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Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method

The technology of anti-cyclic citrullinated peptide and test strip, which is applied in the field of medical immunization, can solve the problems of cumbersome detection steps, long detection time and the like, and achieves the effects of simple operation, low detection cost, and convenient storage and transportation.

Active Publication Date: 2014-06-11
SHANGHAI KEXIN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] 1. The detection steps are cumbersome and the detection time is long;
[0009] 2. Detection reagents need to be refrigerated for transportation and storage;
[0010] 3. Detection requires special equipment
[0011] 4. It is not suitable for single-person operation. For the ELISA method, it is necessary to make standard products and negative and positive reference materials at the same time. Only by testing multiple people at the same time can the detection cost be saved.

Method used

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  • Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method
  • Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method
  • Test strip for detecting anti-cyclic citrullinated peptide antibody in blood and preparation method

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preparation example Construction

[0093] 8) Preparation of coating membrane: Dilute antigen (preferably MAP-carrier protein conjugate) with coating buffer to a concentration of 0.2-2.5 mg / mL, dilute antibody (c) to 0.3-3.0 mg / mL, use Spray the antigen and quality control antibody (c) on the nitrocellulose membrane with a spray volume of 1-10 μl / cm by the BIO-Dot film dispenser, and then place it in an oven at 37°C, and dry it for later use;

[0094]9) Preparation of gold-labeled antibody: adjust the pH value of the colloidal gold solution to 6.0-9.0 with 0.1M potassium carbonate, slowly add 4-25 μg of the antibody to be labeled into each milliliter of the colloidal gold solution, stir for 10-30 minutes, and then add 10 %BSA solution to a final concentration of 0.5 to 5%, continue to stir for 10 to 30 minutes, discard the supernatant after centrifugation, wash the precipitate with washing liquid for 2 to 3 times, and use 1 / 10 of the original volume of colloidal gold solution to preserve the solution. Resuspend ...

Embodiment 1

[0103] Example 1: Cyclic citrullinated peptide structure

[0104] Different cyclic citrullinated peptides (CCPs) have subtle differences and present different antigenic epitopes. The amino acid sequence of one of the cyclic citrullinated peptides is:

[0105]

[0106] Among them, "S --- S" means a disulfide bond, X means citrulline, and other letters are the single-letter symbols of common amino acids, that is, H means histidine, Q means glutamine, C means cysteine, E represents glutamic acid, S represents serine, T represents threonine, G represents glycine, and R represents arginine.

[0107] The branched antigenic peptide (MAP) structure of cyclic citrullinated peptides is based on poly-lysine (poly-Lys, PL) as the core matrix, generally adopts 2 to 8 branches, and these branches can be different For the epitope of cyclic citrullinated peptide, new branched epitopes are added accordingly according to the latest research progress to improve the detection performance. Ta...

Embodiment 2

[0111] Example 2: Antigen Conjugation

[0112] 1. The carrier is bovine serum albumin (BSA)

[0113] Bovine serum albumin was coupled to MAP by the carbodiimide method. Carbodiimide is a very active bifunctional reagent that can condense with both the carboxyl group and the amino group on the hapten. . This method is to mix the amino group of the hapten polypeptide with the carrier protein bovine serum albumin in a certain molecular ratio (1:1 ratio is used in this study) in an appropriate solution, then add carbodiimide, stir for 1 to 2 hours, and place at room temperature React for 24 hours, and finally dialyze to remove the unreacted hapten and carrier protein to obtain the MAP conjugate. Then use ultraviolet spectroscopy to measure its concentration for quantification. The reaction pattern is as follows:

[0114]

[0115] 2. The carrier is avidin

[0116] Avidin was first diluted with ultrapure water to 5-10 mg / ml, then diluted with 0.01M PBS to 1-3 mg / ml, and then...

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Abstract

A test strip for detecting anti-cyclic citrullinated peptide antibody based on colloidal gold immunochromatography and a method of preparing the same are provided. The test strip includes: a cellulose nitrate film (3), a conjugate pad (2), a sample pad (1) and a wicking pad (6), which are stuck on a substrate (7) and overlapping one another in sequence. A detection zone (4) and a control zone (5) are provided on the cellulose nitrate film. Cyclic citrullinated peptide antigen is coated on the detection zone, and antibody as a control is coated on the control zone. Two different antibodies labeled by colloidal gold are coated on the conjugate pad. Branched cyclic citrullinated peptide is used in the test strip. Pretreatment for sample pad and conjugate pad is improved in the preparing method. The test strip for detecting anti-cyclic citrullinated peptide antibody can be applied for diagnosing rheumatoid arthritis (RA).

Description

technical field [0001] The invention belongs to the field of medical immunization applications, and in particular relates to a test strip for detecting anti-cyclic citrullinated peptide antibodies by colloidal gold immunochromatography technology and a preparation method thereof. Background technique [0002] Rheumatoid arthritis, also known as rheumatoid arthritis (RA), is a systemic inflammatory disease characterized by chronic multi-joint damage caused by abnormal growth of joint tissue or pannus, resulting in irregular joint loss of function. Recent studies have shown that joint damage in RA patients occurs two years after the onset of the disease, and early and effective treatment can prevent the subsequent progression of the disease. Therefore, early diagnosis and early treatment have become the focus. However, RA patients do not always show typical symptoms and phenomena in the early stage of the disease, because they may not fully meet the diagnostic criteria for RA...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/551G01N33/558G01N33/577
CPCG01N33/558G01N33/564G01N2800/102G01N2440/18G01N33/54388
Inventor 张玥韩永俊高成秀孙宏彬杨超文司徒维娜左宗宝钱杰
Owner SHANGHAI KEXIN BIOTECH
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