ELISA INHIBITION ASSAY FOR THE QUANTITATION OF ANTIGENIC PROTEIN IN NATURAL RUBBER LATEX

Abstract

ABSTRACT Evaluating allergenicity of natural rubber latex (NRL) products is essential for the successful reduction of the consumer's exposure to potentially allergenic NRL proteins. We have developed an ELISA Inhibition method for the quantitation of extractable proteins from NRL products which has good sensitivity and specificity. The method utilizes rabbit anti-NRL protein serum as a detection mechanism. The source of NRL proteins for immunization and as a reference protein in the assay is ammoniated raw latex (AL). By comparison with the Western blot analysis of the rabbit sera, it appears that the ELISA detects most of the latex proteins present in extracts. To investigate, further, this assumption, we compared the ELISA Inhibition test with two other methods for total protein measurement. We also compared the values generated by the ELISA Inhibition test with other immunological methods for quantitation of antigenic and allergenic proteins. Comparisons were performed with 15 extracts from randomly selected surgical and examination gloves. The samples were coded separately for each test to insure the objectivity of evaluation. The antigenic protein values obtained by the ELISA Inhibition test correlated best with the HPLC amino acid analysis (correlation coefficient (cc) = 0.79) and with the LEAP assay (cc = 0.97). The antigenic protein levels obtained by the ELISA test were 3–10 times lower than those obtained by the HPLC analysis. A lesser correlation was observed with the Modified Lowry assay (cc = 0.45), which is likely due to chemical interference bias in the Lowry method. Our findings suggest that the antigenic proteins measured by the ELISA Inhibition test described here more closely represent the measure of the total protein content in the extracts. It is important to note that the relative values obtained by this method are lower than the values obtained by other total protein methods, possibly due to a large number of small peptides present in NRL products, that would only be measured by the chemical method.