CN106153946A - A kind of mensuration reagent of anti-cyclic citrullinated peptide and preparation method thereof - Google Patents

Abstract

The present invention relates to mensuration reagent of a kind of anti-cyclic citrullinated peptide and preparation method thereof, belong to biochemical apparatus technical field of measurement and test.Including R1 reagent, R2 reagent and anti-cyclic citrulline poly saccharide peptide standard product, described R1 reagent main component is Phosphate/NaCL buffer；Described R2 reagent main component is cyclic citrulline antigen and latex particle/PBS；The main component of described anti-cyclic citrulline poly saccharide peptide standard product is anti-cyclic citrullinated peptide, phosphate buffer, bovine serum albumin and sodium azide.The application is applied to the mensuration of anti-cyclic citrullinated peptide, has that rapid sensitive, accuracy be good, good stability, an advantage such as easy and simple to handle.

Description

A kind of mensuration reagent of anti-cyclic citrullinated peptide and preparation method thereof

Technical field

The present invention relates to mensuration reagent of a kind of anti-cyclic citrullinated peptide and preparation method thereof, belong to biochemical apparatus measuring technology Field.

Background technology

Rheumatoid arthritis (RA) is chronic, the systemic disease based on inflammatory synovitis that a kind of cause of disease is the brightest.Its Feature is hands, the foot multi-joint of Minor articulus, symmetry, aggressive arthritis, is often accompanied by organ outside joint and is involved serum Rheumatoid factor positive, can cause joint deformity and afunction.

The morbidity of RA may be relevant with heredity, infection, gonadal hormone etc..The arthritic pathology of RA mainly has synovial membrane stave cell Hypertrophy, interstitial massive inflammatory cells infiltrated, and the formation of microvascular new life, pannus and cartilage and the destruction of osseous tissue Deng.

This disease has influence on the population in whole world 1-2% at present, and the sickness rate of women is 2～3 times of male, can betide and appoint What, the age occurred frequently is 40～60 years old at age.

The Symptoms of RA is as follows:

(1) morning is stiff: joint motion inflexible subjective sensation when get up in the morning, it is the non-specific table of one of arthritis Existing, its persistent period is directly proportional to the order of severity of inflammation.

(2) performance of joint involvement: 1. multi-joint is got involved: symmetrically property polyarthritis (often >=5 joints).Easily get involved There are hands, foot, wrist, ankle and temporomandibular joint etc. in joint.2. joint deformity: the deformity of hands has fusiformis swelling, ulnar side deflection, sky gooseneck sample Deformity, button style deformity etc..What the deformity of foot had that the downward subluxation of head of metatarsal bone causes face upward microdactyly, valgus deformity, sole of the foot toe close Save subluxation, bend in hammer toe and talipes valgus.3. other: can have canalis carpi that median nerve/posterior tibial nerve pressurized causes/ Tarsal tunnel syndrome, Articular effusion in knee joint clamp-on on rear side of joint formed popliteal cyst (Baker cyst), cervical vertebra get involved (the 2nd, 3 cervical vertebras Common) can there be cervical pain, neck weakness and be difficult to keep its normal position, atlanto-axial subluxation, should there is compression of spinal cord mutually And the performance of vertebro-basilar artery insufficiency.

(3) extra-articular manifestation: the most typically performance: heating, rheumatoid nodules, rheumatoid vasculitis can be had (mainly to involve little The necrotizing arteriolitis of tremulous pulse) and lymphadenectasis.2. Cardiac Involvemant: can have pericarditis, pericardial effusion, visceral pericardium, cardiac muscle And the tuberosity of valve, myocarditis, coronaritis, aortitis, conductive impairment, chronic endocarditis and cardiac valve fibrosis etc. Performance.3. respiratory system is got involved: can have pleuritis, hydrothorax, pulmonary arteritis, interstitial lung disease, nodositas pneumonopathy etc..④ Kidney shows: mainly has primary glomerular and tubulointerstitial nephritis, kidney amyloidosis and is secondary to Drug therapy (gold Preparation, penicillamine and NSAIDs) renal damage.5. nervous system: in addition to the symptom of peripheral nerve pressurized, also can induce neural disease Disease, myelopathy, peripheral neurophaty, it is secondary to vasculitic ischemia neuropathy, muscular hypertrophy and drug-induced nervous system disease Become.6. anemia: be the modal extra-articular manifestation of RA, belong to study on anemia of chronic disease, is often mild to moderate.7. digestive system: Can be because of caused by RA vasculitis, complication or Drug therapy.8. eye: juvenile patient can have uveitis, adult can have scleritis, can Can be by caused by vasculitis.Also can there be the perforation of drying property conjunctivokeratitis, scleromalacia, scleromalacia, corneal solution.

