CN101498715B - Anti-cardiolipin antibody detection kit and use thereof - Google Patents
Anti-cardiolipin antibody detection kit and use thereof Download PDFInfo
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- CN101498715B CN101498715B CN200910047379.0A CN200910047379A CN101498715B CN 101498715 B CN101498715 B CN 101498715B CN 200910047379 A CN200910047379 A CN 200910047379A CN 101498715 B CN101498715 B CN 101498715B
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Abstract
The invention relates to an anti-cardiolipin antibody detecting reagent kit and the application thereof. The anti-cardiolipin antibody detecting reagent kit comprises a pre-coated enzyme-linked plate which is coated by ACA antigen, IgG enzyme label antibody solution which is diluted by enzyme label antibody diluent, scrub solution, sample diluents, negative contrast solution, positive contrast solution, visualization solution and stop solution. The reagent kit has high specificity and sensitivity and good reagent stability and provides a potent and reliable detection basis for clinical diagnosis.
Description
Technical field
The present invention relates to a kind of anti-cardiolipin antibody detection kit and application thereof.
Background technology
It is a kind ofly to using on blood platelet and endothelial cell membrane electronegative cuorin as the autoantibody of target antigen that anticardiolipin antibody (ACL) belongs to.Be common in systemic loupus erythematosus (SLE) and other autoimmune diseases.This antibody and thrombosis, blood platelet, spontaneous abortion or FDIU are in close relations.Studies confirm that, many factors and ACA produce closely related, and common reason has:
(1) autoimmune disease: as chorionitis of systemic loupus erythematosus (SLE), rheumatoid arthritis (RA) etc.;
(2) virus infections: as infection such as adenovirus, rubella virus, varicella virus, mumps viruses;
(3) Other diseases: as mycoplasma systemic disease etc.;
(4) oral some drugs: as chlorpromazine, phenothiazine etc.;
Cuorin is that separate from the cardiac muscle of ox pangborn nineteen forty-one a kind of has antigenic phosphatide, and is named, and its content in mammal flesh is the highest, and each Cardiolipin molecules comprises 4 undersaturated fatty acid, very easily oxidation.ACA is that phosphodiester group electronegative in Cardiolipin molecules is combined, but this combination must have the glyceride part in Cardiolipin molecules, if replace and draw glyceride part with benzyl ring, the antigenicity of cuorin disappears.Fatty acid part in ACA molecule is its antigenic essential composition; The phosphodiester group of ACA on-load in other molecule is not combined, and only with phospholipid molecule in this composition react.In recent years the research such as Hotkko finds, ACA is only combined with already oxidised cuorin, and can not be combined with the cuorin analog of the reductibility being oxidized.
Since nineteen eighty-three Harris etc. has set up the method for survey ACA (anticardiolipin antibody), the research of relevant this antibody worldwide earns widespread respect and develops rapidly.Koike thinks that cuorin is combined with the IgG of ACA needs the existence of the co-factor of ACA beta 2 glycoprotein I (β 2-GPI).The ACA that the ACA solid-phase radioimmunoassay (RLA) that Harris sets up detects now thinks to have higher susceptibility for this method of complex of cuorin and β 2-GPI, to be modified to the ELISA method identical and more convenient with radioimmunology effect by Loizou etc.The method of current existing detection anticardiolipin antibody has gold mark and chemiluminescence detection antibody, can only do simply qualitative, and chemoluminescence method need be with special instrumentation, and for the existing large multiplex enzyme linked immunosorbent assay (ELISA) of the detection of free antibodies in serum, the specificity of this method and susceptibility are all higher and need not specific apparatus, not only can be qualitative but also can be quantitative, detectable antibody class and subclass, be easy to standardization, there is the advantages such as simple to operate, result correct, save time, general institute of Xian Yibei hospital adopts, and becomes the worldwide standard of quantitative measurement ACA.
