CN101281196A - HIV-1 p24 antigen acridine ester chemiluminescence immune analyse detecting method - Google Patents
HIV-1 p24 antigen acridine ester chemiluminescence immune analyse detecting method Download PDFInfo
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- CN101281196A CN101281196A CNA2008101116781A CN200810111678A CN101281196A CN 101281196 A CN101281196 A CN 101281196A CN A2008101116781 A CNA2008101116781 A CN A2008101116781A CN 200810111678 A CN200810111678 A CN 200810111678A CN 101281196 A CN101281196 A CN 101281196A
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Abstract
HIV-1 p24 antigen acridine ester chemiluminescence immunity analyzing testing method pertains to the clinical blood testing analysis method technique field. The prior HIV-1 p24 antigen testing method has problems of low sensitivity, rigmarole operations and the like. The invention uses acridine ester series compounds to mark the HIV-1 p24 antibody, adopts a double antibody/antigen sandwich method to execute immunity combination between the p24 antigen-antibody; uses excitant hydrogen peroxide, nitric acid, Triton X-100, and sodium hydrate to excite acridine ester series compounds to execute chemiluminescence reaction; and tests the number of photon to execute a qualitative and/or quantitative analysis. The invention has good correlativity with the general ELISA testing method, which has a correlation coefficient of 0.951 at the instance of P no more than 0.01; has high sensitivity, wherein, the testing limit is 0.5pg/ml; has wide detection range in 5-6 magnitude order; and the invention also has advantages of short reaction time, easier operation and the like.
Description
Technical field
The invention belongs to clinical blood check and analysis method and technology field, be specifically related to a kind of method clinical and laboratory detection HIV-1p24 antigen that is used for.This method is highly sensitive, sensing range is wide, easy and simple to handle, at aspects such as the monitoring of neonate HIV-1 diagnosis of infection, HIV-1 the infected's PD, drug resistance monitoring, antiviral therapy Evaluation on effect important application is arranged.
Background technology
After HIV-1 (Human immunodeficiency virus) the virus infections human body, in host cell and virus, can produce significant material (the Niel T.Constantine that some HIV infect, Holly Zink.HIV testing technologies after twodecades of evolution.Indian J Med Res 121.2005:519-524), we can carry out in vivo the situation of duplicating of virus examination, monitoring virus, understand the development of infecting the back state of an illness, and grasp the immune system situation of human body in real time by these marks.Behind the virus infections, these marks are along with the virus infection process, and p24 antigen is one of thing as a token of, have been subjected to people's extensive concern aspect viral diagnosis.
Usually to detect be sensitivity and specific method to the nucleic acid of HIV-1 (RNA), but aspect practical application, and the nucleic acid check has and much is not easy to the factor promoted.Detection of nucleic acids is compared with other method of inspection, and required experimental expenses is relatively more expensive, and required experimental apparatus and experiment condition are also relatively harsher.In addition, the complicated operation of nucleic acid check in order to increase the reliability of assay, will the realistic operating personnel of testing have than higher professional operant level.Simultaneously, the nucleic acid experiment is whole consuming time longer.And from global HIV patient's distribution situation (UNAIDS, WHO.AIDS epidemic update 2006.), most the infected is distributed in the area of economics of underdevelopment, and as Africa etc., this has just caused inconvenience for the popularization that the RNA of HIV-1 detects in the under-developed area.
A lot of trials on other method of inspection, have been done in order to solve these problem people on the one hand, as TLC, red blood cell detection, seralbumin detection, p24 Detection of antigen, anti-p24 antibody test etc., attempt to substitute the coherent detection that nucleic acid detection method carries out acquired immune deficiency syndrome (AIDS) with this, wherein the research of HIV-1p24 antigen detection method is the most extensive.All has good effect at neonate HIV-1 diagnosis, the monitoring of HIV-1 the infected's PD, the aspects such as check of antiviral therapy effect, and have good correlativity with nucleic acid detection method, be expected to substitute detection of nucleic acids and extensively promote in vast under-developed area.
