CN103163300B - Anti-human N-acetamido galactosyl transferase 2 double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) kit - Google Patents
Anti-human N-acetamido galactosyl transferase 2 double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) kit Download PDFInfo
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- CN103163300B CN103163300B CN201310065966.9A CN201310065966A CN103163300B CN 103163300 B CN103163300 B CN 103163300B CN 201310065966 A CN201310065966 A CN 201310065966A CN 103163300 B CN103163300 B CN 103163300B
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Abstract
The invention discloses an anti-human N-acetamido galactosyl transferase 2 double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) kit and application. The kit includes diluent, color reagent, terminated liquid, and an elisa plate for coating an antibody, a detected antibody, an enzyme-labeled antibody and a standard control protein. The detection method includes: samples to be detected are put into the elisa plate for coating the antibody, the color reagent is added to develop color after a capture antibody and the enzyme-labeled antibody are added again for reacting, and an optical density (OD) value is detected. A monoclonal antibody and a polyclonal antibody are taken as main reagents of the kit, various nonspecific interferences are effectively ruled out, specificity is strong, and the different epitopes of the same antigen can be detected, and the detected results are more accurate. The detection instrument is simple, the operation sequence is simple and rapid, detection cost is low, and the kit is suitable for detecting multiple samples, and easy to popularize.
Description
Technical field
The present invention relates to a kind of ELISA kit, be specifically related to a kind of anti-human N-acetylamino galactosamine transferase 2 double-antibody sandwich elisa kit and application thereof, belong to virus detection techniques field.
Background technology
N-acetylamino galactosamine transferase (ppGalNAc-T) family is that on catalytic proteins peptide chain, serine or threonine residues, with one of initial enzyme synthesizing O-sugar chain, are played an important role in tumor marker antigen (Tn antigen) synthesis.Wherein ppGalNAc-T2 is responsible for the glycosylation of serum IgA I hinge area (see J Biol Chem, 2003,278 (8): 5613), Recent study shows, there is unconventionality expression in ppGalNAc-T2, such as in squamous cancerous issue, ppGalNAc-T2 expresses rising in various human tumor tissues; In colon cancer, found that the exception that ppGalNAc-T2 expresses raises; The difference of Guo Xianghong application RT PCR method research leukemia cell line k562 and SHI-1 glycosyl transferase mrna expression and inquired into the difference of malignant myeloid cell lines K562 and SHI-1 glycosyltransferases expression, finds to exist the expression of ppGalNAc-T2 higher level in K562 and SHI-1 (see the Chinese Hemorheology magazine 2005 such as Guo Xianghong; 15 (1): 32); Importantly can infer for these discoveries, these unconventionality expression situations being present in ppGalNAc-T2 in tumour can become potential diagnosing tumor foundation.
Takuhiro Kohsaki etc. from Japan detects colon cancer cell and tissue with the anti-human ppGalNAc-T2 polyclonal antibody of rabbit, finds that ppGalNAc-T2 exists obvious high expressed (reference: J Gastroenterol 2000 35:840); Mouse-anti people ppGalNAc-T2 monoclonal antibody has just successfully been prepared and called after UH4 (IgG1) by the Ulla Mandel research group of Denmark, can be used for indirect immunoperoxidase connection adsorption test (ELISA), immunofluorescent flow cellular assay and co-immunoprecipitation method, this research group has also done comparatively careful deep research (reference: Glycobiology 1,999 9 (1): 43) to this antibody.
On the other hand, the method of application double-antibody sandwich elisa kit, to macromolecular detections such as various protein, does not need dilution by inspection sample, can be directly used in mensuration, therefore its susceptibility is relatively higher than all the other method of testings, has simple to operate, quick, sensitive, feature accurately.The glycosyl transferase examination of application double antibody sandwich ELISA to tumor patient has relevant report, the ELISA kit such as like fucosyl transferase, sialyltransferase, for the examination of Serum of Cancer Patients, all show good using value and prospect.But there is not yet about anti-human ppGalNAc-T2 double-antibody sandwich elisa kit and utilize it to the report of anti-human ppGalNAc-T2 albumen vitro detection.
