Low protein latex (LOPROL) developed at the RRIM has been found to be suitable for factory scale production of examination gloves. Both prevulcanised and post vulcanised formulations can give satisfactory tensile properties; the latter requiring modifications of the formulations and compounding techniques. A post vulcanised formulation was used in the factory run.Unaged tensile strength of these gloves was in the region of 25Mpa – 27Mpa and a retention of more than 90% was obtained when they were aged at 70°C for 7 days and 100°C for 1 day.

Although the mechanical stability time of LOPROL has been reported to be lower than 650s, continuous stirring of a compounded LOPROL caused a marginal increase in coagulum content after 7 days compared with HA latex treated in the same manner.

Short on-line wet leaching time of about 1 min gave soluble protein contents of the LOPROL gloves to be <0.1 mg/g film. When these were dry leached for 30s, the soluble protein contents further decreased to between 0.03 mg/g – 0.06 mg/g film.

The high temperature ageing resistance of natural rubber latex films prepared from five different formulations was studied as a function of chlorine concentration and film thickness. Results obtained showed that among other things, formulations that gave films of higher modulus showed better retention of tensile strength after ageing for 22 h at 100° C.

The extractable protein contents of all the chlorinated films, as determined by modified Lowry Microassay against Bovine Serum Albumin, were less than 0.03 mg/g except for the formulation that contained diphenyl guanidine.

It may be concluded that early resolution to these issues would certainly accelerate the process of overcoming this undesirable allergy problem

This paper concerns the study of prevalence of Type 1 latex protein allergy among workers in various latex glove factories in Malaysia. A total of 149 subjects (108 females and 41 males) with a mean age of 30.6 years were examined both by questionnaires and by skin prick test (SPT). In the absence of a standardised SPT latex allergen mixture, the clinical test was carried out using six glove extracts with extractable protein content varying from 0.02 mg/g to 0.75 mg/g of gloves (or 20µg.g to 750µg/g), as measured by the RRIM modified Lowry microassay.

Only three subjects were found to show wheal size ranging from 2-4mm when tested with glove extracts with extractable protein content of >0.6 mg/g. Such reaction, was however not detected in all cases when protein levels were at 0.1 mg/g or lower. This prevalence as compared to those reported in the West is relatively low.

The binding patterns of IgE antibodies to fresh natural rubber latex B- and C-serum proteins were determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis immunoblotting technique. All the IgE samples were from Malaysia rubber tappers who had been skin prick tested with extracts of gloves and food: avocado, potato, tomato and watermelon.

Two of the five IgE samples from tappers reacting to gloves bound to latex proteins, specifically to 35,38 and 40 kD B-serum proteins and to 30 and 75 kD C-serum proteins. The remaining three either did not bind or bound faintly to the latex proteins. Similar binding pattern to only a few latex proteins was shown in one of the three sera of tappers reacting to both gloves and food. The other two, however, exhibited multiple bindings to a wide variety of B-serum proteins of molecular weights less than 20 to greater than 202 kD and to a number of C-serum proteins between 30 to 75 kD. The heterogeneous binding pattern was also demonstrated by eleven of the twenty IgE serum samples of tappers reacting to food and by twenty five of a hundred and thirty six serum samples of tappers reacting negatively to both gloves and food. The fact that only two of thirty eight serum samples that show strong multiple binding pattern corresponded to a positive skin prick test to gloves, indicated that in vitro immunoblotting technique is an unreliable indicator of latex allergy.

Water-soluble proteins in wet natural rubber latex films co-migrated with the evaporation stream to the surface when the film was dried at 100^oC and moisture allowed to evaporate from one surface. When evaporation occurred from both surfaces of the latex film simultaneously, the evaporative pull from both directions appeared to annul each other resulting in little protein migration to either surface. Since unmigrated proteins are not readily extracted, the problem of allergenic proteins in latex films relates essentially to the proteins that migrate to the surface.

Wet-gel leaching of thin latex films (0.15 mm thick when dry) and thicker films (0.25 - 0.35 mm) to remove soluble proteins was investigated by gel-leaching the films for 1 - 3 min with distilled water at room temperature after which they were completely dried at 100^oC. Soluble proteins were reduced in thin films that were gel-leached. However, gel-leaching was counter-productive for the thicker films where extractable proteins increased. The amount of proteins extractable from latex films was also influenced by the d.r.c. of the latex used to prepare the films. Films prepared from 40% d.r.c. latex had significantly higher extractable proteins than films of similar thickness that were prepared from 60% d.r.c. latex.

