CN105467130A - Kit for quantitative detection of anti-Sm antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof - Google Patents

Abstract

The invention relates to the technical field of immunology detection, and particularly relates to a kit for quantitative detection of an anti-Sm antibody IgG by using magnetic particle chemiluminescence, and a preparation method and a detection method thereof. The kit for quantitative detection of the anti-Sm antibody IgG by using magnetic particle chemiluminescence includes an Anti-Sm IgG calibrator, an Anti-Sm IgG reagent No. 1, an Anti-Sm IgG reagent No. 2, an Anti-Sm IgG magnetic separation reagent, an Anti-Sm IgG control material and a cleaning solution. The invention also discloses a preparation method and a detection method of the above kit. Based on the traditional film strip immunoassay and enzyme-linked immunosorbent assay, the detection method increases the sensitivity and linearity range by 3-5 orders of magnitude, achieves the quantitative detection, and has the advantages of high sensitivity and specificity, good accuracy, low cost, simple operation and the objective judgment of results. The method cooperates with an automated chemiluminescence immunoassay analyzer to reach fully automation, and has broad application prospects.

Description

A kind of magnetic microparticle chemiluminescence that uses quantitatively detects kit of anti-Sm antibody IgG and preparation method thereof and detection method

Technical field

The present invention relates to technical field of immunological detection, be specifically related to a kind of magnetic microparticle chemiluminescence that uses and quantitatively detect kit of anti-Sm antibody IgG and preparation method thereof and detection method.

Background technology

Sm is the abbreviation of a patient's name, is diagnosed as in the patients serum of lupus erythematosus at this, have found this antibody first, therefore names.Later a large amount of reports confirms in lupus erythematosus patients serum, there is this kind of antibody, belongs to a class antinuclear antibodies.Main protein antigen in Sm system is B, B ＇, D, E protein, molecular weight 11 ~ 29kD, also has the protein such as A, A ＇, B ＂, C, F, G and 68kD, 150kD in addition.These protein form compound with small nuclear rna s (snRNAs) the i.e. UsnRNAs very abundant containing guanosine.In mammal, find to have 13 kinds of UsnRNAs, i.e. U1 ~ U13snRNAs at least at present.Sm antigen forms micronuclear ribonucleoprotein (U1, U2, U5, U4/U6snRNP) by U1, U2, U5, U4/U6 and above-mentioned protein, and it plays an important role in the conversion of ripe mRNA (mRNA) at heterogeneous nRNA (hnRNA).

Anti-Sm antibody IgG is the specific antibody of systemic loupus erythematosus (SLE), can be used for the diagnosis of SLE together with anti-dsDNA, anti-nucleosome, anti-ribosomal P protein antibody IgG.Anti-Sm antibody can detect in the SLE patient of 5% ~ 40%.Systemic loupus erythematosus (SLE) is the chronic multisystem disease that a kind of incidence of disease is higher, with multiple autoantibodies, is typical autoimmune disease.But up to the present its definite cause of disease and pathogenesis still unclear, this to SLE diagnosis, treatment and prevention progress cause very large obstacle.In patients serum, autoantibody detects the Major Clinical index that oneself becomes SLE laboratory diagnosis.In recent years, in patients serum, the multiple autoantibodies such as anti-ENA antibody, anti-ds-DNA antibody, ANA and anticardiolipin antibody are detected.ENA can be divided into the known and non-principal component totally tens kinds such as Sm, RNP, SSA, SSB, Rib and histone, wherein Sm, RNP and Rib three and SLE in close relations.The anti-Sm antibody positive is considered to the markup antibody of SLE, very at present large to SLE diagnostic significance, uses magnetic microparticle chemiluminescence analytic approach to have not yet to see in the application of anti-Sm antibody IgG immunoassay product.

Chinese patent CN103105489A discloses HP immunoblotting kit detecting autoimmune disease antibody and preparation method thereof, relate to a kind of HP immunoblotting kit detecting various autoimmune disease relevant antibodies, solve and there is no the deficiency in the Western blotting product technology of various autoimmune disease health check-up examination at present: contain on described nitrocellulose filter or nylon membrane: respectively by dsDNA, Sm/RNP, CCP, SSA, SSB, GAD, ICA, IA-2A, TG, natural or the recombinant antigen bag of in totally 10 kinds at least two kinds of AMA-M2 by more than two parallel detection lines, article 1, high concentration quality control band, article 1, middle concentration quality control band and 1 low concentration quality control band.Be a kind of film bar immunization disclosed in this patent, more can illustrate that the sensitivity that film bar immunization exists is lower, the range of linearity is narrow, poor repeatability and reaction time long problem.

Summary of the invention

In order to overcome defect of the prior art, the invention provides a kind of kit using magnetic microparticle chemiluminescence quantitatively to detect anti-Sm antibody IgG, it can prepare with lower cost, and can realize anti-Sm antibody IgG accurately and the quantitative measurement of high precision ground.

The present invention is achieved by the following technical solution: a kind of kit using magnetic microparticle chemiluminescence quantitatively to detect anti-Sm antibody IgG, and described kit comprises: Anti-SmIgG calibration object, No. 1, Anti-SmIgG reagent, No. 2, Anti-SmIgG reagent, Anti-SmIgG Magneto separate reagent, Anti-SmIgG quality-control product and cleaning fluid.

Described Anti-SmIgG calibration object comprises 6 liquid calibration bottles, described liquid calibration bottle internal object concentration is respectively 0,5,20,50,100, the anti-Anti-Sm IgG antibody of 200RU/mL and the Tris damping fluid of bovine serum albumin(BSA);

No. 1, described Anti-SmIgG reagent is 1 bottle containing the biotin labeled Sm antigen of 5mL and the Tris damping fluid of bovine serum albumin(BSA);

No. 2, described Anti-SmIgG reagent is 1 and contains the sheep anti-human polyclonal antibody of alkali phosphatase enzyme mark of 15mL and the bottle of the Tris damping fluid of bovine serum albumin(BSA);

Described Anti-SmIgG Magneto separate reagent is 1 magnetic particle of marked by streptavidin and the Tris damping fluid bottle of bovine serum albumin(BSA) that contain 5mL; Described Anti-SmIgG quality-control product comprises 2 Quality Control bottles, and the scope of the concentration in described Quality Control bottle is respectively: 16-24RU/mL and 80-120RU/mL.

