CN101393217B - Detection kit for schistosomiasis japonica blood serum designated object - Google Patents
Detection kit for schistosomiasis japonica blood serum designated object Download PDFInfo
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- CN101393217B CN101393217B CN200810200612XA CN200810200612A CN101393217B CN 101393217 B CN101393217 B CN 101393217B CN 200810200612X A CN200810200612X A CN 200810200612XA CN 200810200612 A CN200810200612 A CN 200810200612A CN 101393217 B CN101393217 B CN 101393217B
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Abstract
The invention relates to the technical field of biomedical diagnostics and discloses a reagent kit for detecting a serum marker of primary hepatic carcinoma, which consists of an ELISA plate enveloped by antigens, an enzyme-labeled antibody working solution, a sample diluent, a washing liquid, positive and negative control serum, a chromogenic solution and a stopping liquid. The reagent kit is characterized in that the envelope antigens of the ELISA plate are five groups of peptide antigens L4-A and L4-B; L7-B; L11-1, L11-3 and L11-4; L12-A and L12-B; and Suil-A and Suil-B, which correspond to five serum marker antibodies of the primary hepatic carcinoma; and Sui1-A and Sui1-B respectively. Except the L7B which is separately enveloped, each group of all the other groups of peptide antigens is enveloped by proportionally mixing polypeptides in the same group. Proven by evaluation experiments of the reagent kit and clinical trial, the reagent kit has good specificity and sensitivity, and can be used for early warning prompt or early diagnosis before the primary hepatic carcinoma appears or at early stage of the primary hepatic carcinoma, thereby improving the survival rate of carcinoma patients and evaluating treatment effect and disease outcome in time by observing the dynamic change of related indicators of the serum.
Description
Technical field
The present invention relates to the biomedical diagnostic field that learns a skill, is a kind of kit that is used to detect schistosomiasis japonica blood serum designated object.
Background technology
Primary carcinoma of liver (HCC) ranks forefront in the dead rank of malignant tumour, and human life and health in serious threat.China accounts for 40% of whole world PLC mortality number because of suffering from the dead number of liver cancer every year.Liver cancer main cause occurred frequently is that the population of China about 10% is hepatitis B surface antibody (HBsAg) positive, and HBsAg is positive or chronic liver disease medical history person is arranged is the main people at highest risk of liver cancer.At present; Carcinous mark alpha-fetoprotein (the alpha fetoprotein of serology that is used for the primary carcinoma of liver diagnosis; AFP), lower for the susceptibility and the positive rate of diagnosing primary small liver cancer, be respectively 33%～65% and 9.1%～26% when 20 μ g/L (AFP >).And finding early and make a definite diagnosis for the survival rate that improves Patients with Primary of small liver cancer is most important.Therefore, needs are set up efficiently, method is used for primary carcinoma of liver easily, the particularly examination of small liver cancer and early diagnosis.
In recent years research work is found; Hepatitis B virus X antigen (HBx) can bring out the high expressed of 5 kinds of albumen URG4, URG7, URG11, S15a and Suil, the generation of they and primary carcinoma of liver, development closely related (referring to H.L.Hann, J.M Lee; M.A.Feitelson; Et al.Preneoplastic Markers ofHepatitis B Virus-Associated Hepatocellular Carcinoma Cancer Research.2004,64 (15), 7329-35).The appearance of the corresponding antibodies of these 5 kinds of protein induced generations of high expressed is often than using AFP and iconography nuclear magnetic resonance methods such as (MRI) to diagnose out the time of liver cancer to take Zao some months even more than 1 year at present clinically.The inventor filters out 10 polypeptide fragments according to the amino acid sequence of above-mentioned 5 kinds of albumen, is divided into 5 groups with its dietary protein origin, is respectively L4-A and L4-B; L7-B; L11-1, L11-3 and L11-4; L12-A and L12-B; Suil-A and Suil-B, with them as antigen, the corresponding antibodies generation specific reaction that can produce with above-mentioned 5 kinds of albumen respectively, 10 amino acid sequence of polypeptide are as shown in table 1:
The amino acid sequence of table 1,10 polypeptide antigens.
