The awareness of Type I allergy affecting some individuals using NR latex products since the early nineties, has caused great concern in the NR latex industry¹. To address the problem, considerable research activities have been initiated, which include the development of suitable diagnostic tests by the consumer countries in the West, and the improvement of latex products by the manufacturing countries, especially Malaysia. As cause of the problem has been shown to be due to the presence of some residual extractable proteins found in latex products, the status of this fraction is of utmost interest to both the latex product manufacturers and the users. Latest developments of some of the issues involved are being reviewed here. They are: (a) latex allergens, (b) allergic potential of latex products, (c) product improvement and (d) the relevant regulations and requirements set by consumer countries.

It is generally acknowledged that allergenic proteins in the residual extractable protein fraction of latex products are responsible for eliciting the Type allergic reaction in certain individuals. They are mainly derived from raw Hevea latex contains 1%-1.5% of total proteins. The residual extractable protein fraction containing the allergens is, however, very small. It usually does not exceed more than a few percent of the total. To-date, it is still not clear how many allergens are consistently present in the final products, since changes are likely to occur during the manufacturing process. Nonetheless, a number of them have recently been identified in the latex (Table 1).

It may be mentioned that three of them (Hev b II, III & IV) were identified by the RRIM² while the rest by laboratories in various parts of the world³. Molecular weight of the identified allergens varies from as low as about 5kD to as high as more than 100kD. The major ones so far consist of the mature Hevein (4.7kD)⁴ and the Prohevein (20kD)⁵, shown to elicit allergic reaction in a great number of latex hypersensitive people tested from the all the high risk groups. The REF (Hev b I), which was initially thought to be the major one, has now been shown to be not so. Like the 22 kD protein, activity of this allergen appears to be confined to the spina bifida children⁶, and not the other risk groups. The 22kD and Hev b III show somewhat similar molecular weights; it is not certain if they are the same allergen at this stage.

A complete knowledge of the allergens is essential for accurate serological immunoassay assessments of allergenicity of latex products, as well as for more effective diagnosis of latex hypersensitive persons.

Table 1: Latex allergens identified in Hevea latex.

Evaluation of allergic potential of latex products is not only a necessity, but also an important measurement for both the manufacturers and the users. Unfortunately, till to-date, an universally standardized method is still lacking. A number of tests have been introduced and are available for application. Generally, they can be divided into two groups, namely, the determination of total residual extractable proteins, and the assessment of allergen activity/content.

Total Extractable Protein Measurement. Tests available for this category consist of the colorimetric microassays, size-exclusion chromatographic analysis⁷ and the enzyme-linked immunosorbent assay using IgG antibodies⁸. The two employed most frequently for routine analysis are the colorimetric measurements of the RRIM modified Lowry Test⁹ and the ASTM Test (D 5712-95)¹⁰. Both tests are somewhat similar, but because of certain variations adopted in parts of the protocols, the extracted protein content values (EP_RRIM and EP_ASTM) generated by the two tests are strictly not comparable. They are, however, highly correlated (coefficient of correlation r = 0.93, n=90)¹¹. Furthermore, EP_RRIM values are generally higher (mean difference of about 20%) than those of the EP_ASTM. At very low protein levels, as shown by most powderfree gloves, the reverse is often observed. Nevertheless, there are some exceptions.

Total Allergen Activity/Content. Measurement of this parameter usually involves the serological immunoassay technique of either the radio-allergosorbent test with inhibition (RAST-inhibition), or the more recently developed enzyme-linked immunosorbent test (ELISA-inhibition)^12,13. The less frequently used but more direct tests include the histamine release test and the skin-prick test^11,14. The latter test is presently considered to be the "golden standard" for Type I allergy.

In view of the fact that not all proteins in the extract are allergens, these tests may be more appropriate for the evaluation of allergenicity . Their applications however, face certain constraints. While latex hypersensitive persons are necessary for the skin-prick test, the availability of serum pool from these persons, as well as the use of a suitable latex allergen mixture, are required for the serological tests. These requirements are not always easily met, and in addition, a standard serum pool and a standard latex allergen mixture are still lacking, resulting in considerable variability in measurements not only among the different tests, but also the same test done by different laboratories. Furthermore, these tests involve rather tedious procedures not to mention that they are costly to perform.

