first specific test for identifying and quantifying individual NRL allergens

Transcription

1 FITkit The first specific test for identifying and quantifying individual NRL allergens

2 2 FITkit

3 3 Measurement of clinically relevant NRL allergens FITkit - The first specific test for identifying and quantifying individual NRL allergens This novel method is the first ever test for the measurement of clinically relevant natural rubber latex (NRL) allergens, and enables standardization and quantification of individual allergens. It overcomes the significant limitations of current tests by using highly purified and characterized allergens, and specific monoclonal antibodies, against four major latex allergens known to be present in NRL products, such as rubber gloves, condoms, baby teats etc. Latex allergy During the last 15 years NRL allergy has become a serious worldwide health problem. Up to 15% of the healthcare workers and about 1% of the whole population are allergic to NRL. While contact urticaria is the most common clinical manifestation of NRL allergy, the disease may be presented in its most serious form as anaphylaxis, a life threatening systemic reaction. NRL based medical devices (gloves, masks, etc.) are a potential source of sensitisation to both healthcare personnel and patients. Potentially, anyone in contact with objects containing NRL (e.g. gloves, condoms, catheters, balloons, toys etc.) can become sensitised. In spite of that, as several risks threatening healthcare personnel (like HIV etc), the use of medical devices is unavoidable. A long list of latex products are in daily use. Natural rubber latex allergens Liquid latex from the rubber tree, Hevea brasiliensis, is the source of NRL and contains over 200 proteins; only 13 of them have been identified as allergens. Only some allergens retain their allergenic properties through manufacturing processes. Thus when trying to reduce the allergenicity of NRL products, any good testing method must measure all components with clinical relevance present in the final product. The NRL allergens that have been shown to be clinically relevant to NRL allergy, and present in the final NRL products with maintained allergenicity are Hev b 1, Hev b 3, Hev b 5 and Hev b References Turjanmaa, K. et al., Allergy 51 (1996) Liss, G. M. and Sussman, G. L., Am. J. Ind. Med. 35 (1999) ; Poley Jr, G. E. and Slater, J. E., J. Allergy Clin. Immunol. 105 (1997) International Union of Immunological Societies allergens list: Current testing methods Total protein measurement - The Modified Lowry (ASTM D5712) Extracted proteins are precipitated to remove interfering substances. Protein content quantified by colorimetric reaction. Vulnerable to chemical interferences. Amino acid analysis measures the amino acid content of proteins after hydrolysis in hydrochloric acid. Requires expensive and advanced instruments and experienced staff. By measuring total protein level in the sample extract no direct information can be obtained on whether the mixture contains allergens. Antigenic protein measurement - Latex ELISA for Antigenic Proteins and ASTM D6499 These methods use the sera from rabbits immunized with latex proteins. The antibodies produced thus recognize any latex derived antigens. The test result does not directly correlate with the allergen content of the end product because it measures NRL antigenic proteins not allergens. Allergen content measurement - RAST or ELISA Inhibition Assay (IgE) Uses sera from patients diagnosed to have a latex allergy. The binding of IgE antibodies to r e l e v a n t N R L a l l e r g e n s immobilized onto a proper solid phase, such as a micotiter plate, is inhibited by serial dilutions of extracts of gloves or other NRL products. The limitations of this method are the difficulty of standardization, due to the variable antibody reactivity of the given patient pool and the scarce availability of the patient sera. Current tests for measuring the allergenicity of NRL products have several significant drawbacks

