CN101368958A - Chemical luminescence immune assay determination reagent kit for hepatitis B virus core antibody IgM - Google Patents
Chemical luminescence immune assay determination reagent kit for hepatitis B virus core antibody IgM Download PDFInfo
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Abstract
The invention discloses a chemiluminescence immunoassay test kit and a preparing method thereof for hepatitis B virus core antibody IgM, which belongs to the immunodiagnosis technique field. The test kit is composed of positive and negative reference substance of hepatitis B virus core antibody IgM, antihuman IgM (u link) coated carrier, hepatitis B virus core antibody IgM enzyme-labelled compound, neutralizing antigen, chemiluminescence zymolyte liquid and condensed washing liquid. The preparing method of the test kit includes steps as following: the positive and negative reference substance is prepared by using negative and positive human serum of the hepatitis B virus core antibody IgM; the carrier is coated by the antihuman IgM (u link); the hepatitis B virus core antibody is labeled by enzyme; the neutralizing antigen is prepared; the enzyme effected chemiluminescence zymolyte liquid is prepared; the condensed washing liquid is prepared, and then the semi-manufactured products of the positive and negative reference substance, the coating carrier, the enzyme-labelled compound, the neutralizing antigen, the chemiluminescence zymolyte liquid and the condensed washing liquid are filled and packed through separate packing so as to form finished products finally. Through studies in laboratory and clinic, the invention has the advantages of higher specificity, sensitivity and repeatability, therefore providing clinical diagnose, all level blood station, blood-collecting station and scientific research work with a valuable detecting method.
Description
Technical field
The present invention relates to the immune diagnostic technique field, particularly, the invention provides a kind of hepatitis B virus core antibody IgM (anti-HBc-IgM) chemical luminescence immune assay determination reagent kit and preparation method thereof.
Background technology
Hepatitis B is that a kind of incidence of disease is higher, the mankind is had the communicable disease of significant damage.Behind the organism infection hepatitis type B virus (HBV), humoral immune reaction at first produces the immunoglobulin (Ig) based on IgM, and the IgM antibody titer descends subsequently, and IgG tires and rises rapidly.Therefore, the detection of anti-HBc-IgM can be used as the index that HBV infects early diagnosis, especially the negative oxyhepatitis patient of hepatitis B surface antigen (HBsAg) is had significance of differential diagnosis.Anti-simultaneously HBc-IgM and HBV activity in vivo are copied into positive correlation, and the positive prompting of anti-HBc-IgM patient has the hepatitis type B virus of hepatitis b virus infected or chronic hepatitis B patient to have activity to duplicate in the recent period, and blood samples of patients has very strong infectiousness.There is report to point out, the sustainable existence of anti-HBc-IgM for many years behind acute hepatitis b virus infection, in the chronic hepatitis b disease journey, the appearance of anti-HBc-IgM is consistent with the peak of the acute activity of the state of an illness or a little later, and think and take a turn for the better along with the control of the state of an illness, part patient's anti-HBc-IgM may cloudyly change, so the anti-HBc-IgM positive also will be made a concrete analysis of in conjunction with clinical symptoms.Along with the raising of medical test level, the detection of hepatitis B patient being carried out anti-HBc-IgM has great significance for the somatotype and the treatment of hepatitis B.
The immunization method that is used to detect hepatitis B virus core antibody IgM at present mainly contains enzyme immunoassay (EIA) (enzyme immunoassay, EIA), radiommunoassay (radioimmunoassay, RIA), immunofluorescence assay (enzyme-linked fluorescence assay, ELFA), the particulate enzyme is exempted from luminesceence analysis (microparticleenzyme immunoassay, MEIA) and directly the mark chemiluminescent immunoassay(CLIA) (chemiluminescenceimmunoassay, CLIA).The basic theories of these ultramicron detection techniques is identical substantially, but used tracer agent and the signal that sent have nothing in common with each other.According to a large amount of test findings and clinical practice data,, be followed successively by: chemiluminescence immune assay, fluoroimmunoassay, radiommunoassay and EIA enzyme immunoassay from practicality, stability, accuracy and development prospect.
