A number of natural rubber latex proteins has been shown to be "allergenic" as they interact with the IgE antibodies present in the blood sera and on the surface of the mast cells of latex sensitised individuals^1-3. The reaction of these allergenic proteins with the IgE on the masts cells is normally assessed by in vivo skin prick test (SPT). The identity of the allergenic proteins is however, usually characterised by the binding of the proteins to the IgE antibodies present in the individuals’ sera. This is frequently determined by in vitro Western blotting. To date, about 57 latex allergenic proteins/polypeptides have been reported⁴ with 14,20,27,29/30,35/36,45/46 and 75kD proteins being the most commonly cited^5-9. Many of these latex allergens particularly the 20¹⁰,36⁹,46 and 110 kD¹¹ have sequence homology to plant proteins which could partly explain extensive cross-reactivity observed between latex and various food such as avocado, banana, chestnut and potato^11-14. This cross-reactivity between latex and food allergens has been suggested to result in predisposition to latex allergy in patients allergic to food and vise versa¹¹. Thus caution should be exercised when evaluating latex allergy as the clinical response may not be directed to primary sensitising agent but to cross-reacting allergens¹⁴.

Proteins derived from Hevea brasiliensis latex constitute about 1% of the latex. A quarter of the proteins is associated with the rubber particles while the rest are present in the non-rubber fractions, particularly the B-serum (contained in the lutoids) and the C-serum. An immunoblotting study with the IgE from the Finnish patients⁸ shows that majority of the latex allergens are in fact found in the two latex sera, especially the B-serum. There is so far no immunoblotting study of the latex proteins with the IgE antibodies of the Malaysian individuals although there is a paper¹⁵ on the low prevalence of latex allergy among Malaysians frequently exposed to latex and latex products. It is thus the aim of this paper to describe the binding pattern of the fresh latex B- and C-serum proteins with the IgE antibodies from the sera of the Malaysian rubber tappers skin prick tested positive to gloves, to both gloves and food and to food only. The binding pattern with the IgE of tappers reacting negatively to both gloves and food will also be demonstrated. Some refrence would be made as to the reliability of the in vitro immunoblotting test in predicting latex allergy.

Sera were obtained from a hundred and sixty four Malaysian rubber tappers who were all skin prick tested with extracts of NR gloves and food: avocado, potato, tomato and watermelon. Five serum samples were from tappers reacting to gloves, three from those reacting to gloves and food, twenty from those responding to food and a hundred and thirty six from those reacting negatively to both gloves and food.

The B- and C-sera of fresh Hevea brasiliensis latex were obtained by the procedures described by Moir¹⁶. Fresh latex was centrifuged on a Beckman L8-70 ultracentrifuged at 19,000 rpm using rotor 21 for about an hour. The C-serum and the bottom non-rubber particle fraction containing mainly lutoids were isolated. The bottom fraction was freeze-thawed three times, centrifuged at 10,000 rpm using rotor 21 for 45 minutes before the clear B-serum was obtained.

The B- and C-sera, solubilised in the solublising buffer at a ratio 1:4 were first subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for 35 min at a constant voltage of 200 volts (Mini protean 11 cell; Bio-Rad, Richmond, Calif, USA). The proteins were then electroblotted onto 0.2 µm pore size nitrocellulose membrane (Trans-Blot; Bio-Rad) and those proteins which bound to the IgE antibodies in the sera, classified as allergenic proteins, were identified following established by Alenius et al.¹⁷

The latex allergic response is usually assessed by skin prick testing individuals with proteins extracted from gloves. The glove proteins of normally low molecular weights (< 14kD)¹⁸ , however, could not be used in the SDS-PAGE immunoblotting study due to their poor binding affinity to the blotting membrane and their variable composition arising from different ways of producing gloves. The B- and C-serum proteins were thus used as the allergen source in this study. They not only had a good binding affinity to the blotting membrane but also showed a more consistent composition.

Figure 1 shows the binding pattern of B- and C-serum latex proteins with IgE antibodies from tappers skin prick tested positive to glove but not to food extracts. Out of five samples, one showed strong binding to B-serum proteins of approximate molecular weights of 35, 38, and 40 kD and to a 30 kD C-serum protein while another sample bound only to 75 kD C-serum protein. The rest either did not react with any or reacted very faintly to the latex proteins. This however, is not uncommon as there have been reports of negative serologic test with sera of patients skin pricks tested positive to glove proteins^{7,19 .} One of the possible reasons to this could be that fresh latex serum proteins used in the immunoblotting study did not contain specific allergen(s) present in the gloves evoke positive reaction on SPT.A study by Alenius et al¹⁷ showed that certain allergenic proteins in the gloves were not present in the fresh latex. These allergen could result from unfolding or exposure of relevant internal epitopes in the native proteins molecules when the latex is processed into gloves ^5,11.

Figure 1. Immunoblots of B- and C- serum proteins with IgE antibodies of rubber tappers skin prick tested positive to gloves.

It is interesting to note that the IgE of this particular group of Malaysians who are in constant contact with fresh latex did not only bind to a few latex proteins but also showed no response to 14 and 20 kD proteins commonly identified by the Finnish NR latex allergic patients ^6,7 . However, a larger number of serum samples are needed to confirm this.

The selective binding of IgE to a few latex proteins was also observed with the three sera of the tappers reacting to both gloves and food extract (Figure 2). The IgE of this individual recognised three allergenic proteins of molecular weights between 40 to 75 kD in the C-serum but none in the B-serum. The IgE of the other two tappers however, bound to a wide variety of B-and C-serum proteins. Their IgE bound to B-serum proteins of molecular weight less than 20 kD to greater than 202 kD and to C-serum proteins of molecular weights between 30 to 75 kD.

