HISTONE ANTIGEN
AROTEC_Histone_Product_Info.pdf Version/Date: A/00.06.02
ATH01-02 Histone antigen 0.20 mg
ATH01-05 Histone antigen 0.50 mg
ATH01-10 1.0 mg
_________________________________________________________________________________
Description of the Product
Purified from bovine thymus. After coating onto ELISA plates
the product will bind autoantibodies to histone antigen.
Purity: The histone autoantigen 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
Autoantibodies against histones （AHAs） are observed in a
number of autoimmune diseases. AHA are reported in 50-80%
of patients with Systemic Lupus Erythematosus （SLE） being
highest in patients with active disease1
. Although H1 and H2B
are the most common epitopes in SLE, many SLE patients
have conformation-dependent AHA, directed against the
histone complex2
. AHA have particular clinical significance for
drug-induced lupus, particularly in the diagnosis of antinuclear
antibody positive patients receiving proc*, hydralazine
and isoniazide3
. AHA are also prevalent in Felty’s syndrome
（83%）, rheumatoid arthritis （75%） and juvenile arthritis （50-
75%）2
, scleroderma4,5 systemic sclerosis6
and mixed
connective tissue disease7
. AHA （predominantly against H1）
are also observed in approximay 76% of patients with
primary biliary cirrhosis2
.
Histones are small DNA-binding proteins and the major protein
component of the nucleosome. The nucleosome consists of
146 base pairs of DNA wrapped around an octomer of core
histone proteins composed of a central tetramer of two H3-H4
dimers flanked by two H2A-H2B dimers8
. Histone H1 is a
linker histone, present between each nucleosome, and is
responsible for establishing chromatin structure. The
molecular weights of the core histones range from 11,000 to
15,000. Histone H1 is larger, with a molecular weight of
23,000. All of the histones contain many basic amino acids,
with histones H3 and H4 being arginine rich, while H2A and
H2B are slightly lysine-rich8
.
Biochemical and serological studies have revealed a more
complicated picture of histone antigenicity than initially
recognised, particularly highlighted by observations whereby
separation of the histone complex into its individual
components using harsh conditions can result in a loss of
complex formation and antibody reactivity9,10. Histone epitopes
are often located in accessible regions of chromatin （especially
for H1 and H2A/H2B）11 or are conformational determinants
resulting from the association of several histone components.
Acetylation of histone lysine residues may also play a role in
SLE autoantigenicity12. The H2A-H2B dimer is the main
antigen in drug-induce lupus, although also observed in SLE,
whereas linear epitopes are generally only observed for core
histones2
.
Histone amino acid sequences are highly conserved between
species, even between animals and plants13. AROTEC
histone antigen is prepared from calf thymus chromatin and
contains the five main histones, H1, H2A, H2B, H3 and H4,
extracted and purified without the use if harsh denaturing
reagents to ensure maximum reactivity with human
autoantibodies.
Methodology
The following is an ELISA procedure which can be used to
detect anti-histone autoantibodies in human serum using the
ATH01 purified histone antigen:
1. Dilute the purified antigen to 1.0-2.0 ?g/ml in 50 mM
carbonate buffer, pH 9.6 containing 0.5% （w/v） sodium
deoxycholate.
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. Cohen, M.G. et al. （1992） Ann. Rheum. Dis. 51, 61
2. Monestier, M. & Kotzin, B. （1992） In （Ed. Pisetsky, D.） Rheumatic
Disease Clinics of North America, 18, 415
3. Vedove, C.D. et al. （2009） Arch. Dermatol. Res. 301, 99
4. Parodi, A. et al. （1995） Dermatology 191, 16
5. Sato, S. et al. （1993） Arthritis Rheum. 36, 1137
6. Sato, S. et al. （1998） Ann. Rheum. Dis. 57, 470
7. Wayakau, T. et al. （2007） Rheumatol. Int. 28, 113
8. Burlingame, R.W. et al. （1985） Science 228, 546
9. Rodriguez-Collazo, P. et al. （2009） Nucleic Acids Res. 37, e81
10. Feldman, L. & Stollar, B.D. （1977） Biochem. 16, 2767
11. Sato, S. et al. （1994） Arch. Dermatol. 130, 1273
12. Dieker, J.W. et al. （2007） Arthritis Rheum. 56, 1921
13. Baxevanis, A.D. & Landsman, D. （1996） Nucleic Acids Res. 24, 245
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
