阅读说明：本技术 一种稳定、灵敏的抗环瓜氨酸肽抗体检测试剂盒 (A kind of stabilization, sensitive cyclic citrullinated peptid detection kit ) 是由 李静 史建国 包兴艳 李志明 胡晓飞 甘宜梧 董雯 于 2019-08-30 设计创作，主要内容包括：本发明公开了一种稳定、灵敏的抗环瓜氨酸肽抗体检测试剂盒,涉及生物技术领域。所述检测试剂盒包括试剂R1和试剂R2,其中：所述试剂R1由复合缓冲液、无机盐离子、PEG8000、表面活性剂和防腐剂组成；所述试剂R2由复合缓冲液、环瓜氨酸抗原包被胶乳颗粒、表面活性剂和防腐剂组成,环瓜氨酸抗原包被胶乳颗粒采用72nm、126nm、280nm三种不同粒径的羧基胶乳微球联用。本发明能够进一步提高灵敏度和稳定性,并且线性范围宽。(The invention discloses a kind of stabilizations, sensitive cyclic citrullinated peptid detection kit, are related to field of biotechnology.The detection kit includes reagent R1 and reagent R2, in which: the reagent R1 is made of composite buffering liquid, inorganic ion, PEG8000, surfactant and preservative；The reagent R2 is made of composite buffering liquid, cyclic citrulline antigen coat latex particle, surfactant and preservative, and cyclic citrulline antigen coat latex particle is combined using the carboxylated latex microballoon of tri- kinds of different-grain diameters of 72nm, 126nm, 280nm.The present invention can further increase sensitivity and stability, and the range of linearity is wide.)

1. a kind of stabilization, sensitive cyclic citrullinated peptid detection kit, which is characterized in that including reagent R1 and reagent R2, in which:

The ingredient and content of the reagent R1 is as follows:

The ingredient and content of the reagent R2 is as follows:

The cyclic citrulline antigen coat latex particle the preparation method is as follows:

Step 1: be by volume 3 by 72nm Carboxylated latex particles, 126nm Carboxylated latex particles, 280nm Carboxylated latex particles: The ratio of 2:1 is mixed；

Step 2: AMPD buffer being added into the solution that step 1 obtains, oscillation mixes, and then 20000rpm is centrifuged 65min, abandons Supernatant；AMPD buffer is added again later, carries out ultrasonic resuspension；

Step 3: EDC solution is added into the solution that step 2 obtains, the oscillating reactions 45 minutes at a temperature of 20 DEG C；

Step 4: cyclic citrulline antigen being added into the solution that step 3 obtains, mixes, the oscillating reactions 5 hours at a temperature of 30 DEG C；

Step 5: sealer is added into the solution that step 4 obtains, is closed 24 hours at a temperature of 25 DEG C.

Step 6: 20000rpm centrifugation 60min being carried out to the solution that step 5 obtains, supernatant is abandoned, buffer is then added, is surpassed Low voice speaking 5 minutes outstanding, 20000rpm is centrifuged 60min again, removes supernatant, and gained precipitating is cyclic citrulline antigen coat latex Grain.

2. stabilization according to claim 1, sensitive cyclic citrullinated peptid detection kit, which is characterized in that institute Stating buffer is 25 DEG C, and pH is the CAPSO-ADA composite buffering liquid of 7.8-8.2.

3. stabilization according to claim 1, sensitive cyclic citrullinated peptid detection kit, which is characterized in that institute Ingredient and the content for stating surfactant are as follows:

Lauryl carboxymethyl sodium form imidazole quinoline acetate 15g/L

Erucyl amide propyl betaine 10g/L

Polyethylene glycol stearate diester 20g/L.

4. stabilization according to claim 1, sensitive cyclic citrullinated peptid detection kit, which is characterized in that institute In the preparation method for stating cyclic citrulline antigen coat latex particle, the concentration of the AMPD buffer in the step 2 is 3mM, PH Value is 5~6；Buffer in the step 6 is TABS buffer.

5. stabilization according to claim 1, sensitive cyclic citrullinated peptid detection kit, which is characterized in that institute Stating preservative is dimethylol urea.

6. special according to claim 1 to any stabilization, sensitive cyclic citrullinated peptid detection kit in 5 Sign is that the inorganic ion is one of potassium chloride, sodium chloride, magnesium chloride or a variety of.

7. stabilization according to claim 6, sensitive cyclic citrullinated peptid detection kit, which is characterized in that institute The volume ratio for stating reagent R1 and reagent R2 is 3:1.

Technical field

The present invention relates to clinical vitro detection technical fields, and in particular to a kind of stabilization, sensitive anti-cyclic citrullinated peptide are anti- Body detection kit.