Method of Choice. The method of choice, particularly in manufacturing countries where manufacturers often need to monitor a great number of their products during processing, should therefore, be one that is sensitive, reproducible, reliable, simple and fast to perform. Most importantly, the values generated should be indicative of the allergenicity of the product. The RRIM modified Lowry test has been shown to be such a method. The extractable protein values (EP_RRIM) generated have been demonstrated to be very well correlated to not only the allergen activity/content as assessed by the serological tests of ELISA-inhibition and RAST-inhibition immunoassays¹⁵, but also to the allergic response by latex hypersensitive subjects as evaluated by the skin-prick test¹¹. Similar information related to EP_ASTM by the ASTM protein test is, however, not available.

Product Allergenicity. High EP_RRIM values are shown to be associated with positive allergic responses in latex hypersensitive persons, while at EP_RRIM of less than 0.4mg/g (or <400m g/g), more than 60% of the latex hypersensitive subjects tested indicated negative response. At lower levels of 0.1mg/g (or <100m g/g), allergenicity is often very low or negligible¹¹(Figure 1). Products of these EP_RRIM levels can, in fact, be considered to be of very low risk. These findings have provided useful guidelines for the production of low protein latex articles.

Product Improvement

The reduction or removal of the undesirable residual soluble protein fraction in latex products is of importance in the manufacturing of more bio-friendly products that will not give rise to any unwanted sensitization among the users. Various methods and approaches for doing this have been presented and discussed¹⁶. These include the use of low protein latices, the application of suitable leaching protocols, chlorination and polymer coating.

Presently, the two low protein latices introduced by the RRIM, namely, LOPROL¹⁷ and LPPL¹⁸, are being evaluated by some latex producers, particularly LOPROL. More than 80% of reduction can be achieved by the use of these latices. The application of appropriate leaching protocols has been shown to be very effective, particularly when leaching is performed before and after the curing/drying oven (Figure 2), as strongly recommended by the RRIM. The use of cleaner water at elevated temperature and slower flow rate enhances the reducing process^19,20. It is noteworthy that there will be a clear advantage if a combined use of low protein latex with appropriate leaching protocol is adopted, with the resulting EP_RRIM often reaching a very low level of 0.1mg/g or below^17,18. To-date, a number of the manufacturers have already adopted or in the process of adopting some of these techniques for their product improvement.

The most effective reduction by far, is achieved by chlorination. However, the chlorination process must be well controlled, otherwise, inferior physical properties of the final products could result. The RRIM is currently engaged in further research on the production of powder-free latex gloves without chlorination, ie. polymer coating, and also on chemical treatment for protein reduction. One such chemical is the Cab-O-sperse fumed silica, which has been shown to reduce EP as well as increasing the tear strength of latex gloves²¹.

Glove Survey 1996. A survey on the extractable protein content of latex medical gloves has recently been conducted by the RRIM (i) to gauge the present status of our Malaysian gloves, and (ii) to collect relevant data for the consideration of realistic and achievable specifications for the proposed Standard Malaysian Glove (SMG) scheme.

30 manufacturers volunteered for the study, and a total of 77 medical glove samples (powdered and powder-free) were analyzed. Results revealed a drop of nearly 40% in the median values of EP_RRIM when compared to those of earlier survey in 1994. This clearly reflects the successful efforts made by some manufacturers to improve their products. Furthermore, it may be of interest to know that more than 45% of the manufacturers surveyed have shown capability of producing medical gloves (powdered/low powdered) with EP_RRIM content of < 0.4mg/g, about 39% of these companies can produce gloves with EP_RRIM of 0.1mg/g or less. These percentages are expected to rise when more manufacturers are taking measures to improve their products.

Although there are no regulations or requirements implemented in any latex product manufacturing country, there are some being proposed by the consumer countries, particularly the USA, Malaysia's largest latex glove importer. These include:

 1. The removal of the "Hypoallergenic" claim. The use of this term is now considered to be misleading by the FDA since the tests used for this claim do not include the Type I protein allergy.

 2. Labelling of NR latex products - three options have been given :

(a) "This product contains natural rubber latex which may cause allergic reactions in some individuals".

(b) "This product has components that contain natural rubber latex which may cause allergic reactions in some individuals".

(c) "This product is made from natural rubber latex which may cause allergic reactions in some individuals".

The manufacturers may choose the language most appropriate for their products. According to Federal Register by the FDA on 24 June this year, comments are invited to reach them by the 23 September 1996 for both 1 and 2. The final regulation will become effective 180 days after its publication in the Federal Register.