Summary of the invention
The object of this invention is to provide a kind of high specificity, degree of accuracy high, easy and simple to handle, detect anti-human ppGalNAc-T2 double-antibody sandwich elisa kit fast, and disclose its application process.
To achieve the above object of the invention, the technical solution used in the present invention is: a kind of anti-human N-acetylamino galactosamine transferase 2 double-antibody sandwich elisa kit, and comprise cleansing solution, sample diluting liquid, nitrite ion, stop buffer, it is characterized in that, it also comprises:
(1) ELISA Plate of coated antibody: with anti-human N-acetylamino galactosamine transferase 2 monoclonal antibody for coated antibody, with coating buffer, monoclonal antibody is diluted to 10 μ g/ml, every hole adds 100 μ L, 4 DEG C of refrigerator overnight, with cleansing solution washing 2 ~ 4 times, then in ELISA Plate, add confining liquid, 100 μ L/ holes, react 1 ~ 2h at 37 DEG C, cleansing solution washing obtains for 2 ~ 4 times;
Described confining liquid is 1% bovine serum albumin(BSA), 5% bovine serum albumin(BSA), 1% horse serum, 5% horse serum or 5% skimmed milk power;
(2) antibody is detected: rabbit source anti-human N-acetylamino galactosamine transferase 2 polyclonal antibody;
(3) enzyme labelled antibody: the goat-anti rabbit enzyme labelled antibody of horseradish peroxidase-labeled;
(4) standard control albumen: comprise standard positive control N-acetylamino galactosamine transferase 2 albumen, standard negative control coli somatic albumen.
In technique scheme, the hybridoma cell strain LW-5F3 that described anti-human N-acetylamino galactosamine transferase 2 monoclonal antibody is CCTCC NO:C2010106 by deposit number secretes obtained, and preparation method is disclosed by Chinese invention patent CN102061287B.
In technique scheme, the preparation method of described anti-human N-acetylamino galactosamine transferase 2 polyclonal antibody is: anti-human N-acetylamino galactosamine transferase 2 albumen and Freund's complete adjuvant are carried out 1: 1 and mix and carry out purifying, the White Rabbit between 2.0 ~ 2.5 ㎏ is selected to carry out four immunity, time is respectively the 1st day, 14th day, 28th day and the 42nd day, within after the 4th immunity one week, carry out arteria carotis blood sampling, separation of serum, obtains rabbit source anti-human N-acetylamino galactosamine transferase 2 polyclonal antibody.
In technique scheme, the preparation method of described coating buffer is: 1.59g Na
2cO
3with 2.93g NaHCO
3add water after fully dissolving, adding deionized water constant volume is 1L.
The invention also discloses the application of above-mentioned double-antibody sandwich elisa kit in anti-human N-acetylamino galactosamine transferase 2 albumen vitro detection, comprise the following steps:
(1) add measuring samples: the ELISA Plate of getting the coated antibody in kit, add treated measuring samples wherein, 100 μ L/ holes, set up positive control and negative control simultaneously respectively, react 45 ~ 75min at 37 DEG C, cleansing solution washing 2 ~ 4 times;
(2) add detect antibody: get the detection antibody in kit, with sample diluting liquid by 1: 100 volume ratio dilute after join in above-mentioned ELISA Plate, 100 μ L/ holes, at 37 DEG C reaction 25 ~ 35min, cleansing solution wash 2 ~ 4 times;
(3) add enzyme labelled antibody: get the enzyme labelled antibody in kit, with cleansing solution according to 1: 500 ratio carry out diluting and mixing, 100 μ L/ holes, at 37 DEG C react 45 ~ 75min, with cleansing solution washing 2 ~ 4 times;
(4) develop the color: add nitrite ion, 100 μ L/ holes, at room temperature develop the color 10 ~ 15min, uses stop buffer color development stopping;
(5) densitometric value: measure by microplate reader, densitometric value.