The reliability that cuorin antibody ELISA detection kit quantitatively detects ACA result is directly relevant to its quality, how to guarantee specificity and the sensitivity of reagent in ELISA anti-cardiolipin antibody detection kit, improve the stability of reagent, the interference of getting rid of the nonspecific interference factor becomes the main direction of studying of further raising ELISA anti-cardiolipin antibody detection kit quality.
Summary of the invention
The object of this invention is to provide a kind of cardiolipin antibody detection kit and preparation method thereof and use, kit specificity provided by the invention and highly sensitive, reagent stability is good, can detect ACA by qualitative and quantitative, for clinical diagnosis provides strong reliable detection foundation.
Anti-cardiolipin antibody detection kit of the present invention comprises:
(1), with the antigen coated pre-coated elisa plate of ACA;
(2), use the IgG enzyme labelled antibody solution of enzyme labelled antibody diluted;
(3), cleansing solution;
(4), sample diluting liquid;
(5), negative control;
(6), positive control;
(7), nitrite ion;
(8), stop buffer.
Kit of the present invention adopts indirect method to detect cuorin antibody, and specific antigen and solid phase carrier are connect, and forms solid phase antigen; Add and examined sample, the antibody in sample is combined with solid phase antigen, forms solid phase antigen antibody complex; After antibody to be measured is combined with specific antigen, by enzyme labelled antibody further with it reaction, now on solid phase carrier with enzyme amount relevant to the amount of being examined antibody in sample; Substrate for enzymatic activity in solid phase becomes coloured product.By colorimetric, predict the amount of antibody in sample.
The cuorin antigen of ACA antigen in kit for extracting in OX-heart, purity need reach mass percent more than 98%.
IgG enzyme labelled antibody in kit is the IgG antibody of horseradish peroxidase (HRP) mark, and IgG antibody can be various types of IgG antibody, as mouse-anti human IgG, goat anti-human igg or rabbit anti-human igg.
Enzyme labelled antibody stoste is 1 with the part by weight of enzyme labelled antibody diluted: 4000-1: 5000.
In order to make kit have better specificity and sensitivity, the present invention improves on enzyme labelled antibody dilution, and enzyme labelled antibody dilution of the present invention comprises following component: C
6h
10o
4(PO
4)
2hNa, Na
2hPO
4, NaCl and NaH
2pO
4, and the total concentration of above-mentioned each material is 2-9g/L, the pH value of solution is 7.2-7.6.
Preferably, the concentration of each component is:
C
6H
10O
4·(PO
4)
2·HNa 0.5-3g/L
Na
2HPO
4 0.5-3g/L
NaCl 0.1-3g/L
NaH
2PO
4 0.05-2g/L
The solvent of above-mentioned enzyme labelled antibody dilution is water.
In the present invention, cleansing solution can adopt conventional ELISA cleansing solution, as the phosphate buffer that contains tween; Sample diluting liquid also can adopt conventional ELISA sample diluting liquid, as the sample diluting liquid that contains phosphate buffer; Negative control is normal human serum; Positive control is the human serum that contains ACA antibody; Nitrite ion can adopt conventional ELISA nitrite ion, as the nitrite ion that contains hydrogen peroxide and/or nitrite ion of containing tetramethyl benzidine etc.
In kit of the present invention, except comprising above-mentioned substance, needed reagent, instrument or container in the time of also can comprising conventional ELISA test, as distilled water or deionized water, washing lotion drop bottle or test tube etc.
The preparation method of the anti-cardiolipin antibody detection kit that the present invention also further discloses, wherein the preparation of the antigen coated pre-coated elisa plate of ACA comprises the following steps:
The antigen coated elisa plate of A.ACA;
B. confining liquid sealing.
The antigen coated elisa plate of ACA adopts conventional coated mode to prepare, and seals through confining liquid.For further improving sensitivity and the specificity of kit, the present invention also improves confining liquid, and improved confining liquid comprises following component: Tirs, BSA, NaN
3and collagen, and the total concentration of above-mentioned each material is 4-10g/L.
