CN1087997A - The human III type precollagen is put the agent of being excused from an examination, its preparation method and its radioimmunoassay method - Google Patents
The human III type precollagen is put the agent of being excused from an examination, its preparation method and its radioimmunoassay method Download PDFInfo
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Abstract
The present invention relates to from the induced abortion periderm to extract, purifying human III type precollagen, with this antigen preparation the height antiserum of tiring, and with
125I carries out mark, has set up serum II I procollagen type (former PCIII) radio immunoassay.Measured 852 routine patients and normal human serum III procollagen type content with this law, the result: cirrhosis and chronic active hepatitis raise the most remarkable; The fibrosis of liver biopsy and serum II I procollagen type level are close positive correlation (r=0.984; P＜0.001).Assay method of the present invention has very high diagnostic value to liver fibrosis and early-phase hepatocirrhosis.
Description
What the present invention relates to is medical diagnostic reagent, particularly a kind of human III type precollagen hPC III diagnostic reagent of the diagnosing liver fibrosis under measured by radioimmunoassay, its preparation method and its using method in ria-determination.
It is believed that liver fibrosis is the only stage which must be passed bies of various chronic active hepatitis to the cirrhosis development, after cirrhosis further causes the hepatic parenchymal cells grievous injury, could be confirmed by liver function test, but general conventional liver function test can't diagnosing liver fibrosis and early-phase hepatocirrhosis.Clinical main dependence liver biopsy is at present judged, but this is traumatic inspection, and is subjected to the limitation of site of puncture, and is difficult to carry out dynamic observing of liver fibrosis progress.Process according to the formation and development of cirrhosis and even liver cancer, world hepatopathy expert thinks: " stoping or the generation of the liver fibrosis of slowing down; will cure most of chronic hepatitis patients ", therefore, the early diagnosis liver fibrosis reliable markers thing of particularly setting up liver fibrosis clinically has very important significance for the formation and development that prevents cirrhosis.
In recent years, domestic and international many scholar's reports, serum III procollagen type peptide-P III P(Procollagen Type III Peptide) be diagnosing liver fibrosis serological index preferably.Assay method adopts radioimmunoassay more, used antigen extracts from the animal periderm, III procollagen type peptide RIA(Radio Immuno Assay as German Beling company (Behringwerke Co.) release) the critical material P III P in the reagent extracts from the ox periderm, and the report that extracts P III P in the periderms such as sheep, mouse is also arranged.Its foundation as the diagnosing liver fibrosis mark is: show that after deliberation cirrhosis increases based on III Collagen Type VI PC III in early days, cell when synthetic collagen before form of collagen secrete the extracellular, and blood-serum P III P is the polypeptide that III procollagen type PC III takes off through aminoterminal inscribe peptide acid effect, so measure P III P content, can reflect the metabolism of III Collagen Type VI.Yet there is some difference in animals and human beings class tropocollagen molecule some antigenic determinants structurally, must bring influence to assay.In addition, some scholar finds that liver cell inflammation and the downright bad cracking of III Collagen Type VI so that the blood-serum P III P value that exist of all can making raise in liver, thereby the increase of not necessarily pointing out collagen to form of P III P value, that is be to be subjected to the influence of other factors and inaccurate with P III P mensuration liver fibrosis meeting.In addition, the price that the present P III P that introduces is put the agent of being excused from an examination is very expensive, the ria-determination complicated operation that it is concrete, and the process complicated calculations just can obtain the P III P content in patient's test serum.
The objective of the invention is: 1, provide a kind of and comprise that the human III type precollagen (Human Procollagen Type III) that extracts is the hPC III from people's tire of induced abortion, its antibody anti-hPC III, isotope labeling antigen
125I-hPC III put the agent of being excused from an examination; 2, the preparation method of described reagent is provided; 3, the ria-determination method of described reagent is provided.
Because above-mentioned purpose, there is the content of following three aspects in the present invention.
