In view of the latex protein allergy problem relating to NR latex products, especially gloves¹ , there is a need to evaluate the allergic potential of these products. However, till to-date, a universally standardized test for doing so is still lacking. Many different methods^2-9 are being used in various laboratories in different parts of the world. The methods most commonly adopted in manufacturing countries, particularly Malaysia, are the RRIM test (which has recently become a Malaysian Standard Test)⁷ and the ASTM test⁸ , both for the determination of total extractable protein content of latex products.

While the RRIM test has been employed for as long as the beginning of the allergy problem, the ASTM was established a few years later. It is often asked how different are the two sets of values so obtained and if they are correlated. The present study has therefore been undertaken to compare these values and to see if any relationship exists between them. Furthermore, in view of the ageing requirement by the FDA for "Low Protein Labelling Claim" for latex gloves¹⁰, this effect on total extractable proteins is also investigated.

Preparation of gloves pieces.
Square pieces, of dimension 7 cm x 7 cm, were cut from palm area of each glove sample. Care was taken to avoid any contamination.

Accelerated ageing
Glove pieces were subjected to accelerated ageing at 70°C for 7 days, according to ASTM 3578-91.

RRIM modified Lowry test⁷ (Malaysian Standard Protein Test MS 1392-96P)
Cut pieces of each test sample were extracted in 0.01M phosphate buffered saline (pH 7.4) at 23°C for 3 hours. Extract was clarified by centrifugation, to sediment any particulate matter, such as powder. Precipitation of proteins was carried out using trichloroacetic and phosphotungstic acids. This was followed by further centrifugation at 10,000 x g for 30 minutes. The resulting pellet was redissolved in 0.2 M sodium hydroxide, and their concentration determined by the Lowry microassay. Absorbance readings, recorded at 750 nm, were calibrated against standard bovine serum albumin (BSA). Results were expressed as EP_RRIM in µg/g of gloves.

ASTM Protein Test⁸.
Proteins were extracted in distilled water at 37°C for 2 hours. Extract was centrifuged and the proteins were precipitated using deoxycholate, trichloroacetic and phosphotungstic acids. Sedimentation of the protein pellet was carried out by centrifugation at 6000 x g for 15 minutes. The precipitated proteins were redissolved in 0.01 M sodium hydroxide, and their concentration determined by Lowry microassay according to procedure set out in the BioRad DC protein kit. Absorbance was recorded at 750 nm and readings calibrated against standard ovalbumin. Results were presented as EP_ASTM, in µg/g of gloves.

A total of 90 commercial gloves were extracted and tested. Results are summarized in Table 1. It can be seen that total extractable protein content, EP_RRIM, ranged from as high as 1326 µg/g to as low as to 19 µg/g. Their median and overall mean values were 505 µg/g and 519 µg/g respectively. Similarly, values of EP_ASTM, varied from 1377 µg/g to 36 µg/g, with a median of 372 µg/g and a mean of 423 µg/g. Comparison of the overall means indicated EP_ASTM was 18.5% lower than that of the EP_RRIM, while in the case of the medians, the decrease was 26.3%.

Closer examination of the data at different EP_RRIM ranges, revealed that means of EP_ASTM read 13.8% - 25% lower than those of EP_RRIM when the latter values were greater than 100 µg/g. However, at EP_RRIM of 100 µg/g and lower, the mean of EP_ASTM indicated 17.6% higher values than that of EP_RRIM (Table 2.)

Comparison of data based on different EP_ASTM ranges, on the other hand, was found to vary somewhat as shown in Table 3. Although lower EP_ASTM values than those of EP_RRIM was still apparent, the difference was more marked at the low range. Nevertheless, statistical analysis of the two sets of data showed that they are very closely related, with the coefficient of correlation, r = 0.93, P < 0.001 (Figure 1).

TABLE 1. COMPARISON OF EXTRACTABLE PROTEIN CONTENTS BY RRIM AND ASTM METHODS FOR 90 GLOVE SAMPLES.

S.d. - Standard deviation

TABLE 2. MEANS OF EP_RRIM AND EP_ASTM AT DIFFERENT EP_RRIM RANGES FOR 90 GLOVE SAMPLES.

TABLE 3. MEANS OF EP_RRIM AND EP_ASTM AT DIFFERENT EP_ASTM RANGES FOR 90 GLOVE SAMPLES.

