CORRELATION BETWEEN TOTAL EXTRACTABLE PROTEINS AND ALLERGEN LEVELS OF NATURAL RUBBER LATEX GLOVES

Introduction

Type I allergy affecting certain individuals through the use of some NR latex products, has caused great concern among the latex product manufacturers. Emphasis has since been placed on the production of latex devices with better biocompatibility. Many attempts have therefore been made to reduce their total extractable proteins shown to be implicated in the allergy ^1,2. A number of effective methods for such reduction have subsequently been developed in Malaysia. These include the use of low protein latices ^3,4,5, suitable leaching protocols during processing ^4,6, enzyme treatment⁷, chlorination of the finished products ⁸ and polymer coating. When applied under suitable conditions, the extractable protein fraction can be effectively reduced to a very low level.

It is noteworthy that the residual extractable fraction of latex products may consist of both the allergenic proteins (i.e. capable of binding to IgE antibodies) and non-allergenic proteins, the proportion of which may vary from product to product. It is, therefore, of great importance that reduced levels of extractable proteins reflect the reduced allergen level of the final products. The present work was thus undertaken to study the correlation between total extractable proteins and allergen contents of latex gloves. Results were also compared with those reported earlier ⁹ on the relationship between allergic response elicited in latex hypersensitive subjects and total extractable proteins of latex gloves.

Extractable Protein Content - RRIM modified Lowry method [Malaysian Standard Test Method MS 1392 : 1996 (P)] ¹⁰

Protein Extraction. Cut pieces of 7 cm x 7 cm each were extracted in 0.01M phosphate buffered saline at pH 7.4 (1 g/5 ml) at 23°C for 3 hours with agitations at 30 minute intervals. The clear extract was obtained after removing by centrifugation, any insoluble matter that might be present.

Protein Precipitation. Proteins in the extracts were precipitated using trichloroacetic acid (4.4%, w/v) and phosphotungstic acid (0.2%, w/v). The precipitated proteins were then sedimented by centrifugation at 10,000 x g for 30 minutes. The resulting protein pellet was redissolved in minimum volume of 0.2M sodium hydroxide (1-6 ml) after removal of the supernatant containing interfering substances.

Modified Lowry Microassay. 0.8 ml aliquot of each redissolved protein solution was treated with 0.3 ml of a reagent containing 6% sodium carbonate and 1.5% copper sulphate in 3% sodium citrate (carbonate: sulphate = 10 : 0.2). After allowing the mixture to stand for 10 minutes, 0.1ml of 72% of 2M Folin reagent was added. Colour was allowed to develop at room temperature for 30 minutes, and its absorbance at 750 nm was recorded. If precipitation occurred at this stage, further centrifugation was carried out to give clear solution for the colorimetric measurements. Results were read against a standard Bovine Serum Albumin (BSA) calibration curve and converted to µg/g or mg/g of gloves, taking into consideration of the weight of sample extracted and the volume used in each case.

Allergen Content - ELISA-inhibition Test ¹¹

Latex serum proteins containing the allergens. Allergen Mixture Frozen (-70°C) non-ammoniated Hevea brasiliensis latex harvested freshly from the trees under chilled conditions, was thawed and centrifuged to give a clear serum containing latex allergens (NRL serum : protein concentration 10 mg/ml, as measured by Lowry assay). It was diluted to a protein concentration of 20 µg/ml (Lowry assay) in 50 mM carbonate buffer at pH 9.6 and applied onto polystyrene microtitre plate (100 µl per well; Nunc, Denmark), incubated at room temperature for 3 hours. The wells were emptied and post-coated with 1% human serum albumin in 50 mM carbonate buffer.

Inhibition and immunoassay : The IgE serum pool for the inhibition reaction consisted of carefully characterized sera from both latex allergic adults (n=3) and latex allergic children with spina bifida (n=3). Equal volumes of optimally diluted IgE serum pool and serial dilutions of each glove extract were incubated for 1 hour at room temperature, followed by another 2 hours of incubation period in the microtitre wells. After appropriate washes, the bound IgE was detected by biotinylated goat anti-human IgE (Vector) and streptavidin-conjugated alkaline phosphatase (Bio-Rad). Intensity of colour formed upon reaction with the substrate development solution (Sigma) was read at 405 nm.