Therefore radiating immuning analysis technology has certain contaminative to environment, and has complicated operation because of it uses radioelement thing that serves as a mark, measurement result instability, shortcoming such as the reagent holding time is short.Enzyme immunoassay (EIA) sensitivity is low, and influence factor is more, easily causes false negative and false positive.Chemiluminescence immunoassay is a kind of than elder generation and then effective method, can make detection sensitivity reach 10
-18Mol level, and sensing range can reach 6 orders of magnitude, because the enzyme labeling thing is stable, can uses for a long time, thereby obtain increasing concern.
Detecting hepatitis B virus core antibody IgM with enzymatic chemical luminous immunoassay is elder generation and then effective method, helps lend some impetus to application and the development of chemiluminescence immunoassay technology in clinical examination, detection.
Summary of the invention
The present invention combines chemiluminescence with the immunoassay of hepatitis B virus core antibody IgM is effective, show that by a large amount of experimental checks and clinical research kit of the present invention has higher specificity, susceptibility and repeatability, for clinical diagnosis, blood station at different levels, blood-collecting station and research work provide a kind of very valuable detection means.
The purpose of this invention is to provide chemical luminescence immune assay determination reagent kit of a kind of hepatitis B virus core antibody IgM and preparation method thereof.
Kit according to the present invention comprises: hepatitis B virus core antibody IgM yin and yang attribute reference substance; The carrier of anti-people IgM bag quilt; The anti-HBc of enzyme labeling; In and antigen; Chemical luminous substrate that above-mentioned enzyme acted on and concentrated cleaning solution.
According to kit of the present invention, wherein, described hepatitis B virus core antibody IgM positive reference substance is matrix with the NBCS; The carrier of described anti-people IgM bag quilt is microwell plate or plastic tube; The enzyme of described mark anti-HBc is alkaline phosphatase or horseradish peroxidase; In described and antigen be hepatitis B virus core antigen; Described concentrated cleaning solution is the phosphate buffer (PBST) that contains the Tris-HCl damping fluid of polysorbas20 or contain polysorbas20.
According to kit of the present invention, the working concentration of described alkaline phosphatase or horseradish peroxidase-labeled anti-HBc is 1:1000-5000, wherein, what the alkaline phosphatase dilution adopted is to contain 10% NBCS, 0.1% biological preservative Proclin300, the 0.10M Tris-HCl damping fluid (pH7.5) of 150mM NaCl; What the horseradish peroxidase dilution adopted is to contain 20% NBCS, the 0.02M phosphate buffer (pH7.4) of 0.1% biological preservative Proclin300.The working concentration of described hepatitis B virus core antigen is 1:4000-9000, and wherein used diluent is same as above.
According to kit of the present invention, wherein, described chemical luminous substrate is the substrate of alkaline phosphatase effect or the substrate of horseradish peroxidase effect, wherein, the substrate of alkaline phosphatase effect can be (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star; The substrate of horseradish peroxidase effect comprises A liquid and B liquid, and wherein, A liquid is 0.2MpH8.7 boric acid-borate buffer solution, comprises 10mM luminol, 0.3mM 4-xenol, 0.05mM 4-iodobenzene boric acid; B liquid is 0.2M pH7.2 phosphate buffer, comprises 3.5mM urea peroxide, 0.1% Tween20.
The method of mentioned reagent box produced according to the present invention may further comprise the steps: 1) with hepatitis B virus core antibody IgM feminine gender, positive human serum preparation yin and yang attribute reference substance; 2) with anti-people IgM bag suppressed by vector; 3) use the enzyme labeling anti-HBc; 4) preparation in and antigen; 5) the preparation chemical luminous substrate that enzyme acted on; 6) preparation concentrated cleaning solution; 7) the above-mentioned yin and yang attribute reference substance of packing, bag suppressed by vector, enzyme labeling thing, in and antigen, chemical luminous substrate and concentrated cleaning solution; 8) be assembled into finished product.
The method according to this invention, the carrier of anti-people IgM bag quilt is microwell plate or plastic tube;
The method according to this invention, anti-people IgM bag suppressed by vector is will resist people IgM to be coated on the carrier by the direct physical absorption method;
The method according to this invention, the enzyme in the enzyme labeling anti-HBc can be alkaline phosphatase or horseradish peroxidase.As what carry out with alkaline phosphatase that mark then adopts is glutaraldehyde method; As carry out the sodium periodate method that mark then adopts improvement with horseradish peroxidase.