Preliminary ELISA method testing result shows; These 10 polypeptide antigens have specificity and susceptibility preferably; Can detect among the patients serum the protein induced corresponding antibodies that goes out of 5 kinds of high expresseds respectively effectively, therefore, this antibody can be used as schistosomiasis japonica blood serum designated object.But do not see so far with its detection index as primary carcinoma of liver early warning, early diagnosis and curative effect assessment.
Summary of the invention
The schistosomiasis japonica blood serum designated object detection kit that the purpose of this invention is to provide a kind of recall rate height, good reproducibility.
Kit of the present invention; Can bring out the high expressed of 5 kinds of cell protein URG4, URG7, URG11, S15a and Suil and antibody that this 5 kinds of albumen are induced according to hepatitis B virus X antigen HBx and have the characteristics of specificity and susceptibility preferably; The antibody that above-mentioned 5 kinds of albumen are induced is as schistosomiasis japonica blood serum designated object; Utilize above-mentioned 10 polypeptide as the Detection of antigen corresponding antibody; So that before primary carcinoma of liver takes place or generation send early warning in early days or make early diagnosis; Thereby improve the early diagnostic rate and the survival rate of liver cancer patient of small liver cancer, and through the dynamic change of these indexs of observation in liver cancer patient blood serum, in time assess result of treatment and the state of an illness lapses to situation.
Kit of the present invention is formed as follows:
1, antigen coated ELISA Plate
2, enzyme labelled antibody working fluid: 1 bottle
3, sample dilution: 1 bottle
4, cleansing solution A:1 bottle
5, cleansing solution B:1 pipe
6, positive control serum: 1 pipe
7, negative control sera: 1 pipe
8, colour developing liquid A:1 bottle
9, colour developing liquid B:1 bottle
10, stop buffer: 1 bottle
Said antigen coated ELISA Plate, its envelope antigen are respectively above-mentioned 5 groups of polypeptide antigens, L4-A and L4-B; L7-B; L11-1, L11-3 and L11-4; L12-A and L12-B; And Sui1-A and Sui1-B, except that L7B encapsulated separately, all the other all were polypeptide equal proportion hybrid packet quilt on the same group, their amino acid sequence is as shown in table 1; Said enzyme labelled antibody working fluid is that the goat-anti people two of HRP (horseradish peroxidase) mark is anti-; The sample dilution is for containing the Tris-HCl damping fluid of 2.5% (W/V) BSA; Cleansing solution A is the PBS (phosphate buffer) of 0.2M; Cleansing solution B is Tween-20; Positive control serum is to take from liver cancer patient and above-mentioned 5 kinds of serum that indicator antibody is all positive; Negative control sera is the serum of all negative donors with normal of above-mentioned 5 kinds of indicator antibodies; Colour developing liquid A is for containing 0.03% (V/V) H
2O
2The phosphate citrate buffer solution of (oxydol); Colour developing liquid B is for containing the citrate buffer solution of 1.5mM TMB (tetramethyl benzidine); Stop buffer is the H of 2M
2SO
4Solution.
10 peptide species antigens of kit of the present invention can oneself synthesize, and also can entrust relevant unit synthetic, and purity is greater than 95%.
Kit method of application of the present invention is following:
1, preparing washing liquid: cleansing solution A adds 0.1% cleansing solution B more by volume with the deionized water dilution proportion of 1:20 by volume behind the mixing, fully mixing.This is a cleansing solution.
2, serum sample is hatched: (1) respectively with 4 times of dilutions of sample dilution do, joins positive serum contrast, negative serum contrast and patients serum's sample to be measured respectively in the hole of the ELISA Plate that 5 groups of polypeptide antigens encapsulate, and adds a cover, and hatches 45 minutes for 37 ℃; (2) wash with cleansing solution by routine, blot cleansing solution.