Protein removal by wet-gel leaching was found not to be very effective because much of these proteins had not yet migrated to the surface when the film was heated briefly to attain the wet-gel state. When the wet-gel film was completely dried by prolonged heating after the leaching step, more proteins migrated to the surface. In the process of even brief (3 min. or less) dry-film leaching, on the other hand, most of the soluble proteins had evidently migrated to the film surface at the time of leaching and their removal was hence much more effective.

Many proteins derived from the latex of Hevea brasiliensis that remain soluble in trichloroacetic acid (TCA) can be precipitated by phosphotungstic acid (PTA). A combination of 5% TCA and 0.2% PTA precipitates a wide range of proteins effectively even when they are present in low concentrations (below 1 m g ml^-1). Besides its protein purification function, acid precipitation also increases the sensitivity of the subsequent protein assay by allowing the test sample to be concentrated. Another advantage of protein precipitation by TCA and PTA is that very small amounts of protein (of the order of 10 m g) can be repeatably recovered without the use of precipitate-bulking agents such as sodium deoxycholate. This general procedure of protein purification and concentration is simple and rapid, but the use of PTA may not be fully compatible with the Bradford protein assay.

A modified Lowry micro-assay is described which enables about 3 m g ml^-1 to be quantitated at the photometric absorbance of 0.05. When used in conjunction with protein concentration by precipitating with TCA/PTA, approximately 0.4 m g ml^-1 protein present in 6 ml of solution can be assayed.

Two major water-insoluble proteins are located on the surface of rubber particles in Hevea brasiliensis latex. A 14.6 kd protein (Hev b 1) found mainly on large rubber particles (>350 nm diameter) and a 24 kd protein (Hev b 3) found mainly on small rubber particles (average diameter 70 nm) are recognized by IgE from spina bifida patients with latex allergy. While Hev b 1 (also called the "rubber elongation factor", REF) has previously been reported as a major latex allergen, this conclusion has been disputed on the basis of results from other studies. The allergenicity of Hev b 1 is verified in this study by testing the recombinant protein generated from its gene. As allergenicity is confined to spina bifida patients and not observed in adults sensitive to latex, it is not a major latex allergen.

The identification of Hev b 3 as another allergen originating from rubber particles is confirmed by immuno-gold labelling and electron microscopy. Observations using the monoclonal antibody (USM/RC2) developed against Hev b 3 show that the protein has a tendency to fragment into several polypeptides of lower molecular weight (from 24 kd to about 5 kd) when stored at -20^oC. There is also indication of protein aggregation from the appearance of proteins of molecular weight greater than 24 kd. Fragmentation of Hev b 3 is induced immediately upon the addition of latex B-serum which is normally compartmented in the lutoids in fresh latex. In the preparation of ammoniated latex (used for the manufacture of latex products), the lutoids are ruptured and the released B-serum reacts with Hev b 3 on the rubber particles to give rise to an array of low molecular weight polypeptides that are allergenic to spina bifida patients.

The recent protein allergy issue, related to some NR latex-dipped medical devices, has caused certain concern to the use of NR dry rubber products. The processing of Hevea latex into dry rubbers and dry rubber products is different from that of the latex products. Instead of dipping, the process usually involves the use of acid for coagulation and very extensive washing with water throughout the process. Subsequent fabrication of the raw rubbers into dry rubber products is often done at somewhat high temperatures. In view of this, it is not surprising that dry rubbers and their products may have some properties different from those of latex dipped ones, especially the contents of non-rubber substances, such as proteins. Hence the extent of the protein allergy problem confronting the latex-dipped product industry may not be the same as that affecting the dry rubber products.

A study was therefore carried out to evaluate levels of extractable proteins in commercially available dry rubber grades, both raw and their vulcanizates, and some dry rubber products. The extractable proteins, shown to be implicated in the allergic reaction by certain users of latex products, were measured by the RRIM modified Lowry method. The allergenicity and allergen content of the samples, as assessed by both the skin-prick test and the RAST-inhibition test respectively, were also examined. Both these test are the best methods presently available for measuring the allergenic potentials of latex products.

A total of 14 dry rubber samples from various grades, such as the SMR CV,L,5,10,20,RSS and DPNR in the form of raw, compounded and vulcanizated rubbers, were investigated. In addition, rubber products such as cut threads, hot water bottles and diver's flippers were also studied. Extracts of all samples were tested for their allergic response in 31 latex hypersensitive patients and their allergen contents evaluated. Results revealed that the NR dry rubbers and dry rubber products, have indeed very low extractable protein contents (often less than 0.02mg/g or 20µg/g). All samples demonstrated 90% to 100% of negative allergic response in latex hypersensitive patients. Such negligible allergenicity was confirmed by the very low allergen activity shown the same samples when evaluated by radio-immunoassay of RAST-inhibition.