The method according to this invention, described chemical luminous substrate can be the substrate of alkaline phosphatase effect or the substrate of horseradish peroxidase effect, wherein, the substrate of alkaline phosphatase effect can be (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star; The substrate of horseradish peroxidase effect comprises A liquid and B liquid, and wherein, A liquid is 0.2M pH8.7 boric acid-borate buffer solution, comprises 10mM luminol, 0.3mM 4-xenol, 0.05mM 4-iodobenzene boric acid; B liquid is 0.2M pH7.2 phosphate buffer, comprises 3.5mM urea peroxide, 0.1% Tween20.
Concrete mentioned reagent box can comprise anti-HBc-IgM yin and yang attribute reference substance, anti-people IgM bag by plate, enzyme labeling thing, in and antigen, chemical luminous substrate and concentrated cleaning solution etc.Wherein, the raw material of described anti-HBc-IgM positive control is anti-HBc-IgM positive human serum; Anti-people IgM bag is the micropore lath in 48 or 96 holes by plate; The enzyme of the anti-HBc-IgM of mark is a horseradish peroxidase; In and antigen be hepatitis B virus core antigen; Chemical luminous substrate is the luminol system; Concentrated cleaning solution is 20 times of PBST washing lotions.
What the present invention's " chemical luminescence immune assay determination reagent kit for hepatitis B virus core antibody IgM " adopted is the prize law reaction pattern, has not only effectively utilized the chemiluminescence principle, but also has guaranteed the sensitivity that detects.It has high specificity, good reproducibility, advantage such as stable, and every index all reaches the analysis level of similar kit, provides a kind of more accurate, easy, method fast that detects the hepatitis B virus core antibody IgM for clinical, and clinical value is preferably arranged.
Embodiment
Embodiment 1 preparation chemical luminescence immune assay determination reagent kit for hepatitis B virus core antibody IgM of the present invention
One, the preparation of anti-HBc-IgM yin and yang attribute reference substance
Positive reference substance: the anti-HBc-IgM positive human of the high titre serum of determining through definite method is mixed into positive blood plasma (the blood plasma umber is greater than 5), after 56 ℃ of deactivations of heating in 1 hour, use NBCS suitably to dilute then, the Food Red that adds biological preservative and ten thousand/(W/V) makes it to become redness, aseptic filtration, 2-8 ℃ of preservation.
The negative control product: after selecting for use anti-HBc-IgM to detect negative human serum mixing (the blood plasma umber is greater than 10) 56 ℃ of deactivations of heating in 1 hour, aseptic filtration, 2-8 ℃ of preservation.
Two, the preparation of solid-phase coating plate
The carbonate bag of 0.05M pH9.6 is cushioned liquid mixes with anti-people IgM, add then in each hole of microwell plate, every hole 100 μ L place 24h for 4 ℃.It is inferior to give a baby a bath on the third day after its birth with PBST then, pats dry on thieving paper, again with containing 1.0% BSA, 0.1% Proclin300,0.9% NaCl, 3.8mM NaH
2PO
42H
2O, 16.2mMNa
2HPO
412H
2The confining liquid of O seals, and every hole adds confining liquid 180 μ L respectively, and room temperature was placed after 2 hours, got rid of confining liquid, patted dry on thieving paper.After the room temperature removal moisture drying 24 hours, carry out envelope immediately, 2～8 ℃ of preservations of labeling postposition.
Three, the preparation of enzyme mark anti-HBc
Anti-HBc adopts the sodium periodate oxidation labelling method and the horseradish peroxidase of improvement.After the PBS damping fluid is fully dialysed, add equal-volume glycerine, preserve standby below-20 ℃.With containing 20% NBCS, 0.02M phosphate buffer (pH7.4) the enzyme labeling thing dilution of 0.1% biological preservative Proclin300 dilutes according to certain dilutability.Adopting the square formation method to select the working concentration scope of enzyme labeling thing is 1:1000～5000.
Four, in and antigen preparation
When the present invention uses horseradish peroxidase, in the preparation and the used dilution of antigen for containing 20% NBCS, the 0.02M phosphate buffer (pH7.4) of 0.1% biological preservative Proclin300.Working concentration scope with antigen during employing square formation method is selected is 1:4000～9000.
