantage for even if live mites are removed from a sample, mite allergens in the product could still be hazardous to health.

Although the antibody was able to detect mites alone, it could not do so in the presence of either barley or wheat. It is possible that this was because the antibody, which is known to work by recognising a sugar moiety, was at least partly bound to cereal carbohydrates and therefore unavailable to detect the glycoprotein antigen. The problem was overcome by using an additional, polyclonal, antibody in an extra amplification step. This demonstration of an immunoassay for the quantification of L. destructor mites in stored grain is a significant advance on previous immunoassays which have focused on detecting mites in dust.

The work reported here has proven the potential of the immunoassay method for the rapid and sensitive detection of storage mites. It is clear that the existing antibody is not sufficient by itself for detecting mites in cereal grain and, although we have shown how this can be overcome by using an additional polyclonal antibody, it would not be without its own limitations. The supply of polyclonal antibody is not guaranteed and the assay is limited to the detection of the Glycyphagid mites L. destructor and G. domesticus.

It is therefore proposed that future work should raise an antibody against a common storage mite protein rather than crude extracts of mites, with the additional precaution that the selection pressure includes lack of reactivity to cereal grains. This would allow detection of a wider range of mite pests even in the presence of grain. Previous experience in related studies suggests that this approach would be highly successful and the best way to exploit the encouraging results obtained in the present study.