CN105466913A - Kit for quantitative detection of anti-Jo-1 antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof - Google Patents
Kit for quantitative detection of anti-Jo-1 antibody IgG by using magnetic particle chemiluminescence, and preparation method and detection method thereof Download PDFInfo
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- CN105466913A CN105466913A CN201510782564.XA CN201510782564A CN105466913A CN 105466913 A CN105466913 A CN 105466913A CN 201510782564 A CN201510782564 A CN 201510782564A CN 105466913 A CN105466913 A CN 105466913A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
Abstract
The invention relates to the technical field of immunology detection, and particularly relates to a kit for quantitative detection of an anti-Jo-1 antibody IgG by using magnetic particle chemiluminescence, and a preparation method and a detection method thereof. The kit for quantitative detection of the anti-Jo-1 antibody IgG by using magnetic particle chemiluminescence includes an Anti-Jo-1 IgG calibrator, an Anti-Jo-1 IgG reagent No. 1, an Anti-Jo-1 IgG reagent No. 2, an Anti-Jo-1 IgG magnetic separation reagent, an Anti-Jo-1 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 complete automation usage, and has broad application prospects.
Description
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-Jo-1 antibody IgG and preparation method thereof and detection method.
Background technology
Within 1980, reported first anti-Jo-1 antibody finds in the serum of polymyositis (PM) patient, and its antigen is Histidyl-tRNA-synthetase, is present in tenuigenin, and gains the name because patient is called John, is named as anti-Jo-1 antibody.
A large amount of clinical research confirmation, anti-Jo-1 antibody (Anti-Jo-1antibody) is myositis-specific antibodies (Myositis-specificantibodies, MSA) most important antibody in, it is to polymyositis (Polymyositis, the diagnosis of PM)/dermatomyositis (Dermatomyositis, DM), antidiastole, somatotype, therapeutic choice and prognosis evaluation all have important clinical significance.Nineteen eighty-three confirms that its antigen is Histidyl-tRNA synthetase antibody (HRS).The at present film bar immunization that has of the detection of anti-Jo-1 antibody IgG and enzyme linked immunosorbent assay clinically.What film bar immunization was applied is film bar developing technology, its feature measures at same film bar for fixing several project, generally carry out experimental implementation by manual or semi-automatic film bar instrument, qualitatively judge eventually through naked eyes, this sensitivity is low, reaction time is long, and test item can only combine in regular collocation, very flexible.The sensitivity of enzyme linked immunosorbent assay promotes to some extent on film bar immunization basis, but still lower, and the range of linearity is narrow, poor repeatability, and the reaction time is also longer, still can not meet clinical application very well.
Magnetic microparticle chemiluminescence immune assay method, film bar immunization than before and enzyme linked immunosorbent assay, detection sensitivity, sensing range, detection time and automation mechanized operation have had and have greatly improved, and do not pollute, and clinical practice is wide.
At present, magnetic microparticle chemiluminescence analytic approach is used to have not yet to see in the application of anti-Jo-1 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-Jo-1 antibody IgG; it can prepare with lower cost, and can realize anti-Jo-1 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-Jo-1 antibody IgG, and described kit comprises: Anti-Jo-1IgG calibration object, No. 1, Anti-Jo-1IgG reagent, No. 2, Anti-Jo-1IgG reagent, Anti-Jo-1IgG Magneto separate reagent, Anti-Jo-1IgG quality-control product and cleaning fluid.