One, the hPC III is put the agent of being excused from an examination, and it comprises human III type precollagen hPC III, people's III precollagen antibody anti-hPC III, labelled antigen
125I-hPC III, separating agent.Wherein:
1, to reach polyacrylamide gel electrophoresis by Fa Maxi (Pharmacia) company protein test stone pure for the hPC III, with electrophoresis Coomassie brilliant blue R250 dyeing pinkiness, electrophoresis pattern is viewed as single district band, and the visible Liang Tiao of swimming collection of illustrative plates district band is represented peptide chain Pro α respectively behind the β mercapto-ethanol reduction
1(III) and P α
1(III), the former mobility is 0.238, and protein molecular weight is 190,000, and the latter's mobility is 0.310, and the albumen component is 170,000;
2, described anti-hPC III obtains for the hPC immunizing rabbit, its working concentration 0.1%BSA, 2% positive rabbit anteserum NRS and 0.1M, the PBS preparation of PH7.4;
3, described
125I-hPC III 1%BSA, the PBS of 0.1MPH7.4 is formulated as concentration 30000CPm/100ul;
4, described separating agent is 0.1M, and the PBS of PH7.4 prepares 6% polyglycol PEG and is that second antibody obtains with the goat-anti rabbit anteserum.
Two, the preparation method of reagent of the present invention, the purification of (one) described antigen hPC III, it comprises following step:
1, material: get the skin of 4-6 month fetus of the legal induced labor of acrinol ethacridine that, scrape off fat under the periderm, cut into as 1mm
2The fragment of size, and make homogenate with the high speed dispersion device;
2, preparation damping fluid: use 20mMEDTA-Na
2, 20mMC parachloromercuribenzoic acid, 10uM benzyl sulfuryl fluoride be mixed with PH7.4, the Tris-Hcl damping fluid of 150mM;
3, extract skin homogenate: earlier with after described buffer extraction 40-50 hour, with 3000 rev/mins of centrifugal removal of impurities of speed 10 minutes, after getting supernatant adding ammonium sulfate to 20% saturation degree, with 14000xg centrifugal 25 minutes, heavy more molten being deposited in the damping fluid that configures, after adding Nacl to 10%, make III Collagen Type VI and precollagen precipitation, centrifugal 20 minutes with 70000xg speed.
4, get the said extracted liquid precipitate and be dissolved in the desalination of dialysing in the PH7.6Tris-HCL damping fluid;
5, DEAE-32 chromatography, through the 230nm colorimetric, continue to employ the liquid in the chromatography pipe that reaches chromatography elution curve (referring to Fig. 1) first peak peak value, with the dialysis of PH7.6Tris-HCL damping fluid, after concentrating, carry out the DEAE-52 chromatography again, Nacl gradient elution with 0-200mM concentration, get the liquid in the chromatography pipe that reaches chromatography elution curve (referring to Fig. 2) second peak-to-peak value, in 0.1% acetic acid, dialyse again, promptly get the pure product of hPC III after concentrating;
Above-mentioned antigen is kept at below-20 ℃, and more than operation is all carried out in 4 ℃.
(2), the preparation of described antibody anti-hPC III:
Get the healthy male rabbit of 2.5-3 kilogram, carry out immunity.1, fundamental immunity: 0.5ml(contains 200mg with the hPC III), complete Fu Shi helps agent with equivalent, biped toe office hypodermic injection after the emulsification.2, supplementary immunization is got hPC III 100ug and the lymph node of not exclusively helping agent injection armpit or ventral groove enlargement.If lymph node not quite can inject toes or the back, both sides is subcutaneous, behind twice of the supplementary immunization, get the blood test antibody from ear vein and tire.Not ideal enough if tire, can continue supplementary immunization.After waiting to reach satisfactory antibody titer, from the arteria carotis bloodletting, separation of serum, freezing preservation.
(3), labelled antigen
125The preparation of I-hPC III:
Adopt chloramine-t method, wherein make reductive agent mark hPC III with halfcystine, according to the following steps:
The hPC III that 50ulPH7.5PBS and 50ul is contained 12ug is mixed, and adds the 0.5-1mCiNa I again
125Inject the toluene-sodium-sulfonchloramide 20ul that contains 15-25ug behind the mixing fast, vortex vibration 2 minutes, add 20-40ug halfcystine reduction cessation reaction immediately, get 5ul and measure mark rate with the trichloroacetic acid method, all the other footpath SephadexG-25 cross the post purifying, separated free iodine, flow velocity 0.3ml/min detects eluent with the isotope activity meter, clearly two peaks (referring to Fig. 3) of boundary occur, collect leading peak and dialyse, promptly get the pure product of labelled antigen.