3, ELIAS secondary antibody is hatched: (1) adds the enzyme labelled antibody working fluid in each hole of ELISA Plate, add a cover, and hatches 45 minutes for 37 ℃; (2) the same with the cleansing solution washing, blot cleansing solution.
4, substrate colour developing: the ratio according to volume ratio 1:1 adds colour developing liquid A and colour developing liquid B successively in each hole, room temperature lucifuge colour developing 5-10 minute drips the stop buffer cessation reaction.
5, reading: the absorption value (A value) that reads each hole on the ELISA Plate with ELIASA dual wavelength 450/630nm.
6, the result judges: the ratio of the A value of patients serum's sample to be checked and the A value of negative control sera is used the P/N value representation, and the P/N value is positive more than or equal to 2.5; Be less than or equal to 2 negative; Be doubtful sample, need to detect again that when detecting for the second time, the P/N value is positive more than or equal to 2.2 between 2 and 2.5, be less than or equal to 2.2 negative.If the A value of positive control serum is less than or equal to 0.6, or the A value of negative control sera explains that more than or equal to 0.06 kit lost efficacy or misoperation, should detect again.
7, evaluation of result: in 5 kinds of detection of antibodies indexs, if a kind of index positive with " 1+ " expression, is judged to be doubtful the waiting of liver cancer and looks into; 2 kinds of index positives with " 2+ " expression, are judged to be liver cancer and are correlated with, and should follow up a case by regular visits to; Index is positive more than 3 kinds or 3 kinds, and with " >=3+ " expression, it is closely related to be judged to be liver cancer.
Through kit evaluation experimental and clinic trial; This kit specificity and susceptibility are good; Before can be used for primary carcinoma of liver and taking place or early stage early warning or early diagnosis take place; Thereby improve the survival rate of liver cancer patient, and through observing the dynamic change of serum index of correlation, in time assess result of treatment and the state of an illness lapses to situation.
Description of drawings
Fig. 1 is a kit ELISA Plate synoptic diagram of the present invention.
Fig. 2 is used for 200 parts of clinical serum sample positive rate figure for kit of the present invention.
Embodiment
Combine accompanying drawing and embodiment at present, the present invention is described in detail.
One, reagent and material
1, polypeptide antigen: 10 polypeptide L4-A, L4-B, L7-B, L11-1, L11-3, L11-4, L12-A, L12-B, Sui1-A and Sui1-B, entrust Shanghai gill company synthetic, purity>95%.
2, enzyme labelled antibody working fluid: the goat-anti people two of HRP mark is anti-, is the KPL Company products.
3, TMB, Tween20 are that Bioisystech Co., Ltd's product is widely collected in Shanghai.
4, other reagent are chemicals company limited of traditional Chinese medicines group product.
5, enzyme mark capillary strip: 8 * 12 low adsorptive enzyme targets are the Greiner Company products.
Two, the preparation of kit
1, the antigen coated ELISA Plate of preparation
1) preparation coating buffer: get soda mint (NaHCO
3) 2.9g, sodium carbonate (Na
2CO
3) 1.6g, Sodium azide (NaN
3) 0.2g, be dissolved in deionized water 400ml, be settled to 500ml behind the adjust pH to 9.6,4 ℃ are subsequent use.This is the 0.05M carbonate buffer solution.
2) preparation polypeptide antigen solution: purity is dissolved in coating buffer respectively greater than 95% 10 peptide species, abundant mixing, concentration is 10 μ g/ml.
3) coated elisa plate: except that L7B was antigen alone solution, the mark by each group after each equal proportion of all the other 4 groups of antigenic solutions is mixed joined respectively in the corresponding micropore of ELISA Plate, and add a cover in 100 μ l/ holes, placed 2 hours, and spent the night and be placed on 4 ℃ for 37 ℃.