 3. Low protein labelling claim in 510K application - optional.

Manufacturers are allowed to make a low protein labelling claim of their products. Although FDA has not specified any particular protein content to be claimed, maximum level achievable by the process, as determined by the ASTM Test method (D 5712-95), should be provided. In view of the sensitivity limit at 50m g/g by this Test method, values claimed should not be lower than this limit. In addition, the claim should also carry the following caution statement: "Safe use of this glove by or on latex sensitized individuals has not been established"

4. Appointment of US designated Agent for foreign manufacturers. - In view of numerous requests for reconsideration, the FDA has announced a stay of the effective date which was scheduled for July 1996.

Similar rulings have not yet been proposed by CEN in Europe.

Malaysia is presently the world's largest exporter of latex gloves, capturing about 70% of the world's market. Although it has been projected that the future prospect of these products will be bright, it is important that the industry continues to improve the quality of its products so as to maintain its prestigious position. This is particularly the case with their extractable protein content, which warrants further concerted efforts to be made for their reduction or elimination. The availability of more bio-friendly low protein Malaysian latex gloves, marketed under a standardized scheme such as that of the "SMG", could give Malaysian latex gloves not only a quality benchmark, but also a competitive edge in the world market.

The author wishes to thank the Director of Rubber Research Institute of Malaysia for permission to present this paper.

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 4. Alenius H., Kalkkinen N.,Palosuo T., Turjanmaa K. and Reunala T. (1995) Mature Hevein is a major natural rubber latex allergen. (Abstract), Eur. Soc. Dermatol. Res. 25th. Annual Meeting, p.25.

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6. Alenius H., Kalkkinen N., Turjanmaa K., Mäkinen-Kiljunen S., Reunala T. and Palosuo T. (1996) Significance of the rubber elongation factor as a latex allergen. Int. Arch. Allergy Immunol. 109, 362-368.

7. Esah Yip (1993) Determination of extractable proteins in natural rubber products by high performance liquid chromatography (HPLC). Latex and the Glove Industry, Rubber Research Institute of Malaysia, 41-50.

8. Beezhold D.H. (1993) Measurement of latex protein by chemical and immunological methods. Proc. Int. Conf.: "Latex protein allergy: the present position" , Amsterdam, p.25-32.

9. RRIM modified Lowry method for determination of extractable proteins in latex products. (1994), Latex Technology Division, RRIM.

10. ASTM standard test method for analysis of protein in natural rubber and its products. (1995) ASTM designation: D 5712-95, published June 1995.

11. Esah Yip, Turjanmaa K., Ng K.P. and Mok K.L. (1995) Residual extractable proteins and allergenicity of natural rubber products. Proc. Int. Conf.: "Latex Protein Allergy: the latest position" Paris, 9-13.

 12. Yunginger J., Jones R., Fransway A., Kelso J., Warner M., Hunt L. and Reed C. (1994) Extractable latex allergens and proteins in disposable medical gloves and other rubber products. J. Allergy Clin. Immunol. 93, 836.

 13. Turjanmaa K., Mäkinen-Kiljunen S., Alenius H., Reunala T. and Palosuo T. (1996) In-vivo and in-vitro evaluations of allergenicity of natural rubber latex (NRL) gloves used in healthcare: A nation-wide study. (Abstract) J. Allergy Clin. Immunol. 97, 325.

14. Turjanmaa k., Laurila K., Mäkinen-Kiljunen S. and Reunala T. (1988) Rubber contact urticaria: Allergenic properties of 19 brands of latex gloves. Contact Dermatitis, 19, 362- 367.

15. Esah Yip, Palosuo T. and Alenius H. (1996) Extractable proteins and allergenicity of NR latex gloves: an ELISA-inhibition study. To be published in the J. nat. Rubb. Res.

16. Latex Proteins and Glove Industry (1993) Proc. Int. Rubb. Techol. Conf. - Workshop on Latex Proteins, Kuala Lumpur; Rubber Research Institute of Malaysia.

17. Hafsah Mohd. Ghazaly (1994) Factory production of examination gloves from low protein latex. J. nat. Rubb. Res. 9(2), 96-108.

18. Ng K.P. Esah Yip and Mok K.L. (1994) Production of natural rubber latex gloves with low extractable protein content: some practical recommendations. J. nat. Rubb. Res. 9(2), 87-95.

19. Dalryme S.J. and Audley B.G. (1992) Allergenic proteins in dipped products: factors influencing extractable protein levels. Rubber Developments 45(2/3), 51-60.

 20. Amir Hashim Mohd. Yatim (1993) Effect of leaching on extractable protein content. Latex Proteins and Glove Industry; RRIM 51-58.

 21. Jo Anand (1996) Latex protein reduction by fumed silica dispersions. Proc. Int. Conf.: "Latex Protein Allergy: managing the issue", Amsterdam; 31-38.

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