The Quality Identification method and the result of the reagent that said method obtains are as follows:
Through PAGE(Coomassie blue dyeing) identify that the hPC III of acquisition is single district band (photo 1), it is pure to illustrate that purity reaches electrophoresis, and district's band meets the collagen feature for pink; Through the visible Liang Tiao district of β mercapto-ethanol reduction rear electrophoresis (SDS-PAGE) band pro α
1(III) and p α
1(III) (photo 2), but mobility is lower slightly than the mobility of ox III procollagen type.Warp and import (Phamacia Co.) high molecular weight protein standard (HMW) blank determination, the subunit molecular of the hPC III molecule of acquisition is respectively: pro α
1(III) 190,000 dalton (Dolton) and p α
1(III) 170,000 Dolton.And the pro α of ox III procollagen type
1(III) 170,000 dalton (Dolton) and p α
1(III) 150,000(photo 2.Annotate: the PC III is an ox III procollagen type; The HPC III is the hPC III; HPC I behaviour I procollagen type; The c III is the ox Collagen Type VI).By photo 3(SDS-PAGE) can see the purge process of hPC III (before the 1-DEAE-32 chromatography; Before the 2-DEAE-52 chromatography; 3, before the 4-Sephadex G100 chromatography; Behind the 5-Sephadex G100 chromatography), can obtain the pure product of hPC III.
2, the evaluation of anti-hPC III:
The antiserum of above-mentioned preparation is done serial dilution,, do not add competitive antigen, draw the antiserum dilution curve, measure it and tire by putting the method for exempting from; Immunohistochemistry: get each 10 routine patient's of cirrhosis and chronic active hepatitis liver biopsy specimen, the antiserum of above-mentioned preparation is diluted to 1: 100.This high price antiserum is through SABC liver biopsy specimen, and collagenous fibres between the visible cirrhosis pseudolobuli turn out to be specific antibody under the light microscopic.
3, with aforementioned
125I-hPC III is measured with the trichloroacetic acid method, and radiochemical purity reaches 99.6%.
Three, the radioimmunoassay method of the reagent of indication of the present invention system adopts double antibody PEG method, the imbalance method application of sample, and reagent is pressed following capacity packing before the operation:
1, hPC III standard: 7 bottles of (S
0-S
6), the 1ml/ bottle;
Press hPC III content for every bottle: 0,25,50,100,500,1000(ug/e) packing.
2, PBS:1 bottle, 2ml;
3, anti-hPC III: 3 bottles, be total to 22ml;
4, separating agent: 3 bottles, be total to 22ml;
5,
125I-hPC III: 2 bottles, be total to 11ml;
6, quality controlled serum: 1 bottle, 1ml.
The assay method that the present invention is used for radiommunoassay is by following operation steps:
100ul standard or test serum sample → add 100ulanti-hPC III serum (4 ℃ 42-48 hour)/() 100ul
125I-hPC III (30000cPm) (4 ℃ of 6h)/centrifugal 400rPm of () 200ul separating agent (room temperature 30min)/(), 4-10 ℃ of 30min → abandon radioactive activity counting B of supernatant → mensuration precipitation.
The detailed description of aforesaid operations step sees table (unit: ul)
Application of sample order NSB S
0S
1S
2S
3S
4S
5S
6Test serum
Test serum--------100
Titer 100 (S
0) 100 100 100 100 100 100 100-
PBS 200 - - - - - - - -
HPCIII antibody-200 200 200 200 200 200 200 200
Placed 42～48 hours for 4 ℃.
125I-hPCIII 100 100 100 100 100 100 100 100 100
Placed 6 hours for 4 ℃.
Separating agent
Annotate200 200 200 200 200 200 200 200 200
Room temperature was placed 30 minutes; 4～10 ℃, 3500 rev/mins, centrifugal 30 minutes; Abandon supernatant, survey precipitation counting (B).
Annotate: must abundant mixing separating agent before adding!
The calculating of measurement result:
Typical curve is drawn: with B/B.Be ordinate, hPC III standard (5,25,50,100,250,500,1000ug/l) is an abscissa, on semilogarithmic paper with Y=(B
Mark-NSB)/(B
O-NSB) * 100%, X=loghPC III, curve plotting.The test serum sample is made the double parallel pipe and is measured, and looks into above-mentioned typical curve and can measure the result.
It is good that the competition of the typical curve that the data of obtaining with above-mentioned operation steps are drawn suppresses relation, drop is big, the measurement range broad, be 5-1000ug/l, sensitivity 6ng/ml criticizes interior and the differences between batches coefficient respectively is 5.2% and 6.2%, and the recovery is 97.6-102.3%, with other Collagen Type VI no cross reactions, method is stable.Above index has reached the requirement of RIA method standard quality.