Hence, it may be concluded that dry rubbers and dry rubber products not only have extremely low residual extractable proteins, but also negligible allergenicity. The protein allergy problem therefore, is essentially confined to latex-dipped goods.

Extractable protein contents of latex gloves generated by two commonly used methods, the RRIM (MS 1392:96P) and the ASTM (D 5712-95) modified Lowry tests, were examined and their relationship studied. Total extractable proteins, EP_RRIM, determined by the RRIM test, ranged from 1326 µg/g to < 20 µg/g for 90 gloves. Their corresponding EP_ASTM values, obtained by the ASTM test, varied from 1377 µg/g to < 50 µg/g. Statistical analysis showed a very significant correlation between them , with a coefficient of correlation, r = 0.93, P < 0.001 . Generally, EP_RRIM values read higher than those of EP_ASTM. EP_ASTM of 50 µg/g and lower were found to be associated with EP_RRIM values ranging from 267 µg/g to < 20 µg/g, suggesting higher sensitivity of the latter measurements. Relevance of the two sets of EP in relation to the allergenicity/allergic potential of latex gloves was discussed. Accelerated ageing at 70EC for 7 days of latex gloves resulted in the lowering of protein contents. The effect appeared to be more pronounced for EP_RRIM, than for EP_ASTM.

Certain proteins or peptides eluting from natural rubber (NR) latex products can cause immediate hypersensitivity reactions (Type I allergy) in subjects sensitized to them. The amount of total extractable proteins in manufactured latex products is believed to reflect reasonably well their corresponding allergenic protein level, but only a few studies have been published to substantiate this. The aim of the present study is to compare a widely used total protein measurement assay, namely, the RRIM modified Lowry test (EP_RRIM), to latex allergen analysis, carried out by specific IgE-ELISA-inhibition tests. A series of 46 widely marketed medical NR latex gloves was investigated. Their EP_RRIM values ranged from < 20 µg/g to 1290 µg/g, and their allergen content varied from < 1 AU/ml to 570 AU/ml. In the measurement of allergen contents, the reference allergen mixture was prepared from serum proteins of fresh Hevea latex, and IgE antibodies were sourced from both adults and spina bifida children sensitive to latex. Results showed that the allergen levels were very well correlated with the total extractable protein contents (coefficient r = 0.89, P < 0.001, n = 46). With the exception of a few, gloves with high total extractable proteins were generally found to have high allergen contents, and vice versa. Gloves with EP_RRIM levels of 0.1 mg/g or 100 µg/g and below always had very low allergen contents (< 9 AU/ml).

These findings are consistent with those shown by the in-vivo skin-prick test reported earlier. More importantly, they confirm the very low allergen levels observed at EP_RRIM levels of about 100 µg/g and lower. Such information provides useful guideline for the manufacturing of reduced risk NR latex gloves.

Extractable protein content of a total of 39 different gloves samples, determined by the Rubber Research Institute of Malaysia modified Lowry microassay procedure, is shown to range from < 0.020 mg/g to > 1 mg/g. Their allergic responses in latex sensitive persons (a total of 59) are evaluated by means of the skin-prick test. Results demonstrate that higher extractable protein contents are always associated with positive allergic responses, while very low extractable protein levels tend to exhibit weak or no allergic reaction.

Similar studies have also been carried out with 16 dry natural rubbers of various commercial grades and five rubber products including cut threads manufactured via processes quite different from those of latex-dipped articles. Findings reveal that they not only have extremely low extractable protein contents (< 0.020 mg/g - 0.034 mg/g), but also show negligible or no allergic responses when skin-prick tested on a total of 28 latex hypersensitive persons. It may therefore be concluded that dry natural rubber products are free from the protein allergy problem reported for some latex products.

Considerable criticism has been made against the use of natural rubber latex for the manufacture of elastic medical goods due to allergic reactions that can be caused. An analysis of published data reveals that the proportion of the population affected by latex proteins is small, and that the effects are usually not serious. Consideration of the four main groups of alternative materials, namely PVC, polyurethane, nittile and neoprene, and styrene copolymers concludes that the inferior performance of these materials can lead to increased health and safety risks, and that they have also been known to cause toxic and allergic effects.