描述:Identity:Ribosomal P antigen, nRNP antigen.Source Material:Bovine thymus (New Zealand origin).Clinical Indications:Autoantibodies present in systemic lupus erythematosus.Ordering Information:ATR03-02
RIBOSOMAL P ANTIGEN
AROTEC_RiboP_Product_Info.pdf Version/Date: B/04.05.25
ATR03-02 Ribosomal P antigen 0.20 mg
ATR03-05 Ribosomal P antigen 0.50 mg
ATR03-10 1.0 mg
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Description of the Product
Purified from bovine thymus. After coating onto ELISA plates
the product will bind autoantibodies to ribosomal P antigen.
Purity: The ribosomal P antigen subunits P0, P1 and P2 (38,
19 and 17 kDa respectively) are 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
The existence of autoantibodies to ribosomal components has
been known for some time1,2. In 1985, two groups
independently identified the ribosomal P proteins as the major
protein antigens recognised by ribosomal antibodies3,4. Antiribosomal P antibodies are considered to be very specific for
systemic lupus erythematosus (SLE)5,6, and their presence
frequently correlates with disease activity, in particular
psychotic depression6
, hepatitis7-9, and nephritis10,11. Although
anti-ribosomal P antibodies have been reported to occur in
patients with systemic sclerosis12, this would appear to be rare
and normally indicates an overlap with SLE13
.
The P proteins are three of approximay 80 proteins that
make up the largest cytoplasmic ribonucleoprotein, the
ribosome. Since ribosomes are assembled in the nucleoli,
high titre anti-ribosomal P sera will show nucleolar as well as
cytoplasmic immunofluorescent staining14. The exact functions
of the individual P proteins are not fully understood however
studies suggest that they collectively comprise part of a
functional GTPase domain necessary for the binding of factorGTP complexes15,16. This interaction results in catalysis of the
appropriate step of the protein synthesis cycle (initiation,
elongation or release). GTP is hydrolysed and the factor is
released. The ribosomal P proteins are distinctive through
their overall net negative charge, high content of alanine and
predicted secondary structure (approx. 70% helical)14. The Cterminal 17 amino acids of all three P proteins are virtually
identical and are highly conserved between species. Although
the major autoantibody epitope on all three proteins is
believed to be located within the C-terminal 22 amino
acids14,17, there is recent evidence that other individual P
protein-specific epitopes occur18
.
The ribosomal P0, P1 and P2 proteins are all present in
AroTec’s ribosomal P antigen. The antigen typically exhibits a
260/280 nm absorbance ratio of >1.5, suggesting that a
significant rRNA component is present. The sequences of the
bovine P0 and P2 proteins have been determined19, and
found to be very homologous (>99%) to their human
equivalents20. In particular the C-terminal 22 amino acids of
both species were found to be identical.
Methodology
The following is an ELISA procedure which can be used to
detect anti-ribosomal P autoantibodies in human serum using
the ATR03 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. Sturgill, P.H. et al. (1965) Arthritis Rheum. 8, 213
2. Schur, P.H. et al. (1967) Immunochemistry 4, 447
3. Elkon, K.B. et al. (1985) J. Exp. Med. 162, 459
4. Francoeur, A.-M. et al. (1985) J. Immunol. 135, 2378
5. Elkon, K.B. et al. (1992) Rheum. Dis. Clin. 18, 377
6. Teh, L.S. & Isenberg, D.A. (1994) Arthritis Rheum. 37, 307
7. Koren, E. et al. (1993) Arthritis Rheum. 36, 1325
8. Hulsey, M. et al. (1995) Clin. Immunol. Immunopathol. 74, 252
9. Arnett, F.C. & Reichlin, M. (1995), Am. J. Med. 99, 465
10. Martin, A.L. & Reichlin, M. (1996) Lupus 5, 22
11. Sato, T. et al. (1991) J. Rheumatol. 18, 1681
12. Fujimoto, M et al. (1996) J. Dermatol. 23, 33
13. Fujimoto, M. et al. (1995) Br. J. Rheumatol. 34, 908
14. Elkon, K.B. (1994) Man. Biol. Markers Dis. (Kluwer Acad. Publ.)
B2.5/1-11
15. Chu, J.L. et a l. (1991) J. Exp. Med. 174, 507
16. Teh, L.S. et al. (1992) Br. J. Rheumatol. 32, 287
17. Elkon, K. et al. (1986) Proc. Natl. Acad. Sci. USA 83, 7419
18. Fabien, N. et al. (1999) J. Autoimmun. 13, 103
19. SWISS-PROT RLA0_BOVIN primary accession number Q95140 &
RLA2_BOVIN primary accession number P42899
20. Rich, B.E. & Steitz, J.A. (1987) Mol. Cell Biol. 7, 4065
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.