Although latex protein allergy affects only less than one percent of the general population,1,2 there is an increasing need for glove users to identify the low-protein latex gloves with low allergy risk from those with high protein content. Measurement of allergenic potential of latex gloves is therefore an important parameter for manufacturers for product improvement.
Despite such a need, a single universally acceptable test is lacking. There are currently a number of methods available, all attempting to provide the most adequate allergenic potential measurement. Generally, they can be classified into two main categories, namely, (i) measurement of total residual extractable proteins/antigens, and (ii) assessment of allergen content of the residual protein fraction of gloves.
(i) Total residual extractable protein/antigen
Methods for measuring total extractable protein/antigen are non-allergen specific. They include the following:
- the Modified Lowry, the BioRad colorimetric assay, High Performance Liquid Chromatography (HPLC) and total antigen immunoassays (LEAP, ELISA).
Among these, the modified Lowry tests are the most preferred, being sensitive, relatively reproducible and easy to perform. The chemical reagents utilized are readily available in high consistency and purity, making these tests easily standardizable. The BioRad assay is less frequently used because of its great susceptibility to interferences by non-protein substances, while HPLC analysis involves longer testing times and is more suitable for research or validation purposes. The antigen assays, on the other hand, require suitable and consistent reagents that are not easily available, and the procedures are comparatively tedious. Final values produced by the different methods are not comparable, due in part to the use of different calibration standards. The focus of this section will be on the modified Lowry protein and the total antigen measurements.
Modified Lowry Tests
All modified Lowry tests for glove proteins share a common 3-stage protocol, namely, (a) protein extraction, (b) protein precipitation and (c) protein quantification. However, there are variations in the steps of each stage. Variations include the sample piece size, the type and concentration of reagents used and the conditions under which the test is conducted. These variations have led to differences in the sensitivity of the colorimetric assay, which is known to have considerable influence on the final protein values generated. The often-observed relatively higher protein values by the revised protocol of ASTM D5712-99 as compared to that of ASTM D5712-95 may be the result of its higher assay sensitivity. In an attempt to produce a single acceptable Lowry test method, ISO is presently in the process of harmonizing the three existing Lowry tests.
The acid precipitation of protein is an important step as it addresses the problem of chemical interference, which is often associated with all Lowry assays. However, occasional sedimentation of certain glove chemicals has also been observed to occur, leading to a false positive protein reading. Therefore, caution has to be exercised to rectify this, either by adopting the "background subtraction" provided by the ASTM test, or by removing the interfering microsolutes by ultrafiltering the protein test solution prior to the assay.
Relationship with allergenicity and allergen content
Because not all proteins in the residual fraction are allergenic, the question of significance of the measured protein values as associated with allergenicity of the gloves has been frequently asked. A number of studies have in fact demonstrated very significant statistical correlation between the Lowry protein values and the clinical skin test responses by latex-sensitized individuals.3,4,5,6 The correlation coefficient has been shown to vary from r = 0.80 to 0.94 when the MS1392/RRIM method, a Malaysian standard protein test was used, and r = 0.88 and 0.95, using the ASTM D5712-95. Similar significant relationships are also shown by studies of the Lowry protein levels and allergen content; r = 0.81 using allergen specific RAST inhibition, and r = 0.80 and 0.89 using allergen specific ELISA-inhibition have been reported.3,7 It may be mentioned that both MS1392 and ASTM D5712 are essentially the modified Lowry method with however, some variations in the protocol, the former assay has been shown to be relatively more sensitive than the later.8
In all cases, gloves with high Lowry protein levels tend to have high allergenicity and allergen content, while gloves with low protein levels are associated with low allergenicity and allergen content, especially at very low protein levels. These findings have provided some degree of relevance to the Lowry protein values, which can be useful guidelines for the production of gloves with very low allergenic potential.
Total Antigen Measurement
Total antigens are measured using the ELISA protocol. These measurements are also non-allergen specific, utilizing antisera containing non-allergen specific polyclonal antibodies generated by rabbits. As with immunoassays, the test procedures are relatively tedious and the test not easily standardized. The two available test methods are the LEAP test and the ASTM D6499-00 assay.
One advantage of these antigen assays is the absence of chemical interference. Not only are the antigen values generally much lower than those of the Lowry for the same samples tested (regardless of chemical interference, if any), the correlation between these two sets of values is poor.3 The relationship of antigen values by LEAP with allergenicity and allergen content of gloves is somewhat controversial. Two studies indicated a lack of good correlation,3,9 while two others reported otherwise.6,10
As for the newly developed ASTM D6499-00 antigen test, relatively less significant relationship between the antigen and allergen values has also been reported, (r = 0.59, RAST inhibition).11 The inclusion of an antigen limit of 10µg/sq.dm in the ASTM glove standard is of concern. Many feel that its inclusion does not really provide any additional useful information to the consumers regarding the product’s possible potential allergy risk and it certainly incurs higher testing costs for the manufacturers.
(ii) Allergen content
In view of the nature of allergy reactions, assessment of allergen content using serological immunoassays should clearly be the method of choice. Such tests include the latex allergen-specific IgE RAST-inhibition and ELISA-inhibition assays.
Unfortunately, these methods are not yet standardizable due to the lack of suitable standard reference mixtures of both the relevant allergens and IgE antibodies. Although the new FITkitTM, a capture ELISA assay using monoclonal antibodies for four identified latex allergens and the corresponding IgE antibodies, has shown considerable promise, the test kit, as specified, is presently suitable only for research purposes. In addition, all these tests involve somewhat sophisticated procedures and are too costly to be used for routine monitoring of latex products especially by the manufacturers.
At the present time, there is no perfect test method for determining the allergenic potential of latex gloves. The most appropriate method for such a purpose should clearly be one that is latex-allergen specific. Until a suitable
and cost-effective allergen assay has been developed, it appears that total protein quantification by the modified Lowry test may remain the most practical measurement. Being technically easy to perform and capable of standardization, this test offers an interim compromise, particularly when the resulting protein values can be helpful in the development of improved products.
REFERENCES
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- Liss G.M. and Sussman G.L. (1999) Latex sensitization: Occupational versus general population prevalence rates. American Journal of Industrial Medicine, 35, 196-200.
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- Yip E., Turjanmaa K., Ng K. P. and Mok K. L. (1994) Allergic responses and levels of extractable proteins in NR latex gloves and dry rubber products. J. nat. Rubber Research, 9(2), 79-86.
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- Beezhold D., Pugh B., Liss G. and Sussman G.L. (1996) Correlation of protein levels with skin prick test reactions in patients allergic to latex. J. Allergy Clin. Immunol. 98, 1097-1102.
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- Yip E. (2000) Harmonization of Standards: A case study on extractable proteins of natural rubber latex gloves. SIRIM Standards & Quality News, May-June, 1-3.
- Wrangsio K. and Lundberg M. (1996) Prevention of latex allergy. Allergy, 51, 65-67.
- Beezhold D., Swanson M., Zehr B.D. and Kostysi D. (1996) Measurement of natural rubber proteins in latex glove extracts: comparisons of the methods. Ann. Allergy, 76, 20-26.
- Tomazic-Jezic V.J. and Beezhold D.H. (2000) Evaluation of the ELISA inhibition assay for natural rubber latex (NRL) proteins: comparison with other methods for protein and allergen measurements. J. Allergy Clin. Immunol. 104:S56.
