Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1989-03-29
1995-10-10
Saunders, David
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 693, 435 697, 435975, 436508, 436518, 436543, G01N 33543, G01N 33564
Patent
active
054570298
DESCRIPTION:
BRIEF SUMMARY
This invention relates to nuclear antigens, in particular the human autoantigen La, and to the use of a synthetic polypeptide displaying the antigenicity of the human autoantigen La, antigenic fragments thereof or fused polypeptides containing the antigenic polypeptide or fragments in diagnostic tests for the detection of the antinuclear antibody, anti La, in serum.
Autoantibodies reactive with nuclear antigens characterise many human autoimmune diseases (1). The antinuclear antibody (ANA) anti La is very strongly associated with Sjogrens Syndrome, one of the multisystem rheumatic diseases, and serves as a diagnostic marker of that disease (2). The nuclear antigen La, with which anti La reacts, has been extensively studied to determine its nature and possible relationship to the etiology of Sjogrens Syndrome, and, as reported by several groups, La is a protein of MW 48-50kD which associates with a series of small nuclear RNAs (3).
It will be appreciated that polypeptides displaying antigenicity characteristics of the human autoantigen La are of particular utility in highly specific and sensitive diagnostic immunoassays, such as an ELISA for the detection of the antinuclear antibody (ANA), anti-La.
Sensitive immunoassays have previously only been available for the detection of antibodies to autoantigens that are abundant in cells and which can be readily purified biochemically (5,6,7). Biochemical purification of extractable nuclear antigens (ENA) results in extremely small yields of pure autoantigen (8). While the source of tissue for the biochemical purification of La has been bovine or rabbit thymus (9,10,11,12,13), differences have been demonstrated between La from human, bovine and murine tissue (14,15) suggesting that La of human origin would be the preferred source of autoantigen for diagnostic assays.
In accordance with the present invention, there is provided a diagnostic test for the detection of the antinuclear antibody anti-La in a serum sample, which comprises the steps of: synthetic polypeptide displaying the antigenicity of all or a portion of the human autoantigen La or an antigenically active fragment thereof, the amino acid sequence of said polypeptide or fragment comprising or including the sequence GluAspGlnGlnGluSerLeuAsnLysTrpLysSerLysGly ArgArgPheLysGlyLysGlyLysGlyAsnLysAlaAlaGln ProGlySerGlyLysGlyLysValGlnPheGlnGlyLysLys ThrLysPheAlaSerAspAspGluHisAspGluHisAspGlu AsnGlyAlaThrGlyProValLysArgAlaArgGluGluThr AspLysGluGluProAlaSerLysGlnGlnLysThrGluAsn GlyAlaGlyAspGln
(b) detecting the presence of anti-La antibody in said serum bound to said synthetic polypeptide or fragment.
This invention also provides a diagnostic test kit for detection of antinuclear antibody anti-La in a serum sample, which comprises:
(a) a support having immobilised thereon a synthetic polypeptide displaying the antigenicity of all or a portion of the human autoantigen La or an antigenically active fragment thereof, the amino acid sequence of said polypeptide or fragment comprising or including the sequence GluAspGlnGlnGluSerLeuAsnLysTrpLysSerLysGly ArgArgPheLysGlyLysGlyLysGlyAsnLysAlaAlaGln ProGlySerGlyLysGlyLysValGlnPheGlnGlyLysLys ThrLysPheAlaSerAspAspGluHisAspGluHisAspGlu AsnGlyAlaThrGlyProValLysArgAlaArgGluGluThr AspLysGluGluProAlaSerLysGlnGlnLysThrGluAsn GlyAlaGlyAspGln
(b) means for detecting the presence of anti-La antibody in said serum bound to said synthetic polypeptide or fragment.
A partial cDNA sequence encoding the putative carboxyl-terminal 12% of the human La protein has been published (4), with the prediction that this 55 amino acid region contains an antigenic determinant. Following further investigations in this regard, including the use of probes derived from the published sequence to isolate homologous human cDNA clones which have been used to express portions of the La protein and to map regions of antibody reactivity, significant anomalies have now been shown to exist in the previously published sequence.
In work leading to the present invention, a recombinant DNA molecule has been constructe
REFERENCES:
patent: 4751181 (1988-06-01), Keene
Chambers et al, Proc. Natl. Acad. Sci. USA, 82, 2115-2119, 1985.
Chambers et al, Jour. Biol. Chem. 263, 18043-18051, 1988.
Maggio, Enzyme-Immunossay, CRC Press Inc., Boca Raton, Fla., 1980, pp. 39 and 181-192.
St Clair et al, Arth. Rheum., 30, (4), 534, 1987.
St Clair et al, Arth. Rheum., 31, 506-514, 1988.
Vi Mehra et al, Proc. Natl. Acad. Sci. USA, 83, 7013-7017, 1986.
Coppel Ross L.
Sturgess Allan D.
Amrad Corporation Limited
Saunders David
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