HIV-1p24 antigen detection method commonly used is enzyme-linked immunosorbent assay (Enzymelinked immuno-sorbent assay, ELISA), and adopt the double antibodies sandwich method to detect more, and the sensitivity of detection is at 3.5-10pg/ml, and detection width is at 2-3 the order of magnitude.In order to improve detection sensitivity, make the HIV-1p24 antigen detection method can be aspect detection sensitivity and specificity constantly near the level of detection of nucleic acids, people have done various improvement in twenties years in the past on common ELISA basis, and wherein successful method has ELASTELISA method and IPCR (Immune polymerase chain reaction)-ELISA method etc. the most.Ledergerber et al reports that in the literature (Perkin-Elmer Life Sciences, Boston MA) carry out HIV-1 type p24 detection of antigens, and its detectability can reach 0.5pg/ml to use ELAST ELISA method.Janet M.Barletta uses the immuno-PCR method, and (NY) combination detects lower bound and can reach 1000 p24 antigen molecules for Zeptomertrix, Buffalo with the ELISA method.
But analyze above two kinds of methods from practical application: the applied amplification system of first method mainly is the biotin-streptavidin amplification system, the nonspecific reaction of appearance in the blood serum sample check of this system regular meeting, operation steps increases after introducing amplification system simultaneously, reaction time also can prolong, and also will increase in order to reduce the nonspecific reaction washing times; The immuno-PCR reaction of introducing in second kind of system is increasing on reaction time and the operation steps outside the difficulty, has also increased inconvenience in the operation of experimental system in the nucleic acid marking reaction.
Its principle of chemiluminescence immune analysis method is similar to enzyme-linked immunoassay method, is that chemiluminescent substance and immunological response are combined, and uses up the tested immunizing composition concentration of reaction performance.Promptly high-sensitive light reaction is combined with specific immunological response, come materials such as antigen, antibody are carried out the method for quantitative test with luminous intensity.Wherein chemiluminescence is an emission phenomenon of following the light that chemical reaction process produces.Some material has absorbed the chemical energy that is produced in the course of reaction when carrying out chemical reaction, make reaction product be energized into excited electronic state from molecular state.When electronics produces radiation when the lowest vibration energy level of excited state is got back to each vibrational energy level of ground state, unnecessary energy discharges with the form of photon, this phenomenon be called chemiluminescence (Wu Jianmin. clinical chemistry robotization immunoassay. Science Press, 2000).
Chemiluminescence immune analysis method self has the very high sensitivity (C.Dodeigne of sensitivity, L.Thunus, R.Lejeune.Chemiluminescence asDiagnostic Tool.Talanta 2000,51:415～439), owing to do not need external light source, avoided noises such as Rayleigh scattering and Raman scattering, thereby background is low and have a signal to noise ratio (S/N ratio) higher than fluorescence method, high 1 to 2 order of magnitude of its remolding sensitivity enzyme-linked immuno assay or radioimmunoassay method; Chemical illuminating reagent itself also has the advantage A.C.Calokerinos that detects linear wide ranges in addition, N.T.Deftereos, W.R.G.Baeyens.Chemiluminescence in Drug Assay.Journal of Pharmaceutical and BiomedicalAnalysis.1995,13:1063-1071.), can reach 6-7 the order of magnitude.
The acridinium ester compounds has obtained paying close attention to widely in the research of chemiluminescence immune assay system as a kind of common chemical luminescence reagent.It has the advantage of highly sensitive, easy mark on albumen, polypeptide and the micromolecule.And because acridinium ester or acridine sulfamide compound are having H
2O
2Dilute alkaline soln in chemiluminescence can take place, need not catalytic process, do not need reinforcing agent yet, thereby reduced background luminescence, improved signal to noise ratio (S/N ratio), therefore interference effect is few, is the desirable luminous substrate of chemiluminescence immune assay.For example, the ACS-180 immunoassay kits system of U.S. CibaCorning company development adopts the direct mark of dimethyl acridinium ester, and sensitivity can reach 10
-15G/l.Being used for carrying out solid-phase immunity learns the acridinium ester compounds of reaction and can select 4-(2-succinimide base carboxyl) phenyl-10-methylacridine-9-carboxylate fluoro sulfonate (Acridinium C
2NHS Ester) etc., clinically at present be used for thyroid function, reproductive physiology, tumor markers, drug surveillance and a plurality of projects such as cardiovascular are applied.
But yet there are no the report that acridinium ester chemistry luminescence immunoassay detection method is used for the HIV-1p24 Detection of antigen.
Summary of the invention
The object of the present invention is to provide a kind of highly sensitive, sensing range is wide, simple, quick, the HIV-1p24 antigen acridine ester of being convenient to robotization is learned the luminescence immunoassay detection method.