EP_RRIM and EP_ASTM contents of latex gloves were determined with and without accelerated ageing. Of the 77 lots of commercial gloves tested, 61 lots were powdered gloves, and 16 lots were powder-free of which 4 were siliconized, 3 polymer coated and the rest chlorinated. Results are summarized in Table 4 below.

It is apparent that accelerated ageing resulted in a lowering of 25.2% in the overall means of EP_RRIM and 16.5% reduction in the EP_ASTM measurements. Further analysis of the data showed that the magnitude of changes was relatively greater in the case of the powdered gloves (reduction of 25.4% in EP_RRIM, and 16.9% in EP_ASTM), and less so in the powder-free gloves (10.6% and 4.2% respectively).

The aged EP values were very well related to those of the unaged in both cases, especially in the case of EP_RRIM, as illustrated in Figures 2 & 3. The coefficients of correlation are 0.98 for EP_RRIM and 0.92 for EP_ASTM, P < 0.001.

Total Extractable Proteins

Although both the RRIM and ASTM tests involved the Lowry colorimetric assay technique, their protocols are not the same. Briefly, the general procedure comprises three parts: (1) Protein extraction, (2) Protein precipitation and (3) Protein quantitation. A number of variations throughout have been adopted by both tests (Appendix). These have consequently resulted in differences not only in the sensitivity of the tests, but also in the extractable protein (EP) values so generated. Sensitivity limits of the methods are, in fact, 20 µg/g and 50 µg/g for the RRIM and the ASTM tests respectively. In agreement with this are also the fact that the EP_RRIM values read higher than those of the corresponding EP_ASTM, for the same latex glove samples tested. Furthermore, it is noteworthy that, as shown by the present findings, low EP_RRIM of up to 100 µg/g corresponded to EP_ASTM of similar range, i.e. up to 107 µg/g (Table 2), while low EP_ASTM values of 100 µg/g and less were found to be associated with a much wider range of EP_RRIM , ie. 267 µg/g and lower (Table 3). Of special interest is the fact that gloves with EP_ASTM of 50 µg/g (sensitivity limit of the ASTM test) and less, were shown to have EP_RRIM varying from 206 µG/g to < 20 µg/g. This could mean that the RRIM test is capable of yielding more sensitive measurements than the ASTM test. On the other hand, it could also be argued that such behavior might be due to differences in response to interference by the two tests. However, there is presently no data to substantiate this. The two sets of EP data are, in fact, very well correlated, coefficient of correlation, r = 0.93, P < 0.001 and n = 90.

Although both tests measure only the total extractable proteins, and not the allergens alone, the EP data so obtained could be of relevance if they relate well to the allergen contents or allergenicity of the test samples. This has, in fact, been shown to be the case for EP_RRIM^11,12. Gloves with EP_RRIM contents varying from > 1000 µg/g to < 20 µg/g were both evaluated for their allergen levels using the IgE ELISA-inhibition test¹¹ , as well as tested by the skin prick test (SPT) on latex hypersensitive subjects from a European population¹² . Highly significant relationships were obtained in both cases. More importantly, EP_RRIM contents of about 100 µg/g and lower were shown to be associated with not only very low allergen levels, but also with very little allergic response by the sensitive persons. Such findings have rendered the EP_RRIM measurements useful as indicators of allergic potential of latex gloves.

There are no similar studies reported on EP_ASTM, but according to Sussman et.al.¹³ who recently SPT tested some latex sensitive persons in Canada with gloves of EP^ASTM contents up to 50 µg/g , more than 50% of the subjects responded positively. In the light of the present observations, it may not be impossible that the gloves tested included some with EP_RRIM levels of as high as 200 µg/g, hence contributing to the relatively high positive responses observed. To confirm this, more studies are needed.

TABLE 4. EFFECT OF ACCELERATED AGEING ON EP_RRIM AND EP_ASTM OF LATEX GLOVES

The Food and Drug Administration (FDA) of the USA, in addressing the latex allergy issue, allowed a "Low Protein Labelling Claim" for latex medical gloves¹⁰ in May 1995. As one of the requirements relating to the claim, accelerated ageing is necessary prior to analysis of the test sample for total extractable protein content. This is in view of the fact that the EP content might increase during storage.