4) sealing: sucking-off polypeptide antigen solution from each hole; Add confining liquid more respectively, said confining liquid is added a cover for containing the 0.02M Tris-HCl damping fluid of 5% (W/V) BSA; Hatched 2 hours for 37 ℃; The sucking-off confining liquid, it is subsequent use that the sealing freeze-drying is placed on 4 ℃ of preservations, is antigen coated ELISA Plate.
2, enzyme labelled antibody working fluid: the goat-anti people two of HRP mark is anti-, and the ratio according to 1:1000 before using forms with above-mentioned confining liquid dilution.
3, sample dilution: get Tris0.6g, be dissolved in deionized water 400ml, with concentrated hydrochloric acid adjust pH to 7.4, add the BSA of 12.5g, fully be settled to 500ml behind the mixing, 4 ℃ subsequent use.This is for containing the 0.01M Tris-HCl damping fluid of 2.5% (W/V) BSA.
4, cleansing solution A: get NaCl16.0g, KCl0.4g, Na
2HPO
4.12H
2O 6.96g, KH
2PO
40.48g, being dissolved among the deionized water 90ml, adjust pH to 7.4 is settled to 100ml, and 4 ℃ are subsequent use.This is the PBS of 0.2M.
5, cleansing solution B:Tween-20.
6, positive control serum: through detecting 5 liver cancer patient blood serums that indicator antibody is all positive.
7, negative control sera: through detecting 5 donors with normal serum that indicator antibody is all negative.
8, colour developing liquid A: get Na
2HPO
40.73g, citric acid 0.46g, 30%H
2O
21ml adds deionized water and is settled to 1000ml, and 4 ℃ subsequent use.This is for containing 0.03% (V/V) H
2O
2The phosphate citrate buffer solution.
9, colour developing liquid B: get TMB39mg, citric acid 0.11g, EDTA (ethylenediamine tetraacetic acid) 18.6mg, glycerine 20ml, DMSO (dimethyl sulfoxide (DMSO)) 3ml, adding distil water is to 100ml after the heating for dissolving, and 4 ℃ are subsequent use.This is the citrate buffer solution that contains 1.5mM TMB.
10, stop buffer: be the H of 2M
2SO
4Solution.
Embodiment 1: a kind of detection kit of schistosomiasis japonica blood serum designated object
The composition of kit of the present invention is following:
1. 1 of antigen coated ELISA Plate, this ELISA Plate is made up of the enzyme in 10 1 * 8 holes mark bar, and per 2 is 1 group, and being divided into is 5 groups, is labeled as URG4, URG7, URG11, S15a and Sui1 respectively, with corresponding to the corresponding antigens that encapsulates, referring to Fig. 1.As be labeled as the enzyme mark bar of URG4, that encapsulate is L4A and L4B; Be labeled as URG7, that encapsulate is L7-B; Be labeled as URG11, that encapsulate is L11-1, L11-2 and L11-3; Be labeled as S15a, that encapsulate is L12-A and L12-B; Be labeled as Sui1, that encapsulate is Sui1-A and Sui1-B.
2, the enzyme labelled antibody working fluid is 1 bottle, contains enzyme labelled antibody working fluid 20ml.
3, the sample dilution is 1 bottle, contains sample dilution 20ml.
4, cleansing solution A1 bottle contains cleansing solution A50ml.
5, cleansing solution B1 pipe contains cleansing solution B1ml.
6, positive control serum 1 pipe contains serum 500 μ l.
7, negative control sera 1 pipe contains serum 500 μ l.
8, colour developing liquid A1 bottle contains colour developing liquid A10ml.
9, colour developing liquid B1 bottle contains colour developing liquid B10ml.
10, stop buffer is 1 bottle, contains stop buffer 10ml.
This kit can detect 14 person-portion serum samples simultaneously.
Kit of the present invention can be as required, the ELISA Plate of configurable different size, model and quantity, and each reagent reagent corresponding amount.
Embodiment 2: the kit with the embodiment of the invention 1 detects patients serum's sample to be checked
1, with antigen coated ELISA Plate order label URG4, URG7, URG11, S15a and Sui1, fixes with adhesive tape.
