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Kit for in vitro detection of anti-cyclic citrullinated peptide (CCP) antibody and preparation method thereof

An anti-cyclic citrullinated peptide, in vitro detection technology, applied in the field of bioengineering, can solve the problems of long concentration time, complicated procedures, etc., and achieve the effect of significant technological progress, accurate and convenient detection results

Inactive Publication Date: 2012-01-18
上海精臻生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The object of the present invention is to provide an anti-cyclic citrullinated peptide antibody in vitro detection kit and its preparation method. The anti-cyclic citrullinated peptide antibody in vitro detection kit and its preparation method adopt colloidal gold diafiltration The concentration of anti-cyclic citrullinated peptide antibody in human serum is detected by the principle of method and chromatography, which solves the technical problems of long time and complicated procedures in the prior art for detecting the concentration of anti-cyclic citrullinated peptide antibody in serum

Method used

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  • Kit for in vitro detection of anti-cyclic citrullinated peptide (CCP) antibody and preparation method thereof
  • Kit for in vitro detection of anti-cyclic citrullinated peptide (CCP) antibody and preparation method thereof
  • Kit for in vitro detection of anti-cyclic citrullinated peptide (CCP) antibody and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1 colloidal gold preparation condition selection

[0018] 1.1 Selection of the dosage of trisodium citrate

[0019] Group A: add 2.0 ml of 5% trisodium citrate to 100 ml of 0.05% gold chloride solution.

[0020] Group B: add 1.5 ml of 5% trisodium citrate to 100 ml of 0.05% gold chloride solution.

[0021] Group C: add 1.0 ml of 5% trisodium citrate to 100 ml of 0.05% gold chloride solution.

[0022] Group D: add 0.5 ml of 5% trisodium citrate to 100 ml of 0.05% gold chloride solution.

[0023] Experimental results:

[0024] Wavelength λ: 540 535 530 525 520 510nm.

[0025] ABS value: Group A 0.471 0.608 0.791 1.044 1.426 1.063

[0026] Group B 0.601 0.746 1.093 1.411 0.942 0.711

[0027] Group C 0.623 0.803 1.318 0.952 0.714 0.635

[0028] Group D 0.998 1.431 1.091 0.801 0.678 0.600

[0029] Visually inspect the color of the colloidal gold solution: Group A: orange-red. Group B: red. Group C: red, Group D: purple.

[0030] Acc...

Embodiment 2

[0031] Embodiment 2 colloidal gold conjugate preparation condition selection

[0032] 2.1 pH selection of colloidal gold-labeled SPA

[0033] Select the pH value of colloidal gold-labeled SPA, use 1.0% anhydrous sodium carbonate solution to adjust the pH value of colloidal gold-labeled SPA, and compare the appropriate pH range of colloidal gold-labeled SPA by detection sensitivity.

[0034] Experimental results:

[0035]

[0036] The experimental results show that the pH value range of colloidal gold-labeled SPA is negative when the critical value sample is at pH6.0~6.4, and the effect of colloidal gold-labeled SPA is similar when pH≥6.4, and there is no significant difference. Therefore, it is suggested that colloidal gold-labeled SPA The pH range is controlled at pH 6.4 to 7.2, that is, for every 100 milliliters of colloidal gold, add 1.2 milliliters of 1.0% anhydrous sodium carbonate solution.

[0037] 2.2.2 Concentration selection of colloidal gold-labeled SPA

[003...

Embodiment 3

[0041] Embodiment 3 Detection plate preparation condition selection

[0042] 3.3.1 Comparison of the pH conditions of the coating medium at the detection point

[0043] Experimental Materials:

[0044] (1) Nitrocellulose membrane with a pore size of 0.45 μm.

[0045] (2) Coating buffer: 1.0mol / L NaHCO 3

[0046] NaHCO 3 (Analytical pure) 8.401 g, put into a container, measure process water with a graduated cylinder and pour into the container, wait to dissolve, then take process water to make up the volume to 100 ml, and mix well.

[0047] (3) Checkpoint antigen: cyclic citrullinated peptide (CCP) antigen (1.0mg / mL)

[0048] experiment procedure:

[0049] (1) Detection point antigen processing:

[0050] Cyclic citrullinated peptide (CCP) antigen (1.0 mg / mL) stock solution, pH=7.0-7.5.

[0051] Add 1.0mol / L NaHCO to 0.05ml of cyclic citrullinated peptide (CCP) antigen (1.0mg / mL) stock solution 3 0.005ml, mix well, pH≥8.0.

[0052] (2) Coating: Take 2.0uL of the detect...

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Abstract

The invention belongs to the field of biological engineering, and provides a kit for in vitro detection of an anti-cyclic citrullinated peptide (CCP) antibody, which comprises a detection plate, a CCP antigen, a colloidal gold-labeled recombinant gold staphylococcus aureus A protein conjugate, a confining liquid, a washing liquid, a positive reference product and a negative reference product. The invention also provides a preparation method of the kit for the in vitro detection of the anti-CCP antibody. The kit adopts an indirect method immunosorbent assay principle to detect the anti-CCP antibody in a human serum, the CCP antigen is coated on a nitrocellulose membrane to be made into a solid-phase antigen for capturing the anti-CCP antibody in the human serum, and then colloidal gold is labeled on staphylococcus aureus A protein (SPA) to form the colloidal gold conjugate as a tracer; and if the detected human serum contains the anti-CCP antibody, then a solid phase antigen-anti-CCP antibody-colloid gold conjugate is formed and has red spots. The invention can be applied to the auxiliary diagnosis of rheumatoid arthritis.

Description

technical field [0001] In the field of bioengineering, the present invention particularly relates to a kit and a preparation method thereof, in particular to a qualitative in vitro detection kit and a preparation method of an anti-cyclic citrullinated peptide antibody. Background technique [0002] Rheumatoid arthritis (Rheumatoid Arthritis, RA) is a chronic inflammatory systemic autoimmune disease with multi-joint involvement. The diagnosis of RA is currently based on the classification criteria revised by the American Rheumatism Association (ARA) in 1987. The laboratory index is rheumatoid factor (RF). In 2000, it was first reported abroad that Cyclic Citrulinated Peptide (CCP) was synthesized based on the cDNA sequence of Filaggrin, which was used as an antigen to establish an enzyme-linked immunosorbent assay (ELISA), and successfully Anti-CCP antibody was detected in RA serum, and it was found that the antibody had high sensitivity and specificity in the diagnosis of R...

Claims

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Application Information

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IPC IPC(8): G01N33/544G01N33/532
Inventor 朱晓敏王泉龙朱慧琳刘颖成杨杰刘颖冰张瑞镐
Owner 上海精臻生物科技有限公司
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