Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – 8 to 10 amino acid residues in defined sequence
Reexamination Certificate
1999-01-22
2001-05-01
Nolan, Patrick J. (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
8 to 10 amino acid residues in defined sequence
C530S352000, C530S868000, C436S506000, C436S509000, C514S012200, C514S015800
Reexamination Certificate
active
06225442
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method of immunologically-detecting rheumatism and a reagent of immunologically detecting therefor. In more detail, it relates to a method of immunologically detecting antibodies which react with autoantigenic proteins present in the serum of a rheumatic patient and a reagent therefor.
2. Description of the Prior Art
Rheumatism is a chronic systemic inflammatory autoimmune disease that causes swelling and pain in the multi-joints and malaise, infirmity, weight loss, febricula and anorexia in other body organs. Criteria for the classification of rheumatism established by American Rheumatoid Association have been currently used in clinical diagnoses (Arnet, F. C. et al. Arthritis Rheum. vol. 305, Abstract 45, 1987); (1) morning stiffness, (2) arthritis of 3 or more joint areas, (3) arthritis of hand joints, (4) symmetric arthritis, (5) radiographic changes, (6) serum rheumatoid factor, (7) rheumatoid nodules. For the classification purposes, a patient shall be diagnosed to have rheumatism if he/she has satisfied at lease 4 of these 7 criteria. Moreover, by examining blood, there are chronic inflammatory symptoms such as acceleration of erythrocyte sedimentation rate and increase of C-reative protein reaction in the sera. There are also special symptoms such as anemia, increase of white blood cells, increase of thrombocytes, increase of &ggr;-globulin in sera. However, all the diagnosis are applied to patients after the occurrence of some symptoms. Therefore, it has long been desired that a new method should be established enabling to diagnose patients before the occurrence of the clinical symptoms or with early staged symptoms. The present invention attempts to find an early diagnosis of rheumatism before the obvious occurrence of the symptoms. This invention relates to a method of detecting a serum based on rheumatic specific antigens at the early stage and it is considered highly useful for its convenience and reliability.
DETAILED DESCRIPTION OF THE INVENTION
From the point of view that rheumatism is an autoimmune disease and that focusing at autoantibodies present in the serum of a rheumatic patient, the present invention attempts to detect autoantibodies which reacts with specific autoantigens. The inventors of the present invention found that ezrin, radixin and moesin were antigen substances which reacted with the autoantibodies present in the serum of a rheumatic patient, which led to the completion of the invention.
Therefore, the present invention provides for a method of detecting autoantibodies present in the serum of a rheumatic patient by reacting one or more proteins selected from the group consisting of ezrin, radixin and moesin of mammalian origin and/or one or more peptides comprising at least 9 or more consecutive amino acid residues in an amino acid sequence derived from ezrin, radixin and moesin with human sera. Although ezrin, radixin, moesin which are derived from mammalian origin are applicable, those derived from human are specially more preferable.
Autoantigen substances containing one or more than two various kinds can be used for the detection.
The present invention provides for a method of detecting autoantibodies present in the serum of a rheumatic patient by reacting radixin with human sera, a method of detecting autoantibodies present in the serum of a rheumatic patient by reacting radixin and moesin with human sera, and a method of detecting autoantibodies present in the serum of a rheumatic patient by reacting ezrin, radixin and moesin with human sera.
Further, the present invention relates to a reagent for immunologically detecting rheumatism containing as an antigen at least one mammalian protein selected from the group of ezrin, radixin and moesin and/or a peptide composed of at least nine consecutive amino acid residues
Further, the present invention provides for a reagent for immunologically detecting rheumatism containing radixin as an antigen, a reagent for immunologically detecting rheumatism containing radixin and moesin as an antigen, and a reagent for immunologically detecting rheumatism containing ezrin, radixin and moesin as an antigen.
Further, the present invention provides for a reagent for immunologically detecting rheumatism containing a peptide composed of at least nine consecutive amino acid residues found in the amino acid sequences of radixin as an antigen, a reagent for immunologically detecting rheumatism containing a peptide composed of at least nine consecutive amino acid residues found in the amino acid sequences of moesin as an antigen, and a reagent for immunologically detecting rheumatism containing a peptide composed of at least nine consecutive amino acid residues found in the amino acid sequences of ezrin as an antigen.
The present invention has a great advantage in that it enables to detect rheumatism by a convenient method of isolating the serum and measuring the amount of antibodies. On the other hand, the conventional diagnosis of rheumatism mainly depends on the occurrence of the clinical symptoms.
The autoantibodies against various nuclear antigens are detected in the sera of patients with autoimmune diseases, and they are known to be closely related to the clinical symptoms. Recently, it has been disclosed that corresponding antigens (autoantigens) against many autoantibodies are giant molecules in the cells and that they are proteins which are conserved beyond species. The present invention at first attempted to detect tissues which reacted with the serum of a rheumatic patient by using a frozen mouse whole body section for the purpose of isolating and identifying unknown autoantigens which reacted with autoantibodies in the serum of a rheumatic patient. As a result, there found an reactive portion in cytoplasm in hypertrophic chondrocytes of mouse cartilage. Then, in order to identify the reactive portion in a molecular level, a method of isolating proteins were established. By applying the method, it was found that not only mouse cartilage but also mouse brain, mouse spleen and mouse liver had a relevant protein. Moreover, it became clear that the cell lines such as MG63 cell which was derived from human osteosarcoma, HepG2 cell which was derived from human liver cancer or 3T6 cell which was derived from mouse embryo had a relevant protein. It was demonstrated that these proteins were conserved beyond the species. It was demonstrated that these proteins were molecules with 80 kD and 77 kD by Coomassie brilliant blue staining. By Western blot analysis with the serum of a rheumatic patient, a band with 80 kD reacted with the serum of a rheumatic patient by about 33% out of the total sera of rheumatic patients. It was found that a band with 77 kD reacted with the serum of a rheumatic patient by 30% out of those sera which reacted with 80 kD. It was further found that the band with 80 kD was separated into two bands with 81 kD and 80 kD when the concentration of the gel of the band was reduced to 7%. It was further identified that by analysing amino acid sequences of the proteins, the 81 kD protein was radixin which was derived from a mammalian protein, the 80 kD protein was ezrin and the 77 kD protein was moesin. These proteins belong to a same protein family (Ezrin Radixin Moesin: ERM family) with linking to cytoskeleton.
Ezrin is a protein which was identified in an intestinal brush border microvilli by Bretsher et al in 1983 (J. Cell Biol., vol. 97, p.425-432, 1983), and its full nucleotide sequences were determined by the cloning of the cDNA in 1989 (Gould, K. L. et al., EMBO J., vol., p.4133-4142, 1989; Turunen, O. et al., J. Biol. Chem., vol. 264, p.16727-16732, 1989).
Radixin is a protein which adheres to a barbed end of an actin and localized in the undercoat of a cell-cell adhesions junction or in a border in the period of cytokinesis, but its function remains unknown (Tsukita, S. et al., J. Cell Biol, vol. 108, p.2369-2382, 1989). It was demonstrated by the cloning of the cDNA that it consisted of 4,241 basic pairs
Kimrua Michio
Takeuchi Fujio
Wagatsuma Masako
Watanabe Hiroshi
Dade Behring Marburg GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Nolan Patrick J.
LandOfFree
Method of detecting autoantibody present in the serum of... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method of detecting autoantibody present in the serum of..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of detecting autoantibody present in the serum of... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2564825