CN106018829A - Testing reagent for serum amyloid protein A and preparation method of testing reagent - Google Patents

Abstract

The invention relates to a testing reagent for serum amyloid protein A and a preparation method of the testing reagent and belongs to the technical field of testing of biochemical means. The testing reagent comprises a reagent R1, a reagent R2 and a serum amyloid protein A standard substance, wherein main ingredients of the reagent R1 are trihydroxymethyl aminomethane and NaN3; main ingredients of the reagent R2 are latex granules enveloping antibodies resisting to human serum amyloid protein A as well as NaN3; main ingredients of the serum amyloid protein A standard substance are serum amyloid protein A, bovine serum albumin, a phophate buffer solution and NaN3. The testing reagent is used for testing serum amyloid protein A and has the advantages of fastness, sensitivity, good accuracy, good stability, simplicity and convenience in operation and the like.

Description

A kind of mensuration reagent of serum amyloid A protein and preparation method thereof

Technical field

Mensuration reagent that the present invention relates to a kind of serum amyloid A protein and preparation method thereof, belongs to biochemical apparatus test Technical field.

Background technology

Serum amyloid A protein (serum amyloid A, SAA) is the polymorphic protein family of a class polygenes coding, The precursor substance of tissue amyloid A, belongs to Acute reaction protein.Inflammation or acute stage of infection its in 48-72h i.e. Raise rapidly, and decline rapidly in the convalescent period of disease.At present, antibacterial, virus infection, atherosclerosis, coronary heart disease, urgency The property disease such as graft-rejection, tumor all detects that Serum SA A raises.Some disease, as virus infect, transplant rejection anti- Should, coronary heart disease etc., the sensitivity of SAA is higher than CRP, can be that clinic provides more preferable reference value.Refer to as a new detection Mark, SAA is just more and more paid close attention to by people.

Urieli etc. find, SAA virus and antibacterial infect in all raise, and virus infect in CRP raise hardly or Raise inconspicuous.Therefore, in the normal patients with viral infections of CRP, Non-Invasive or early stage invasive bacterial infection patient, SAA is a index the most useful.At infant infectious diseases in early days, owing to body constitution and the symptom of pediatric patient are indefinite, It is difficult to discriminate between antibacterial to infect or virus infection.So differentiating that pediatric patient antibacterial infects and virus infects the most rapidly, Prevention for treatment and various complication timely and effectively has great significance.

Amyloidosis is to cause amyloid filaments to be deposited on tissue and device due to the false folding of protein Threatening a class disease of human life in official, the amyloid filaments of deposition is produced by corresponding precursor protein partial hydrolysis fracture Raw, different types of amyloid filaments has the most different precursor proteins.At present, it has been found that tens of kinds of precursor proteins and shallow lake Powder sample degenerative disease is relevant.SAA is the precursor substance of AA, and it plays key in the pathogenic course of secondary amyloidosis Effect.In almost all of AA type (reactive) amyloidosis patients serum, SAA can be that the diagnosis of this disease, treatment and prognosis are commented The preferable reference information of offer is provided.

Along with inflammation effect played in the evolution of coronary heart disease is elucidated with further, traditional inflammatory protein, as CRP, also receives more and more attention with the relation of coronary heart disease.CRP, the highest quick CRP concentration raise with healthy population with The dangerous one-tenth positive correlation of rear generation cardiovascular event.SAA is the apolipoprotein being incorporated on HDL.In serum raise SAA with What HDL combined increases the metabolism that can affect body cholesterol, and increases the Ca in coronary blood tube wall smooth muscle cell²⁺Dense Degree, thus play a crucial role in the formation and development of coronary atherosclerosis.After discharging other risk factors, SAA can make It it is a medium independent risk factor of cardiovascular event.

When there is graft-rejection after renal transplant recipients application immunosuppressant, A-SAA concentration raises, and CRP concentration Change less, the graft-rejection of transplant patient and infection or inflammation can be made a distinction in conjunction with other indexs.Before one Look forward or upwards Journal of Sex Research to find, one of index raised the earliest when Serum SA A is rejection.In view of sensitivity, specificity and first The time of rejection, Serum SA A can be used for the monitoring of kidney transplantation exclusion reaction as first-selected index.It addition, SAA can be as head Select index for the monitoring of kidney transplantation exclusion reaction.It addition, SAA is alternatively arranged as a sensitive mark for liver transplantation in early days Rejection and the monitoring of acute graft versus host reaction.