Described Anti-Jo-1IgG calibration object comprises 6 liquid calibration bottles, described liquid calibration bottle internal object concentration is respectively 0,5,20,50,100, the anti-Anti-Jo-1 IgG antibody of 200RU/mL and the Tris damping fluid of bovine serum albumin(BSA);
No. 1, described Anti-Jo-1IgG reagent is 1 bottle containing the Tris damping fluid of the biotin labeled Jo-1 antigen of 5mL and bovine serum albumin(BSA);
No. 2, described Anti-Jo-1IgG reagent is 1 bottle containing the sheep anti-human polyclonal antibody of 15mL alkali phosphatase enzyme mark and the Tris damping fluid of bovine serum albumin(BSA);
Described Anti-Jo-1IgG Magneto separate reagent is 1 and contains the magnetic particle of 5mL marked by streptavidin and the Tris damping fluid bottle of bovine serum albumin(BSA); Described Anti-Jo-1IgG 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-Jo-1IgG reagent mixing incubation, anti-Jo-1 antibody IgG in sample is combined with Jo-1 antigentic specificity, add described Anti-Jo-1IgG Magneto separate reagent again, the immune complex of anti-Jo-1 antibody IgG and Jo-1 antigen is adsorbed to magnetic particle surface, unconjugated antibody and impurity are removed in washing, add No. 2, Anti-Jo-1IgG reagent and mix incubation.Goat anti-human igg antibody and the anti-Jo-1 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-Jo-1 antibody IgG concentration is proportional within the specific limits, just can be calculated the anti-Jo-1 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-Jo-1 antibody IgG, and the preparation method of described kit comprises the following steps:
Step 1. is prepared described Anti-Jo-1IgG calibration object and is comprised: Anti-Jo-1IgG calibration object diluent preparing step and Anti-Jo-1IgG calibration object preparation steps:
The preparation steps of described Anti-Jo-1IgG 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-Jo-1IgG calibration object comprises:
With above-mentioned Anti-Jo-1IgG calibration object dilution anti-Jo-1 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-Jo-1IgG reagent and is comprised: Anti-Jo-1IgG reagent No. 1 diluent preparing step and Anti-Jo-1IgG reagent No. 1 preparation steps:
The preparation steps of described Anti-Jo-1IgG 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-Jo-1IgG 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 Jo-1 antigen and biotin solution mass ratio joins the biotin solution of above-mentioned 0.5mg/mL in Jo-1 antigenic solution, mix, room temperature leaves standstill 18h, reaction generates the reactant liquor of Jo-1 antigen-biotin conjugate, the Jo-1 obtained antigen-biotin conjugate reactant liquor is separated by G-25 gel column, remove unreacted biotin, obtain Jo-1 antigen-biotin conjugate, with described reagent No. 1 dilution, Jo-1 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-Jo-1IgG reagent and is comprised: the preparation steps of Anti-Jo-1IgG reagent No. 2 diluent preparing steps and No. 2, Anti-Jo-1IgG reagent:
The preparation steps of described Anti-Jo-1IgG 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 0.2 μm of frit, by obtain described reagent No. 2 dilutions be kept at 2-8 DEG C stand-by;
The preparation steps of No. 2, described Anti-Jo-1IgG 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-Jo-1IgG Magneto separate reagent and is comprised: Anti-Jo-1IgG Magneto separate reagent dilutions preparation steps and Anti-Jo-1IgG Magneto separate preparation of reagents step:
Described Anti-Jo-1IgG 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 0.2 μm of frit, by obtain described Magneto separate reagent dilutions be kept at 2-8 DEG C stand-by;
Described Anti-Jo-1IgG 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-Jo-1IgG Magneto separate reagent.
Further, the present invention also provides 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 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, the described cleaning concentrate obtained is kept at 2-8 DEG C stand-by, before using, described cleaning fluid is added purified water to 100mL dilution proportion according to every 7.5mL and mixes.