The present invention adopts the method for double antibodies sandwich, and is luminous with the acridinium ester of exciting agent excitation labeling on anti-HIV-1 p24 antibody, carries out the qualitative and quantitative analysis of HIV-1p24 antigen, and concrete steps are as follows:
(1) with anti-HIV-1 p24 antibody solid phase carrier is wrapped quilt;
(2) handle the HIV-1 sample, the HIV-1 sample that promptly dissociates separates HIV-1p24 antigen;
(3) reaction system in the anti-HIV-1 p24 pairing antibody adding step (1) of HIV-1 sample after handling in the step (2) and acridinium ester compounds mark is carried out immune response;
(4) with the reaction system in exciting agent 1 and the exciting agent 2 adding steps (3), carry out chemiluminescence reaction;
(5) detect the photon number that chemiluminescence reaction produces in the step (4), carry out qualitative or/and quantitative test.
Wherein, the anti-HIV-1 p24 antibody in the step (1) can wrap quilt to various solid phase carriers, as polystyrene latex, polystyrene plastics goods, polyvinyl chloride plastic material products, liposome, immunity magnetic micropearls etc.
HIV-1 sample described in the step (2) is the HIV-1p24 antigen of originating arbitrarily.Arbitrarily the source is meant, HIV-1 HIV patient's blood, is the p24 recombinant antigen that expression vector obtains according to genetic engineering with the cellular incubation product that obtains behind the HIV-1 virus infection cell, with prokaryotes or eucaryote.
Wherein, blood need be by the centrifugal serum that obtains of physical method.The cellular incubation product needs physical method, chemical method and biological method to make its cracking, its supernatant of centrifuging and taking.Recombinant antigen need make its purifying by physical method, chemical method and biological method.
The p24 antigen in blood source and cell culture source, form with a kind of or combination in any of the compound of free p24 antigen molecule and p24 antigen and antibody thereof exists, and need add thermal dissociation in advance or acidity is dissociated so that antigen dissociates out from antigen antibody complex.
HIV-1 sample described in the step (2) comprises the HIV-1 type virus of any type.Any type is meant: a kind of or combination in any of M subgroup and O subgroup.Comprise 10 hypotypes such as A, B (B '), C, D, E, F, G, H, I and J in the described M subgroup.
Adopt acridinium ester compounds marker ligand antagonist in the inventive method.Mainly by chemical reaction that a kind of molecule is covalently bound to another kind of molecule, two kinds of materials that participate in coupling reaction are called label and are labeled thing.The purpose of chemical labeling is to make the character (as immunological properties) that is labeled thing maintenance self and have some character (as luminosity) of label again.The acridinium ester compounds makes acridinium ester combine by the amino of covalent bond with the albumen that is labeled, polypeptide or nucleic acid by chemical reaction.
Nitric acid involved in the present invention and Qu Latong 100 are the luminous exciting agent of acridinium ester compounds, reinforcing agent, and making that the acridinium ester compounds is instantaneous under the effect of exciting agent can be luminous, with enzyme-linked immuno assay reacting phase in the past than the time that has shortened operation greatly.
The exciting agent of acridinium ester compounds involved in the present invention detects with the instant method that detects of instant application of sample, luminously can detect photon number, does not need to add to stop extra reaction such as reagent, signal amplification system, has also reduced operation steps.
The automaticity of common chemiluminescence immune analysis method is very high, present full-automatic chemical light-emitting appearance both domestic and external is a lot, as the ACS:180 of Ciba Corning company, the Multilabel Counter VICTOR 1420 of PerkinElmer company etc.The method of the instant adding exciting agent that the inventive method adopts for automation mechanized operation, reduce the manual operation error, to improve detection sensitivity all helpful.
Compared with prior art, the present invention has following beneficial effect:
1) sensitivity is higher, can reach 0.5pg/ml, reaches advanced world standards.
2) sensing range broad can reach 5-6 the order of magnitude, and the sample of higher concentration be need not dilution can be detected.
3) easy and simple to handle, the reaction time is short, and the acridinium ester compounds can be luminous in two seconds under the exciting agent effect.
4) help the automation mechanized operation of reaction system.
Description of drawings:
The Hooke effect of Fig. 1, acridinium ester chemistry luminescence immunoassay HIV-1p24 antigen.