In the evolution of tumor, the especially invasion and attack of tumor and transfer, due to its distinctive 26S Proteasome Structure and Function, SAA The effect of key can be played.As suppressed the combination of tumor cell and stromatin；The expression of inducer substance metalloproteases and The sticking, shift and infiltrate of cell；The invasion and attack of tumor cell, transfer and revascularization can also be suppressed.Research shows, hepatocarcinoma, The kinds of tumors SAA in the patient such as pulmonary carcinoma, breast carcinoma, carcinoma of prostate, carcinoma of endometrium all have and raise in various degree, and its level Obvious dependency, the curative effect of reaction tumor patient and prognosis is had with the active stage of tumor, grade malignancy and Invasion and Metastasis.

Finding so far from 1974, many detection methods have been applied to the detection of patients serum SAA.It is presently used for clinic The method of Serum SA A detection mainly includes radioimmunoassay (RIA), elisa (ELISA), immunity speed Rate scattered light urbidmetry, microsphere trapped enzyme immunization (MEIA) etc..The method of current domestic detection serum amyloid A protein (SAA) Mainly there is elisa (ELISA).Colloidal gold method is easy and simple to handle, quick, result judges directly perceived, but detection is sensitive Spend relatively low；The detection sensitivity of ELISA method is of a relatively high, but complex operation step, time-consuming, and automaticity is low, inapplicable In a large amount of detections.

Based on this, make the application.

Summary of the invention

In order to overcome existing serum amyloid A protein (SAA) drawbacks described above present in the test process, the present invention is first There is provided that a kind of rapid sensitive, accuracy be good, good stability, easy and simple to handle, it is adaptable to clinical full-automatic or semiautomatic biochemistry analysis Serum amyloid A protein (SAA) measure reagent.

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

The mensuration reagent of a kind of serum amyloid A protein (SAA), including R1 reagent, R2 reagent and serum amyloid A protein Standard substance, described R1 reagent main component is Tris (trishydroxymethylaminomethane), NaN₃；Described R2 reagent main component It is to be coated with the latex particle of AHS's amyloid A antibody, NaN₃；Described serum amyloid A protein standard substance Main component is serum amyloid A protein, bovine serum albumin (BSA), phosphate buffer, NaN₃。

Serum amyloid A protein as characterized above (SAA) measures the preparation method of reagent, comprises the steps:

(1) preparation of R1 reagent: join in container by Tris (trishydroxymethylaminomethane) buffer, opens stirring, Mixing speed is 300-500 rev/min；Add NaN₃, continue to stir to NaN₃It is completely dissolved；With Tris buffer constant volume.

(2) preparation of R2 reagent: purified water is joined in container, open stirring, mixing speed be 300-500 turn/ Point；Adding the latex particle being coated with AHS's amyloid A antibody, stirring to this latex particle is completely dissolved；Add NaN₃, stir to NaN₃It is completely dissolved；Use purified water constant volume.

(3) preparation of serum amyloid A protein standard substance: joined by phosphate buffer in container, opens stirring, stirs Mix speed and be 300-500 rev/min；Adding serum amyloid A protein, stirring to serum amyloid A protein is completely dissolved；Add Bovine serum albumin, stirring to bovine serum albumin is completely dissolved；Adding sodium azide, stirring to sodium azide is completely dissolved；Use phosphorus Phthalate buffer constant volume.

The operation principle of the present invention is as follows:

It is coated with the latex particle of amyloid A (SAA) antibody, when with sample mix containing SAA antigen, can send out Raw agglutination, thus cause the change of absorbance, its size is in direct ratio with SAA antigenic content in sample.Absorbance is become Change and compare with the calibration object of concentration known, can quantitatively draw the content of amyloid A in sample.

Detailed description of the invention

The mensuration reagent of the present embodiment one serum amyloid A protein (SAA), forms sediment including R1 reagent, R2 reagent and serum Powder sample protein A standard, described R1 reagent main component is Tris (trishydroxymethylaminomethane), NaN₃；Described R2 examination Agent main component is to be coated with the latex particle of AHS's amyloid A antibody, NaN₃；Described serum amyloid protein The main component of A standard substance is serum amyloid A protein, bovine serum albumin (BSA), phosphate buffer, NaN₃。

Serum amyloid A protein as characterized above (SAA) measures the preparation method of reagent, comprises the steps:

(1) preparation of R1 reagent:

1. 2.0L Tris (trishydroxymethylaminomethane) buffer is joined in appropriate containers；

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

3. by 2.375g NaN₃Adding in above-mentioned solution, stirring ten minutes, until being completely dissolved；

4. it is settled to 2.5L with Tris buffer.