In the present invention, cleansing solution, sample diluting liquid, nitrite ion, stop buffer belong to prior art, and those skilled in the art can configure as required, and the present invention is preferred:
Cleansing solution: get NaCl 8g, KH
2pO
40.27g, Na
2hPO
41.42g, KCl 0.2g, adds 0.5mL Tween-20 after dissolving, fully mix with 800mL distilled water, and adjustment PH to 7.2, is settled to 1000mL;
Sample diluting liquid: get NaCl 8g, KH
2pO
40.27g, Na
2hPO
41.42g, KCl 0.2g, adds 10g standard bovine albumin BSA after dissolving, fully dissolve mixing with 800mL distilled water, and adjustment PH to 7.2, is settled to 1000mL;
Nitrite ion comprises substrate solution A and substrate solution B, wherein substrate solution A: get 3,3
,, 5
,, 5-tetramethyl biphenyl diamines 0.2g, absolute ethyl alcohol 100mL, adds distilled water and is settled to 1000mL; Substrate solution B: get Na
2hPO
414.6g, citric acid 9.3g, 0.75% hydrogen peroxide urea solution 6.4mL, adds distilled water 800mL, and regulate PH to 5.2, supplementary distilled water is settled to 1000mL;
Stop buffer: 2M H
2sO
4solution.
The present invention utilizes anti-human ppGalNAc-T2 monoclonal antibody and anti-human ppGalNAc-T2 polyclonal antibody, prepares anti-human ppGalNAc-T2 double-antibody sandwich elisa kit; Adopt DASELISA immunoadsorption assay (ELISA) method, detect the content of anti-human ppGalNAc-T2 in patients serum with this kit, especially leukaemic.By the content to ppGalNAc-T2 anti-human in dissimilar serum of leukaemia, and ppGalNAc-T2 comparision contents anti-human with normal human serum, provide an important reference frame by these differences to leukaemia early detection.
Because technique scheme is used, the present invention compared with prior art has following advantages:
1. the present invention adopts anti-human ppGalNAc-T2 monoclonal antibody and anti-human ppGalNAc-T2 polyclonal antibody as the main agents of kit, effectively eliminate multiple nonspecific interference, high specificity, and the different epi-positions that can detect same antigen, make testing result more accurate.
2. detecting instrument of the present invention is simple, and simple operating steps, fast, testing cost is low, is applicable to the detection of a large amount of sample, is easy to popularize.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
Embodiment one: the preparation of the anti-human ppGalNAc-T2 polyclonal antibody in rabbit source
With recombinant anti human ppGalNAc-T2 albumen (1mg/mL) disclosed in Chinese invention patent CN102061287B as immunogene, get this antigen 1 00 μ L, carry out 1:1 with Freund's complete adjuvant and mix.Antigen and adjuvant are mixed to form stable emulsion (Water-In-Oil) by vortex shaker completely, hypodermic injection will be carried out and rear thigh carries out intramuscular injection under the skin of this emulsion around rabbit both shoulders, the immunogene of 1/4 is approximately used in each region, and such immunogene can exist lastingly thus improve immune response.
Need before antigen injection to collect some normal serums, as negative control during for detecting antibody.Inject and carry out rabbit ear arterial blood extracting after four days, amount for taking blood 3ml.First immunisation FCA, uses FIA later.Adopt the injecting method of initial immunity, interval 2 Zhou Houzai selects difference to inject (do not select to close on site, otherwise ulcer healing being bad) in above-mentioned position.The antigen amount of each immunity is identical, immunity four times.