Fig. 2, the match of acridinium ester chemistry luminescence immunoassay HIV-1p24 antigen typical curve.
The invention will be further described below in conjunction with the drawings and specific embodiments.
Embodiment
Material and reagent:
Acridinium ester (Acridinium C
2NHS Ester) available from Assay designs company;
Solid phase carrier (white 96 microwell plate Immuno
TMMaxiSorp
TM) available from Nunc company;
Bag is cushioned liquid: Na
2CO
3-NaHCO
30.05M
pH 9.5
Lavation buffer solution: Na
2HPO
4-NaH
2PO
40.01M
NaCl 0.9％
pH 7.4
Sealing damping fluid: Na
2HPO
4-NaH
2PO
40.05M
NaCl 0.9％
pH 7.4
Analysis buffer: Na
2HPO
4-NaH
2PO
40.05M
NaCl 0.9％
Tween-20 0.05％
BSA 0.5％
pH 7.0
Exciting agent 1:HNO
30.1M
H
2O
2 0.08M
Exciting agent 2:Triton X-100 2%
NaOH 0.2M
The HIV-1 sample:
HIV-1p24 recombinant antigen (virus provides);
HIV-1p24 antigen country's reference material (available from Nat'l Pharmaceutical ﹠ Biological Products Control Institute) comprising: 20 HIV-1p24 antigen negative reference materials are numbered N1-N20; 10 HIV-1p24 antigen positive reference materials are numbered P1-P10; 10 branch lines personality sensitivity reference material is numbered L1-L10; HIV-1p24 Detection of antigen precision reference material is numbered AgCV;
Negative control: human serum (Vironostika
HIV-1Antigen, bioMerieuxInc., Durham, NC);
Positive control: reorganization p24 antigen (Vironostika
HIV-1Antigen, bioMerieux Inc., Durham, NC).
Embodiment
1) be cushioned liquid with bag and will wrap quilt with behind anti-p24 antibody dilution to the 6 μ g/ml, in white 96 microwell plates, every hole adds 100 μ l, and 4 ℃ were sealed 16 hours;
2) get rid of liquid in the clear opening, the every hole of lavation buffer solution is added 300 μ l washing, washed 3 minutes at every turn, wash altogether 3 times;
3) every hole adds sealing damping fluid 300 μ l, 37 ℃ of isothermal vibrations 1 hour;
4) get rid of liquid in the clear opening, the every hole of lavation buffer solution is added 300 μ l washing, washed 3 minutes at every turn, wash altogether 3 times, dry back 4 ℃ of preservations;
5) with concentration be 80,40,20,10 respectively, five positive control sample of 5pg/ml add in the hand-hole, 2 multiple holes of each concentration, every hole adds 100 μ l, the negative control sample is added respectively in 4 holes, and every hole adds 100 μ l, and HIV-1p24 antigen country's 40 samples of reference material (N1-N20, P1-P10 and L1-L10) are respectively added 1 hole, every hole adds 100 μ l, the AgCV sample is added respectively in 4 holes, and every hole adds 100 μ l, and 37 ℃ of constant temperature vibrated 1 hour;
6) get rid of liquid in the clear opening, the every hole of lavation buffer solution is added 300 μ l washing, washed 3 minutes at every turn, wash altogether 3 times;
7) with analysis buffer the anti-p24 antibody of acridinium ester mark is carried out 1: 200 times of dilution after, every hole adds 100 μ l;
8) the anti-HIV-1 p24 antibody of HIV-1 sample and acridinium ester mark is fully mixed after, in 37 ℃ of constant temperature vibrations 1 hour;
9) get rid of liquid in the clear opening, the every hole of lavation buffer solution is added 300 μ l washing, washed 3 minutes at every turn, wash altogether 3 times;
10) with 50 μ l exciting agents 1 and 50 μ l exciting agents 2, add immediately in the hand-hole by multi-functional analyzer respectively, two reagent joining days were separated by 1 second;
11) detect luminous value immediately behind the adding exciting agent, testing result is as shown in table 1.
| Evaluation index | Testing result |
| Negative reference material (20) | 20/20 |
| Positive reference material (10) | 10/10 |
| Sensitivity (U/ml) | 0.625U/ml |
| Aberration rate (CV%) | 9.56 |
| R(L1-L5)(P＜0.01) | 0.95 |
Table 1HIV-1p24 antigen country reference material testing result
Evaluation criterion:
Positive reaction (20/20) must not appear in 20 HIV-1p24 antigen negative reference materials.