Described kit Cleaning Principle is by testing sample, No. 1, described Anti-SmIgG reagent mixing incubation, anti-Sm antibody IgG in sample is combined with Sm antigentic specificity, add described Anti-SmIgG Magneto separate reagent again, the immune complex of anti-Sm antibody IgG and Sm antigen is adsorbed to magnetic particle surface, unconjugated antibody and impurity are removed in washing, add No. 2, Anti-SmIgG reagent and mix incubation.Goat anti-human igg antibody and the anti-Sm antibody IgG specific binding be combined on magnetic bead of alkali phosphatase enzyme mark (ALP).Washing adds chemical luminous substrate after removing unconjugated antibody and impurity, and ALP catalytic substrate is luminous, measures relative luminous intensity (RLU).In RLU and sample, anti-Sm antibody IgG concentration is proportional within the specific limits, just can be calculated the anti-Sm antibody IgG content of testing sample by RLU from typical curve.

The another technical scheme that the present invention takes is: the preparation method of the magnetic microparticle chemiluminescence immue quantitative detection reagent box of a kind of anti-Sm antibody IgG, and the preparation method of described kit comprises the following steps:

Step 1. is prepared described Anti-SmIgG calibration object and is comprised: Anti-SmIgG calibration object diluent preparing step and Anti-SmIgG calibration object preparation steps:

The preparation steps of described Anti-SmIgG calibration object dilution comprises:

Add the Tris of 800mL purified water and 11.2g in container, be uniformly mixed, add the sodium chloride of 8.5g and the Proclin300 of 2mL again, be stirred to and dissolve completely, pH value is adjusted to 7.0-7.5, add the bovine serum albumin(BSA) of 40g in container, be stirred to and dissolve completely, then pH value is adjusted to 7.0-7.5, be settled to 1L by purified water, use 0.2 μm of frit, by obtain described calibration object dilution be kept at 2-8 DEG C stand-by;

The preparation steps of described Anti-SmIgG calibration object comprises:

With above-mentioned Anti-SmIgG calibration object dilution anti-Sm antibody IgG is formulated as respectively 0,5,20,50,100,200RU/mL totally 6 concentration point;

Step 2. is prepared No. 1, described Anti-SmIgG reagent and is comprised: Anti-SmIgG reagent No. 1 diluent preparing step and Anti-SmIgG reagent No. 1 preparation steps:

The preparation steps of described Anti-SmIgG reagent No. 1 dilution:

Add 800mL purified water, 12.1g the NaCl of Tris and 5.8g in 1L container, be stirred to and dissolve completely, add the Proclin300 of 2mL again, be stirred to and dissolve completely, then add the bovine serum albumin(BSA) of 5g, be stirred to and dissolve completely, pH value is adjusted to 7.0-7.5, be settled to 1L by purified water, use 0.2 μm of frit, by obtain described reagent No. 1 dilution be kept at 2-8 DEG C stand-by;

The preparation steps of No. 1, described Anti-SmIgG reagent:

By the biotin solution of the pH9.0 carbonate buffer solution preparation 0.5mg/mL of 0.2mol/L, the ratio being 10:1 according to Sm antigen and biotin solution mass ratio joins the biotin solution of above-mentioned 0.5mg/mL in Sm antigenic solution, mix, room temperature leaves standstill 18h, reaction generates the reactant liquor of Sm antigen-biotin conjugate, the Sm obtained antigen-biotin conjugate reactant liquor is separated by G-25 gel column, remove unreacted biotin, obtain Sm antigen-biotin conjugate, with described reagent No. 1 dilution, Sm antigen-biotin conjugate is diluted to 0.1-0.3 μ g/mL again, obtain No. 1, described reagent,

Step 3. is prepared No. 2, described Anti-SmIgG reagent and is comprised: the preparation steps of Anti-SmIgG reagent No. 2 diluent preparing steps and No. 2, Anti-SmIgG reagent:

The preparation steps of described Anti-SmIgG reagent No. 2 dilutions:

The NaCl adding 800mL purified water, the 4-hydroxyethyl piperazine second sulphur of 6.06g and 8.5g, in container, is stirred to and dissolves completely, then add the bovine serum albumin(BSA) of 5g, the ZnCl of 0.1g ₂, 0.2mL the MgCl of Proclin300 and 0.1g ₂, be stirred to and dissolve completely, pH value is adjusted to 7.5-8.0, be settled to 1L by purified water, use 0.2um frit, described reagent No. 2 dilutions obtained are kept at 2-8 DEG C stand-by;

The preparation steps of No. 2, described Anti-SmIgG reagent:

Be 2-imines thiophane solution by the coupling agent of the 10mg/mL of the goat anti-human antibody of 1mg and 2-4 μ L, room temperature leaves standstill 20min, add the glycine solution 10 μ L of 0.1mol/L, room temperature leaves standstill 5min, with G-25 gel column desalination, collect the rear antibody of activation, by activation after antibody be kept at 2-8 DEG C for subsequent use, get the alkaline phosphatase of 1.5mg, add 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution 10-20 μ L of 5mg/mL, room temperature leaves standstill 30min, with G-25 gel column desalination, collect the rear alkaline phosphatase of activation, by activation after alkaline phosphatase be kept at 2-8 DEG C for subsequent use, again the goat anti-human antibody of above-mentioned activation is mixed with the alkaline phosphatase of activation, 12-24h is left standstill under 2-8 DEG C of condition, purify with Supperdex200 gel-purified post, obtain goat anti-human antibody-alkaline phosphatase connector strong solution, be placed in 2-8 DEG C to save backup, by goat anti-human antibody-alkaline phosphatase connector strong solution by described reagent No. 2 diluted to 0.02-0.1 μ g/mL, obtain No. 2, described reagent,

