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La （SSB） ANTIGEN
AROTEC_La-SSB_Product_Info.pdf Version/Date: B/04.05.20
ATL01-02 La （SSB） antigen 0.20 mg
ATL01-05 La （SSB） antigen 0.50 mg
ATL01-10 1.0 mg
_________________________________________________________________________________
Description of the Product
Purified from bovine thymus. After coating onto ELISA plates
the product will bind autoantibodies to La （SSB）.
Purity: The La autoantigen （45-50 kDa） is more than 90%
pure, as assessed 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 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
Sjögren's syndrome （SS） is a common systemic autoimmune
inflammatory disorder characterised by lymphocyte-mediated
destruction of exocrine glands leading to diminished or absent
glandular secretion1-4. SS may present as a primary disease
or in association with other systemic autoimmune diseases
（referred to as secondary SS）. Autoantibodies to the La （SSB）
antigen can be detected in the sera of up to 87% of patients
with primary or secondary SS5,6. The presence of anti-La
（SSB） autoantibodies usually coincides with the presence of
anti-Ro （SSA） autoantibodies7
, however the fact that anti-Ro
autoantibodies are far more common in other rheumatological
conditions such as systemic lupus erythematosis （SLE） and
mixed connective tissue disease （MCTD） suggests that anti-La
is more specific for primary and secondary SS than anti-Ro8,9
.
Anti-La autoantibodies have also been reported to be present
in other clinical conditions, most notably in the sera of mothers
of infants with neonatal lupus syndrome10, but also in 10 to
15% of SLE patients11,12
.
binds to the oligo（U） 3' termini of nascent
RNA polymerase III transcripts and facilitates transcriptional
termination and reinitiation by this enzyme13,-17. It has also
been reported to function as an ATP-dependent helicase able
to melt RNA-DNA hybrids18. La （SSB） may be involved in
other processes as well such as maturation and/or nuclear
export of RNA polymerase III products and some aspects of
translation19,20. La （SSB） is a highly phosphorylated protein
which migrates at about 50 kDa in SDS-polyacrylamide gel
electrophoresis21. Phosphorylated residues are present at the
carboxy-terminal part of the protein22. At least 8 isoelectric
forms （pI range 6 to 7） have been identified23
.
The amino acid sequences of both human and bovine La
（SSB） antigen have been determined by cDNA cloning and
sequencing19,28. Comparison of the two sequences shows 22
largely conservative amino acid substitutions with a total of
95% identity. Three regions of the La molecule （amino acids
1-107, 111-242 and 346-408） are thought to contain the major
epitopes reactive with human anti-La sera19,24. The broad
cross-reactivity of patient sera with La （SSB） from diverse
mammalian species indicates the presence of conserved
epitopes25. The use of bovine for the
detection of human anti-La （SSB） antibodies has been
described by several authors25-27
.
Methodology
The following is an ELISA procedure which can be used to
detect anti-La （SSB） autoantibodies in human serum using the
ATL01 purified autoantigen:
1. Dilute the purified antigen to 0.5-1.0 µg/ml in PBS （10 mM
potassium 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 +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 µl PBS
containing 1% BSA per well. Cover and incubate at +4o
C
overnight.
5. Empty plates and apply 100 µl of serum samples diluted
1:100 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 per
well. Incubate 5 minutes then empty plates. Repeat this step
twice.
7. Apply 100 µl anti-human IgG-enzyme conjugate
（horseradish peroxidase or alkaline phosphatase） diluted in
PBS / 1% BSA / 1% casein / 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. Molina, R. et al. （1986） Am. J. Med. 80, 23
2. Bloch, K.J. et al. （1965） Medicine 44, 187
3. Fox, R.I. et al. （1986） Arthritis Rheum. 29, 577
4. Aziz, K.E. et al. （1992） Aust. NZ J. Med. 22, 671
5. Manoussakis, M.N. et al. （1986） Scan. J. Rheumatol. 61, 89
6. Harley, J.B. et al. （1986） Arthritis Rheum. 29, 196
7. Craft, J.E. & Hardin, J.A. （1987） J. Rheumatol. 14 S13, 106
8. St. Clair, E.W. （1992） Rheum. Dis. Clin. N. America 18, 359
9. Harley, J.B. （1989） J. Autoimmun. 2, 383
10. Buyon, J.P. et al. （1989） J. Clin. Invest. 84, 627
11. Reichlin, M. （1986） J. Clin. Immunol. 6, 339
12. Wiascewk, C.A. & Reichlin, M. （1982） J. Clin. Invest. 69, 835
13. Stefano, J.E. （1984） Cell 36, 145
14. Gottlieb, E. & Steitz, J.A. （1989） EMBO J. 8, 841
15. Gottlieb, E. & Steitz, J.A. （1989） EMBO J. 8, 851
16. Maraia, R.J. et al. （1994） Mol. Cell Biol. 14, 2147
17. Maraia, R.J. （1996） Proc. Natl. Acad. Sci. USA 93, 3383
18. Bachmann, M. et al. （1990） Cell 60, 85
19. Chambers, J.C. et al. （1988） J. Biol. Chem. 263, 18043
20. Bachmann, M. et al. （1989） Mol. Cell Biochem. 85, 103
21. Pruijn, G.J.M. （1994） Man. Biol. Markers Dis. （Kluwer Acad.
Publ.） B4.2/1-14
22. Pfeifle, J. et al. （1987） Biochim. Biophys. Acta 928, 217
23. Francoeur, A.M. et al. （1985） mol. Cell Biol. 5, 586
24. McNeilage, L.J. （1990） J. Immunol. 145, 3829
25. Chan, E.K.L. & Tan, E.M. （1987） J. Exp. Med. 166, 1627
26. Zhang, W. & Reichlin, M. （1996） Arthritis Rheum. 39, 522
27. Chan, E.K.L. et al. （1986） J. Immunol. 136, 3744
28. Chan, E.K.L. et al. （1989） Nucleic Acids Res. 17, 2233
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.
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