Negative reaction (10/10) must not appear in 10 HIV-1p24 antigen positive reference materials.
10 branch lines personality sensitivity reference material is to the HIV-1p24 antigen detecting agent, limit of identification must not be higher than 1.25U/ml (detecting L1-L5 at least), when carrying out the p24 quantitative measurement, totally 5 sample determination primary system score of L1-L5 analyses necessary 〉=0.95 of linear relationship coefficient (R value), back.
CV≤15% after the statistical study of precision mensuration.
In conjunction with evaluation criterion as can be known the inventive method testing result meet the requirement of HIV-1p24 antigen country reference material standard.
Determining of relevant parameter
1, sensitivity for analysis
The HIV-1p24 recombinant antigen is carried out gradient dilution with analysis buffer, obtain 0 respectively, 0.05pg/ml, 0.1pg/ml, 0.2pg/ml, 0.5pg/ml, 1pg/ml, 2pg/ml, 5pg/ml, 10pg/ml, 20pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, 500pg/ml isoconcentration gradient, 3 multiple holes of each concentration, every hole adds 100 μ l.0 standard and each sample are made replication in 10 times batches, ask every group average (M), standard deviation (SD) and the coefficient of variation (CV%).
Reacting applied acridinium ester labelled antibody is 1: 1000 times of dilution. Exciting agent 1 and 2 is the instant application of sample of multi-functional analyzer, the instant detection.
With each concentration gradient gained (M-3SD)
x(M+3SD) of value and negative control
NCValue compares, and chooses (M-3SD)
x＞(M+3SD)
NCThe time the Cmin value be the detectability of this system, be limited to 0.5pg/ml through the detection of the inventive method relatively.
2, detection width
2.1 Hooke effect
The HIV-1p24 recombinant antigen is carried out gradient dilution with analysis buffer, obtain 0 respectively, 1ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, 100ng/ml, 200ng/ml, 500ng/ml, 1000ng/ml, 2000ng/ml, 5000ng/ml isoconcentration gradient, 3 multiple holes of each concentration, every hole adds 100 μ l.4 times batches of interior replications of 0 standard and each sample are asked every group average.
Reacting applied acridinium ester labelled antibody is 1: 100 times of dilution. Exciting agent 1 and 2 is the instant application of sample of multi-functional analyzer, the instant detection.
The pairing luminous value of the concentration gradient of 100ng/ml is the highest.When detected HIV-1p24 antigen concentration during greater than 100ng/ml, its luminous value does not only raise and sharply descends on the contrary, is significantly hook-shaped, as shown in Figure 1.
2.2 detection width
The HIV-1p24 recombinant antigen is carried out gradient dilution with analysis buffer, obtain 0 respectively, 10pg/ml, 20pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, 500pg/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, the sample of 14 concentration such as 100ng/ml, 2 multiple holes of each concentration, every hole adds 100 μ l.0 standard and each sample are made replication in 4 times batches, ask every group the coefficient of variation.
Reacting applied acridinium ester labelled antibody is 1: 1000 times of dilution. Exciting agent 1 and 2 is the instant application of sample of multi-functional analyzer, the instant detection.
In selected concentration range, the Variation Lines number average of each gradient is less than 20%, so with the upper limit of 100ng/ml as sensing range.Lower limit for sensing range, usually choose function sensitivity, the function sensitivity of the inventive method is 0.5pg/ml, and the range of linearity that draws HIV-1p24 acridinium ester chemistry luminescence immunoassay method of the present invention check thus is 0.5pg/ml-100ng/ml, totally 6 orders of magnitude.
3, typical curve match
The HIV-1p24 recombinant antigen is carried out gradient dilution with analysis buffer, obtain 0 respectively, 0.1pg/ml, 0.2pg/ml, 0.5pg/ml, 1pg/ml, 2pg/ml, 5pg/ml, 10pg/ml, 20pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, 500pg/ml, 1ng/ml, 2ng/ml, 5ng/ml, 10ng/ml, 20ng/ml, 50ng/ml, the sample of 20 concentration such as 100ng/ml, 4 multiple holes of each concentration, every hole adds 100 μ l, asks the photon number average of each concentration.
Reacting applied acridinium ester labelled antibody is 1: 1000 times of dilution. Exciting agent 1 and 2 is the instant application of sample of multi-functional analyzer, the instant detection.