(4) Felty syndrome: the RA patient of 1% can have splenomegaly, Neutrophilic granulocytopenia (and thrombocytopenia, erythrocyte Counting reduces), often there is RF and the ANA positive of serious arthropathy, high titre, belong to a kind of Severe RA.

(5) RA of Late-onset: often > 65 years old onset, sex difference is little, and how in acute onset, development is very fast, and (part is with OA For initially performance, after several years, occur that typical RA shows).It is prominent with brothers' edema, canalis carpi and tarsal tunnel syndrome and polymyalgia Performance, morning is stiff substantially.The complication such as patient's Chang Yin cardiovascular, infection and impaired renal function and dead.

The diagnosis of rheumatoid arthritis (RA) currently mainly depends on clinical symptoms and judges, only Serologic detection is Judge whether serum contains the existence of rheumatoid factor.Cyclic citrullinated peptid (CCP) is the polypeptide of ring-type Filaggrin Fragment, is based on the antibody of IgG type, rheumatoid arthritis (RA) is had good Sensitivity and Specificity, and anti-CCP Patient's RA osteoclasia of antibody positive is serious compared with antiCCP antibody negative patient.Early diagnosis RA and give alleviate the state of an illness medicine control Treat, extremely important to symptom management.1998, abroad, the cyclic citrullinated peptid (anti-cyclic of reported first Cirullinated peptide antibodies, antiCCP antibody) detection, RA is had higher sensitivity and special Property, it is blood serum designated object new for RA.Can be used for the early diagnosis of RA, examination of curative effect and prognosis evaluation.

The main method of the anti-cyclic citrullinated peptide of current domestic mensuration (CCP) is ELISA method, although the method compares into Ripe, but shortcoming is also evident from: and dosing accuracy is poor, and the operating time is long, and automaticity is low, is normally only used for qualitative inspection Survey.

Based on this, make the application.

Summary of the invention

In order to overcome existing anti-cyclic citrullinated peptide (CCP) drawbacks described above present in the test process, first the present invention carries , good stability good for a kind of rapid sensitive, accuracy, easy and simple to handle, it is adaptable to clinical full-automatic or semiautomatic biochemistry is analyzed Anti-cyclic citrullinated peptide (CCP) measures reagent.

For achieving the above object, the technical scheme that the present invention takes is as follows:

A kind of anti-cyclic citrullinated peptide (CCP) measures reagent, including R1 reagent, R2 reagent and anti-cyclic citrulline poly saccharide peptide standard product, Described R1 reagent main component is Phosphate/NaCL buffer；Described R2 reagent main component is cyclic citrulline antigen With latex particle/PBS；The main component of described anti-cyclic citrulline poly saccharide peptide standard product be anti-cyclic citrullinated peptide, phosphate buffer, Bovine serum albumin (BSA) and sodium azide.

The preparation method of anti-cyclic citrullinated peptide (CCP) as characterized above mensuration reagent:

(1) R1 preparation of reagents: Phosphate/NaCL buffer is joined in container；Opening stirring, mixing speed is 300-500 rev/min, stir.

(2) R2 preparation of reagents: add in purified water with latex particle/PBS by cyclic citrulline antigen, opens stirring, stirring Speed is 300-500 rev/min；Add sodium azide, continue stirring ten minutes, until sodium azide is completely dissolved；Add purified water fixed Hold.

(3) anti-cyclic citrulline poly saccharide peptide standard product: add phosphate buffer in a reservoir, opens stirring, and mixing speed is 300-500 rev/min；Adding anti-cyclic citrullinated peptide, stirring ten minutes, until being completely dissolved；Adding bovine serum albumin, stirring is very Clock, until being completely dissolved；Add sodium azide, stir ten minutes, until being completely dissolved, constant volume.

The operation principle of the present invention is as follows:

Use anti-cyclic citrullinated peptide (CCP) antibody in latex enhancing immune turbidimetry detection by quantitative human serum.By ring melon ammonia Acid peptide combines BSA and is adsorbed on latex particle, ties with antigen by hatching specific antibody in the human serum or blood plasma making dilution Closing, after hatching, the luminance delta of inhaling of generation is directly proportional to the specific antibody concentrations in detection sample.

Detailed description of the invention

A kind of anti-cyclic citrullinated peptide (CCP) of the present embodiment measures reagent, including R1 reagent, R2 reagent and anti-cyclic citrullinated peptide Standard substance, described R1 reagent main component is Phosphate/NaCL buffer；Described R2 reagent main component is ring melon Propylhomoserin antigen and latex particle/PBS；The main component of described anti-cyclic citrulline poly saccharide peptide standard product is anti-cyclic citrullinated peptide, phosphoric acid Salt buffer, bovine serum albumin (BSA) and sodium azide.