Step 4. is prepared described Anti-SmIgG Magneto separate reagent and is comprised: Anti-SmIgG Magneto separate reagent dilutions preparation steps and Anti-SmIgG Magneto separate preparation of reagents step:

Described Anti-SmIgG Magneto separate reagent dilutions preparation steps:

Add 800mL purified water, 12.1g the NaCl of Tris and 8.5g in container, be stirred to and dissolve completely, add the Proclin300 of the bovine serum albumin(BSA) of 5g, the NBCS of 50mL and 0.2mL again, be stirred to and dissolve completely, pH value is adjusted to 7.9-8.1, be settled to 1L by purified water, use 0.2um frit, the described Magneto separate reagent dilutions obtained is kept at 2-8 DEG C stand-by;

Described Anti-SmIgG Magneto separate preparation of reagents step:

Get 100mg magnetic particle, Magneto separate removes supernatant, the pH value of taking 0.025mol/L is that the MES damping fluid 10mL of 4.5-6 is resuspended, 2 ~ 3h is mixed under room temperature, adding the concentration that 0.5-1.0mL newly prepares is the carbodiimide of 10mg/mL and the MES damping fluid of N-hydroxy thiosuccinimide, shaken at room temperature suspendible 30-60min, magnetic bead is fully activated, Magneto separate, remove supernatant, be that the MES damping fluid 10mL of 4.5-6 is resuspended by the pH value of 0.025mol/L, add the Streptavidin of 4-8mg, room temperature suspendible 16-20h, carry out Magneto separate again, remove supernatant, with the bovine serum albumin(BSA) of percent concentration 0.5%, the MES damping fluid 10mL of percent concentration 0.5% skimmed milk power and percent concentration 0.05% Tween-20 is resuspended, 2 ~ 3h is mixed under room temperature condition, Magneto separate again, remove supernatant, 0.5mg/mL is resuspended to the dilution of magnetic particle damping fluid, 16 ~ 24h is balanced at 2 ~ 8 DEG C, after mixing 2 ~ 3h under room temperature condition, sonic oscillation instrument is used to be dispersed into individual particle state by assembling agglomerating magnetic bead in suspension, obtain described Anti-SmIgG Magneto separate reagent.

Further, the present invention also provides the preparation process of described cleaning:

Add 800mL purified water, 12.1g the NaCl of Tris and 8.5g in 1L container, be stirred to and dissolve completely, add the Tween-20 of 5g and the Triton100 of 5g again, stir, until mix completely, pH value is adjusted to 7.5-8.0, constant volume 1L, use 0.2 μm of frit, by obtain described cleaning fluid be kept at 2-8 DEG C stand-by, use before by described cleaning fluid according to every 7.5mL add purified water to 100mL dilution proportion and mixing after re-use.

Present invention also offers a kind of detection method using magnetic microparticle chemiluminescence quantitatively to detect the kit of anti-Sm antibody IgG, the detection method of described kit comprises the following steps:

Anti-SmIgG calibration object is put in Full-automatic chemiluminescence immunoassay analysis meter reagent cabin by step 1., obtains the matched curve exported by Full-automatic chemiluminescence immunoassay analysis meter;

Anti-SmIgG quality-control product is put into above-mentioned analyser test position by step 2., obtains the test luminous value of the described quality-control product exported by Full-automatic chemiluminescence immunoassay analysis meter and is obtained the concentration value of Anti-SmIgG quality-control product by the matched curve matching that step 1 obtains;

Testing sample is put into above-mentioned analyser test position by step 3., automatically presses 1:20 by Sample Dilution, obtain the concentration value of the testing sample exported by Full-automatic chemiluminescence immunoassay analysis meter by described analyser.

Further, the invention provides described a kind of detection method using magnetic microparticle chemiluminescence quantitatively to detect the kit of anti-Sm antibody IgG, described step 1, step 2 and step 3 include the fully-automated synthesis step of Full-automatic chemiluminescence immunoassay analysis meter:

Step 1) add the sample to be measured after 20 μ L calibration objects or quality-control product or 1:20 dilution in detector tube;

Step 2) add No. 1, the Anti-SmIgG reagent of 50 μ L to step 1) in described detector tube, after mixing, 37 ± 0.5 DEG C of incubation 10min;

Step 3) add the Anti-SmIgG Magneto separate reagent of 50 μ L to step 2) in described detector tube, after mixing, 37 ± 0.5 DEG C of incubation 5min, carry out Magneto separate, remove supernatant;

Step 4) add the cleaning fluid of 300 μ L in detector tube, mixing, carries out Magneto separate and removes supernatant;

Step 5) repeat step 4) twice;

Step 6) add No. 2,150 μ LAnti-SmIgG reagent in detector tube, after mixing, 37 ± 0.5 DEG C of incubation 10min, carry out Magneto separate and remove supernatant;

Step 7) add the cleaning fluid of 300 μ L in detector tube, mixing, carries out Magneto separate and removes supernatant;

Step 8) repeat step 7) twice;

Step 9) add the chemical luminous substrate of 200 μ L in detector tube, mixing, detect luminous intensity.

