Novel test method for rheumatoid arthritis and kit for rheumatoid arthritis test
A rheumatoid, inspection method technology, applied in biochemical equipment and methods, microbial determination/inspection, biological testing, etc.
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Embodiment 1
[0045] The blood of each subject was collected into an EDTA tube, and centrifuged at 2500 rpm for 10 minutes at room temperature to obtain plasma.
[0046] The amount of talin in the above plasma was measured by sandwich ELISA method.
[0047] First, the H-18 antibody (Santa Cruz Biotechnology Co., Ltd.) that recognizes the N-terminus of talin was diluted to 1 μg / mL with phosphate buffered solution (PBS), and then added to a 96-well microwell plate in an amount of 100 μL / well. Incubate overnight at 4°C, and then wash 3 times with 200 μL / well of washing solution. Next, 100 μL / well of plasma from each subject was added to a 96-well microplate, incubated at 25° C. for 1 hour, and then washed 3 times with 200 μL / well of washing solution. Next, the H-300 antibody (Santa Cruz Biotechnology Co., Ltd.) that recognizes the N-terminus of talin as the primary antibody was diluted with PBS to make it 2 μg / mL, and then added to a 96-well microplate in an amount of 100 μL / well Incubate at...
Embodiment 2
[0054] The amount of talin in plasma was measured by the sandwich ELISA method in the same manner as in Example 1, except that the M54246M antibody (Biodesign) recognizing the C-terminus of talin was used as the primary antibody.
[0055] The ROC curve of embodiment 2 is as figure 2 shown. The results of the ROC analysis, figure 2 The area under the ROC curve (AUC) was 0.819. In addition, the positive and negative results when OD=0.05 was used as the cutoff value are shown in Table 2 below.
[0056] [Table 2]
[0057]
[0058] From these results, the sensitivity of rheumatoid arthritis diagnosis by the sandwich ELISA method using the H-18 antibody and the M54246M antibody was 15 / 17×100=88.2%, and the specificity was 11 / 14×100=78.6%. .
Embodiment 3
[0067] The therapeutic effects of rheumatoid arthritis therapeutic agents on 5 RA patients were confirmed by measuring the amount of talin. As in Example 1, the amount of talin was measured by measuring the OD value by the sandwich ELISA method using the H-18 antibody and the H-300 antibody. In addition, the CRP level and the MMP-3 level were also measured by the same method as the usual clinical examination. Furthermore, the DAS (Disease Activity Score) 28 score recommended by the European Union for the Prevention and Treatment of Rheumatism (EULAR) was also calculated. In terms of DAS28 scores, 5.1 or more is judged as high disease activity, 3.2 or more and less than 5.1 is judged as moderate disease activity, and less than 3.2 is judged as low disease activity. The results are shown in Table 4 below.
[0068] [Table 4]
[0069]
[0070] Case 1 is an example of a good responder (DAS28: 5.43→2.62) when EULAR response criteria were applied by using ADA (adalimumab) in co...
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