The preparation method of anti-cyclic citrullinated peptide (CCP) mensuration reagent:

(1) R1 preparation of reagents:

1. 3L Phosphate/NaCL buffer is joined in appropriate containers；

2. opening motor stirrer, mixing speed is 450 revs/min, stirs ten minutes.

(2) R2 preparation of reagents:

1. 10mL cyclic citrulline antigen is joined in about 0.8L purified water with latex particle/PBS, open electric stirring Device, stirs ten minutes；

2. 2g sodium azide being added in above-mentioned solution, continuing stirring ten minutes, until being completely dissolved；

3. add purified water and be settled to 1L.

(3) anti-cyclic citrulline poly saccharide peptide standard product:

1. in appropriate containers, add the phosphate buffer of about 150ml；

2. opening motor stirrer, mixing speed is 450 revs/min；

3. in above-mentioned solution, adding the anti-cyclic citrullinated peptide of 0.02001g, stirring ten minutes, until being completely dissolved；

4. in above-mentioned solution, adding 0.0091g bovine serum albumin, stirring ten minutes, until being completely dissolved；

5. in above-mentioned solution, adding 0.0842g sodium azide, stirring ten minutes, until being completely dissolved；

6. 200ml it is settled to.

(4) reagent test

1. instrument configuration: instrument uses Hitachi 7100 automatic clinical chemistry analyzer.

Location parameter sets in instrument in strict accordance with product description, and basic location parameter is as follows:

Method: end-point method；The Direction of Reaction: be incremented by；Wavelength: 546nm (leading), 800nm (secondary)；Temperature: 37 DEG C.

Cuvette optical path: 1cm；R1 reagent: 150 μ l, R2 reagent: 50 μ l, sample: 5 μ l.

First light-metering point: add latter 5 minutes at R1 reagent and read；Second light-metering point: add latter 5 minutes at R2 reagent and read Take.

2. reagent visual examination: visual inspection.

3. loading quantity inspection: use general gage measuring.

4. reagent blank measures:

With distilled water or deionized water as blank sample test kit, under test dominant wavelength, record test starting Time absorbance (A1 reagent) and the absorbance (A2 reagent) after about 5 minutes, A2 is the blank absorbency of working reagent.

5. the range of linearity measures:

Take the high level sample normal saline doubling dilution close to the range of linearity upper limit and be configured to the sample of variable concentrations gradient This series.Desired value is calculated with H-number and dilution ratio.Dilution process (is adjusted according to the big I of high level accordingly with reference to shown in table 1 Whole dilution gradient).

Table 1 Sample Dilution table

Embodiment	1	2	3	4	5
						Normal saline (ml)	0	0.5	0.5	0.5	0.5
High level specimen H (ml)	1	0.5	0.5	0.5	0.5
						Dilution ratio	Former times	1/2	1/4	1/8	1/16

In table: the concentration of embodiment 1 sample is known definite value CH, embodiment 2-5 concentration of specimens presses formula: concentration of specimens= CH × dilution ratio, calculates as desired value, the series of samples diluted fully is mixed, mensuration system after calibration is pressed Measuring 2 times from low value to high level sequential parallel, as corresponding embodiment sample measured value, (such as 2 times, measurement result has substantially average Deviation should be rejected and resurvey).

Statistical analysis: with desired value as abscissa, sample measured value is vertical coordinate mapping and linear regression analysis, determines Linear interval calculates linear equation y=a+bx and correlation coefficient, is linear good when correlation coefficient r >=0.990.

Data Analysis Services uses Microsoft Excel software.

Correlation formula is as follows:

$b=\frac{{\mathrm{n\ΣX}}_i{Y}_i-{\mathrm{\ΣX}}_i\·{\mathrm{\ΣY}}_i}{{{\mathrm{n\ΣX}}_i}^2-{\left({\mathrm{\ΣX}}_i\right)}^2}\mathrm{...}\left(1\right)$

$\left|a\right|=\frac{\left|{\mathrm{\ΣY}}_i-{\mathrm{b\ΣX}}_i\right|}{n}\mathrm{...}\left(2\right)$

$r=\frac{{\mathrm{n\ΣX}}_i{Y}_i-{\mathrm{\ΣX}}_i\·{\mathrm{\ΣY}}_i}{\sqrt{\[n\·\mathrm{\Σ}Xi-\left(\mathrm{\Σ}Xi\right)\]\[n\mathrm{\Σ}Yi-\left(\mathrm{\Σ}Yi\right)\]}}\mathrm{...}\left(3\right)$

In formula, C: concentration；V: volume；The slope of b: the regression line；A: the absolute value of regression line intercept；R: correlation coefficient； X_i: each pipe desired value；Y_i: each pipe measured value；I:1,2,3. ..., n；N: measure sample number.