Present invention also offers a kind of detection method using magnetic microparticle chemiluminescence quantitatively to detect the kit of anti-Jo-1 antibody IgG, the detection method of described kit comprises the following steps:
Anti-Jo-1IgG 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-Jo-1IgG 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-Jo-1IgG 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-Jo-1 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-Jo-1IgG 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-Jo-1IgG 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, the Anti-Jo-1IgG reagent of 150 μ L 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-Jo-1IgG reagent 1, in described kit, No. 2, Anti-Jo-1IgG reagent, Anti-Jo-1IgG 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-Jo-1IgG Magneto separate reagent participation reaction time in described kit is only 5 minutes, can avoid non-specific adsorption to limits;
3, the Anti-Jo-1IgG Magneto separate reagent in described kit is the super paramagnetic microsphere of polymolecularity, ensures that magnetic particle can not be assembled completely in preservation and use procedure;
Alkaline phosphatase in No. 2, the Anti-Jo-1IgG 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-Jo-1IgG 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 cross reaction with anti-nRNP/Sm antibody, anti-Sm antibody, Anti SS-A antibody, Anti SS-B antibody, Anti-Scl-70, anti-centromere antibody;
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-Jo-1 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-Jo-1 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-Jo-1 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-Jo-1 antibody IgG, this kit comprises: Anti-Jo-1IgG calibration object, No. 1, Anti-Jo-1IgG reagent, No. 2, Anti-Jo-1IgG reagent, Anti-Jo-1IgG Magneto separate reagent, Anti-Jo-1IgG 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-Jo-1 antibody IgG sample A join in the sample B of serum or other corresponding matrix, 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: 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 sample A liquid;
C
0: the concentration measuring sample 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 concentration-RLU value result 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 range of linearity of this method is 2 ~ 200RU/mL, and correlation coefficient r answers >=0.9900.Data are shown in Fig. 2 see table 3 and curve.
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 | 6.84％ |
| 100 | 30 | 4.20％ |
6) Evaluation on specificity
Getting 6 parts of Anti-Jo-1IgG content is the sample of 0, add anti-nRNP/Sm antibody, anti-Sm antibody, Anti SS-A antibody, Anti SS-B antibody, Anti-Scl-70, anti-centromere antibody respectively, cross reaction substrate concentration in sample is made to be 200RU/mL, this kit is used to detect this sample, the Anti-Jo-1IgG content in working sample.The results are shown in Table 6, this method and anti-nRNP/Sm antibody, anti-Sm antibody, Anti SS-A antibody, Anti SS-B antibody, Anti-Scl-70, anti-centromere antibody no cross reaction.
Table 6 specificity experiments
| Test cross reaction thing | Concentration (RU/mL) | RLU | Degree of disturbance (RU/mL) |
| Anti-nRNP/Sm antibody | 200 | 5007 | ＜1 |
| Anti-Sm antibody | 200 | 4340 | ＜1 |
| Anti SS-A antibody | 200 | 4762 | ＜1 |
| Anti SS-B antibody | 200 | 4529 | ＜1 |
| Anti-Scl-70 | 200 | 4735 | ＜1 |
| Anti-centromere antibody | 200 | 4592 | ＜1 |
7) relativity evaluation
By the kit of embodiment and commercial anti-Jo-1 antibody IgG detection kit enzyme linked immunosorbent assay, 240 parts of human serum samples are detected simultaneously.Its testing result as shown in Figure 3, with the result of anti-Jo-1 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, and dependent equation is: y=0.9809x+0.6682, and related coefficient is R
2: 0.9869.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 and carry out 4 DEG C of 12 months and the experiments of 37 DEG C of accelerated stabilities of 7 days respectively, 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, and 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
Use magnetic microparticle chemiluminescence quantitatively to detect a preparation method for the kit of anti-Jo-1 antibody IgG, comprise the following steps:
Step 1. is prepared described Anti-Jo-1IgG calibration object and is comprised: Anti-Jo-1IgG calibration object diluent preparing step and Anti-Jo-1IgG calibration object preparation steps:
