Nucleosome antigen

Identity:Core histones (H2A, H2B, H3, H4) and DNA (approximay 146 bp)Source Material:Bovine thymus (New Zealand origin).Clinical Indications:Autoantibodies present in systemic lupus erythematosus a

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NUCLEOSOME ANTIGENAROTEC_Nucleosome_Product_Info.pdf Version/Date: A/00.08.18ATN02-02 Nucleosome antigen 0.20 mgATN02-05 Nucleosome antigen 0.50 mgATN02-10 1.0 mg_________________________________________________________________________________Description of the ProductPurified from bovine thymus. After coating onto ELISA platesthe product will bind autoantibodies to nucleosome antigen.Purity: The nucleosome antigen is more than 90% pure, asassessed by SDS gel electrophoresis.Concentration: 0.5 -2.0 mg protein/ml.Storage: The product is stabilised with 0.1% Micr-O-protectTM.Store at -20 oC or below （long term） or at +4oC （short term）.Avoid repeated freezing and thawing. Mix thoroughly beforeuse.Clinical and Biochemical DataAnti-nucleosome antibodies （also referred to as anti-chromatinantibodies） constitute one of the first autoantibody specificitiesfound to be present in systemic lupus erythematosus （SLE）having been detected as early as 1948 using the LE celltest1,2. The prevalence of anti-nucleosome antibodies in SLE isof the order of 50-100%2, and the nucleosome has beenidentified as the initiating and driving immunogen in SLE3,4.Antinucleosome antibodies are now considered to be a moresensitive marker for SLE than anti-dsDNA antibodies5and ahigh anti-nucleosome antibody titre has been reported to beindicative of lupus nephritis6-8. Anti-nucleosome antibodieshave been found （with a lower prevalence than in SLE） in anumber of other autoimmune diseases6,9,10,19 such as systemicsclerosis, Sjogren's syndrome and mixed connective tissuedisease and are also found in 40-50% of patients withautoimmune hepatitis type I11,12. Anti-ribosomal P antibodieshave also been reported to bind to nucleosomes13,14.The nucleosome is the basic structural subunit of chromatin,the native complex of histones and DNA found in the nucleusof eukaryotic cells. It is composed of about 200 base pairs ofDNA wrapped twice around a （H2A-H2B-H3-H4）2 histoneoctamer with histone H1 bound on the periphery2,15,16,19.Nucleosomes can be isolated by digesting the linker DNAholding them together in chromatin with micrococcal nuclease.The most effective form of nucleosomes for detecting patientautoantibodies are prepared in this way and stripped of H1histone to yield a nucleosome core particle in which 146 basepairs of DNA are wrapped around an octamer of two H2A-H2Bdimers that surround an H3-H4 tetramer2,17. Autoantibodiesagainst subnucleosome particles occur in SLE, they arehowever less frequent3.AROTEC nucleosome antigen is prepared from calf thymuschromatin and contains the four core histones H2A, H2B, H3and H4 bound to DNA of about 146 base pairs in length.Histone amino acid sequences are highly conserved betweenspecies, even between animals and plants18. Homologybetween human and bovine amino acid sequences is asfollows （ExPASy accession numbers human:bovine areshown）:H2A （P0C0S5:P0C0S4） 128 aa: 128 identicalH2B （B2R4S9:A5D7N2） 126 aa: 125 identical, 1 conservativesubsititionH3 （P84243:Q5E9F8） 136 aa: 136 identicalH4 （P62805:P62803） 103 aa: 103 identicalMethodologyThe following is an ELISA procedure which can be used todetect anti-nucleosome autoantibodies in human serum usingthe ATN02 purified nucleosome antigen:1. Dilute the purified antigen to 1.0-2.0 g/ml in 20 mMTris/HCl buffer, pH 8.0; containing 0.15 M NaCl.2. Coat ELISA plates with 100 l 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 l PBScontaining 1% BSA per well. Cover and incubate at +4oCovernight.5. Empty plates and apply 100 l of serum samples diluted1:100 in PBS / 1% BSA / 0.1% Tween20. Incubate at roomtemperature for 1 hour.6. Empty plates and add 200 l PBS / 0.1% Tween20 perwell. Incubate 5 minutes then empty plates. Repeat this steptwice.7. Apply 100 l anti-human IgG-enzyme conjugate（horseradish peroxidase or alkaline phosphatase） diluted inPBS / 1% BSA / 0.1% Tween20 per well and incubate for 1hour.8. Repeat step 6.9. Add enzyme substrate and stop the reaction whenappropriate.10. Read absorbance in an ELISA spectrophotometer.References1. Hargraves, M.M. et al. （1948） Proc. Mayo Clin. 23, 252. Gomez-Puerta, J.A. et al. （2008） Autoimmunity Rev. 7, 6063. Burlingame, R.W. et al. （1994） J. Clin. Invest. 94, 1844. Muller, S. et al. （2008） Lupus 17, 4315. Putova, I et al. （2007） Annals N.Y. Acad. Sci. 1109, 2756. Cervera, R. et al. （2003） Ann. Rheum. Dis. 62, 4317. Manson, J.J. （2009） Arthritis Res. Ther. 11, R1548. Kiss, E. et al. （2009） Autoimmunity 42, 3939. Amoura, Z. et al. （2000） Arthritis Rheum. 43, 7610. Schett, G. et al. （2002） Lupus 11, 70411. Ghillani-Dalbin, P. et al. （2003） Lupus 12, 83312. Koutouzov, S. et al. （2004） Rheum. Dis. Clin. North Am. 30, 52913. Chindalore, V. et al. （1998） Clin. Immunol. Immunopathol. 87, 29214. Caponi, L. et al. （2002） Clin. Exp. Immunol. 130, 54115. Lutter, L.C. （1978） J. Mol. Biol. 124, 39116. Luger, K. et al. （1997） Nature 389, 25117. Burlingame, R.W. & Rubin, R.L. （1990） J. Immunol. Meth. 134, 18718. Baxevanis, A.D. & Landsman, D. （1996） Nucleic Acid Res. 24, 24519. Decker, P. （2006） Clin. Chim. Acta 366, 48Micr-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.