Controlling the levels of proteins in natural rubber latex (NRL) products to lower user exposure was found to reduce the rate of allergic sensitization to NRL.1 Measuring protein levels on NRL products poses some unique difficulties because of the complex nature of the manufactured product. Extracts of NRL products contain variable levels of hydrolyzed latex protein but also powder, processing chemicals, and nonlatex proteins (casein) that cause false positive reactions in total protein tests. With respect to NRL products, total protein typically refers to protein that is measured using a chemical method such as Lowry, Bradford or BCA to quantify protein. The American Society of Testing and Materials (ASTM) standard method D57122 is a modified Lowry method that measures total protein content by a relatively simple chemical means but incorporates a precipitation step to help eliminate interfering substances. Antigenic protein refers to proteins that are measured using IgG antibodies specific for the latex protein. Typically, rabbits are immunized with NRL protein and their sera is used to measure antigenic protein extracted from a product. Allergens are antigenic proteins that induce adverse immune reactions usually mediated through IgE antibodies. Thus, for NRL products, allergenic protein is defined as that protein fraction that can be quantified with IgE antibodies from latex-allergic individuals, by RAST or ELISA inhibition assays.
Latex-specific ELISA methods using animal antisera to measure antigenic proteins have been established in both indirect and inhibition formats.3,4 The LEAP assay is an indirect ELISA that was available for use by manufacturers since 1993.3 The ELISA assays have few problems with chemical interference or the detection of nonlatex proteins because the animals are immunized with latex proteins only. Recently the ASTM established a standardized ELISA assay for latex proteins.
While similar to the LEAP assay, the ASTM ELISA differs in that it uses an inhibition format. The protocol was approved as an ASTM standard (D6499-00) in December of 2000.5
The D6499 assay was optimized and has a linear working range between 30 and 2000 ng/ml.6,7,8 The method shows good reproducibility (intra-laboratory 5%, inter-laboratory 10%) and a sensitivity of less than 0.5 µg/g6. The source of protein for the immunization of the rabbits was found to be critical for the assay performance.7 By Western blot analysis, the antigenic profile of rabbit anti-AL (ammoniated latex) but not anti-NAL appeared similar to allergenic protein profiles (human IgE). Since all dipped latex products are made from AL, AL reagents were chosen. The choice of adjuvant (substance that is injected with the antigen to stimulate the immune system) for immunization of the animals was also found to be an important factor. The use of Freund’s adjuvant produced higher titer antisera and appeared to have a more complete repertoire of antibodies than when a synthetic adjuvant was used. The D6499 assay results were compared to results obtained by HPLC amino acid analysis, Lowry (D5712), LEAP, and RAST inhibition methods. By comparison, the D6499 method correlated well with the LEAP (r=0.89) and the HPLC method (r=0.77), and lesser with the D5712 (r=0.48) or RAST inhibition (r=0.59) methods. The antigen content appeared to more closely represent total NRL protein. Protein levels derived by the ELISA methods were considerably lower than the values obtained by the other methods. Recently, ASTM standards for medical gloves (D3577 and D3578) were revised and now recommend <10 µg/dm2 of antigenic protein (D6499) on exam and surgical gloves.
The D6499 method is the first ELISA assay to be developed as a national standard by the ASTM. The ELISA assay has a distinct advantage over the total protein assays in that it can discriminate NRL protein from nonlatex protein. The use of a latex-specific antibody produced a test that was 100 times more sensitive than the D5712 and overcomes the problem of interferences due to processing chemicals. The specificity for NRL protein and lack of interferences makes the assay useful, particularly for measuring antigenic protein levels on low protein products. The use of polyclonal antisera also meets the objective to measure all latex protein capable of inducing an immune response (antigen). Although all latex proteins do not appear to be allergens for all latex-allergic individuals, theoretically
any antigen can be an allergen.9 Antigenic NRL proteins represent proteins that potentially could induce IgE production. Thus, the antigen level should estimate "potential" allergenic protein content because the immunological processes whereby proteins are recognized as antigens or allergens are similar. This makes the D6499 method a suitable aid for product selection and the recommendation by ASTM that products be below 10 µg/dm2 by this assay should further increase the trend towards lower protein products.
REFERENCES
- Tarlo SM. Easty A, Eubanks K, Parsons CR, Min F, Juvet S, Liss G. Outcomes of a natural latex control program in an Ontario teaching hospital. J Allergy Clin Immunol. 2001; 108: 628-633.
- Standard Test Method for Analysis of Protein in Natural Rubber and its Products, ASTM D5712, Annual Book of ASTM Standards, vol. 14, June 1995.
- Beezhold D. LEAP: Latex ELISA for Antigenic Protein, a preliminary report. Guthrie Journal, 1992; 61: 77-81.
- Chabane H, Barbara J, Hrabina M, Leynadier F. Competitive immunoassay for antigenic latex protein measurement: rabbit antiserum-based assay compared to modified Lowry and human IgE-inhibition methods. J Investig Allergol Clin Immunol. 1999; 9: 372-379.
- Standard test method for the immunological measurement of antigenic protein in natural rubber and its products, D6499. Annual Book of ASTM Standards, vol. 9.02, January 2000.
- Tomazic-Jezic VJ, Woolhiser MR, Beezhold DH. ELISA inhibition assay for the quantitation of antigenic protein in natural rubber latex. Journal of Immunoassay & Immunochemistry. (submitted), 2001.
- Tomazic-Jezic, VJ, Woolhiser MR, Beezhold, DH, Quantification of latex proteins by inhibition ELISA. J Allergy Clin. Immunol 1999; 103: S162.
- Tomazic-Jezic VJ, Beezhold DH, Evaluation of the ELISA inhibition assay for natural rubber latex (NRL) proteins: comparison with other methods for protein and allergen measurements. J Allergy Clin. Immunol. 2000; 104: S56.
- Aalberse RC. Structural biology of allergens. J Allergy Clin Immunol 2000, 106: 228-238.