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5 5 Dedicated tests for direct determination of NRL allergens FITkit - Unique tests for measurement of clinically relevant allergens FITkit was developed in 2001 by the leading scientists working in the area of NRL allergy. FITkit tests are reliable immunological tests for direct determination of clinically relevant latex allergens in NRL products that overcome the limitations of previous methods. These highly sensitive tests use specific monoclonal antibodies developed against four clinically relevant latex allergens (Hev b 1, Hev b 3, Hev b 5 & Hev b 6.02) present in NRL products, such as latex gloves, condoms, baby teats etc. The highly purified and characterized allergens and monoclonal antibodies allow sensitive and specific results without interference or crossreactions. FITkit tests show excellent correlation to the allergenicity of gloves measured by currently available patient IgE based methods. Quattromed Ltd. provides a test kit dedicated to each allergen individually. If desired, by combining the results, they provide a reliable and accurate IEMA (immuno-ensymometric assay) method for determining the presence of four major NRL allergens. Each test provides all reagents, ready to use, for testing up to 40 duplicate samples. FITkit advantages The use of highly purified and characterized allergens and monoclonal antibodies enables standardization and quantification of individual allergens. The first specific method for identifying and quantifying individual NRL allergens Direct measurement of relevant allergens individually High sensitivity Consistent and reliable results Convenience and short assay time (< 2 hours) No interference from other substances References Timo Palosuo, Vladimir Ovod, Tytti Kärkkäinen, Nisse Kalkkinen, Markku Kulomaa, Timo Reunala, Kristiina Turjanmaa, Hely Reinikka-Railo: The Major Latex Allergens Hev b 6.02 (hevein) and Hev b 5 are Regularly Detected in Medical Gloves with Moderate or High Allergen Content. J Allergy Clin Immunol 2001; 107:S32 Timo Palosuo, Harri Alenius, Kristiina Turjanmaa: Quantification of Latex Allergens. Methods ; Please note that the FITkit tests are intended For Research Use Only. They should not be used for diagnostic purposes. Performance characteristics Hev b 1 Hev b 3 Hev b 5 Hev b 6.02 Detection limit 1.2 µg/l 2.3 µg/l 0.5 µg/l 0.1 µg/l Repeatability (N=16) Low 5.6% 5.3% 4.4% 4.6% Medium 4.6% 3.4% 2.5% 4.1% High 2.8% 4.6% 5.1% 5.8% Reproducibility (N=5) Low 6.8% 6.9% 5.2% 5.6% Medium 5.6% 6.0% 2.6% 4.3% High 4.6% 7.6% 5.4% 6.9% Recovery % Linearity %

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7 7 FITkit meets previously unmet needs Various applications FITkit -s can be used to detect NRL allergens in various materials, such as NRL gloves, catheters, condoms, baby teats, balloons etc. The highly purified and characterized allergens and monoclonal antibodies allow sensitive and specific results without interference or crossreactions. In addition, FITkit -s have been used to quantify NRL allergens in foods. This has been reported by FSA (Food Standard Agency), United Kingdom Reference Joanna Topping, Megan Gibbons, John Haines, Sian Knellar, Fioana Angus, Pradip Patel: Assessment and quantification of latex protein (LP) transfer from LP containing contact materials into food and drink products. Food Standards Agency Contract A03043; FITkit Hev b 1 Standard Curve FITkit Hev b 3 Standard Curve 1 1 A414 nm A414 nm Hev b 1 (µg/l) Hev b 3 (µg/l) 10 FITkit Hev b 5 Standard Curve FITkit Hev b 6.02 Standard Curve A414 nm A414 nm Hev b 5 (µg/l) Hev b 6.02 (µg/l) Cat # K K K K Product name FITkit Hev b 1 FITkit Hev b 3 FITkit Hev b 5 FITkit Hev b 6.02 (Hevein) Test for 40 duplicate samples 40 duplicate samples 40 duplicate samples 40 duplicate samples

8 FITkit Preparative and test procedures Preparative steps Bring all reagents to room temperature Test procedure Reconstitute test control with 500 µl distilled water. Let it stand at least for 30 min. Mark the wells to be used. Dilute 50 ml Wash Concentrate with 450 ml of distilled water Dispense 100 µl Assay Buffer Add 25 µl calibrators, controls and product extract into appropriate wells Incubate for 60 min with shaking at RT Wash 4 times Dispense 100 µl Enzyme Conjugate Incubate for 30 min with shaking at RT Wash 4 times Dispense 100 µl Substrate Solution Incubate for 15 min with shaking at RT Dispense 100 µl Stopping Solution Shake for 1 2 minutes and measure at 414 nm (or 405 nm). Fit the curve and read off results.