Preferably, in above-mentioned confining liquid, the concentration of each component is:
Tirs 0.5-3g/L
BSA 0.5-5g/L
NaN
3 0.001-1.5g/L
Collagen 0.5-5g/L
The solvent of above-mentioned confining liquid is water.
The use of anti-cardiolipin antibody detection kit of the present invention comprises the steps:
A, application of sample: take the antigen coated pre-coated elisa plate of ACA, blank hole, negative serum control wells are established in each experiment
With positive control hole, in each hole, first add sample diluting liquid, then add successively negative serum contrast, positive serum contrast and
Sample, mixes, and puts 37 ℃ of incubation 15-30 minute;
B, wash plate: abandon or adopt liquid in each hole, adopt cleansing solution washing well;
C, enzyme-added: add the IgG enzyme labelled antibody solution of enzyme labelled antibody diluted in each hole, blank hole does not add, and puts 37 ℃ of incubation 15-30 minute;
D, wash plate: abandon or adopt liquid in each hole, adopt cleansing solution washing well;
E, colour developing: in each hole, add developer successively, mix, put 18 ℃-25 ℃ of room temperatures, 10-15 minute;
F, termination; In each hole, add stop buffer successively, mix;
G, mensuration: by microplate reader, blank well is returned to zero, measure the absorbance (OD value) in each hole.
As the anticardiolipin antibody in qualitative detection sample, can, according to the comparison of measuring OD value and Cut off value (negative control mean light absorbency (OD value)+3SD), learn that serum sample anticardiolipin antibody is negative or positive.
By qualitative judgement method, also can obtain semi-quantitative results, by titre, represent the intensity of reaction.In this semiquantitative determination, it is to test after example dilution that sample is done to one, and the high dilution being positive is titre, according to the height of titre, can judge the reactive power of sample, obtains by experiment the threshold value of P/N.
Test confirms, anti-cardiolipin antibody detection kit specificity of the present invention and highly sensitive, and kit good stability, can be used for qualitative and quantitative and detects ACA.
Embodiment
Below enumerate specific embodiment to further illustrate the present invention, should understand embodiment not for limiting the scope of the invention.
The preparation of embodiment 1 kit
1. cuorin antigen (source: Sigma company), through thin-layer chromatography and efficient liquid phase chromatographic analysis comparison, it is consistent that its RF value and peak shape and standard items cuorin meet, and purity > 98%.
The preservation of cuorin antigen: purity has been identified to standard compliant cuorin antigen puts-20 ℃ and save backup.
2. use the preparation of the antigen coated pre-coated elisa plate of cuorin:
2.1 is coated: the cuorin antigen that step 1 is made is to carbonate buffer solution (pH9.6, Na
2cO
31.6g/L, NaHCO
3h
2o3.2g/L formula) in, mix, add in enzyme linked plate holes every hole 100 microlitres, overnight incubation, with the phosphate buffer (20X, the Nacl 85g/L that contain tween, T-200.5ml/L, formula) detersive enzyme yoke plate, complete the preparation with the antigen coated pre-coated elisa plate of ACA;
2.2 sealings: the every hole of pre-coated elisa plate is added to confining liquid 150 microlitres, and overnight incubation, abandons confining liquid, dries up.
Adopt listed confining liquid sealing in following table formula:
| Formula | 1#(g/L) | 2#(g/L) | 3#(g/L) | 4#(g/L) |
| Tirs | 1.8 | 3 | 0.5 | 0.9 |
| BSA (bovine serum albumin(BSA)) | 2.0 | 0.5 | 5 | 1 |
| NaN 3 | 0.001 | 1 | 0.5 | 1.5 |
| Collagen | 2.0 | 5 | 0.5 | 1 |
Contrast confining liquid 1:5% skimmed milk power
Comparison to different confining liquids: (only change confining liquid formula, other reagent and detection method are all identical)
Select the sample of identical serum, with dilution in 1: 100, adopt the coated lath of different confining liquids to contrast detection; The difference that compares the absorbance (OD value) of its positive sample, ' negative ' specimens, blank well;
Result data shows by experiment:
The average OD value 0.03 of confining liquid blank well of the present invention, and the average OD value 0.09 of contrast confining liquid 1 blank well will exceed 3 times, and the OD value of confining liquid positive sample of the present invention is higher, the OD value of ' negative ' specimens is lower, these further illustrate confining liquid of the present invention can effectively suppress non-specific binding, guarantees to detect the accuracy of reagent.In addition, the confining liquid in this kit also can prevent growing of bacterium; Effectively extend use and the holding time of pre-coated plate.
