Ribosomal P antigen

Identity:Ribosomal P antigen, nRNP antigen.Source Material:Bovine thymus (New Zealand origin).Clinical Indications:Autoantibodies present in systemic lupus erythematosus.Ordering Information:ATR03-02

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RIBOSOMAL P ANTIGENAROTEC_RiboP_Product_Info.pdf Version/Date: B/04.05.25ATR03-02 Ribosomal P antigen 0.20 mgATR03-05 Ribosomal P antigen 0.50 mgATR03-10 1.0 mg_________________________________________________________________________________Description of the ProductPurified from bovine thymus. After coating onto ELISA platesthe product will bind autoantibodies to ribosomal P antigen.Purity: The ribosomal P antigen subunits P0, P1 and P2 （38,19 and 17 kDa respectively） are more than 90% pure, asassessed by SDS-polyacrylamide gel electrophoresis.Concentration: 0.1-1.0 mg protein/ml.Storage: The product is stabilised with 20% glycerol and 0.1%Micr-O-protectTM. Store at -20 oC or below （long term） or at+4oC （short term）. Avoid repeated freezing and thawing. Mixthoroughly before use.Clinical and Biochemical DataThe existence of autoantibodies to ribosomal components hasbeen known for some time1,2. In 1985, two groupsindependently identified the ribosomal P proteins as the majorprotein antigens recognised by ribosomal antibodies3,4. Antiribosomal P antibodies are considered to be very specific forsystemic lupus erythematosus （SLE）5,6, and their presencefrequently correlates with disease activity, in particularpsychotic depression6, hepatitis7-9, and nephritis10,11. Althoughanti-ribosomal P antibodies have been reported to occur inpatients with systemic sclerosis12, this would appear to be rareand normally indicates an overlap with SLE13.The P proteins are three of approximay 80 proteins thatmake up the largest cytoplasmic ribonucleoprotein, theribosome. Since ribosomes are assembled in the nucleoli,high titre anti-ribosomal P sera will show nucleolar as well ascytoplasmic immunofluorescent staining14. The exact functionsof the individual P proteins are not fully understood howeverstudies suggest that they collectively comprise part of afunctional GTPase domain necessary for the binding of factorGTP complexes15,16. This interaction results in catalysis of theappropriate step of the protein synthesis cycle （initiation,elongation or release）. GTP is hydrolysed and the factor isreleased. The ribosomal P proteins are distinctive throughtheir overall net negative charge, high content of alanine andpredicted secondary structure （approx. 70% helical）14. The Cterminal 17 amino acids of all three P proteins are virtuallyidentical and are highly conserved between species. Althoughthe major autoantibody epitope on all three proteins isbelieved to be located within the C-terminal 22 aminoacids14,17, there is recent evidence that other individual Pprotein-specific epitopes occur18.The ribosomal P0, P1 and P2 proteins are all present inAroTec’s ribosomal P antigen. The antigen typically exhibits a260/280 nm absorbance ratio of >1.5, suggesting that asignificant rRNA component is present. The sequences of thebovine P0 and P2 proteins have been determined19, andfound to be very homologous （>99%） to their humanequivalents20. In particular the C-terminal 22 amino acids ofboth species were found to be identical.MethodologyThe following is an ELISA procedure which can be used todetect anti-ribosomal P autoantibodies in human serum usingthe ATR03 purified autoantigen:1. Dilute the purified antigen to 0.5-1.0 µg/ml in PBS （10 mMpotassium phosphate, pH 7.4, 0.15 M NaCl）.2. Coat ELISA plates with 100 µl of diluted antigen per well.Cover and incubate 24 hours at +4oC.3. Empty the plates and remove excess liquid by tapping on apaper towel.4. Block excess protein binding sites by adding 200 µl PBScontaining 1% BSA per well. Cover and incubate at +4oCovernight.5. Empty plates and apply 100 µl of serum samples diluted1:100 in PBS / 1% BSA / 1% casein / 0.1% Tween20.Incubate at room temperature for 1 hour.6. Empty plates and add 200 µl PBS / 0.1% Tween20 perwell. Incubate 5 minutes then empty plates. Repeat this steptwice.7. Apply 100 µl anti-human IgG-enzyme conjugate（horseradish peroxidase or alkaline phosphatase） diluted inPBS / 1% BSA / 1% casein / 0.1% Tween20 per well andincubate for 1 hour.8. Repeat step 6.9. Add enzyme substrate and stop the reaction whenappropriate.10. Read absorbance in an ELISA spectrophotometer.References1. Sturgill, P.H. et al. （1965） Arthritis Rheum. 8, 2132. Schur, P.H. et al. （1967） Immunochemistry 4, 4473. Elkon, K.B. et al. （1985） J. Exp. Med. 162, 4594. Francoeur, A.-M. et al. （1985） J. Immunol. 135, 23785. Elkon, K.B. et al. （1992） Rheum. Dis. Clin. 18, 3776. Teh, L.S. & Isenberg, D.A. （1994） Arthritis Rheum. 37, 3077. Koren, E. et al. （1993） Arthritis Rheum. 36, 13258. Hulsey, M. et al. （1995） Clin. Immunol. Immunopathol. 74, 2529. Arnett, F.C. & Reichlin, M. （1995）, Am. J. Med. 99, 46510. Martin, A.L. & Reichlin, M. （1996） Lupus 5, 2211. Sato, T. et al. （1991） J. Rheumatol. 18, 168112. Fujimoto, M et al. （1996） J. Dermatol. 23, 3313. Fujimoto, M. et al. （1995） Br. J. Rheumatol. 34, 90814. Elkon, K.B. （1994） Man. Biol. Markers Dis. （Kluwer Acad. Publ.）B2.5/1-1115. Chu, J.L. et a l. （1991） J. Exp. Med. 174, 50716. Teh, L.S. et al. （1992） Br. J. Rheumatol. 32, 28717. Elkon, K. et al. （1986） Proc. Natl. Acad. Sci. USA 83, 741918. Fabien, N. et al. （1999） J. Autoimmun. 13, 10319. SWISS-PROT RLA0_BOVIN primary accession number Q95140 &RLA2_BOVIN primary accession number P4289920. Rich, B.E. & Steitz, J.A. （1987） Mol. Cell Biol. 7, 4065Micr-O-protect is from Roche Diagnostics GmbH （Mannheim,Germany）.Tween20 is a registered trademark of ICI Americas Inc.NOTE: No patented technology has been used by AroTecduring the preparation of this product.