9 FITkit Frequently asked questions Why it is not enough to measure the total protein level? Only some of the proteins are allergenic; total protein content does not give reliable information about the allergenicity of a product. Why it is not enough to measure only one allergen? Patients can be sensitised to one or more allergens; different gloves and other NRL products can contain different allergens. Are all proteins and antigens from the rubber tree equally allergenic? No. During rubber manufacturing processes many of the proteins degrade, denature and lose their activity, but some proteins retain their allergenic properties throughout the process. Gloves can have high protein but low allergen levels and vice versa. Why have we chosen Hev b 1, Hev b 3, Hev b 5 and Hev b 6.02 to be included in the kits? According to the current literature, these four allergens are the ones that have been unequivocally demonstrated to be present in the glove extracts and to resist glovemanufacturing processes. [1] Why do we have four separate kits? It enables the direct measurement of each individual allergen and thus the definition of product based reference values for the customer. Alternatively, the results from the four tests can be combined. How well does the selection of these four allergens cover the total allergenicity of the gloves? Convincing results have been obtained showing high correlation to gloves' allergenicity measured by currently available patient IgE-based methods. In comparison, the gloves' allergenicity has correlated only moderately or poorly to the total protein measurement method (Lowry). [1] Which allergens are relevant for health care workers? The two most important latex allergens are Hev b 5 and Hev b 6.02 (hevein). These allergens can resist the process of glove manufacturing and they have been demonstrated in extracts of medical gloves. [2, 3, 4] What is the relationship between Hev b 6 and Hev b 6.02? Hev b 6 (prohevein or hevein preprotein) is the 20 kd precursor protein of Hev b 6.02 (4.72 kd polypeptide known as mature hevein). Hevein carries the major IgE epitope of prohevein. What is the significance of Hev b 1 and Hev b 3 in latex allergy? Hev b 1 and Hev b 3 are the two most important latex allergens for patients with spina bifida. [5, 6, 7] How do I prepare the product extract? NRL products can be extracted in PBS (phosphate buffered saline). For instance, 1 g of rubber product can be cut into pieces and extracted in 5 mls PBS. After extraction, the rubber products are removed and the extract is centrifuged. References 1. Timo Palosuo, Harri Alenius, Kristiina Turjanmaa: Quantification of Latex Allergens. Methods ; Akasawa A, Hsieh S-H, Martin BM, Liu T, Lin Y. A novel acidic allergen, Hev b 5, in latex. J Biol Chem 1996; 271: Alenius H, Kalkkinen N, Reunala T, Turjanmaa K, Palosuo T. The main IgE-binding epitope of a major latex allergen, prohevein, is present in its N-terminal 43 amino acid fragment, hevein. J Immunol 1996; 156: Timo Palosuo, Vladimir Ovod, Tytti Kärkkäinen, Nisse Kalkkinen, Markku Kulomaa, Timo Reunala, Kristiina Turjanmaa, Hely Reinikka-Railo: The Major Latex Allergens Hev b 6.02 (hevein) and Hev b 5 are Regularly Detected in Medical Gloves with Moderate or High Allergen Content. J. Allergy Clin Immunol 2001; 107:S Czuppon AB, Chen Z, Rennert S, Engelke T, Meyer HE, Heber M, Baur X. The rubber elongation factor of rubber trees (Hevea brasiliensis) is the major allergen in latex. J Allergy Clin Immunol 1993; 92: Alenius H, Palosuo T, Kelly K, Kurup V, Reunala T, Mäkinen- Kiljunen S, Turjanmaa K, Fink J. IgE reactivity to 14-kD and 27-kD natural rubber proteins in latex-allergic children with spina bifida and other congenital anomalies. Int Arch Allergy Immunol 1993; 102: Yeang HY, Cheong KF, Sunderasan E, Hamzah S, Chew NP, Hamid S, Hamilton RG, Cardosa J:The 14.6 kd rubber elongation factor (Hev b 1) and 24 kd (Hev b 3) rubber particle proteins are recognized by IgE from patients with spina bifida and latex allergy. J Allergy Clin Immunol 1996; 98:

10 Quattromed is an innovative biotech company providing medical diagnostics services and performing custom research programs for biopharmaceutical industry. Our main focus of activities is in protein expression and production. In-house proprietary technologies are used combined with extensive process know-how and partnership with academic and industrial groups. Quattromed Ltd Nooruse Tartu Estonia Phone: Fax: quattromed@quattromed.com