Can at initial immunity after one week, rabbit ear edge venous blood sampling 1mL detects antibody titer.Adopt ELISA method, concrete operation step: get immunize rabbit serum and each 50 μ L of normal rabbit serum as antibody to be checked, detect and the previous day ppGalNAc-T2 recombinant protein coating buffer press 1:20 dilution and wrap by ELISA Plate with the 100 every holes of μ L, 4 DEG C of bag quilts that spend the night, then 3 times are washed with 0.01 PBS cleansing solution, then close and hatch 2h, 100 μ L(1:500 are added after washing) goat antirabbit of horseradish peroxidase-labeled two resists, cessation reaction after colour developing 15min, microplate reader surveys OD450, positive criteria is made with negative and positive hole OD ratio, P/N > 2.1 is positive, wherein P is the OD value of measuring samples, N is the OD value of negative control.
Get blood after last immunity and can adopt arteria carotis bloodletting, concrete operations are as follows: rabbit lies on the back fixing head on rabbit frame, use gauze fixing limbs, head is slightly lowerd to expose neck, shave hair and skin of sterilizing, cut skin along cervical midline scalpel and be about 10cm, be separated subcutaneous connective tissue, until expose the nutator of tracheae both sides, the neck trigonum open texture between nutator and tracheae is separated with straight head hemostatic forceps, make it free with elbow ophthalmic tweezers after exposing arteria carotis communis, peel off neural and connective tissue, two black silk threads are inserted under artery, be placed in the heart far away and proximal part respectively, the silk thread of ligation distal end, the artery blood vessel clip of proximal part is clamped, with little finger pad under blood vessel, the artery ancient piece of jade, round, flat and with a hole in its centre cutting between two rhizoid lines with tip ophthalmologic operation cuts an osculum (not cutting off), insert plastics bloodletting pipe, again the silk thread ligation of proximal part is fixed on bloodletting pipe, in case the slippage of bloodletting pipe, unclamp hemostatic forceps, blood is flowed in container.Blood sample room temperature is left standstill 2h, then the centrifugal 20min of 3000rpm, draw the transparent serum in upper strata, by serum packing ,-80 DEG C of preservations.
Embodiment two: the preparation of enzyme labelled antibody
Get 40ml rabbit anteserum and be placed in beaker, add 40m1 0.O1M pH7.2 PBS and mix, then dropwise add 80ml saturated ammonium sulfate solution, limit edged stirs, and room temperature leaves standstill 30 minutes, and centrifugal 30 minutes of 3000rpm, goes supernatant to stay precipitation.In precipitation, add 80mlPBS dissolution precipitation, then add saturated ammonium sulfate solution and reach 33% saturation degree, limit edged stirs, and room temperature leaves standstill 30 minutes, and 3000rpn goes supernatant to stay precipitation in centrifugal 20 minutes, 2 ~ 3 times so repeatedly.Again sediment is dissolved in 12ml water. to 0.07M pH6.3 phosphate buffer 4 DEG C dialysis, until outer liquid and Nessler's reagent or 1% BaCl react and be negative as IP.Dialyse complete, centrifugal 5 minutes of 3000rpm, the IgG solution (enzyme labelled antibody) going supernatant to be slightly to carry.
Embodiment three: ELISA Plate reaction homogeneity detects
Stochastic choice 3 pieces of ELISA Plate, IgM(G according to 1 μ g/ml rabbit), 100 μ L/ holes add, 4 DEG C are spent the night, discard and wash, repeated washing 3 times, ELISA Plate is patted dry, add cow's serum confining liquid (10%) 200 μ L/ hole carry out 37 DEG C close 1 hour, wash away confining liquid (the same), then 100 μ L/ holes add the goat anti-rabbit antibody (1:500) of horseradish peroxidase-labeled, hatch 1h for 37 DEG C, washing; Then add 100 μ L/ hole nitrite ion lucifuge 15min, add 50 μ L/ hole stop buffers, microplate reader surveys OD value, calculates the coefficient of variation between each hole, detects ELISA Plate homogeneity.