The relation (referring to Fig. 4) of aforementioned calculation result and liver biopsy fibrosis dyes through H-E through 37 routine liver biopsy specimens, by fiberization distribution range and fibroblast proliferation degree divide light, in, weigh three grades, no fibroplasia person is 0 grade.Its blood-serum P C III measurement result: 0 grade of 11 example is 89.5 ± 18.1; Light level 10 examples are 135.5 ± 20.5; Middle rank 8 examples are 260.8 ± 39.2; Heavy duty 8 examples are 369.8 ± 68.0.Blood-serum P C III is along with degree of hepatic fibrosis increases the weight of to increase progressively, and both are close positive correlation (r=0.984, P ∠ 0.001).
Photo 4-7 is the microphoto of the pathological section of 4 routine liver biopsy specimens wherein, proves that measurement result of the present invention and disease match clearly.
Reagent of the present invention and ria-determination method warp are in seven large hospital clinic trial of the Chongqing City tumour hospital and the whole nation, and its effect sees table:
Tumour Inst. of Chongqing City
Annotate 1The seven tame results of hospital are comprehensive
Group
Example number average number ± standard deviation example number average number ± standard deviation
Oxyhepatitis 22 131.1 ± 30.2 53 111.2 ± 30.1
Chronic persistent hepatitis 18 94.9 ± 26.5 143 93.6 ± 26.2
Chronic active hepatitis 13 188.2 ± 73.9 122 174.0 ± 42.6
Compensatory cirrhosis 10 277.4 ± 91.0 13 210.3 ± 86.6
Lose compensatory cirrhosis 20 228.2 ± 157.1 129 211.1 ± 67.9
Cirrhosis merges liver cancer 46 288.7 ± 101.4 62 269.7 ± 66.6
Primary carcinoma of liver (simple form) 22 168.5 ± 39.3
Cholelithiasis 12 88.0 ± 18.6 15 87.9 ± 16.6
Other: hepatopathy 7 88.2 ± 26.0 23 93.4 ± 19.7
Non-hepatic disorder 11 88.6 ± 10.5
The result who measures 100 routine normal persons and 181 routine various hepatopathys and other patients serum PC III by the method for above-mentioned foundation in the table shows, each group of cirrhosis and chronic active hepatitis group blood-serum P C III raise the most remarkable, anxious liver and simple property liver cancer slightly raise, chronic persistent hepatitis, cholelithiasis and other diseases all do not have remarkable rising, and the scorching sick necrosis of this explanation liver cell may be less to the influence of PC III.
Brief summary
1, extractions from the periderm of your induced abortion of acrinol ethacridine, purifying human III type precollagen, confirmation hPC III such as subunit's mensuration is pure product through polyacrylamide gel electrophoresis and after reducing.
2, obtain the high price antiserum with hPC III immunizing rabbit, and confirm the collagenous fibres pigmentable through SABC.
3, use Nal
125Mark hPC III, radiochemicsl purity reaches 93-98%.
4, the blood-serum P C III of Jian Liing is put the method for exempting from-double antibody PEG method, and every quality index all meets the mensuration requirement.
5, measure the result of 281 routine blood-serum P C III clinically: 100 routine normal control mean value SD omit under the 85.0 ± 17.5ug/l(unit), upper limits of normal is 120; Liver cirrhosis group rises the highest; Compensatory, mistake compensatory and merging liver cancer component are not 227.4 ± 91.0,266.2 ± 157.5 and 288.7 ± 101.4; The chronic active hepatitis group has remarkable liter 188.2 ± 73.9; Anxious liver and liver cancer (simple form) group slightly raise, and respectively are 131.1 ± 30.2 and 168.5 ± 39.3; Chronic persistent hepatitis, cholelithiasis and other groups all do not have obviously rising.
6,37 routine liver biopsies are divided into by fibrosis: 0, light, in and heavy duty, its blood-serum P C III is respectively 89.5 ± 18.1,135.5 ± 20.5,260.8 ± 39.2 and 369.8 ± 68.0.Both are close positive correlation (r=0.984, P ∠ 0.001).
Good effect of the present invention is:
1, is that the ria-determination method that the serum index is set up can reflect the liver tissue fibrosis degree objectively with the hPC III, the treatment of diagnosing liver fibrosis and early-phase hepatocirrhosis is had significant values.