6. precision measures:

A. repeatability measures:

By clinical samples or quality controlled serum test kit, retest 10 times, the meansigma methods of computation and measurement valueWith Standard deviation (s).It is calculated as follows the coefficient of variation (CV).

With coefficient of variation CV (%)

$\stackrel{\‾}{X}={\mathrm{\ΣX}}_1/n\mathrm{...}\left(4\right)$

$CV=\sqrt{\frac{\mathrm{\Σ}(\stackrel{\‾}{X}-Xi)}{{\left(\stackrel{\‾}{X}\right)}^2(n-1)}}\mathrm{...}\left(5\right)$

In formula:The average of test serum sample；

X_iMeasure the measurement result of serum sample；

N measures number of times

The CV coefficient of variation

B. difference between batch measures

Taking three lot number censorship reagent, each lot number takes 3 bottles, measures 1 part of clinical sample or quality controlled serum respectively, can be selected for Roche quality-control product, calculates 9 parts of reagent respectively and measures averageMensuration average with each 3 parts of reagent of lot numberAnd the coefficient of variation measured with three lot number reagent of Microsoft Excel software statistics.

Correlation formula is as follows:

In formula:

——In maximum；

——In minima；

Grand mean.

7. accuracy measures:

Take calibration object (having card reference material, CRM) test kit is tested, duplicate detection 3 times, take test result average (M), relative deviation (B) is calculated by formula (7).

Relative deviation (B)=(M-T)/T × 100% ... ... ... ... ... ... ... ... (7)

In formula: T has card reference material (CRM) sign value；M sample determination result average；

8. sensitivity for analysis:

With the sample test test kit of concentration known or activity, the absorbance that record produces under test kit specifies parameter changes Become, be scaled the absorbance difference (Δ A) of n unit.

(5) reagent measurement result is as follows:

The range of linearity: in the range of linearity (5-100U/ml), correlation coefficient r >=0.990 of linear regression.Concentration≤ During 45U/ml, absolute deviation is less than ± 5U/ml；As concentration ＞ 45U/ml, relative deviation is in the range of ± 10%.

Accuracy: Comparability test: correlation coefficient r >=0.990.Relative deviation≤15%.

Precision of measurement: coefficient of variation CV≤10%, relative deviation R≤10%.

Blank absorbency: blank absorbency (A) 0.20 is (temperature 37 DEG C；Wavelength 546nm；Cuvette optical path 1.0cm).

Sensitivity for analysis: during test kit test 20U/ml measured object, absorbance difference (Δ A) >=0.006ABS.

Above content be the preferred implementation combining the invention provided technical scheme is made further in detail Describe in detail bright, it is impossible to assert that the invention is embodied as being confined to these explanations above-mentioned, for technology belonging to the invention For the those of ordinary skill in field, without departing from the concept of the premise of the invention, it is also possible to make some simple deductions Or replace, all should be considered as belonging to the protection domain of the invention.

Claims (3)

1. the mensuration reagent of an anti-cyclic citrullinated peptide, it is characterised in that: include R1 reagent, R2 reagent and anti-cyclic citrullinated peptide mark Quasi-product, described R1 reagent main component is Phosphate/NaCl buffer；Described R2 reagent main component is ring melon ammonia Acid antigen and latex particle/PBS；The main component of described anti-cyclic citrulline poly saccharide peptide standard product is anti-cyclic citrullinated peptide, phosphate Buffer, bovine serum albumin and sodium azide.

A kind of anti-cyclic citrullinated peptide the most as claimed in claim 1 measure reagent preparation method, it is characterised in that include as Lower step:

(1) R1 preparation of reagents: Phosphate/NaCl buffer is joined in container, stirs；

(2) R2 preparation of reagents: added in purified water with latex particle/PBS by cyclic citrulline antigen, stir, adds nitrine Sodium, continues stirring and is completely dissolved to sodium azide, add purified water constant volume；

(3) anti-cyclic citrulline poly saccharide peptide standard product: add phosphate buffer in a reservoir, stir；Add anti-cyclic citrullinated peptide, Stirring to anti-cyclic citrullinated peptide is completely dissolved；Adding bovine serum albumin, stirring to bovine serum albumin is completely dissolved；Add nitrine Sodium, stirring to sodium azide is completely dissolved, constant volume.

The preparation method measuring reagent of a kind of anti-cyclic citrullinated peptide the most as claimed in claim 2, it is characterised in that: described Mixing speed is 300-500 rev/min.