Embodiment four: the anti-the best bag of Sheet is determined by concentration and how anti-best effort concentration
Dilute by the anti-human ppGalNAc-T2 monoclonal antibody of coating buffer by purifying, ratio is followed successively by: 1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400,1:10000,1:12800,1:25600; Wrap respectively and spent the night (4 DEG C) by plate.With the PBS dilution containing 1% hyclone, the rabbit of purifying resisting is diluted according to 1:100,1:200,1:400,1:800,1:1600,1:3200,1:6400,1:10000 morely, carry out application of sample according to square formation.With L929-ppGalNAc-T2 cytolytic proteins do positive, L929-MOCK does negative control, carries out sandwich ELISA, often group establishes 3 multiple holes.According to yin and yang attribute ratio P/N > 2.1, the OD mean value of relatively more each group, determines that the suitableeest monoclonal antibody bag is by concentration and suitable much the most anti-concentration.
Embodiment five: confining liquid and off-period are optimized
With the suitableeest monoclonal antibody concentration 4 DEG C of coated elisa plates that spend the night, after washing, close with the confining liquid that 1%BSA, 5%BSA, 1% horse serum, 5% horse serum, 5% skimmed milk power 5 kinds are different respectively, 200 μ l/ holes, close 2 hours for 37 DEG C, carry out double crush syndrome, respectively organize the OD value of yin and yang attribute sample, to determine the sealing effect of different confining liquid.
With the suitableeest monoclonal antibody concentration 4 DEG C of coated elisa plates that spend the night, after washing, 200 μ l/ holes add best confining liquid 37 DEG C and close, and select 0.5,1.0,1.5,2,3 hour off-period, carry out ELISA respectively, survey OD value.Respectively organize the OD value of yin and yang attribute sample, to determine best off-period.
Embodiment six: antibody diluent optimization
With the suitableeest monoclonal antibody concentration and bag by condition coated elisa plate, yin and yang attribute sample is added after closing, many anti-and ELIAS secondary antibody three each dilution proportion liquid 1:100,1:500 and 1:3000 dilutions respectively, l group 1%BSA PBST, the 2nd group with 5%, 3rd group with 5% horse serum PBST, 37 " react after 2 hours under C, through washing, add substrate and to develop the color 15 minutes cessation reactions; microplate reader measures OD450 value, to determine optimum antibody dilution.
Embodiment seven: detection limit is determined
With the monoclonal antibody bag of 5pg/ml by good ELISA Plate, 8 dilutabilitys clinical serum being carried out 1:10,1:50,1:100,1:500,1:1000,1:2000,1:5000,1:10000 carry out ELISA mensuration, set up yin and yang attribute to contrast simultaneously.
Embodiment eight: replica test is identified
Repeatability checking in plate: be placed in 37 DEG C of 1h by plate with monoclonal antibody bag, then 4 DEG C are spent the night, washing rear enclosed, the sample that detection 6 parts is different in same plate: 3 parts of positive reference substances and 3 parts of negative samples, every increment originally repeats 3 holes, carry out ELISA detection according to the top condition optimized, calculate the coefficient of variation CV% of same this OD450 of increment value, be used for evaluating the repeatability detecting sample in plate.
Repeatability checking between plate: wrap by good ELISA Plate under the same conditions with 6 pieces, different time points duplicate detection 6 increment product: 3 parts of positive reference substances and 3 parts of negative samples, every increment originally repeats 3 holes, ELISA detection is carried out according to the top condition optimized, calculate the coefficient of variation CV% of same this OD450 of increment value, be used for evaluating the repeatability detecting sample in plate.With CV% lower than 10%, and there was no significant difference is critical criterion.