A standard curve for the inhibition reaction was based on serial 10-fold dilutions of the NRL serum, the stock solution of which was assigned to contain 100,000 arbitrary allergen units (AU/ml). OD values obtained each test sample analyzed was transformed to these units from the standard curve.

In the present study, relationship between total extractable proteins (EP _RRIM), as determined by the RRIM modified Lowry Test, and allergen content/activity, as assessed by the ELISA-inhibition test, was investigated. The reference allergen mixture consisted of serum proteins from fresh Hevea latex, while IgE antibodies were sourced from both adults and spina bifida children who showed sensitivity to latex. Allergen measurements so generated using these reference mixtures have been shown to be highly correlated to the allergic response by the skin prick test (correlation coefficient r = 0.94, P < 0.001, n = 20) ¹², indicating the reliability of the test for allergen quantitation. Accordingly, allergen content of < 10 AU/ml is low, 10-100 AU/ml is moderate, and > 100 AU/ml is high.

A total of 46 commercially available brands of medical latex gloves of which 11 were powder-free, were examined. Their extractable protein (EP _RRIM) content varied from as low as less than 20 µg/g (or < 0.02 mg/g) to as high as 1290 µg/g (or 1.290 mg/g), and their allergen content ranged from < 1 AU/ml to 570 AU/ml (Table 1). Median values of the gloves samples were 485 µg/g and 117 AU/ml respectively. Generally, gloves with high protein values had high allergen contents and vice versa. However, there were some exceptions at higher protein levels. These could well be attributed to variability in protein composition of the residual fraction in the samples concerned, due to marked differences in the processing conditions employed. It is most apparent that at EP _RRIM levels of about 100 µg/g or 0.1 mg/g and less, the corresponding allergen contents are consistently remarkably low at < 9 AU/ml (Figure 1). This latter group of gloves consisted of all the 11 powderfree gloves examined, in addition to 3 powdered ones. Statistical analysis revealed that the two parameters are well correlated, with the coefficient of correlation r = 0.89, P < 0.001 (Figure 2).

Discussion

Allergenicity or allergic potential of latex products with reference to latex protein allergy has recently become a parameter of importance in the manufacturing of safer latex articles of low allergen quality, particularly the medical devices. There are, however, no standardised methods for such measurement to-date. Several tests, most of them competitive immunoassays, are commonly employed by various laboratories in the West. These include mainly the radioallergosorbent test with inhibition (RAST-inhibition) ^13,14 and the enzyme-linked immunosorbent test with inhibition (ELISA-inhibition)¹². In these in-vitro tests, latex allergens are quantified by allowing the soluble latex allergens in the sample extract to compete with a reference allergen mixture on a solid phase, for the binding sites of the latex-specific human IgE antibodies. The amount of the latex-specific antibodies bound to the solid phase is determined, and is inversely proportional to the quantity of latex allergens in the test sample. Resulting measurements are expressed as allergen content/activity in AU/ml. However, absolute values of these measurements of the same sample between tests may vary when either or both the reference allergen mixture and the IgE antibodies used differ. The most representative result should be one that is derived from a reference allergen mixture containing all the relevant allergenic proteins, and an IgE serum pool comprising the complementary specific IgE antibodies. Unfortunately, such standard mixture and serum pool have yet to be developed. Hence, there is a need to correlate results obtained between tests as well as between laboratories when latex allergens and IgE antibodies from different sources are used.

On the other hand, allergenicity can be specifically evaluated by the in-vivo clinical skin prick test, by assessing the allergic response elicited by the protein extract in latex hypersensitive subjects ^9,12,14,15. This test is both sensitive and specific. But the availability of latex sensitive persons is essential. This requirement is not always easily met, especially in latex product manufacturing countries such as Malaysia where prevalence even among the high risk groups has been shown to very low ¹⁶.