Background technique

It is aobvious that cyclic citrullinated peptid (Anti-CCP) is that the polypeptide piece of cyclic annular Filaggrin has the diagnosis of myocardial damage The specific fragment of work is the antibody based on IgG type, to rheumatoid arthritis (RA) with good sensibility and specifically Property, and patient's RA osteoclasia of the antiCCP antibody positive is serious compared with antiCCP antibody negative patient.There is scholar to think antiCCP antibody to RA Diagnostic sensitivity is 50%~78%, and specificity is 96%, and early stage patient positive rate is up to 80%.In addition, antiCCP antibody is not only It is RA early diagnosis diagnosis index, or identifies invasion and the sensitive indexes of Non-Invasive RA, antibody positive patient is than anti- The more serious joint destruction of bone of the patient Yi Fazhan of body feminine gender.Therefore, the detection of Anti-cyclic critrullinated polypeptide antibody is to RA's Diagnosis is of great significance.

Currently, measuring the common method of anti-cyclic citrullinated peptide (CCP) antibody has radioimmunology analytic approach, ELISA method, exempts from Epidemic disease turbidimetry etc..Wherein, time-consuming for the detection of radioimmunology analytic approach, and has the pollution of radioactive element, use by Limitation；ELISA method is cumbersome, and sensitivity is low, and testing result is mostly qualitative；Immunoturbidimetry is easy to operate, easy to use, but Existing immunoturbidimetry detection kit is there are sensitivity and stability are poor, the narrow disadvantage of the range of linearity.

Summary of the invention

The present invention, which provides one kind, can further increase sensitivity and stability, and the wide stabilization of the range of linearity, sensitive Cyclic citrullinated peptid detection kit.

In order to solve the above technical problems, present invention offer technical solution is as follows:

The present invention provides a kind of stabilization, sensitive cyclic citrullinated peptid detection kit, including reagent R1 and reagent R2, in which:

The ingredient and content of the reagent R1 is as follows:

The ingredient and content of the reagent R2 is as follows:

The cyclic citrulline antigen coat latex particle the preparation method is as follows:

Step 1: by volume by 72nm Carboxylated latex particles, 126nm Carboxylated latex particles, 280nm Carboxylated latex particles It is mixed for the ratio of 3:2:1；

Step 2: AMPD buffer being added into the solution that step 1 obtains, oscillation mixes, and then 20000rpm is centrifuged 65min abandons supernatant；AMPD buffer is added again later, carries out ultrasonic resuspension；

Step 3: EDC solution is added into the solution that step 2 obtains, the oscillating reactions 45 minutes at a temperature of 20 DEG C；

Step 4: cyclic citrulline antigen being added into the solution that step 3 obtains, mixes, the oscillating reactions 5 at a temperature of 30 DEG C Hour；

Step 5: sealer is added into the solution that step 4 obtains, is closed 24 hours at a temperature of 25 DEG C.

Step 6: 20000rpm centrifugation 60min is carried out to the solution that step 5 obtains, supernatant is abandoned, buffer is then added, into Row ultrasound is resuspended 5 minutes, and 20000rpm is centrifuged 60min again, removes supernatant, and gained precipitating is cyclic citrulline antigen coat glue Newborn particle.

Further, the buffer is 25 DEG C, and pH is the CAPSO-ADA composite buffering liquid of 7.8-8.2.

Further, the ingredient of the surfactant and content are as follows:

Lauryl carboxymethyl sodium form imidazole quinoline acetate 15g/L

Erucyl amide propyl betaine 10g/L

Polyethylene glycol stearate diester 20g/L.

Further, in the preparation method of the cyclic citrulline antigen coat latex particle, the AMPD in the step 2 is slow The concentration of fliud flushing is 3mM, and pH value is 5~6；Buffer in the step 6 is TABS buffer.

Further, the preservative is dimethylol urea.

Further, the inorganic ion is one of potassium chloride, sodium chloride, magnesium chloride or a variety of.

Further, the volume ratio of the reagent R1 and reagent R2 is 3:1.

Compared with prior art, the invention has the following advantages:

1) present invention uses latex enhancing immune turbidimetry, uses composite buffering liquid by reagent R1 and reagent R2, and excellent The proportion for changing each component, significantly improves the stability of reagent.

2) present invention uses lauryl carboxymethyl sodium form imidazole quinoline acetate, erucyl amide propyl betaine, polyethylene glycol The compound surfactant solution of three kinds of novel surfactants of double stearates, can promote and maintain antibody stabilization, prevent System is muddy, significantly enhances the stability and anti-interference ability of reagent.

3) preparation method of the invention by optimization cyclic citrulline antigen coat latex particle, and use 72nm, The carboxylated latex microballoon of tri- kinds of different-grain diameters of 126nm, 280nm is combined, greatly strengthen reagent reaction sensitivity and linear model It encloses, and the repeatability and anti-interference ability of reagent are stronger.

In conclusion detection kit of the invention is easy to operate quickly, it is suitable for automated analysis, is a kind of more steady The cyclic citrullinated peptid detection kit fixed, sensitive, the range of linearity is wide, the accuracy of kit and has good stability, and resists Interference is strong, easy to use, can satisfy clinical needs completely.

Detailed description of the invention

Fig. 1 is the stability contrast curve graph of the kit of kit and comparative example of the invention.

Specific embodiment

To keep the technical problem to be solved in the present invention, technical solution and advantage clearer, below in conjunction with specific implementation Example and attached drawing are described in detail.