(2) preparation of R2 reagent:

1. purified water is joined in appropriate containers；

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

The latex particle that 3. 3.0g is coated with AHS's amyloid A antibody adds in above-mentioned solution, stirs ten Minute, until being completely dissolved；

4. by 0.475g NaN₃Adding in above-mentioned solution, stirring ten minutes, until being completely dissolved；

5. it is settled to 500mL by purified water.

(3) preparation of serum amyloid A protein standard substance:

1. 80mL phosphate buffer is joined in appropriate containers；

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

3. 16mg serum amyloid A protein being added in above-mentioned solution, stirring ten minutes, until being completely dissolved；

4. 4.5mg bovine serum albumin being added in above-mentioned solution, stirring ten minutes, until being completely dissolved；

5. 42mg sodium azide being added in above-mentioned solution, stirring ten minutes, until being completely dissolved；

6. it is settled to 100mL with phosphate buffer.

The operation principle of the present invention is as follows:

It is coated with the latex particle of amyloid A (SAA) antibody, when with sample mix containing SAA antigen, can send out Raw agglutination, thus cause the change of absorbance, its size is in direct ratio with SAA antigenic content in sample.Absorbance is become Change and compare with the calibration object of concentration known, can quantitatively draw the content of amyloid A in sample.

(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: upwards；Wavelength: 546nm；Temperature: 37 DEG C.

Cuvette optical path: 1cm；R1 reagent: 250 μ l, R2 reagent: 50 μ l, sample: 8 μ l.

First light-metering point: read immediately after R2 reagent adds；Second light-metering point: add latter 5 minutes at R2 reagent and read.

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:

$b=\frac{n\ΣX_i{Y}_i-\ΣX_i\·\ΣY_i}{n\Σ{X_i}^2-{(\ΣX_i)}^2}\mathrm{...}\left(1\right)$

$\left|a\right|=\frac{|\ΣY_i-b\ΣX_i|}{n}\mathrm{...}\left(2\right)$

$r=\frac{n\ΣX_i{Y}_i-\ΣX_i\·\ΣY_i}{\sqrt{\[n\·\ΣXi-(\ΣXi)\]\[n\ΣYi-(\ΣYi)\]}}\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 value

With standard deviation (s).It is calculated as follows the coefficient of variation (CV).

With coefficient of variation CV (%)

$\stackrel{\‾}{X}=\ΣX_1/n\mathrm{...}\left(4\right)$

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

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 (5～100mg/L), correlation coefficient r >=0.990 of linear regression.

As concentration≤10mg/L, absolute deviation is less than ± 1mg/L；As concentration ＞ 10mg/L, relative deviation ± In the range of 10%.

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

Precision of measurement: coefficient of variation CV≤8%, relative deviation R≤10%.

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

Sensitivity for analysis: during test kit test 10mg/L unit measured object, absorbance difference (Δ A) >=0.0075ABS.

Calibration object accuracy: Comparability test: correlation coefficient r >=0.990, relative deviation≤10%.

Between calibration object bottle poor: CV≤10% between bottle.

Claims (3)

1. the mensuration reagent of a serum amyloid A protein, it is characterised in that: include R1 reagent, R2 reagent and serum amyloid sample Protein A standard, described R1 reagent main component is trishydroxymethylaminomethane, NaN₃；Described R2 reagent main component It is to be coated with the latex particle of AHS's amyloid A antibody, NaN₃；Described serum amyloid A protein standard substance Main component is serum amyloid A protein, bovine serum albumin, phosphate buffer, NaN₃。

The preparation method measuring reagent of a kind of serum amyloid A protein the most as claimed in claim 1, it is characterised in that include Following steps:

(1) preparation of R1 reagent: by Tris(trishydroxymethylaminomethane) buffer joins in container, stirs；Add NaN₃, continue to stir to NaN₃It is completely dissolved；With Tris buffer constant volume；

(2) preparation of R2 reagent: purified water is joined in container, stir；Add and be coated with AHS's amyloid egg The latex particle of white A antibody, stirring to this latex particle is completely dissolved；Add NaN₃, stir to NaN₃It is completely dissolved；Use purification Water constant volume；

(3) preparation of serum amyloid A protein standard substance: phosphate buffer is joined in container, stir；Add blood Clear amyloid A, stirring to serum amyloid A protein is completely dissolved；Adding bovine serum albumin, stirring is to bovine serum albumin It is completely dissolved in vain；Adding sodium azide, stirring to sodium azide is completely dissolved；Use phosphate buffer constant volume.

The preparation method measuring reagent of a kind of serum amyloid A protein the most as claimed in claim 2, it is characterised in that: institute The mixing speed stated is 300-500 rev/min.