By far, the colorimetric measurement of total extractable proteins in latex devices offers a relatively simple procedure, involving standard chemicals which can be obtained easily. It has therefore, been adopted for routine monitoring of protein reduction during manufacturing. The modified Lowry microassay protocol which is often used consists of three parts, namely, protein extraction, protein precipitation and the colorimetric microassay. Only the commercially available chemicals are used in this test, and the testing time is 2-3 hours (excluding extraction time), as compared to 1-2 days for the immunoassays. Results are expressed in mg/g or µg/g of test sample, with reference to a standard protein. One drawback of this test concerns the fact that it measures all the extractable proteins some of which may not be allergenic. Therefore, for the values generated to be meaningful, they should be related to the allergenicity or the allergen levels.

There had been some speculations that total extractable proteins were not correlated to their allergenicity or allergen contents. This however, has been shown to be not so. An earlier study by Yip et.al. ⁹ of total extractable protein content (EP _RRIM) of 39 NR latex gloves and skin prick test (SPT) allergic response elicited in a total of 59 latex hypersensitive subjects clearly indicated that the two parameters are well correlated (coefficient of correlation, r = 0.83, P < 0.001). This is consistent with the report by Yunginger et.al.¹³ who showed a significant correlation between extractable protein content of 71 latex gloves, as determined by a modified ninhydrin method, and their allergen levels as assessed by IgE-RAST inhibition immunoassay. Further substantiation is now obtained in the present study which demonstrated a highly significant correlation between the total extractable proteins and the allergen levels of latex gloves, as evaluated by the IgE-ELISA-inhibition immunoassay.

It has been shown that soluble proteins migrate towards the surface of a latex film^17,18 during the manufacturing process of latex-dipped products. The degree of removal of these proteins from the surface is very much influenced by the processing conditions employed, some being more effective than others¹⁹. In view of the fact that the residual extractable proteins may not all be allergenic, the doubt often arises as to whether there is any preference in the removal between the non-allergenic and allergenic proteins or among the various allergenic proteins from this fraction. The relationships observed between total extractable proteins and allergen levels and allergenicity⁹ suggest that reduction affects both types of proteins, and the existence of some preferences has also been implied.

It is also of interest to note that when reduction reaches a very low EP level of about 100 µg/g and less, evidence strongly indicated that the amount of allergens present, if any, is often too little to facilitate substantial binding with latex-specific IgE or elicit any allergic response in latex hypersensitive persons, as demonstrated by the tests conducted. This is regardless of whether the gloves are powdered or powderfree. Hence latex products with such low extractable protein levels can be considered to be of low risk to users. The availability and use of such latex products are expected to reduce or even to prevent further sensitization. However, it should be stressed that there is a small number of highly atopic people who are sensitive to a great number of allergens. For these subjects, even minute amounts of allergens can elicit hypersensitivity reactions, implying avoidance of all the relevant allergens should be recommended.

It may be mentioned that since absolute values of total extractable proteins generated by different colorimetric methods are not fully comparable²⁰, results of the present study are relevant only to those determined by the RRIM modified Lowry test. This has consequently rendered the test a convenient and a useful one for monitoring purposes in the manufacture of low protein latex products.

Total extractable protein contents of latex gloves (EP_RRIM), as determined by the RRIM modified Lowry test, were found to correlate well with their allergen contents as assessed by the IgE-ELISA-inhibition test. Gloves with high total extractable protein contents are generally associated with high allergen contents, while those with low total extractable proteins tend to have low allergen contents. Protein levels with minimal allergen content/activity have been identified to be about 100 µg/g and lower. These findings are highly consistent with those reported for EP_RRIM values and allergic responses by the skin prick test. Such information provides very useful guidelines not only for the manufacture of the more bio-friendly low protein latex products, but also for users in their selection of gloves.

The authors wish to thank the Director of the Rubber Research Institute of Malaysia for permission to publish this paper, Dr. P.F. Lai and Dr. Tan Hong for their heplful comments. The capable technical assistance of Miss Vijayalakshmi K. Rasiah and Mr. Ng Chong Seng is gratefully acknowledged.

Figure 1. Total extractable protein (EP_RRIM), as determined by RRIM modified Lowry test, and the corresponding allergen content, as assessed by the ELISA-inhibition test, for 46 brands of medical latex gloves.

Figure 2. Correlation between total extractable protein contents as determined by the RRIM modified Lowry test, and their corresponding allergen levels as assessed by the IgE ELISA-inhibition assay, for 46 commercially available medical NR latex gloves

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