Five, chemical luminous substrate
The compound method of the chemical luminous substrate liquid of horseradish peroxidase used in the present invention:
A liquid is 0.2M pH8.7 boric acid-borate buffer solution, comprises 10mM luminol, 0.3mM 4-xenol, 0.05mM 4-iodobenzene boric acid;
B liquid is 0.2M pH7.2 phosphate buffer, comprises 3.5mM urea peroxide, 0.1% Tween20.
Using method: A liquid mixes according to the use amount equal-volume with B liquid before using.
Six, concentrated cleaning solution
Cleansing solution is to contain 0.1～0.5%Tween-20, the 0.02M phosphate buffer (PBST) of 0.2%Proclin-300 biological preservative, and the pH value is 7.4, uses 20 times of distilled water dilutings during use.
Seven, semi-manufacture and finished product are formed
Said components is carried out packing be semi-manufacture.Semi-manufacture are carried out quality arbitration, just can be assembled into finished product, i.e. chemical luminescence immune assay determination reagent kit for hepatitis B virus core antibody IgM after qualified.Finished product box also need be inspected by random samples, can dispatch from the factory after index tests such as its specificity, susceptibility, accuracy and stability are qualified.
The anti-HBc-IgM chemical luminescence immune assay determination reagent kit of embodiment 2 preparation the present invention
Divided by glutaraldehyde method hepatitis B core antibody and alkaline phosphatase are marked coupling, with AMPPD as chemical luminous substrate, with containing 10% NBCS, 0.1% biological preservative Proclin300, the 0.10MTris-HCl damping fluid (pH7.5) of 150mM NaCl as enzyme mark thing and in and the dilution of antigen, with containing 0.5%Tween-20,0.2% Proclin-300 biological preservative, the 0.05M Tris-HCl damping fluid (pH7.5) of 150mM NaCl is outside the cleansing solution, and all the other all prepare anti-HBc-IgM chemical luminescence immune assay determination reagent kit with the method identical with embodiment 1.
The anti-HBc-IgM chemical luminescence immune assay determination reagent kit of embodiment 3～4 preparation the present invention
As outside the carrier, all the other all prepare anti-HBc-IgM chemical luminescence immune assay determination reagent kit with the method identical with embodiment 1,2 divided by plastic tube.
The using method of embodiment 5 kits of the present invention
The concrete operations that the anti-HBc-IgM chemical luminescence immune assay determination reagent kit for preparing with embodiment 1 experimentizes are as follows:
1. in 4 ℃ of refrigerators, take out kit, equilibrium at room temperature 15 minutes;
2. blank one hole is set, and positive and negative contrast each two hole, and every hole adds each 100 μ L of reference substance, the test serum sample is carried out the 1:1000 dilution with physiological saline, and every hole adds the sample to be measured 100 μ L after the dilution, fully mixing, shrouding was put 37 ℃ of incubations 30 minutes;
3. abandon liquid in the hole, wash plate 5 times, on clean filter paper, buckle at last and do with the cleansing solution after the dilution;
4. every hole add the enzyme labelled antibody bond, in and each 50 μ L (except the blank hole) of antigen, abundant mixing, shrouding was put 37 ℃ of incubations 30 minutes;
5. abandon liquid in the hole, wash plate 5 times, on clean filter paper, buckle at last and do with the cleansing solution after the dilution;
6. every hole adds the mixed chemical luminous substrate 100 μ L of equal-volume, measures the luminous intensity (RLU) in each hole in must the 5-30 minute after adding chemical luminous substrate liquid, 1 second/hole of Measuring Time;
7. RLU value and the CUT OFF with each hole compares, and the average RLU value in the sample determination value 〉=positive control hole/average RLU value of 30+ negative control hole then is judged as the positive, otherwise negative.
The methodology calibrating of embodiment 6 kits of the present invention
According to manufacturing conventional in this area and vertification regulation the kit of preparation among the embodiment 1 is examined and determine, the result is as follows:
1, kit specific assay
Kit according to embodiment 1 detects Nat'l Pharmaceutical ﹠ Biological Products Control Institute's anti-HBc-IgM reference material serum dish, investigates the coincidence rate of negative and positive reference material, and the result is as shown in table 1, shows that the coincidence rate of the kit yin and yang attribute reference material of embodiment 1 reaches 100%.