Compared with prior art, superior effect is:

No. 1, the Anti-SmIgG reagent 1, in described kit, No. 2, Anti-SmIgG reagent, Anti-SmIgG Magneto separate reagent are the more excellent formulas under this reaction system, provide powerful guarantee to the use of this kit effect phase and detection perform;

2, the Anti-SmIgG Magneto separate reagent participation reaction time in described kit is only 5 minutes, can avoid non-specific adsorption to limits;

3, the Anti-SmIgG Magneto separate reagent in described kit is the super paramagnetic microsphere of polymolecularity, ensures magnetic

Particulate can not be assembled completely in preservation and use procedure;

Alkaline phosphatase in No. 2, the Anti-SmIgG reagent 4, in described kit can catalytic luminescence substrate luminous, and namely reach luminescence-producing reaction plateau, and the light signal of held stationary in 5 seconds, ensure high measurement accuracy and efficiency.

No. 2, the Anti-SmIgG reagent 5, in described kit is the sheep anti-human polyclonal antibody of alkali phosphatase enzyme mark, and the use of sheep anti-human polyclonal antibody, reduces the cost of kit;

6, this kit successfully avoids the no cross reaction with human serum albumins;

7, the sample requirement to be measured in described kit is 20 μ L samples after 1:20 doubly dilutes, and reduces the cost of kit;

8, described detection method is on the basis of traditional film bar immunization and enzyme linked immunosorbent assay, by sensitivity and linear measurement range improving 3-5 the order of magnitude, realizing the quantitative detection of real meaning, has the features such as highly sensitive, high specificity, accuracy are good;

9, described detection method coordinates Full-automatic chemiluminescence immunoassay analysis meter to realize full-automatic use, and easy and simple to handle, result judges the advantages such as objective, has important value for clinical diagnosis, has a extensive future.

Accompanying drawing explanation

Fig. 1 is that in the blank limit evaluation test of the kit of anti-Sm antibody IgG of the present invention, A, B point connects some matched curve schematic diagram;

Fig. 2 is the curve map that the kit range of linearity of anti-Sm antibody IgG of the present invention is evaluated;

Fig. 3 is the magnetic microparticle chemiluminescence quantitative measurement Virus monitory result of kit of the present invention and the relativity evaluation of commercial anti-Sm antibody IgG detection kit enzyme linked immunosorbent assay testing result;

Horizontal ordinate x in Fig. 3 is the kit sample measured value of control methods, and concentration unit is RU/mL, and ordinate y is described kit sample measured value, and concentration unit is RU/mL.

Embodiment

Embodiment 1

Use magnetic microparticle chemiluminescence quantitatively to detect a kit of anti-Sm antibody IgG, this kit comprises: Anti-SmIgG calibration object, No. 1, Anti-SmIgG reagent, No. 2, Anti-SmIgG reagent, Anti-SmIgG Magneto separate reagent, Anti-SmIgG quality-control product and cleaning fluid.

The analytical performance evaluation of kit of the present invention:

1) accuracy estimating

Concentration is about 200RU/mL, allow its concentration deviation be ± 20% anti-Sm antibody IgG sample A join in blood serum sample B liquid, add A volume be no more than 10% of cumulative volume A+B, recovery R is calculated according to formula (1), the recovery of this method is within the scope of 85-115%, and data are see table 1.

$R=\frac{C\×(V_0+V)-C_0\×V_0}{V\×C_S}\×100\%\mathrm{......}\left(1\right)$

R: the recovery;

The volume of V: sample A liquid;

V ₀: the volume of sample B liquid;

C: sample B liquid adds the detectable concentration after A liquid;

C ₀: the concentration measuring B liquid;

The concentration of Cs: sample A liquid.

Table 1-accuracy estimating-interpolation recovery experiment data

2) blank limit is evaluated

Detect as sample with zero-dose calibration object, replication 20 times, draw the RLU of 20 measurement results, RLU is relative light unit, calculates its mean value M and standard deviation SD, draws the RLU value corresponding to M+2SD, carry out 2 regression fits according to the result of the concentration-RLU value between zero-dose calibration object and adjacent calibration object and draw linear function, RLU value corresponding to M+2SD is brought in above-mentioned equation, obtains corresponding concentration value, be blank limit.The blank limit of this method is not more than 1RU/mL.Concrete data are see table 2, and A, B point connects some matched curve as shown in Figure 1.

Table 2 blank limit is evaluated

3) range of linearity evaluation

Dilute at least 5 kinds of concentration by a certain percentage by the high level sample close to the range of linearity upper limit being 200RU/mL, wherein the sample of low value concentration must close to the lower limit of the range of linearity.Operate by kit instructions, to the equal duplicate detection of the sample of each concentration 2 times, calculate its mean value, result mean value and dilution ratio least square method are carried out fitting a straight line, and calculate linearly dependent coefficient r, the measurement range of this method is 2 ~ 200RU/mL, and correlation coefficient r answers >=0.9900.Data are see table 3.Curve is shown in Fig. 2.

The evaluation of table 3 range of linearity

4) reproducibility

The kit duplicate detection concentration of getting in the present embodiment is each 10 times of the sample of 20 ± 4RU/mL and 100 ± 20RU/mL, calculate mean value M and the standard deviation SD of 10 measurement results, draw coefficient of variation CV according to formula CV=SD/M × 100%, this method coefficient of variation (CV) is not more than 8%.Data are see table 4.

CV＝SD/M×100％......(2)

In formula: CV-coefficient of variation; The standard deviation of SD-10 time measurement result; The mean value of M-10 time measurement result.

Table 4 reproducibility

Measure serum-concentration (RU/mL)	Measure number of times	CV (%) between analysis
			20	10	6.66％
100	10	6.40％

5) difference between batch evaluation

The kit of embodiment is got three batches, often criticize kit and all measure the sample of concentration within the scope of 20 ± 4RU/mL and 100 ± 20RU/mL, often criticize replication 10 times, calculate mean value (M) and the standard deviation (SD) of 30 measurement results, calculate the coefficient of variation (CV) according to formula (3), this method coefficient of variation (CV) is not more than 15%.Data are see table 5.