The preparation steps of described Anti-Jo-1IgG 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-Jo-1IgG calibration object comprises:
With above-mentioned Anti-Jo-1IgG calibration object dilution anti-Jo-1 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-Jo-1IgG reagent and is comprised: Anti-Jo-1IgG reagent No. 1 diluent preparing step and Anti-Jo-1IgG reagent No. 1 preparation steps:
The preparation steps of described Anti-Jo-1IgG 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-Jo-1IgG 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 Jo-1 antigen and biotin solution mass ratio joins the biotin solution of above-mentioned 0.5mg/mL in Jo-1 antigenic solution, mix, room temperature leaves standstill 18h, reaction generates the reactant liquor of Jo-1 antigen-biotin conjugate, the Jo-1 obtained antigen-biotin conjugate reactant liquor is separated by G-25 gel column, remove unreacted biotin, obtain Jo-1 antigen-biotin conjugate, with described reagent No. 1 dilution, Jo-1 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-Jo-1IgG reagent and is comprised: the preparation steps of Anti-Jo-1IgG reagent No. 2 diluent preparing steps and No. 2, Anti-Jo-1IgG reagent:
The preparation steps of described Anti-Jo-1IgG 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-Jo-1IgG 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-Jo-1IgG Magneto separate reagent and is comprised: Anti-Jo-1IgG Magneto separate reagent dilutions preparation steps and Anti-Jo-1IgG Magneto separate preparation of reagents step:
Described Anti-Jo-1IgG 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-Jo-1IgG 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-Jo-1IgG 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, Tris12.1g and NaCl8.5g in 1L container, be stirred well to and dissolve completely; Add the Tween-20 of 5g and the Triton100 of 5g again, abundant stirring, until mix completely, adjust pH controls at 7.5-8.0, constant volume 1L, with 0.2 μm of metre filter, temperature is that 2-8 DEG C of scope is preserved, and before using, described cleaning fluid is added purified water to 100mL dilution proportion according to every 7.5mL and mixes.
Embodiment 4
Described kit is adopted to be applied to the detection method of a kind of anti-Jo-1 antibody IgG, the detecting step comprised:
1) by the Anti-Jo-1IgG-R1 in kit, Anti-Jo-1IgG-R2, Anti-Jo-1IgG-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-Jo-1IgG calibration object is put into the test position in instrument reagent cabin, 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-Jo-1IgG 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 in instrument reagent cabin, 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-Jo-1IgG reagent to step 1) in described detector tube, after mixing, 37 ± 0.5 DEG C of incubations 10 minutes;
Step 3) add 50 μ LAnti-Jo-1IgG Magneto separate reagent 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 and remove supernatant;
Step 5) repeat step 4) twice;
Step 6) add No. 2, the Anti-Jo-1IgG reagent of 150 μ L in detector tube, after mixing, 37 ± 0.5 DEG C of incubations 10 minutes; Carry out Magneto separate and remove supernatant;
Step 7) add 300 μ L cleaning fluids in detector tube, mixing; Carry out Magneto separate and remove 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-Jo-1 antibody IgG; described kit comprises: Anti-Jo-1IgG calibration object, No. 1, Anti-Jo-1IgG reagent, No. 2, Anti-Jo-1IgG reagent, Anti-Jo-1IgG Magneto separate reagent, Anti-Jo-1IgG quality-control product and cleaning fluid, is characterized in that:
Described Anti-Jo-1IgG calibration object comprises 6 liquid calibration bottles, described liquid calibration bottle internal object concentration is respectively 0,5,20,50,100, the anti-Anti-Jo-1 IgG antibody of 200RU/mL and the Tris damping fluid of bovine serum albumin(BSA);
No. 1, described Anti-Jo-1IgG reagent is 1 bottle containing the Tris damping fluid of biotin labeled Jo-1 antigen and bovine serum albumin(BSA);
No. 2, described Anti-Jo-1IgG reagent is the bottle containing the sheep anti-human polyclonal antibody of alkali phosphatase enzyme mark and the Tris damping fluid of bovine serum albumin(BSA);
Described Anti-Jo-1IgG Magneto separate reagent is the Tris damping fluid bottle of magnetic particle containing marked by streptavidin and bovine serum albumin(BSA);
Described Anti-Jo-1IgG 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.