3. the preparation of enzyme labelled antibody dilution
Adopt following formulated enzyme labelled antibody dilution, the pH value of solution is 7.2-7.6:
| Formula | 5#(g/L) | 6#(g/L) | 7#(g/L) | 8#(g/L) |
| C 6H 10O 4·(PO 4) 2·HNa | 2 | 0.5 | 0.7 | 3 |
| Na 2HPO 4 | 1.2 | 3 | 0.5 | 2.2 |
| NaCl | 0.5 | 0.1 | 1 | 3 |
| NaH 2PO 4 | 0.2 | 2 | 0.05 | 0.4 |
Comparison with conventional enzyme labelled antibody dilution:
Contrast enzyme labelled antibody dilution 1 formula: pH7.4; Na
2hPO
4.12H
2o*2.9g/L; NaCL2g/L gram; KCL0.2g/LKH
2pO
40.2g/L; NaN
30.2g/L
Two kinds of different diluent vigour to enzyme labelled antibody: (only change dilution formula, other reagent and detection method are all identical) selects the sample of identical serum, with dilution in 1: 100, relatively its absorbance (OD value)
Result shows: the enzyme labelled antibody dilution of kit of the present invention has improved the release of enzymatic activity greatly, has improved the acting force of enzyme.
| 5# enzyme labelled antibody dilution | 1∶500 | 1∶1000 | 1∶2000 | 1∶3000 | 1∶4000 | 1∶5000 | 1∶6000 | 1∶7000 | 1∶8000 |
| Positive serum 1: 100 | 1.225 | 0.973 | 0.811 | 0.584 | 0.423 | 0.242 | 0.110 | 0.085 | 0.030 |
| Negative serum 1: 100 | 0.210 | 0.154 | 0.083 | 0.056 | 0.059 | 0.048 | 0.033 | 0.005 | 0.009 |
| Contrast enzyme combination diluent 1 | |||||||||
| Positive serum 1: 100 | 0.358 | 0.298 | 0.107 | 0.043 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
| Negative serum 1: 100 | 0.127 | 0.098 | 0.030 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
As seen from the above table, normal enzyme labeling antibody dilution more than 1: 4000 just cannot detect OD value, but adopt the enzyme labelled antibody dilution of kit of the present invention can also reach more than 0.4 at 1: 4000 o'clock, that is to say, the enzyme labelled antibody dilution of employing kit of the present invention can improve the acting force of enzyme, obtain same OD value, the enzyme amount needing is wanted much less, also the cost that can further economize in raw materials, the in the situation that of same enzyme amount, sensitivity is higher.
Get mouse-anti human IgG enzyme labelled antibody, with the dilution in 1: 4500 of enzyme labelled antibody dilution, encapsulation.
4. the preparation of other component:
Cleansing solution: for containing the phosphate buffer of tween, formula: 170 grams of NaCl; T-2010ml; H
2o1000ml,
Nitrite ion: formula substrate A: Na
2hPO
4.12H
2o18.5g/L; Citric acid 5g/L; H
2o
2; 1.ml/L;
Substrate B: TMB0.2g/L; Citric acid 1g/L; EDTA0.15g/L
Stop buffer: be 2M sulfuric acid
The phosphate buffer of sample diluting liquid: 10mM includes 0.5% BSA
Negative control: the negative human serum that contains < 0.1%NaN3.
Positive control: the anti-cuorin serum of people that contains < 0.1%NaN3.