Figure 2. Immunoblots of B- and C- serum proteins with IgE antibodies of rubber tappers skin prick tested positive to gloves and food.

The multiple binding pattern of IgE to latex proteins was observed to be more common with IgE from tappers reacting to avocado, potato, tomato and watermelon (Figure 3). Fourteen of twenty (70%) IgE samples from this group showed multiple bindings to B- and C-serum proteins, more with the B- than the C-serum proteins. The remainder either did not show any binding or bound to only a few proteins. This clearly demonstrated that IgE from individuals reacting to avocado, potato, tomato and watermelon can cross-react with latex proteins on immunobloting.

The 35, 38 and 40 kD B-serum proteins and the 30 kD C-serum proteins were again among the major proteins identified by the IgE of tappers reacting to food. Other prominent B-serum proteins were the three proteins around 20 to 30 kD, a 30 kD protein, two proteins of 68 to 75 kD and a high molecular weight protein of greater than 202 kD. The 14 and 20 kD proteins were not significant allergens. As a high proportion of latex proteins reacted with this group oh IgE antibodies, a large number of the latex proteins may have sequence homology to plant proteins. The 35kD B-serum protien could be the 36 kD protein characterised by Alenius et al.⁹ to have a considerable structure homologies to several plant 1,3-ß-glucosidases.

Although some tappers reacted specifically to certain food on SPT, their IgE binding pattern did not show any characteristic to distinguish them from the rest (Figure 3). Based on the twenty serum samples it was generally observed that the IgE from tappers responding to watermelon alone bound to more proteins than the IgE of tappers reacting to potato only. The least was with the IgE from tappers reacting to only avocado. There was no IgE sample specific to tomato as the tappers’ reaction to tomato was always associated with reaction to other food.

Figure 3. Immunoblots of B- and C- serum proteins with IgE antibodies of rubber tappers skin prick tested positive to food

1,3,5	-	tappers reacting to potato (P)
2	-	tappers reacting to watermelon (W)
4	-	tappers reacting to avocado
6,7	-	tappers reacting to P & W
8
-	tappers reacting to tomato & W

Binding Pattern of IgE from Rubber Tappers with Negative SPT to Gloves and Food.

A similar multiple binding pattern was observed with twenty five of a hundred and thirty six IgE serum samples from tappers with negative response to both food and glove extracts (Figure 4). The remaining sample showed none to faint binding to the latex proteins. This indicated that some tappers contained IgE which could be induced by allergens other than gloves, avocado, potatoes, tomatoes and watermelons which could cross-react with fresh latex serum proteins.

Figure 4. Immunoblots of B- and C- serum proteins with IgE antibodies of rubber tappers skin prick tested negative to gloves and food

Multiple binding pattern with fresh latex proteins seemed to occur mainly with IgE antibodies of rubber tappers reacting to food and probably to other non-latex allergens. The fact that these IgE could bind to latex proteins, implied that there are other non-latex sensitising agents to latex allergy. Since twenty of twenty three rubber tappers who reacted to food responded negatively on SPT with glove extracts, it showed that reaction to food need not necessarily result in adverse reaction to latex gloves. This is in agreement with Levy¹⁹, who reported that majority of the patients who were primarily allergic to food were less likely to be clinically allergic to latex goods.

The study also showed that immunoblotting test is not a good indicator of latex allergy as out of thirty eight cases of positive immunoblots with multiple binding pattern to latex proteins only two or 5.3% corresponded to positive SPT to gloves. On the other hand six cases of few to no bands showed positives SPT to gloves. This evidence supports the general view that medical history and good clinical allergic reactions, such as that SPT, provide more reliable predictions for latex protein allergy.

4. Conclusion

The binding pattern of IgE antibodies of Malaysians rubber tappers with fresh latex B- and C-serum proteins depended strongly on the source of the IgE. It was observed that IgE from tappers who were skin prick tested positive to gloves showed no to limited binding to a few latex proteins particularly to 35, 38 and 40 kD C-serum proteins and to 35 and 75 kD C-serum proteins. On the other hand IgE from tappers who did not react to gloves either showed no to few binding or multiple bindings to latex proteins. The variety of proteins recognised by the latter group of IgE ranges from less than 20kD to greater than 202 kD B-serum proteins and 30 to 75 kD C-serum proteins. It is noteworthy that the 14 and 20 kD common latex allergens were not significantly recognised by all the IgE of the rubber tappers understudy. As the IgE from tappers reacting to avocado, potato, tomato and watermelon could bind to latex proteins, these food could be another sensitising agent to latex allergy. However, majority of the rubber tappers who reacted to these food did not respond to latex gloves.

Although immunoblotting is known to be a good technique to identify allergenic proteins it was found to be an unreliable test to predict latex allergy as its result did not correspond well with the result of SPT.

Acknowledgement

The authors would like to convey special thanks to the Management of Kirby Estate, Seremban for allowing the rubber tappers to volunteer for SPT and to donate blood for the study. Thanks are also due to Dr Lai Pin Fah, Head of Latex Technology Division and Dr. Timo Palosuo of National Public Health Institute of Finland for their valuable comments. The excellent technical assistance of Encik Mohd. Yusof Rais and the co-operation of Dr. Harri Alenius of National Public Health Institute, Finland in sharing the technique of immunoblotting are deeply appreciated.

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