The present study has in fact shown that decreases in the extractable protein contents of latex gloves were detected after the ageing process. The drop in EP could likely be due to denaturation of some extractable proteins when the dry gloves were subjected to prolonged heating, leading to certain amount of insolubilization of these proteins. Such decreases were more apparent in the case of the powdered gloves than the powder-free gloves, suggesting that some loss of powder during the ageing process might have partly contributed to this difference.

Very occasional increases in EP have also been observed. The presence of water in the not so dry test sample could be the cause. In such cases, heating during the ageing process could have facilitated further migration of the soluble proteins towards to surface of the film along side with the water, resulting in higher EP contents.

It may be concluded that both the RRIM and the ASTM tests are suitable for measuring total extractable proteins of latex products. Protein values by the former test are generally higher than those of the latter test, and the two sets of data are significantly correlated. However, it should be pointed out that the EP values in both cases are not absolute, being referred to arbitrary protein standards. As such, their comparison could only be deemed more precise if done in reference to a standardized method, which is currently not yet available . But in view of the good correlations between EP_RRIM and allergenicity / allergen content, the EP_RRIM may be considered to be a useful indicator for allergic potential of latex gloves, particularly for the manufacture of low protein gloves. On the other hand, more data is required in the case of EP_ASTM.

Accelerated ageing of latex gloves generally has no adverse effect of their extractable protein contents.

Figure 1. Correlation between EP _RRIM and correlation EP_ASTM values of latex gloves.
Coefficient of correlation, r= 0.93, P<0.001, n=90

Figure 2. Relationship between aged and unaged EP _RRIM values for 77 lots of latex gloves.
Correlation coefficient, r= 0.98, P<0.001

Figure 3. Relationship between aged and unaged EP_ASTM values for 77 lots of latex gloves.
Coefficient of correlation, r= 0.92, P<0.001

The author wishes to thank the Director of RRIM for permission to present this paper. She also likes to acknowledge the very capable assistance of Miss R. Vijalakshmi, Mr. Ng Chong Seng and En. Abd. Aziz Awang. Contribution by Miss Mok Kok Lang during the initial stage of the study is much appreciated.

1. Proceedings of "International Latex Conference: Sensitivity to latex medical devices", (1992), November 5 - 7, Baltimore, Maryland, USA.

2. Turjanmaa K., Lauren K., Mäkinen- Kiljunen S. and Reunala T. (1988) Rubber contact urticaria: Allergenic properties of 19 brands of latex gloves. Contact Derm., 19, 362.

3. Beezhold D.H. (1993) Measurement of latex protein by chemical and immunological methods. Proceedings of "Latex Protein Allergy: the present position", Amsterdam, 25.

4. Yunginger J.W., Jones R.T., Faraway A.F., Kelso J.M., Warner M.A. and Hunt L.W. (1994) Extractable latex allergens and proteins in disposable medical gloves and other rubber products. J. Allergy Clin. Immunol., 93, 836.

5. Hamilton R.G. and Adkinson N.F. (1994) Serologic assays for antigens and antibodies. Immunol. Allergy Clin. N. Amer., 14, 351.

6. Faridah Yusof and Yeang H.Y. (1992) Quantitation of proteins from natural rubber latex gloves. J. nat. Rubb. Res., 7(3), 206.

7. Test method for the analysis of extractable proteins in natural rubber products. (1996) Malaysian Standard Test, MS 1392 : 96P.

8. Standard test method for analysis of protein in natural rubber and its products (1995) ASTM Designation 5712 - 95.

9. Palosuo T., Mäkinen-Kiljunen S., Alenius H., Reunala T., Esah Yip and Turjanmaa K. (1997) Measurement of natural rubber latex allergen levels in medical gloves by allergen-specific IgE ELISA-inhibition, RAST-inhibition and skin prick testing. Submitted for publication.

10. Interim guidance on protein content labelling claim for latex medical gloves (1995) Food and Drug Administration (FDA), Centre for Devices and Radiological Health (CDRH) and Office of Device Evaluation (ODE) Document.

11. Esah Yip, Palosuo T., Alenius H. and Turjanmaa K. (1997) Correlation between total extractable proteins and allergen levels of natural rubber latex gloves. To be published.

12. Esah Yip, Turjanmaa K., Ng K.P. and Mok K.L. (1995) Proceedings of International Conference on "Latex Protein Allergy: the latest position", Paris, 33.

13. Sussman G.L. and Beezhold D.H. (1997) Safe use of natural rubber latex. Allergy and Asthma Proc. 17(2), 101.

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