Above-mentioned each solution encapsulates respectively, makes kit with IgG enzyme labelled antibody and the elisa plate assembly of enzyme labelled antibody diluted.
The qualitative detection of embodiment 2ACA
Adopt following method to use the kit of embodiment 1 preparation:
A, application of sample: take the antigen coated pre-coated elisa plate of ACA, each experiment is established blank hole, negative serum control wells, in each hole, is first added sample diluting liquid 100 microlitres, add successively again negative serum contrast, positive serum control wells and each 100 microlitres of sample, mix, stick adhesive sticker bar, put 37 ℃ of incubations 30 minutes;
B, wash plate: abandon or adopt liquid in each hole, cleansing solution, with after 20 times of distilled water dilutings, is filled with to each hole, standing 30 seconds, get rid of cleansing solution, repeat to wash plate, finally pat dry;
C, enzyme-added: add IgG enzyme labelled antibody 100 microlitres by enzyme labelled antibody diluted, blank hole does not add, and sticks adhesive sticker bar, puts 37 ℃ of incubations 30 minutes;
D, wash plate: the same b of method
E, colour developing: in each hole, add successively developer A liquid, each 50 microlitres of B liquid, mix, put room temperature;
F, termination; In each hole, add successively 50 microlitre stop buffers, mix;
G, mensuration: by microplate reader, blank well is returned to zero, measure the absorbance (OD value) in each hole;
According to the average OD value of the result Cut off value=negative control X 2.1 that 500 normal persons' blood sample is detected, serum sample and ratio >=2.1 positive (+) negative control OD value; Serum sample and ratio < 2.1 negative (-) negative control OD value.
By the kit of embodiment 1 (elisa plate of the IgG enzyme labelled antibody of 5# enzyme labelled antibody diluted and 1# confining liquid sealing) for detection of 504 normal persons with 204 through the positive patient of clinical method confirmation ACA, measurement result sees the following form:
| Patient (+) | Normal person (-) | Add up to | |
| Positive (+) | 200 | 4 | 204 |
| Negative (-) | 4 | 500 | 504 |
| Add up to | 204 | 504 | 708 |
Adopt the interpretation of result of SPSS statistical software: susceptibility: 200/204=98%
Specificity: 500/504=99.2%
Accuracy: (200+500)/708=98.87%
Embodiment 3 kit stability tests
The stability experiment of kit, selects five batches of different batches, and every batch of 2 boxes are done stability test, wherein 2 batches of kits are placed in 37 ℃-3 days, three batches are placed in 4 ℃ and keep in Dark Place for-6 months
The physical property testing result of table one, kit
Table two, main performance index testing result
Definite (the determining of threshold value) of embodiment 4 kit sensitivity for analysis
The result judgement of qualitative determination:
In sample, whether to be contained determined antigen or antibody is made to examining:, the simple answer of " having " or " nothing ", use respectively " positive ", " feminine gender " to represent." positive " represents that this sample responds in this mensuration system." feminine gender " is reactionless.
By qualitative judgement method, also can obtain semi-quantitative results, by titre, represent the intensity of reaction.In this semiquantitative determination, it is to test after example dilution that sample is done to one, and the high dilution being positive is titre, according to the height of titre, can judge the reactive power of sample, obtains by experiment the threshold value of P/N.
One. material:
(1) with the kit of embodiment 2
(2) positive serum sample: from clinical typical patient blood serum sample.
Two. calibrating procedure: the method for pressing embodiment 2
1) serum samples diluted liquid was done to two-fold dilution to 1 since 1: 10: 2560, each dilutability is done 2 holes, and every hole application of sample 100ul does the positive simultaneously, each 2 holes of negative control, blank well 1 hole, 37 ℃ of incubations 30 minutes, wash plate 4 times.
2) every hole drips enzyme conjugates (the anti-human IgG McAb of HRP-) 2 (100ul), and 37 ℃ of incubations 30 minutes, wash plate 4 times.
3) every hole drips substrate A, each 1 (50ul) 37 ℃ of incubations of B 15 minutes, and every hole drips 1 of stop buffer (50ul).