2, can obtain the pure product of hPC with method of the present invention, make the reductive agent gained with halfcystine during with chloramine-t method mark hPC III
125Stability＞1.5 of I-hPC III month, obviously than Sodium Metabisulfite for well, very little to hPC III molecular damage, and radiochemical purity reaches 99.6%.
3, simple to operate, all compositions all are mixed with using liquid, add respectively to get final product, and needn't remake other additional work; Patients serum to be measured needn't do any dilution can obtain III procollagen type content.Measurement range is enough clinical uses (as 25-1000ug/l), and sensitivity reaches 6ug/l; Reaching interassay coefficient of variation in batch only is respectively 5.2% and 6.2%, and all indexs all reach PIA method quality requirements.
Claims (5)
1, a kind of reagent that is used for the measured by radioimmunoassay liver fibrosis is characterized in that: it comprises human III type precollagen hPC III, human III type precollagen antibody anti-hPC III, labelled antigen
125I-hPC III and separating agent; Wherein:
(1), the hPC III by Fa Maxi (Pharmacia) but that public affairs protein test stone reaches polyacrylamide gel electrophoresis is pure, with electrophoresis Coomassie brilliant blue R250 dyeing pinkiness, electrophoresis pattern is viewed as single district band, and the visible Liang Tiao of swimming collection of illustrative plates district band is represented peptide chain Pro α respectively behind the β mercapto-ethanol reduction
1(III) and P α
1(III), the former mobility is 0.238, and protein molecular weight is 190,000, and the latter's mobility is 0.310, and molecular weight of albumen is 170,000;
(2), the anti-hPC III is that hPC III immunizing rabbit obtains, its working concentration 0.1%BSA, 2% positive rabbit anteserum NRS and 0.1M, the PBS of PH7.4 prepares;
(3),
125I-hPC III 1%BSA, the PBS of 0.1MPH7.4 is formulated as concentration 30000CPm/100ul;
(4), separating agent is 0.1M, the PBS of PH7.4 prepares 6% polyglycol PEG and is that second antibody obtains with the goat-anti rabbit anteserum.
2, a kind of method that is used to prepare the hPC III is characterized in that it carries out according to the following steps:
(1), get the skin of 4-6 month fetus of the legal induced labor of Fo Nuoer, scrape off the periderm subcutaneous fat, be cut into fragment, make homogenate with high speed rejector;
(2), use 20mMEDTA-Na
2, the 20mM parachloromercuribenzoic acid, 10uM benzyl sulfuryl fluoride is mixed with PH7.4, the Tris-HCL damping fluid of 150mM;
(3), with above-mentioned buffer extraction skin homogenate after 40-50 hour, with 3000 rev/mins of centrifugal removal of impurities 10 minutes, after getting supernatant adding ammonium sulfate to 20% saturation degree, with 14000xg centrifugal 25 minutes, heavy more molten being deposited in the damping fluid that configures, after adding Nacl to 10%, with 70000 * g speed centrifugal 20 minutes;
(4), get the said extracted liquid precipitate and be dissolved in the desalination of dialysing in the PH7.6Tris-HCL damping fluid;
(5), DEAE-32 chromatography, through the 230nm colorimetric, continue to employ the liquid in the chromatography pipe that reaches chromatography elution curve first peak peak value, with the dialysis of PH7.6Tris-HCL damping fluid, after concentrating, carry out the DEAE-52 chromatography again, Nacl gradient elution with 0-200mM concentration, get the liquid in the chromatography pipe that reaches chromatography elution curve second peak-to-peak value, in 0.1% acetic acid, dialyse again, promptly get the pure product of hPC III after concentrating;
(6), above operation is all carried out in 4 ℃.
3, a kind of method for preparing the anti-hPC III is characterized in that with the hPC III healthy male rabbit being carried out immunity, and method comprises:
(1), fundamental immunity: 0.5ml(contains 200mg with the hPC III), complete Fu Shi helps agent with equivalent, biped toe office hypodermic injection after the emulsification;
(2) supplementary immunization, get hPC III 100ug and the lymph node of not exclusively helping agent injection armpit or ventral groove enlargement, perhaps inject toes or the back, both sides is subcutaneous, behind twice of the supplementary immunization, getting test antibody from ear vein tires, after supplementary immunization reaches satisfactory antibody effect value, from the arteria carotis bloodletting, distinguish, freezing preservation gets final product.
