Development of immunoassay for the detection of protein allergens in natural rubber products

Abstract

Natural rubber latex has been identified as a source of protein allergens in many countries. A number of current information suggests that soluble proteins in latex cause hypersensitivity in sensitized individuals. The IgE-mediated sensitization to latex protein has been reported from mild local urticaria to fatal anaphylaxis. The health care worker and children with multiple surgery, especially spina bifida, have been shown as having increased risk for latex allergy. Little is known about the prevalence of latex allergy in Thai population and the population at risk. The objective of this studies was to determine the prevalence of the anti-latex protein IgE-antibodies. When relative risk between 352 healthy blood donors and 200 general allergic patients were compared, it had been found that 4.5% of the blood donor group had anti-latex protein IgE-antibodies whereas 11.0% of the general allergic group had such IgE. This result indicated that the allergic patients had 2.6 time higher risk than the healthy ones. By using the specific IgE positive sera, the molecular weight of protein allergens could be identified in four rubber clones: RRIM600, GT1, PB5/51 and KRS165. There was no difference among the clones observed. The identified-allergens were 14, 18, 25.5, 30, 38 and 52 kD polypeptides. They were fractionated from other soluble proteins in rubber latex clear sera by gel permeation chromatography. Pooled fractions of protein allergen could be used to immunize rabbits to produce allergen specific antisera (IgG) of titer 8-16 in the 7th week of immunization. An indirect competitive Enzyme-linked immunosorbent assay had been developed by the rabbits IgG antibodies and used for quantitative analysis of allergens in latex of the four. Rubber clones and in the extracted-soluble proteins of natural rubber gloves and tires. The coefficient of variation was lower than 5% in the intra-assay, and lower than 10% in the inter-assay. Concentration of the allergens in the extractable protein could be determined in the range of 10-100 ng/well. A dot blot method had been modified to reduce interference by background color of the specimens through transfer of protein onto nitrocellulose film. In addition a latex particle agglutination inhibition method had been devised to detect protein allergens effectively. However, the latex particles were stable for a limited period. It was concluded that both healthy and allergic Thai people had a risk to allergy by latex protein with the same prevalence as reported in other countries. Therefore, a specific latex allergen detection kit was required for the quality control of natural rubber products.