The kit of table 1 embodiment 1 detects reference material serum dish result
2, kit sensitivity determination
Use the sensitivity reference material serum of 3 parts of serial dilutions in Nat'l Pharmaceutical ﹠ Biological Products Control Institute's anti-HBc-IgM reference material serum dish, investigate the sensitivity of kit.The result is as shown in table 2, shows that the sensitivity of kit of embodiment 1 is good.
The sensitivity of the kit of table 2 embodiment 1
3, the kit accuracy is measured
With the precision serum in the national reference material in Nat'l Pharmaceutical ﹠ Biological Products Control Institute's anti-HBc-IgM reference material serum dish, investigate the accuracy of kit.Repeat at least 10 holes according to the method for embodiment 5 and detect, calculate the coefficient of variation.After testing, accuracy serum luminous intensity mean value is 266246, and standard deviation is 23681, and calculating coefficient of variation CV% is 9%, shows that the accuracy of prepared chemical luminescence immune assay determination reagent kit for hepatitis B virus core antibody IgM is good.
4, kit stability experiment
With the kit of embodiment 1 in 37 ℃ place 3 days, 7 days, 15 days after, the result shows that every index of kit all can conformance with standard.
More than sensitivity, precision, accuracy, specificity and the stability of explanation " chemical luminescence immune assay determination reagent kit for hepatitis B virus core antibody IgM " are to meet clinical requirement fully.
Embodiment 7 kit clinical practices of the present invention and enzyme are exempted from kit relatively
Use anti-HBc-IgM mensuration kit and the commercially available hepatitis B core antibody IgM enzyme linked immunological kit of embodiment 1 that 1350 routine clinical blood serum samples are detected simultaneously, kit of the present invention is carried out the clinical detection assessment.
One, the source of clinical serum specimen
Hepatitis B great three positive patients serum 150 examples through clinical definite; "small three positive" patients serum 200 examples; Positive blood sample 78 examples of hepatitis C virus (HCV); Positive blood sample 54 examples of hepatitis A virus IgM (anti-HAV-IgM); Positive blood sample 23 examples of hepatitis E virus IgM (anti-HEV-IgM); Chronic persistant hepatitis patients serum 36 examples; Rheumatoid arthritis patients serum 43 examples; HBsAg carrier serum 200 examples; Normal human serum 566 examples.
Two, experimental result
It is more as shown in table 3 that kit of the present invention and enzyme are exempted from kit detection hepatitis B and other diseases and normal population serum result:
Table 3 kit of the present invention and the clinical blood sample measured value of enzyme linked immunological kit comparison result
Test findings shows: 1350 routine clinical serum samples after testing, " chemical luminescence immune assay determination reagent kit for hepatitis B virus core antibody IgM " of the present invention is 99.78% with the total coincidence rate of result that enzyme joins anti-HBc-IgM detection kit, and enzyme joins and occurs 3 parts of gray area positive findingses when anti-HBc-IgM detection kit detects 566 routine normal human serums.
To sum up, utilize kit of the present invention to detect, highly sensitive, high specificity, good reproducibility, "dead" pollution can be used for clinical hepatitis B patients serum and learns inspection, for the propagation of the clinical diagnosis of hepatitis B, treatment effectiveness evaluation, strict screening blood supply blood source, blocking hepatitis B etc. certain reference value is arranged.
Claims (6)
1. a hepatitis B virus core antibody IgM chemical luminescent detecting kit is characterized in that, described kit is mainly by hepatitis B virus core antibody IgM yin and yang attribute reference substance; The carrier of anti-people IgM bag quilt; The anti-HBc of enzyme labeling; In and antigen; The chemical luminous substrate that above-mentioned enzyme acted on; And concentrated cleaning solution is formed.
2. kit as claimed in claim 1 is characterized in that, described hepatitis B virus core antibody IgM positive reference substance is matrix with the NBCS; The carrier of described anti-people IgM bag quilt is microwell plate or plastic tube; The enzyme of described mark anti-HBc is alkaline phosphatase or horseradish peroxidase; In described and antigen be hepatitis B virus core antigen; Described chemical luminous substrate is the substrate of alkaline phosphatase effect or the substrate of horseradish peroxidase effect; Described concentrated cleaning solution is the phosphate buffer (PBST) that contains the Tris-HCl damping fluid of polysorbas20 or contain polysorbas20.