CV＝SD/M×100％......(3)

In formula: CV-coefficient of variation; The standard deviation of SD-30 time measurement result; The mean value of M-30 time measurement result.

The evaluation of table 5 difference between batch

Measure serum-concentration (RU/mL)	Measure number of times	CV (%) between analysis
			20	30	6.89％
100	30	5.25％

6) Evaluation on specificity

Getting 1 part of Anti-SmIgG levels is the sample of 0RU/mL, adds human serum albumins, makes human serum albumins concentration in sample be 5000ng/mL, uses this kit to detect this sample, the Anti-SmIgG content in working sample.The results are shown in Table 6, this method and human serum albumins no cross reaction.Data are see table 6.

Table 6 specificity experiments

Test cross reaction thing	Concentration (ng/mL)	RLU	Measurement result (RU/mL)
				Human serum albumins	5000	2468	<1

7) relativity evaluation

By the kit of embodiment and commercial anti-Sm antibody IgG detection kit enzyme linked immunosorbent assay, 240 parts of human serum samples are detected simultaneously.As shown in Figure 3, with the result of anti-Sm antibody IgG detection kit enzyme-linked immunosorbent assay for horizontal ordinate, the result of mensuration is in the process of the present invention that ordinate does regretional analysis to its testing result, and dependent equation is y=0.9986x-0.2855, and related coefficient is R ²: 0.9843.Show through statistical procedures result, this method is good with the kit clinical sample measured value correlativity of additive method.

8) estimation of stability

Get described kit respectively at 4 DEG C 12 months and at 37 DEG C 7 days carry out stability experiment, result show the accuracy of kit standard product, blank limit, linear, repeatability and quality-control product the index such as assignment validity all within normal range, the kit term of validity can reach 12 months.Data are in table 7 and table 8.

Table 7 kit 4 DEG C of stability experiments

Table 8 kit 37 DEG C of stability experiments

Embodiment 2

A kind of preparation method using magnetic microparticle chemiluminescence quantitatively to detect the kit of anti-Sm antibody IgG comprises the following steps:

Step 1. is prepared described Anti-SmIgG calibration object and is comprised: Anti-SmIgG calibration object diluent preparing step and Anti-SmIgG calibration object preparation steps:

The preparation steps of described Anti-SmIgG calibration object dilution comprises:

Add the Tris of 800mL purified water and 11.2g in container, be uniformly mixed, add the sodium chloride of 8.5g and the Proclin300 of 2mL again, be stirred to and dissolve completely, pH value is adjusted to 7.0-7.5, add the bovine serum albumin(BSA) of 40g in container, be stirred to and dissolve completely, then pH value is adjusted to 7.0-7.5, be settled to 1L by purified water, use 0.2 μm of frit, by obtain described calibration object dilution be kept at 2-8 DEG C stand-by;

The preparation steps of described Anti-SmIgG calibration object comprises:

With above-mentioned Anti-SmIgG calibration object dilution anti-Sm antibody IgG is formulated as respectively 0,5,20,50,100,200RU/mL totally 6 concentration point;

Step 2. is prepared No. 1, described Anti-SmIgG reagent and is comprised: Anti-SmIgG reagent No. 1 diluent preparing step and Anti-SmIgG reagent No. 1 preparation steps:

The preparation steps of described Anti-SmIgG reagent No. 1 dilution:

Add 800mL purified water, 12.1g the NaCl of Tris and 5.8g in 1L container, be stirred to and dissolve completely, add the Proclin300 of 2mL again, be stirred to and dissolve completely, then add the bovine serum albumin(BSA) of 5g, be stirred to and dissolve completely, pH value is adjusted to 7.0-7.5, be settled to 1L by purified water, use 0.2 μm of frit, by obtain described reagent No. 1 dilution be kept at 2-8 DEG C stand-by;

The preparation steps of No. 1, described Anti-SmIgG reagent:

By the biotin solution of the pH9.0 carbonate buffer solution preparation 0.5mg/mL of 0.2mol/L, the ratio being 10:1 according to Sm antigen and biotin solution mass ratio joins the biotin solution of above-mentioned 0.5mg/mL in SM antigenic solution, mix, room temperature leaves standstill 18h, reaction generates the reactant liquor of Sm antigen-biotin conjugate, the Sm obtained antigen-biotin conjugate reactant liquor is separated by G-25 gel column, remove unreacted biotin, obtain Sm antigen-biotin conjugate, with described reagent No. 1 dilution, Sm antigen-biotin conjugate is diluted to 0.1-0.3 μ g/mL again, obtain No. 1, described reagent,

Step 3. is prepared No. 2, described Anti-SmIgG reagent and is comprised: the preparation steps of Anti-SmIgG reagent No. 2 diluent preparing steps and No. 2, Anti-SmIgG reagent:

The preparation steps of described Anti-SmIgG reagent No. 2 dilutions:

The NaCl adding 800mL purified water, the 4-hydroxyethyl piperazine second sulphur of 6.06g and 8.5g, in container, is stirred to and dissolves completely, then add the bovine serum albumin(BSA) of 5g, the ZnCl of 0.1g ₂, 0.2mL the MgCl of Proclin300 and 0.1g ₂, be stirred to and dissolve completely, pH value is adjusted to 7.5-8.0, be settled to 1L by purified water, use 0.2um frit, described reagent No. 2 dilutions obtained are kept at 2-8 DEG C stand-by;

The preparation steps of No. 2, described Anti-SmIgG reagent:

Be 2-imines thiophane solution by the coupling agent of the 10mg/mL of the goat anti-human antibody of 1mg and 2-4 μ L, room temperature leaves standstill 20min, add the glycine solution 10 μ L of 0.1mol/L, room temperature leaves standstill 5min, with G-25 gel column desalination, collect the rear antibody of activation, by activation after antibody be kept at 2-8 DEG C for subsequent use, get the alkaline phosphatase enzyme solutions of 1.5mg, add 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution 10-20 μ L of 5mg/mL, room temperature leaves standstill 30min, with G-25 gel column desalination, collect the rear alkaline phosphatase of activation, by activation after alkaline phosphatase be kept at 2-8 DEG C for subsequent use, again the goat anti-human antibody of above-mentioned activation is mixed with the alkaline phosphatase of activation, 12-24h is left standstill under 2-8 DEG C of condition, purify with Supperdex200 gel-purified post, obtain goat anti-human antibody-alkaline phosphatase connector strong solution, be placed in 2-8 DEG C to save backup, by goat anti-human antibody-alkaline phosphatase connector strong solution by described reagent No. 2 diluted to 0.02-0.1 μ g/mL, obtain No. 2, described reagent,

Step 4. is prepared described Anti-SmIgG Magneto separate reagent and is comprised: Anti-SmIgG Magneto separate reagent dilutions preparation steps and Anti-SmIgG Magneto separate preparation of reagents step:

Described Anti-SmIgG Magneto separate reagent dilutions preparation steps:

Add 800mL purified water, 12.1g the NaCl of Tris and 8.5g in container, be stirred to and dissolve completely, add the Proclin300 of the bovine serum albumin(BSA) of 5g, the NBCS of 50mL and 0.2mL again, be stirred to and dissolve completely, pH value is adjusted to 7.9-8.1, be settled to 1L by purified water, use 0.2um frit, the described Magneto separate reagent dilutions obtained is kept at 2-8 DEG C stand-by;

Described Anti-SmIgG Magneto separate preparation of reagents step:

Get 100mg magnetic particle, Magneto separate removes supernatant, the pH value of taking 0.025mol/L is that the MES damping fluid 10mL of 4.5-6 is resuspended, 2 ~ 3h is mixed under room temperature, adding the concentration that 0.5-1.0mL newly prepares is the carbodiimide of 10mg/mL and the morpholino b acid damping fluid of N-hydroxy thiosuccinimide, shaken at room temperature suspendible 30-60min, magnetic bead is fully activated, Magneto separate, remove supernatant, be that the MES damping fluid 10mL of 4.5-6 is resuspended by the pH value of 0.025mol/L, add the Streptavidin of 4-8mg, room temperature suspendible 16-20h, carry out Magneto separate again, remove supernatant, with the bovine serum albumin(BSA) of percent concentration 0.5%, the MES damping fluid 10mL of percent concentration 0.5% skimmed milk power and percent concentration 0.05% Tween-20 is resuspended, 2 ~ 3h is mixed under room temperature condition, Magneto separate again, remove supernatant, 0.5mg/mL is resuspended to the dilution of magnetic particle damping fluid, 16 ~ 24h is balanced at 2 ~ 8 DEG C, after mixing 2 ~ 3h under room temperature condition, sonic oscillation instrument is used to be dispersed into individual particle state by assembling agglomerating magnetic bead in suspension, obtain described Anti-SmIgG Magneto separate reagent.

Embodiment 3

On the basis of embodiment 2, described kit also comprises cleaning fluid, the preparation process of described cleaning fluid:

Add 800mL purified water, 12.1g the NaCl of Tris and 8.5g in 1L container, be stirred well to and dissolve completely; Add the Tween-20 of 5g and the Triton100 of 5g again, fully stir, until mix completely, adjust pH controls at 7.5-8.0, constant volume 1L, and with 0.2 μm of metre filter, temperature is that 2-8 DEG C of scope is preserved.

Embodiment 4

Described kit is adopted to be applied to the detection method of a kind of anti-Sm antibody IgG, the detecting step comprised:

1) by the Anti-SmIgG-R1 in kit, Anti-SmIgG-R2, Anti-SmIgG-M tri-components are positioned in the Full-automatic chemiluminescence immunoassay analysis meter reagent cabin of our company's development;

2) on instrument software, carry out calibration curve, Anti-SmIgG calibration object is put into instrument test position, obtain exporting matched curve by Full-automatic chemiluminescence immunoassay analysis meter;

3) if instrument prompting matching is passed through, then on instrument software, carry out quality-control product test application, Anti-SmIgG quality-control product is put into instrument assigned address.Obtain exporting by Full-automatic chemiluminescence immunoassay analysis meter the concentration value of quality-control product that quality-control product tested luminous value and obtained by standard curve fit.

4) if Quality Control measured value is in preset range, carry out sample test application, sample is put into instrument test position, the concentration value being exported sample test luminous value by Full-automatic chemiluminescence immunoassay analysis meter and obtained by standard curve fit.

5) Full-automatic chemiluminescence immunoassay analysis meter detecting step is as follows:

Step 1) add the sample to be measured after 20 μ L calibration objects or quality-control product or 1:20 dilution in detector tube;

Step 2) add No. 1,50 μ LAnti-SmIgG reagent to step 1) in described detector tube, after mixing, 37 ± 0.5 DEG C of incubations 10 minutes;

Step 3) add the Anti-SmIgG Magneto separate reagent of 50 μ L to step 2) in described detector tube, after mixing, 37 ± 0.5 DEG C of incubations 5 minutes; Carry out Magneto separate, remove supernatant;

Step 4) add 300 μ L cleaning fluids in detector tube, mixing; Carry out Magneto separate, remove supernatant;

Step 5) repeat step 4) twice;

Step 6) add No. 2,150 μ LAnti-SmIgG reagent in detector tube, after mixing, 37 ± 0.5 DEG C of incubations 10 minutes; Carry out Magneto separate, remove supernatant;

Step 7) add the cleaning fluid of 300 μ L in detector tube, mixing, carries out Magneto separate, removes supernatant;

Step 8) repeat step 7) twice;

Step 9) add 200 μ L chemical luminous substrates in detector tube, mixing, detect luminous intensity.