2. use magnetic microparticle chemiluminescence quantitatively to detect a preparation method for the kit of anti-Jo-1 antibody IgG, it is characterized in that, described preparation method comprises:
Step 1. is prepared described Anti-Jo-1IgG calibration object and is comprised: Anti-Jo-1IgG calibration object diluent preparing step and Anti-Jo-1IgG calibration object preparation steps:
The preparation steps of described Anti-Jo-1IgG 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-Jo-1IgG calibration object comprises:
With above-mentioned Anti-Jo-1IgG calibration object dilution anti-Jo-1 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-Jo-1IgG reagent and is comprised: Anti-Jo-1IgG reagent No. 1 diluent preparing step and Anti-Jo-1IgG reagent No. 1 preparation steps:
The preparation steps of described Anti-Jo-1IgG 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-Jo-1IgG 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 Jo-1 antigen and biotin solution mass ratio joins the biotin solution of above-mentioned 0.5mg/mL in Jo-1 antigenic solution, mix, room temperature leaves standstill 18h, reaction generates the reactant liquor of Jo-1 antigen-biotin conjugate, the Jo-1 obtained antigen-biotin conjugate reactant liquor is separated by G-25 gel column, remove unreacted biotin, obtain Jo-1 antigen-biotin conjugate, with described reagent No. 1 dilution, Jo-1 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-Jo-1IgG reagent and is comprised: the preparation steps of Anti-Jo-1IgG reagent No. 2 diluent preparing steps and No. 2, Anti-Jo-1IgG reagent:
The preparation steps of described Anti-Jo-1IgG 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 0.2 μm of frit, by obtain described reagent No. 2 dilutions be kept at 2-8 DEG C stand-by;
The preparation steps of No. 2, described Anti-Jo-1IgG 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 S μ pperdex200 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-Jo-1IgG Magneto separate reagent and is comprised: Anti-Jo-1IgG Magneto separate reagent dilutions preparation steps and Anti-Jo-1IgG Magneto separate preparation of reagents step:
Described Anti-Jo-1IgG 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 0.2 μm of frit, by obtain described Magneto separate reagent dilutions be kept at 2-8 DEG C stand-by;
Described Anti-Jo-1IgG 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-Jo-1IgG Magneto separate reagent.
3. a kind of preparation method using magnetic microparticle chemiluminescence quantitatively to detect the kit of anti-Jo-1 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, the described cleaning concentrate obtained is kept at 2-8 DEG C stand-by, before using, described cleaning fluid is added purified water to 100mL dilution proportion according to every 7.5mL and mixes.
4. use magnetic microparticle chemiluminescence quantitatively to detect a detection method for the kit of anti-Jo-1 antibody IgG, comprise the following steps:
Anti-Jo-1IgG 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-Jo-1IgG 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-Jo-1IgG 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-Jo-1 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-Jo-1IgG 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-Jo-1IgG 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, removes supernatant;
Step 5) repeat step 4) twice;
Step 6) add No. 2, the Anti-Jo-1IgG reagent of 150 μ L in detector tube, after mixing, 37 ± 0.5 DEG C of incubation 10min, 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 the chemical luminous substrate of 200 μ L in detector tube, mixing, detect luminous intensity.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105929165A (en) * | 2016-04-20 | 2016-09-07 | 北京中航赛维生物科技有限公司 | Magnetic particle-based quantitative chemiluminescent assay kit for anti-SLA/LP antibody IgG, and preparation and detection methods thereof |