4) microplate reader wavelength 450nm place surveys OD value.
5) result is judged: P/N > 2.1, is judged to the positive (N < 0.1, by 0.1 calculating, > 0.1 is by calculated with actual values).
(positive hole OD value/negative control hole OD value > 2.1, is judged to the positive)
Three. result:
| Serum diluting multiple | 1∶10 | 1∶20 | 1∶40 | 1∶80 | 1∶160 | 1∶320 | 1∶640 | 1∶1280 | 1∶2560 | S + | S - | Blank |
| 1 hole | 1.625 | 1.410 | 1.234 | 1.005 | 0.732 | 0.566 | 0.348 | 0.220 | 0.105 | 1.050 | 0.065 | |
| 2 holes | 1.593 | 1.426 | 1.221 | 0.987 | 0.741 | 0.558 | 0.344 | 0.231 | 0.096 | 0.964 | 0.057 | |
| Average | 1.609 | 1.418 | 1.128 | 0.996 | 0.737 | 0.562 | 0.346 | 0.226 | 0.102 | 1.007 | 0.061 |
Four, conclusion:
Serum is diluted at 1: 1280 o'clock, and OD value is 0.226, is greater than 0.21 positively, i.e. the sensitivity of kit can reach 1: 1280, and it is positive that typical patient serum 0.008ul just can measure IgG antibody.
Claims (3)
1. an anti-cardiolipin antibody detection kit, comprising:
(a), with the antigen coated pre-coated elisa plate of ACA;
(b), use the IgG enzyme labelled antibody solution of enzyme labelled antibody diluted; The IgG antibody that described IgG enzyme labelled antibody is horseradish peroxidase-labeled;
(c), cleansing solution;
(d), sample diluting liquid;
(e), negative control;
(f), positive control;
(g), nitrite ion;
(h), stop buffer;
The antigen coated pre-coated elisa plate of described ACA is made by the preparation method who comprises the following steps:
The antigen coated elisa plate of A.ACA;
B. confining liquid sealing;
Confining liquid in described step B comprises following component: Tris, BSA, NaN
3and collagen, and the total concentration of above-mentioned each component is 4-10g/L, in described confining liquid, the concentration of each component is:
The solvent of confining liquid is water.
2. anti-cardiolipin antibody detection kit as claimed in claim 1, is characterized in that, described ACA antigen is cuorin antigen, and purity reaches mass percent more than 98%.
3. anti-cardiolipin antibody detection kit as claimed in claim 1, is characterized in that, in the described IgG enzyme labelled antibody solution by enzyme labelled antibody diluted, the part by weight of IgG enzyme labelled antibody and enzyme labelled antibody dilution is 1:4000-1:5000.
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| CN103163292A (en) * | 2013-03-28 | 2013-06-19 | 天津市宝坻区人民医院 | Secreting type IgA anticardiolipin antibody detection kit and use method |
| WO2018000339A1 (en) * | 2016-06-30 | 2018-01-04 | 深圳市亚辉龙生物科技股份有限公司 | Modified cardiolipin-coated magnetic nanobead and preparation method therefor |
| CN106443005A (en) * | 2016-06-30 | 2017-02-22 | 深圳市亚辉龙生物科技股份有限公司 | Anticardiolipin antibody IgM chemiluminiscence immunoassay kit and preparation method thereof |
| CN109613270A (en) * | 2019-01-10 | 2019-04-12 | 江苏三联生物工程有限公司 | A kind of protein chip and preparation method thereof for systemic loupus erythematosus marker detection |
| CN110982866A (en) * | 2019-12-31 | 2020-04-10 | 莫纳(连云港)生物科技有限公司 | Preparation and sealing method of protein powder for sealing Western blotting |
| CN112285343A (en) * | 2020-10-16 | 2021-01-29 | 黑龙江迪安医学检验所有限公司 | Application method of anti-cardiolipin antibody lgA/G/M detection kit |
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