4, a kind of preparation
125The method of I-hPC III, employing be chloramine-t method, it is characterized in that making reductive agent mark hPC III with halfcystine in the method, carry out according to the following steps:
The hPC III that 50ulPH7.5PBS and 50ul is contained 12ug is mixed, and adds the 0.5-1mCiNa I again
125Inject the toluene-sodium-sulfonchloramide 20ul that contains 15-25ug behind the mixing fast, vortex vibration 2 minutes, add 20-40ug halfcystine reduction cessation reaction immediately, get 5ul and measure mark rate with the trichloroacetic acid method, all the other cross the post purifying through SephadexG-25, separated free iodine, flow velocity 0.3ml/min detects eluent with the isotope activity meter, clearly two peaks of boundary occur, collect leading peak and dialyse, promptly
125I-pure the product of hPC III.
5, a kind of method with the measured by radioimmunoassay liver fibrosis is characterized in that method is capable suddenly by following progress:
100ul standard or test serum sample → add 100ulanti-hPC III serum (4 ℃ 42-48 hour)/() 100ul
125I-hPC III (30000cPm) (4 ℃ of 6h)/() 200ul separating agent (room temperature, 30min)/() centrifugal 4000rPm, the radioactive activity counting B of 4-10 ℃ of 30min → abandon supernatant → mensuration precipitation → with B/B is an ordinate, hPC III (5,25,50,100,250,500,1000ug/l) is an abscissa, on semilogarithmic paper with Y=(B.-NSB)/(B.-NSB) * 100%, X=loghPC III drawing standard curve → test serum sample is made the double parallel pipe and is measured, and finds the result on typical curve.
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|---|---|---|---|
| CN 93114698 CN1045820C (en) | 1993-11-18 | 1993-11-18 | Human third-type precollagen radioimmuno-reagent, its prepn. and radioimmunoassay |
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|---|---|---|---|
| CN 93114698 CN1045820C (en) | 1993-11-18 | 1993-11-18 | Human third-type precollagen radioimmuno-reagent, its prepn. and radioimmunoassay |
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| CN1045820C CN1045820C (en) | 1999-10-20 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1306272C (en) * | 2000-11-17 | 2007-03-21 | 罗切斯特大学 | In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells |
| CN100416273C (en) * | 2000-08-21 | 2008-09-03 | 法国公立援助医院 | Diagnosis method of fibrotic disease using biochemical markers |
| CN1662817B (en) * | 2002-04-16 | 2012-06-27 | 福拉姆斯大学生物技术研究所 | Marker for measuring liver cirrhosis |
| CN103076452A (en) * | 2012-12-30 | 2013-05-01 | 上海市内分泌代谢病研究所 | Method for testing adiponectin and application thereof |
| CN103399158A (en) * | 2013-08-02 | 2013-11-20 | 上海市同济医院 | Liquid protein chip kit for detecting liver fibrosis degree |
| CN103399156A (en) * | 2013-07-30 | 2013-11-20 | 中国农业科学院北京畜牧兽医研究所 | Green fluorescent protein radio-immunity kit as well as preparation method and detection method thereof |
-
1993
- 1993-11-18 CN CN 93114698 patent/CN1045820C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100416273C (en) * | 2000-08-21 | 2008-09-03 | 法国公立援助医院 | Diagnosis method of fibrotic disease using biochemical markers |
| CN1306272C (en) * | 2000-11-17 | 2007-03-21 | 罗切斯特大学 | In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells |
| CN1662817B (en) * | 2002-04-16 | 2012-06-27 | 福拉姆斯大学生物技术研究所 | Marker for measuring liver cirrhosis |
| CN103076452A (en) * | 2012-12-30 | 2013-05-01 | 上海市内分泌代谢病研究所 | Method for testing adiponectin and application thereof |
| CN103076452B (en) * | 2012-12-30 | 2015-02-25 | 上海市内分泌代谢病研究所 | Method for testing adiponectin and application thereof |
| CN103399156A (en) * | 2013-07-30 | 2013-11-20 | 中国农业科学院北京畜牧兽医研究所 | Green fluorescent protein radio-immunity kit as well as preparation method and detection method thereof |
| CN103399158A (en) * | 2013-08-02 | 2013-11-20 | 上海市同济医院 | Liquid protein chip kit for detecting liver fibrosis degree |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1045820C (en) | 1999-10-20 |
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