3. kit as claimed in claim 2, it is characterized in that, the working concentration of described alkaline phosphatase or horseradish peroxidase-labeled anti-HBc is 1:1000-5000, wherein, what the alkaline phosphatase dilution adopted is to contain 10% NBCS, 0.1% biological preservative Proclin300, the 0.10M Tris-HCl damping fluid (pH7.5) of 150mM NaCl; What the horseradish peroxidase dilution adopted is to contain 20% NBCS, the 0.02M phosphate buffer (pH7.4) of 0.1% biological preservative Proclin300.
4. kit as claimed in claim 2, it is characterized in that, the working concentration of described hepatitis B virus core antigen is 1:4000-9000, wherein, when the enzyme labeling thing is alkaline phosphatase, what the dilution of hepatitis B virus core antigen adopted is to contain 10% NBCS, 0.1% biological preservative Proclin300, the 0.10M Tris-HCl damping fluid (pH7.5) of 150mM NaCl; When the enzyme labeling thing was horseradish peroxidase, what the dilution of hepatitis B virus core antigen adopted was to contain 20% NBCS, the 0.02M phosphate buffer (pH7.4) of 0.1% biological preservative Proclin300.
5. kit as claimed in claim 2, it is characterized in that, the substrate of described alkaline phosphatase effect is (diamantane)-1,2-two oxidative ethanes, 3-(2 '-the spiral diamantane)-4-methoxyl-4-(3 " the phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star; The substrate of described horseradish peroxidase effect comprises A liquid and B liquid, and wherein, A liquid is 0.2M pH8.7 boric acid-borate buffer solution, comprises 10mM luminol, 0.3mM 4-xenol, 0.05mM 4-iodobenzene boric acid; B liquid is 0.2M pH7.2 phosphate buffer, comprises 3.5mM urea peroxide, 0.1% Tween20.
6. a method for preparing the described kit of claim 1 is characterized in that, with hepatitis B virus core antibody IgM feminine gender, positive human serum preparation yin and yang attribute reference substance; With anti-people IgM (μ chain) bag suppressed by vector; Use the enzyme labeling anti-HBc; In the preparation and antigen; The preparation chemical luminous substrate that enzyme acted on; The preparation concentrated cleaning solution, then the above-mentioned yin and yang attribute reference substance of packing, bag suppressed by vector, enzyme labeling thing, in and antigen, chemical luminous substrate and concentrated cleaning solution semi-manufacture, be assembled into finished product at last.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN 200810103267 CN101368958A (en) | 2008-04-02 | 2008-04-02 | Chemical luminescence immune assay determination reagent kit for hepatitis B virus core antibody IgM |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810103267 CN101368958A (en) | 2008-04-02 | 2008-04-02 | Chemical luminescence immune assay determination reagent kit for hepatitis B virus core antibody IgM |
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| CN101368958A true CN101368958A (en) | 2009-02-18 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103969447A (en) * | 2014-05-21 | 2014-08-06 | 张从从 | Vascular endothelial growth factor quantitative determination kit and light-emitting substrate solution thereof |
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2008
- 2008-04-02 CN CN 200810103267 patent/CN101368958A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103969447A (en) * | 2014-05-21 | 2014-08-06 | 张从从 | Vascular endothelial growth factor quantitative determination kit and light-emitting substrate solution thereof |
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Owner name: BEIJING KEMEI BIOLOGICAL TECHNOLOGY CO., LTD. Free format text: FORMER OWNER: KEMEI DONGYA BIOLOGICAL TECHNOLOGY CO., LTD., BEIJING Effective date: 20111028 |
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Effective date of registration: 20111028 Address after: 100094 Beijing city Haidian District Yongfeng base Feng Xian Road No. 7 North Park Applicant after: Beijing Kemei Biological Technology Co., Ltd. Address before: 100094 Beijing city Haidian District Yongfeng base Feng Xian Road No. 7 North Park Applicant before: Kemei Dongya Biological Technology Co., Ltd., Beijing |
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Application publication date: 20090218 |