| CN106706896A (en) * | 2016-06-30 | 2017-05-24 | 深圳市亚辉龙生物科技股份有限公司 | Anti-Jo-1 antibody IgG chemiluminescent immunoassay kit and preparation method thereof |
| CN108051586A (en) * | 2017-11-29 | 2018-05-18 | 北京美联泰科生物技术有限公司 | A kind of magnetic microparticle chemiluminescence quantitatively detects kit of GFAP and preparation method thereof |
| CN108490196A (en) * | 2018-05-16 | 2018-09-04 | 江苏维尔生物科技有限公司 | Test-strips of filaria volvulus metabolin NATOG and preparation method thereof, test card in a kind of detection urine |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202141726U (en) * | 2011-01-30 | 2012-02-08 | 上海科新生物技术股份有限公司 | Anti-Jo-1 antibody test paper with colloidal gold immunochromatography |
| CN103076458A (en) * | 2012-12-16 | 2013-05-01 | 北京利德曼生化股份有限公司 | Dissociate human chorionic gonadotrophin beta-subunit magnetic particle chemiluminescence quantitative assay kit and its preparation method |
| CN103185777A (en) * | 2011-12-30 | 2013-07-03 | 深圳市亚辉龙生物科技有限公司 | Reagent device and method for detecting anti-Jo-1 antibody |
| CN103777025A (en) * | 2012-10-18 | 2014-05-07 | 上海铭源数康生物芯片有限公司 | Preparation method of critical-value first-grade reference for protein micro-array chip, and critical value coefficient determination and experiment result decision method |
| CN103901203A (en) * | 2014-04-11 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Chemiluminescence quantitative detection kit for procalcitonin, and preparation method and detection method thereof |
-
2015
- 2015-11-16 CN CN201510782564.XA patent/CN105466913A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202141726U (en) * | 2011-01-30 | 2012-02-08 | 上海科新生物技术股份有限公司 | Anti-Jo-1 antibody test paper with colloidal gold immunochromatography |
| CN103185777A (en) * | 2011-12-30 | 2013-07-03 | 深圳市亚辉龙生物科技有限公司 | Reagent device and method for detecting anti-Jo-1 antibody |
| CN103777025A (en) * | 2012-10-18 | 2014-05-07 | 上海铭源数康生物芯片有限公司 | Preparation method of critical-value first-grade reference for protein micro-array chip, and critical value coefficient determination and experiment result decision method |
| CN103076458A (en) * | 2012-12-16 | 2013-05-01 | 北京利德曼生化股份有限公司 | Dissociate human chorionic gonadotrophin beta-subunit magnetic particle chemiluminescence quantitative assay kit and its preparation method |
| CN103901203A (en) * | 2014-04-11 | 2014-07-02 | 苏州浩欧博生物医药有限公司 | Chemiluminescence quantitative detection kit for procalcitonin, and preparation method and detection method thereof |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105929165A (en) * | 2016-04-20 | 2016-09-07 | 北京中航赛维生物科技有限公司 | Magnetic particle-based quantitative chemiluminescent assay kit for anti-SLA/LP antibody IgG, and preparation and detection methods thereof |
| CN106706896A (en) * | 2016-06-30 | 2017-05-24 | 深圳市亚辉龙生物科技股份有限公司 | Anti-Jo-1 antibody IgG chemiluminescent immunoassay kit and preparation method thereof |
| CN108051586A (en) * | 2017-11-29 | 2018-05-18 | 北京美联泰科生物技术有限公司 | A kind of magnetic microparticle chemiluminescence quantitatively detects kit of GFAP and preparation method thereof |
| CN108490196A (en) * | 2018-05-16 | 2018-09-04 | 江苏维尔生物科技有限公司 | Test-strips of filaria volvulus metabolin NATOG and preparation method thereof, test card in a kind of detection urine |
| CN109085343A (en) * | 2018-10-15 | 2018-12-25 | 江苏浩欧博生物医药股份有限公司 | A kind of kit and detection method measuring anti-Jo-1 antibody |
| CN109085343B (en) * | 2018-10-15 | 2022-02-15 | 江苏浩欧博生物医药股份有限公司 | Kit for determining anti-Jo-1 antibody and detection method |
| CN111944773A (en) * | 2020-08-17 | 2020-11-17 | 珠海丽珠试剂股份有限公司 | Jo-1 antigen coupling magnetic particle and preparation method, application and product thereof |
| CN111944773B (en) * | 2020-08-17 | 2022-03-25 | 珠海丽珠试剂股份有限公司 | Jo-1 antigen coupling magnetic particle and preparation method, application and product thereof |
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