Above-described embodiment is only for illustrating technical conceive of the present invention and feature; its object is to person skilled in the art can be understood content of the present invention and implement according to this; can not limit the scope of the invention with this; all equivalences done according to Spirit Essence of the present invention change or modify, and all should be encompassed within protection scope of the present invention.

Claims (5)

1. the kit using magnetic microparticle chemiluminescence quantitatively to detect anti-Sm antibody IgG; described kit comprises: Anti-SmIgG calibration object, No. 1, Anti-SmIgG reagent, No. 2, Anti-SmIgG reagent, Anti-SmIgG Magneto separate reagent, Anti-SmIgG quality-control product and cleaning fluid, is characterized in that:

Described Anti-SmIgG calibration object comprises 6 liquid calibration bottles, described liquid calibration bottle internal object concentration is respectively 0,5,20,50,100, the anti-Anti-Sm IgG antibody of 200RU/mL and the Tris damping fluid of bovine serum albumin(BSA);

No. 1, described Anti-SmIgG reagent is 1 bottle containing the biotin labeled Sm antigen of 5mL and the Tris damping fluid of bovine serum albumin(BSA);

No. 2, described Anti-SmIgG reagent is 1 and contains the sheep anti-human polyclonal antibody of alkali phosphatase enzyme mark of 15mL and the bottle of the Tris damping fluid of bovine serum albumin(BSA);

Described Anti-SmIgG Magneto separate reagent be 1 containing 5mL containing the magnetic particle of marked by streptavidin and the Tris damping fluid bottle of bovine serum albumin(BSA);

Described Anti-SmIgG quality-control product comprises the Quality Control bottle of 2, and the scope of the concentration in described Quality Control bottle is respectively: 16-24RU/mL and 80-120RU/mL.

2. use magnetic microparticle chemiluminescence quantitatively to detect a preparation method for the kit of anti-Sm antibody IgG, it is characterized in that, described preparation method comprises:

Step 1. is prepared described Anti-SmIgG calibration object and is comprised: Anti-SmIgG calibration object diluent preparing step and Anti-SmIgG calibration object preparation steps:

The preparation steps of described Anti-SmIgG calibration object dilution comprises:

Add the Tris of 800mL purified water and 11.2g in container, be uniformly mixed, add the sodium chloride of 8.5g and the Proclin300 of 2mL again, be stirred to and dissolve completely, pH value is adjusted to 7.0-7.5, add the bovine serum albumin(BSA) of 40g in container, be stirred to and dissolve completely, then pH value is adjusted to 7.0-7.5, be settled to 1L by purified water, use 0.2 μm of frit, by obtain described calibration object dilution be kept at 2-8 DEG C stand-by;

The preparation steps of described Anti-SmIgG calibration object comprises:

With above-mentioned Anti-SmIgG calibration object dilution anti-Sm antibody IgG is formulated as respectively 0,5,20,50,100,200RU/mL totally 6 concentration point;

Step 2. is prepared No. 1, described Anti-SmIgG reagent and is comprised: Anti-SmIgG reagent No. 1 diluent preparing step and Anti-SmIgG reagent No. 1 preparation steps:

The preparation steps of described Anti-SmIgG reagent No. 1 dilution:

Add 800mL purified water, 12.1g the NaCl of Tris and 5.8g in 1L container, be stirred to and dissolve completely, add the Proclin300 of 2mL again, be stirred to and dissolve completely, then add the bovine serum albumin(BSA) of 5g, be stirred to and dissolve completely, pH value is adjusted to 7.0-7.5, be settled to 1L by purified water, use 0.2 μm of frit, by obtain described reagent No. 1 dilution be kept at 2-8 DEG C stand-by;

The preparation steps of No. 1, described Anti-SmIgG reagent:

By the biotin solution of the pH9.0 carbonate buffer solution preparation 0.5mg/mL of 0.2mol/L, the ratio being 10:1 according to Sm antigen and biotin solution mass ratio joins the biotin solution of above-mentioned 0.5mg/mL in Sm antigenic solution, mix, room temperature leaves standstill 18h, reaction generates the reactant liquor of Sm antigen-biotin conjugate, the Sm obtained antigen-biotin conjugate reactant liquor is separated by G-25 gel column, remove unreacted biotin, obtain Sm antigen-biotin conjugate, with described reagent No. 1 dilution, Sm antigen-biotin conjugate is diluted to 0.1-0.3 μ g/mL again, obtain No. 1, described reagent,

Step 3. is prepared No. 2, described Anti-SmIgG reagent and is comprised: the preparation steps of Anti-SmIgG reagent No. 2 diluent preparing steps and No. 2, Anti-SmIgG reagent:

The preparation steps of described Anti-SmIgG reagent No. 2 dilutions:

The NaCl adding 800mL purified water, the 4-hydroxyethyl piperazine second sulphur of 6.06g and 8.5g, in container, is stirred to and dissolves completely, then add the bovine serum albumin(BSA) of 5g, the ZnCl of 0.1g ₂, 0.2mL the MgCl of Proclin300 and 0.1g ₂, be stirred to and dissolve completely, pH value is adjusted to 7.5-8.0, be settled to 1L by purified water, use 0.2um frit, described reagent No. 2 dilutions obtained are kept at 2-8 DEG C stand-by;

The preparation steps of No. 2, described Anti-SmIgG reagent:

