Histone antigen

Identity:Histone H1, H2A, H2B, H3 and H4Source Material:Bovine thymus (New Zealand origin).Clinical Indications:Autoantibodies present in systemic lupus erythematosus, drug-induced lupus erythematosus

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HISTONE ANTIGENAROTEC_Histone_Product_Info.pdf Version/Date: A/00.06.02ATH01-02 Histone antigen 0.20 mgATH01-05 Histone antigen 0.50 mgATH01-10 1.0 mg_________________________________________________________________________________Description of the ProductPurified from bovine thymus. After coating onto ELISA platesthe product will bind autoantibodies to histone antigen.Purity: The histone autoantigen 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 DataAutoantibodies against histones （AHAs） are observed in anumber of autoimmune diseases. AHA are reported in 50-80%of patients with Systemic Lupus Erythematosus （SLE） beinghighest in patients with active disease1. Although H1 and H2Bare the most common epitopes in SLE, many SLE patientshave conformation-dependent AHA, directed against thehistone complex2. AHA have particular clinical significance fordrug-induced lupus, particularly in the diagnosis of antinuclearantibody positive patients receiving proc*, hydralazineand isoniazide3. AHA are also prevalent in Felty’s syndrome（83%）, rheumatoid arthritis （75%） and juvenile arthritis （50-75%）2, scleroderma4,5 systemic sclerosis6and mixedconnective tissue disease7. AHA （predominantly against H1）are also observed in approximay 76% of patients withprimary biliary cirrhosis2.Histones are small DNA-binding proteins and the major proteincomponent of the nucleosome. The nucleosome consists of146 base pairs of DNA wrapped around an octomer of corehistone proteins composed of a central tetramer of two H3-H4dimers flanked by two H2A-H2B dimers8. Histone H1 is alinker histone, present between each nucleosome, and isresponsible for establishing chromatin structure. Themolecular weights of the core histones range from 11,000 to15,000. Histone H1 is larger, with a molecular weight of23,000. All of the histones contain many basic amino acids,with histones H3 and H4 being arginine rich, while H2A andH2B are slightly lysine-rich8.Biochemical and serological studies have revealed a morecomplicated picture of histone antigenicity than initiallyrecognised, particularly highlighted by observations wherebyseparation of the histone complex into its individualcomponents using harsh conditions can result in a loss ofcomplex formation and antibody reactivity9,10. Histone epitopesare often located in accessible regions of chromatin （especiallyfor H1 and H2A/H2B）11 or are conformational determinantsresulting from the association of several histone components.Acetylation of histone lysine residues may also play a role inSLE autoantigenicity12. The H2A-H2B dimer is the mainantigen in drug-induce lupus, although also observed in SLE,whereas linear epitopes are generally only observed for corehistones2.Histone amino acid sequences are highly conserved betweenspecies, even between animals and plants13. AROTEChistone antigen is prepared from calf thymus chromatin andcontains the five main histones, H1, H2A, H2B, H3 and H4,extracted and purified without the use if harsh denaturingreagents to ensure maximum reactivity with humanautoantibodies.MethodologyThe following is an ELISA procedure which can be used todetect anti-histone autoantibodies in human serum using theATH01 purified histone antigen:1. Dilute the purified antigen to 1.0-2.0 g/ml in 50 mMcarbonate buffer, pH 9.6 containing 0.5% （w/v） sodiumdeoxycholate.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. Cohen, M.G. et al. （1992） Ann. Rheum. Dis. 51, 612. Monestier, M. & Kotzin, B. （1992） In （Ed. Pisetsky, D.） RheumaticDisease Clinics of North America, 18, 4153. Vedove, C.D. et al. （2009） Arch. Dermatol. Res. 301, 994. Parodi, A. et al. （1995） Dermatology 191, 165. Sato, S. et al. （1993） Arthritis Rheum. 36, 11376. Sato, S. et al. （1998） Ann. Rheum. Dis. 57, 4707. Wayakau, T. et al. （2007） Rheumatol. Int. 28, 1138. Burlingame, R.W. et al. （1985） Science 228, 5469. Rodriguez-Collazo, P. et al. （2009） Nucleic Acids Res. 37, e8110. Feldman, L. & Stollar, B.D. （1977） Biochem. 16, 276711. Sato, S. et al. （1994） Arch. Dermatol. 130, 127312. Dieker, J.W. et al. （2007） Arthritis Rheum. 56, 192113. Baxevanis, A.D. & Landsman, D. （1996） Nucleic Acids Res. 24, 245Micr-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 AROTEC