Ever since the problem of latex allergies escalated in the late eighties, the question about the most efficient method for the determination of the soluble proteins has been raised. Several methods were being used and consumers were confused by the differing results. ASTM and CEN started activities to standardize the method for the determination of proteins in NRL. It was understood that these methods should be relatively easy to perform, have good correlation to glove allergenicity and be utilized worldwide. In 1995 ASTM published standard ASTM D5712 and the European Commission started a study to develop suitable methods for the determination of proteins and accelerators.
The results of the European study were used in creating the European standard EN 455-3, which was published in 2000. This European study clearly showed that amino acid analysis is the method with the best correlation to allergological data. However, this method is very expensive and complex and therefore not useful for routine testing, but it is recommended as a reference method to validate the results of the modified Lowry. The results of the European study led to the use of the modified Lowry, which showed a good correlation in most of the tested gloves - the same method which ASTM used. Beside the Lowry method, three other methods presently are known and used - ELISA-inhibition (based on RAST-inhibition), ASTM D6499-00 (measuring antigenic protein) and the new FIT method, measuring four latex-specific proteins.
Limits of protein content
Whereas the FDA recommends a limit of 200 µg protein / dm2 in medical gloves, the European standard has set no limit yet. In Germany, the BGW (Berufsgenossenschaft für Gesundheitsdienst und Wohlfahrtspflege) recommends a limit of 30 µg/g and since 1997 has published a list of powder free gloves with a protein content below this limit. In 2000, this recommendation and the ban of powdered gloves in Germany led to a decrease of new occupationally- caused latex allergies, to less than half of the cases reported in 1998. This clearly indicates that forcing a decrease in the protein content in the gloves sold in Germany proved advantageous and shall be continued.
Differences between CEN and ASTM
Although an attempt was made to harmonize ASTM D5712 and EN 455-3, some differences exist, making harmonization difficult. EN 455-3 is a standard for medical gloves only, while ASTM D5712 is intended for all rubber devices. The greatest differences are in the calculation and presentation of the results. The concentration of the standard protein is calculated differently and the results are given in either µg/dm2 (ASTM D5712-00) or µg/g (EN 455-3). The determination of the exact surface area of a glove is difficult, so the average dimensions of every glove size are used for the ASTM calculation. This may lead to considerable differences between the final values for both methods. In the meantime, ISO has finished a proposal that tries to combine the ASTM and CEN methods.
Description of the methods
1. Modified Lowry
In the Modified Lowry, extracted proteins are acid-precipitated to separate them from most water-soluble, interfering substances. The precipitated proteins are redissolved in alkali and quantified colorimetrically (by spectrophotometer) after copper/Folin reactions lead to a characteristic blue color. Some chemicals can interfere with the color development during the determination, but in recent years, improved glove processing and the introduction of powder free gloves has decreased protein levels and interferences by chemical substances. In 2001, 83.8 % of 130 different brands of gloves (powdered and powder free) had protein concentrations of less than 30 µg/g, meeting German specifications.
The Modified Lowry, with a quantification limit of 1.0 µg/ml of extract, shows clinical relevance, based on its correlation to skin prick tests. The Lowry is a good universal method for routine protein testing due to its simplicity, reliability and usefulness.
2. Amino acid analysis
AA analysis involves hydrolyzing water-soluble proteins in hydrochloric acid. The amount of each amino acid is then measured by HPLC (liquid chromatography). The sum of all amino acids equals the protein content, if the amount of free amino acids (measured prior to hydrolysis) has been subtracted. Silicone, which may disturb the column, is the only known interfering substance for this test.
The limit of quantification for this test is 10µg/g and its clinical relevance (skin prick test) is excellent, proving to be the best of all of the protein tests studied in the 1998 European research project. Unfortunately, this test is expensive and highly technical, so is not useful for routine testing. It is best used as a reference to validate the results of other protein tests.
3. Latex ELISA for Antigenic protein ASTM D6499-00
This method is a sandwich ELISA - E(nzyme) - L(inked) I(mmunoad) S(orbent) A(ssay), described in ASTM D6499-00. It uses polyclonal antibodies created by rabbits after latex protein injections. This assay is therefore dependent on the availability of these antibodies, which are limited in supply. The assay is further dependent on the standard latex proteins used, usually created from a mix of various latex batches. The assay shows a very high day-to-day variation, even in a laboratory highly experienced in ELISA technique.
Limits of quantification and any clinical relevance have yet to be determined for this test. The results, which are low when compared to total protein tests, may be misinterpreted as very low protein content, which can instill a false sense of security in the consumer. Due to its complexity, cost and variations in latex pools and antibody sera, this assay is not useful in routine analyses.
4. RAST-inhibition or ELISA inhibition
This assay uses the IgE-antibodies of latex-allergic patients. The standard allergens are fixed on a solid phase (cellulose sponge in RAST-inhibition or polystyrene in ELISA-inhibition). The IgE-antibodies from the pooled serum of patients are pre-incubated and herewith preloaded with the latex proteins of the glove extract. Not-loaded antibodies are then measured by RAST or another equivalent method. This assay cannot be standardized world-wide because it needs serum from latex-allergic patients, which cannot be produced for all laboratories in the world in the same composition. Therefore there will be varying results from lab to lab when testing the same gloves.
A limit of quantification has yet to be determined for this assay and clinical relevance is debatable. Good correlation was shown in Finland but could not be confirmed by the 1998 European research project, probably due to the use of different sera. Because the sera pool (latex-allergic individuals) is limited and because global standardization is not possible, this assay is not very useful.
5. ELISA inhibition with monoclonal antibodies
The same principle as ELISA inhibition is used, but instead of patients’ antibodies, monoclonal antibodies to four different hevein latex proteins are used. This assay can only detect a few known allergens since many unidentified allergens are ignored and new allergens will not be detected. However, this assay measures the specific allergens known to be most clinically significant, which often is discussed as an advantage. Some feel that an assay measuring total protein will be more suitable for the prevention of allergies than an assay that ignores a large number of potentially known and new allergens.
The test has been shown to be clinically relevant and the limits of detection for the four proteins range from 0.1 up to 2.3 ug/ml.
6. Conclusion
The determination of proteins should be done by the methods described in EN 455-3 and in ASTM D5712. Because EN 455-3 describes a method for medical gloves only it shall be used for gloves to be sold in Europe. WG 3 CEN TC 205 is still working on the improvement of the standard. Limits for powder and protein will be defined and new assays will be checked for suitability. If necessary, the standard method will be changed. All values from methods other than described in the standards could confuse the consumer and should be avoided.