Claims (4)
1. a people N-acetylamino galactosamine transferase 2 double-antibody sandwich elisa kit, comprise cleansing solution, sample diluting liquid, nitrite ion, stop buffer, it is characterized in that, it also comprises:
(1) ELISA Plate of coated antibody: with anti-human N-acetylamino galactosamine transferase 2 monoclonal antibody for coated antibody, with coating buffer, monoclonal antibody is diluted to 10 μ g/ml, every hole adds 100 μ L, 4 DEG C of refrigerator overnight, with cleansing solution washing 2 ~ 4 times, then in ELISA Plate, add confining liquid, 100 μ L/ holes, react 1 ~ 2h at 37 DEG C, cleansing solution washing obtains for 2 ~ 4 times;
Described confining liquid is 1% bovine serum albumin(BSA), 5% bovine serum albumin(BSA), 1% horse serum, 5% horse serum or 5% skimmed milk power;
(2) antibody is detected: rabbit source anti-human N-acetylamino galactosamine transferase 2 polyclonal antibody;
(3) enzyme labelled antibody: the goat-anti rabbit enzyme labelled antibody of horseradish peroxidase-labeled;
(4) standard control albumen: comprise standard positive control N-acetylamino galactosamine transferase 2 albumen and standard negative control coli somatic albumen.
2. double-antibody sandwich elisa kit according to claim 1, is characterized in that: the hybridoma cell strain LW-5F3 that described anti-human N-acetylamino galactosamine transferase 2 monoclonal antibody is CCTCC NO:C2010106 by deposit number secretes obtained.
3. double-antibody sandwich elisa kit according to claim 1, it is characterized in that: the preparation method of described anti-human N-acetylamino galactosamine transferase 2 polyclonal antibody is, people N-acetylamino galactosamine transferase 2 albumen and Freund's complete adjuvant are carried out 1: 1 to mix and carry out emulsification, the White Rabbit between 2.0 ~ 2.5 ㎏ is selected to carry out four immunity, time is respectively the 1st day, 14th day, 28th day and the 42nd day, within after the 4th immunity one week, carry out arteria carotis blood sampling, separation of serum, obtains rabbit source anti-human N-acetylamino galactosamine transferase 2 polyclonal antibody.
4. double-antibody sandwich elisa kit according to claim 1, is characterized in that, the preparation method of described coating buffer is: 1.59g Na
2cO
3with 2.93g NaHCO
3add water after fully dissolving, adding deionized water constant volume is 1L.
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| CN201310065966.9A CN103163300B (en) | 2013-03-01 | 2013-03-01 | Anti-human N-acetamido galactosyl transferase 2 double-antibody sandwich enzyme-linked immuno sorbent assay (ELISA) kit |
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| CN105510585A (en) * | 2015-12-01 | 2016-04-20 | 苏州市博力生物科技有限公司 | Double-antibody sandwich ELISA kit for human beta3 acetylglucosaminyltransferase 8 (beta3GnT8) |
| CN109825579B (en) * | 2019-01-23 | 2020-01-14 | 山东大学齐鲁医院 | Application of GALNT2 as biomarker in glioma diagnosis and/or treatment |
| CN114740205A (en) * | 2022-05-24 | 2022-07-12 | 君研生物科技(山西)有限公司 | Double-antibody sandwich ELISA (enzyme-Linked immuno sorbent assay) detection kit for dispase II |
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| US5861318A (en) * | 1994-11-16 | 1999-01-19 | Pharmacia & Upjohn Company | Scintillation proximity assay for N-acetylgalactosaminyltransferase activity |
| JP2008512085A (en) * | 2004-06-03 | 2008-04-24 | ネオス テクノロジーズ インコーポレイティッド | Cleaved GalNAcT2 polypeptides and nucleic acids |
| CN101042403A (en) * | 2007-04-25 | 2007-09-26 | 华南农业大学 | ELISA kit for detecting EPSPS gene in herbicide-tolerance soybeans and method of use thereof |
| CN101386650A (en) * | 2008-10-30 | 2009-03-18 | 上海交通大学 | Method for preparing ppGalNAc-T18 specific polyclonal antibody |
| CN102061287B (en) * | 2010-11-23 | 2012-10-03 | 苏州大学 | Anti-human ppGa1NAc-T2 monoclonal antibody and application thereof |
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