Obtain curve by curve fitting, as shown in Figure 2, drawing fit equation is four parameter l ogistic curve forms:
CPS/CPSmax is the ratio between the highest luminous value in the pairing acridinium ester luminous value of variable concentrations p24 antigen and this detection; Four parameter: a, b are respectively 0.9953,0.04764, and c is 2.934, and d is 1.209, R
2Be 0.9990, have the very good statistical significance that gets.
The function sensitivity of HIV-1p24 acridinium ester chemistry luminescence immunoassay method is 0.5pg/ml, than low 1-2 the order of magnitude of common ELISA method; Sensitivity for analysis is 0.5pg/ml; The range of linearity is at 0.5pg/ml-100ng/ml, than the common high 1-2 of an ELISA method order of magnitude.
Claims (8)
1, a kind of HIV-1p24 antigen acridine ester is learned the luminescence immunoassay detection method, and concrete steps are as follows:
(1) with anti-HIV-1 p24 antibody solid phase carrier is wrapped quilt;
(2) handle the HIV-1 sample;
(3) reaction system in the anti-HIV-1 p24 pairing antibody adding step (1) of HIV-1 sample after handling in the step (2) and acridinium ester compounds mark is carried out immune response;
(4) with the reaction system in exciting agent 1 and the exciting agent 2 adding steps (3), carry out chemiluminescence reaction;
(5) detect the photon number that chemiluminescence reaction produces in the step (4), carry out qualitative or/and quantitative test.
2, method according to claim 1 is characterized in that, the solid phase carrier described in the step (1) is polystyrene latex, polystyrene plastics goods, polyvinyl chloride plastic material products, liposome or immunity magnetic micropearls.
3, method according to claim 1 is characterized in that, the HIV-1 sample described in the step (2) is selected from one or more of serum, blood plasma, HIV-1 cell culture cracking supernatant or HIV-1p24 recombinant antigen.
According to claim 1 or 3 described methods, it is characterized in that 4, the HIV-1 type virus that described HIV-1 sample comprises is a kind of or combination in any of M subgroup and O subgroup.
5, method according to claim 4 is characterized in that, comprises A, B, B ', C, D, E, F, G, H, I and J hypotype in the described M subgroup.
6, method according to claim 1 is characterized in that, the exciting agent 1 described in the step (4) is the mixed liquor of hydrogen peroxide and nitric acid, and wherein the concentration of hydrogen peroxide is 0.05-0.1M, and the concentration of nitric acid is 0.1-0.5M.
7, method according to claim 1 is characterized in that, the exciting agent 2 described in the step (4) is the mixed liquor of Qu Latong 100 and NaOH, and wherein the concentration of Qu Latong 100 is 0.1-0.5M, and the massfraction of NaOH is 2-5%.
8, method according to claim 1 is characterized in that, adds exciting agent 1 in the step (4), and 0.5-1 adds exciting agent 2 again after second.
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| CN104280542A (en) * | 2014-10-21 | 2015-01-14 | 南京基蛋生物科技有限公司 | Dual-enhanced chemiluminescent immunoassay method based on metal enhanced luminescence and nano particle labelled amplification |
| CN104697830A (en) * | 2015-02-10 | 2015-06-10 | 深圳市新产业生物医学工程股份有限公司 | Acid treating agent for HIV detection, sample pretreatment method, kit and detection method |
| CN105652018A (en) * | 2016-02-04 | 2016-06-08 | 广州科方生物技术有限公司 | C-peptide quantitative determination kit |
| CN106645756A (en) * | 2016-12-30 | 2017-05-10 | 广州市达瑞生物技术股份有限公司 | Kit for detecting NMP22 (Nuclear Matrix Protein 22) and preparation method thereof |
| CN107247135A (en) * | 2017-08-11 | 2017-10-13 | 太原瑞盛生物科技有限公司 | A kind of chemiluminescence detection kit of sulfamethazine and preparation method thereof |
| CN108226483A (en) * | 2018-01-19 | 2018-06-29 | 辽宁迈迪生物科技股份有限公司 | A kind of DAS detection kits and its preparation method and application |
| CN108226483B (en) * | 2018-01-19 | 2020-06-26 | 辽宁迈迪生物科技股份有限公司 | DAS detection kit and preparation method and application thereof |
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