Nucleosome antigen

NUCLEOSOME ANTIGEN 
AROTEC_Nucleosome_Product_Info.pdf Version/Date: A/00.08.18 
ATN02-02 Nucleosome antigen 0.20 mg 
ATN02-05 Nucleosome antigen 0.50 mg 
ATN02-10 1.0 mg 
_________________________________________________________________________________
Description of the Product
Purified from bovine thymus. After coating onto ELISA plates 
the product will bind autoantibodies to nucleosome antigen. 
Purity: The nucleosome antigen is more than 90% pure, as 
assessed 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 o
C or below （long term） or at +4o
C （short term）. 
Avoid repeated freezing and thawing. Mix thoroughly before 
use. 
Clinical and Biochemical Data 
Anti-nucleosome antibodies （also referred to as anti-chromatin 
antibodies） constitute one of the first autoantibody specificities 
found to be present in systemic lupus erythematosus （SLE） 
having been detected as early as 1948 using the LE cell 
test1,2. The prevalence of anti-nucleosome antibodies in SLE is 
of the order of 50-100%2
, and the nucleosome has been 
identified as the initiating and driving immunogen in SLE3,4
. 
Antinucleosome antibodies are now considered to be a more 
sensitive marker for SLE than anti-dsDNA antibodies5
 and a 
high anti-nucleosome antibody titre has been reported to be 
indicative of lupus nephritis6-8. Anti-nucleosome antibodies 
have been found （with a lower prevalence than in SLE） in a 
number of other autoimmune diseases6,9,10,19 such as systemic 
sclerosis, Sjogren's syndrome and mixed connective tissue 
disease and are also found in 40-50% of patients with 
autoimmune hepatitis type I11,12. Anti-ribosomal P antibodies 
have 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 nucleus 
of eukaryotic cells. It is composed of about 200 base pairs of 
DNA wrapped twice around a （H2A-H2B-H3-H4）2 histone 
octamer with histone H1 bound on the periphery2,15,16,19
. 
Nucleosomes can be isolated by digesting the linker DNA 
holding them together in chromatin with micrococcal nuclease. 
The most effective form of nucleosomes for detecting patient 
autoantibodies are prepared in this way and stripped of H1 
histone to yield a nucleosome core particle in which 146 base 
pairs of DNA are wrapped around an octamer of two H2A-H2B 
dimers that surround an H3-H4 tetramer2,17. Autoantibodies 
against subnucleosome particles occur in SLE, they are 
however less frequent3
. 
AROTEC nucleosome antigen is prepared from calf thymus 
chromatin and contains the four core histones H2A, H2B, H3 
and H4 bound to DNA of about 146 base pairs in length. 
Histone amino acid sequences are highly conserved between 
species, even between animals and plants18. Homology 
between human and bovine amino acid sequences is as 
follows （ExPASy accession numbers human:bovine are 
shown）: 
H2A （P0C0S5:P0C0S4） 128 aa: 128 identical 
H2B （B2R4S9:A5D7N2） 126 aa: 125 identical, 1 conservative 
subsitition 
H3 （P84243:Q5E9F8） 136 aa: 136 identical 
H4 （P62805:P62803） 103 aa: 103 identical 
Methodology
The following is an ELISA procedure which can be used to 
detect anti-nucleosome autoantibodies in human serum using 
the ATN02 purified nucleosome antigen: 
1. Dilute the purified antigen to 1.0-2.0 ?g/ml in 20 mM 
Tris/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 +4o
C. 
3. Empty the plates and remove excess liquid by tapping on a 
paper towel. 
4. Block excess protein binding sites by adding 200 ?l PBS 
containing 1% BSA per well. Cover and incubate at +4o
C 
overnight. 
5. Empty plates and apply 100 ?l of serum samples diluted 
1:100 in PBS / 1% BSA / 0.1% Tween?
 20. Incubate at room 
temperature for 1 hour. 
6. Empty plates and add 200 ?l PBS / 0.1% Tween?
 20 per 
well. Incubate 5 minutes then empty plates. Repeat this step 
twice. 
7. Apply 100 ?l anti-human IgG-enzyme conjugate 
（horseradish peroxidase or alkaline phosphatase） diluted in 
PBS / 1% BSA / 0.1% Tween?
 20 per well and incubate for 1 
hour. 
8. Repeat step 6. 
9. Add enzyme substrate and stop the reaction when 
appropriate. 
10. Read absorbance in an ELISA spectrophotometer. 
References 
1. Hargraves, M.M. et al. （1948） Proc. Mayo Clin. 23, 25 
2. Gomez-Puerta, J.A. et al. （2008） Autoimmunity Rev. 7, 606 
3. Burlingame, R.W. et al. （1994） J. Clin. Invest. 94, 184 
4. Muller, S. et al. （2008） Lupus 17, 431 
5. Putova, I et al. （2007） Annals N.Y. Acad. Sci. 1109, 275 
6. Cervera, R. et al. （2003） Ann. Rheum. Dis. 62, 431 
7. Manson, J.J. （2009） Arthritis Res. Ther. 11, R154 
8. Kiss, E. et al. （2009） Autoimmunity 42, 393 
9. Amoura, Z. et al. （2000） Arthritis Rheum. 43, 76 
10. Schett, G. et al. （2002） Lupus 11, 704 
11. Ghillani-Dalbin, P. et al. （2003） Lupus 12, 833 
12. Koutouzov, S. et al. （2004） Rheum. Dis. Clin. North Am. 30, 529 
13. Chindalore, V. et al. （1998） Clin. Immunol. Immunopathol. 87, 292 
14. Caponi, L. et al. （2002） Clin. Exp. Immunol. 130, 541 
15. Lutter, L.C. （1978） J. Mol. Biol. 124, 391 
16. Luger, K. et al. （1997） Nature 389, 251 
17. Burlingame, R.W. & Rubin, R.L. （1990） J. Immunol. Meth. 134, 187 
18. Baxevanis, A.D. & Landsman, D. （1996） Nucleic Acid Res. 24, 245 
19. Decker, P. （2006） Clin. Chim. Acta 366, 48 
Micr-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 
during the preparation of this product.