Be 2-imines thiophane solution by the coupling agent of the 10mg/mL of the goat anti-human antibody of 1mg and 2-4 μ L, room temperature leaves standstill 20min, add the glycine solution 10 μ L of 0.1mol/L, room temperature leaves standstill 5min, with G-25 gel column desalination, collect the rear antibody of activation, by activation after antibody be kept at 2-8 DEG C for subsequent use, get the alkaline phosphatase of 1.5mg, add 4-(N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester solution 10-20 μ L of 5mg/mL, room temperature leaves standstill 30min, with G-25 gel column desalination, collect the rear alkaline phosphatase of activation, by activation after alkaline phosphatase be kept at 2-8 DEG C for subsequent use, again the goat anti-human antibody of above-mentioned activation is mixed with the alkaline phosphatase of activation, 12-24h is left standstill under 2-8 DEG C of condition, purify with Supperdex200 gel-purified post, obtain goat anti-human antibody-alkaline phosphatase connector strong solution, be placed in 2-8 DEG C to save backup, by goat anti-human antibody-alkaline phosphatase connector strong solution by described reagent No. 2 diluted to 0.02-0.1 μ g/mL, obtain No. 2, described reagent,

Step 4. is prepared described Anti-SmIgG Magneto separate reagent and is comprised: Anti-SmIgG Magneto separate reagent dilutions preparation steps and Anti-SmIgG Magneto separate preparation of reagents step:

Described Anti-SmIgG Magneto separate reagent dilutions preparation steps:

Add 800mL purified water, 12.1g the NaCl of Tris and 8.5g in container, be stirred to and dissolve completely, add the Proclin300 of the bovine serum albumin(BSA) of 5g, the NBCS of 50mL and 0.2mL again, be stirred to and dissolve completely, pH value is adjusted to 7.9-8.1, be settled to 1L by purified water, use 0.2um frit, the described Magneto separate reagent dilutions obtained is kept at 2-8 DEG C stand-by;

Described Anti-SmIgG Magneto separate preparation of reagents step:

Get 100mg magnetic particle, Magneto separate removes supernatant, the pH value of taking 0.025mol/L is that the MES damping fluid 10mL of 4.5-6 is resuspended, 2 ~ 3h is mixed under room temperature, adding the concentration that 0.5-1.0mL newly prepares is the carbodiimide of 10mg/mL and the MES damping fluid of N-hydroxy thiosuccinimide, shaken at room temperature suspendible 30-60min, magnetic bead is fully activated, Magneto separate, remove supernatant, be that the MES damping fluid 10mL of 4.5-6 is resuspended by the pH value of 0.025mol/L, add the Streptavidin of 4-8mg, room temperature suspendible 16-20h, carry out Magneto separate again, remove supernatant, with the bovine serum albumin(BSA) of percent concentration 0.5%, the MES damping fluid 10mL of percent concentration 0.5% skimmed milk power and percent concentration 0.05% Tween-20 is resuspended, 2 ~ 3h is mixed under room temperature condition, Magneto separate again, remove supernatant, 0.5mg/mL is resuspended to the dilution of magnetic particle damping fluid, 16 ~ 24h is balanced at 2 ~ 8 DEG C, after mixing 2 ~ 3h under room temperature condition, sonic oscillation instrument is used to be dispersed into individual particle state by assembling agglomerating magnetic bead in suspension, obtain described Anti-SmIgG Magneto separate reagent.

3. a kind of preparation method using magnetic microparticle chemiluminescence quantitatively to detect the kit of anti-Sm antibody IgG according to claim 2, it is characterized in that, described preparation method comprises the preparation process of cleaning fluid:

Add 800mL purified water, 12.1g the NaCl of Tris and 8.5g in 1L container, be stirred to and dissolve completely, add the Tween-20 of 5g and the Triton100 of 5g again, stir, until mix completely, pH value is adjusted to 7.5-8.0, constant volume 1L, use 0.2 μm of frit, by obtain described cleaning fluid be kept at 2-8 DEG C stand-by, use before by described cleaning fluid according to every 7.5mL add purified water to 100mL dilution proportion and mixing after re-use.

4. use magnetic microparticle chemiluminescence quantitatively to detect a detection method for the kit of anti-Sm antibody IgG, comprise the following steps:

Anti-SmIgG calibration object is put into Full-automatic chemiluminescence immunoassay analysis meter test position by step 1., obtains the matched curve exported by Full-automatic chemiluminescence immunoassay analysis meter;

Anti-SmIgG quality-control product is put into above-mentioned analyser test position by step 2., obtains the test luminous value of the described quality-control product exported by Full-automatic chemiluminescence immunoassay analysis meter and is obtained the concentration value of Anti-SmIgG quality-control product by the matched curve matching that step 1 obtains;

Sample to be tested is put into above-mentioned analyser test position by step 3., automatically presses 1:20 by Sample Dilution, obtain the concentration value of the sample to be tested exported by Full-automatic chemiluminescence immunoassay analysis meter by described analyser.

5. a kind of detection method using magnetic microparticle chemiluminescence quantitatively to detect the kit of anti-Sm antibody IgG according to claim 4, is characterized in that: described step 1, step 2 and step 3 include the fully-automated synthesis step of Full-automatic chemiluminescence immunoassay analysis meter:

Step 1) add the sample to be measured after 20 μ L calibration objects or quality-control product or 1:20 dilution in detector tube;

Step 2) add No. 1, the Anti-SmIgG reagent of 50 μ L to step 1) in described detector tube, after mixing, 37 ± 0.5 DEG C of incubation 10min;

Step 3) add the Anti-SmIgG Magneto separate reagent of 50 μ L to step 2) in described detector tube, after mixing, 37 ± 0.5 DEG C of incubation 5min, carry out Magneto separate, remove supernatant;

Step 4) add the cleaning fluid of 300 μ L in detector tube, mixing, carries out Magneto separate and removes supernatant;

Step 5) repeat step 4) twice;

Step 6) add No. 2,150 μ LAnti-SmIgG reagent in detector tube, after mixing, 37 ± 0.5 DEG C of incubation 10min, carry out Magneto separate and remove supernatant;

Step 7) add the cleaning fluid of 300 μ L in detector tube, mixing, carries out Magneto separate and removes supernatant;

Step 8) repeat step 7) twice;

Step 9) add the chemical luminous substrate of 200 μ L in detector tube, mixing, detect luminous intensity.