2, get each 1 part of negative control sera, positive control serum, 14 parts in patients serum's sample to be checked, totally 16 parts of serum; Add sample dilution 75 μ l in the every hole of ELISA Plate earlier, from 16 parts of serum, respectively get 25 μ l respectively by group again and add in the hand-hole mixing; Add a cover, place 37 ℃ to hatch 45min.
3, washing agent A50ml is diluted to 900ml with deionized water, adds washing agent B1ml, be settled to 1000ml after fully mixing, wash each hole respectively 5 times, each 3 minutes, clap and do.
4, add enzyme labelled antibody working fluid 100 μ l in every hole, add a cover, place 37 ℃ to hatch 45min, take out each hole of washing, back 5 times, each 3 minutes, clap and do.
5, add colour developing liquid A and each 50 μ l of colour developing liquid B, room temperature lucifuge colour developing 10 minutes in every hole successively.
6, add stop buffer 50 μ l in every hole.
7, measure each hole A value with ELIASA with wavelength 450nm/630nm, the result is as shown in table 2.
Table 2, pattern detection A value.
9, the result judges and estimates: according to judging and evaluation criterion (referring to the 5th, 6 of kit method of application of the present invention); 5 A values that detect index of positive control are all greater than 0.6; And 5 A values that detect index of negative control are all less than 0.06, and it is effective that this detects data; Calculate 5 P/N values that detect index of 14 serum samples to be checked respectively, as shown in table 3; 7 P/N values are arranged in the table 3 between 2 and 2.5, detect again, calculate the P/N value again after recording the A value, as shown in table 4,2 P/N values are arranged greater than 2.2, be judged to be the positive; As shown in table 5 according to table 3, table 4 judgement and evaluation result.
The P/N value of table 3, sample.
The P/N value that table 4, part sample detect for the second time.
Table 5, sample are judged and evaluation result.
Three, kit evaluation
1, specificity experiment
Choose respectively 8 parts in primary carcinoma of liver, liver cirrhosis patient and hepatitis B, third liver, cancer of the stomach, the carcinoma of the rectum, lung cancer, cholangiocarcinoma patients serum sample, carry out ELISA and detect, detection method is with embodiment 2, and the result sees table 6.Table 6, kit specificity experimental result of the present invention.
Visible by table 6,2 above positive rates of index of Patients with Primary serum are 100% (8/8); 2 above positive rates of index of liver cirrhosis patient serum are 87.5% (7/8); 2 above positive rates of index of hepatitis B and cholangiocarcinoma patients serum are respectively 37.5% (3/8) and 12.5% (1/8).Explain that kit of the present invention has susceptibility and specificity preferably.
2, accuracy experiment
(1) criticize in A value coefficient of variation accuracy test: the ELISA Plate that encapsulates with the polypeptide antigen L4A and the L4B of same batch of preparation; Get 3 parts of serum to be detected and detect, every duplicate samples is established 10 multiple holes, and the A value detection method is with embodiment 2; Calculate the variation within batch coefficient according to each hole A value, the result sees table 7.
Table 7, kit of the present invention are criticized interior A value coefficient of variation accuracy experimental result
Visible by table 7, A value coefficient of variation average is 4.6% in batch.Detect by the same method, batch interior A value coefficient of variation average of the ELISA Plate that other 4 groups of polypeptide antigens encapsulate all is no more than 5.5%.
(2) criticize between A value coefficient of variation accuracy experiment: the ELISA Plate that polypeptide antigen L4A for preparing with 10 different batches and L4B encapsulate; Getting 3 parts of serum to be detected detects; Every duplicate samples is established 10 multiple holes; The A value detection method calculates the variation within batch coefficient with embodiment 2 according to each hole A value, and the result sees table 8.
Table 8, kit of the present invention are criticized an A value coefficient of variation accuracy experimental result
Visible by table 8, A value coefficient of variation average is 9.3% between batch.Detect by the same method, the ELISA Plate that other 4 groups of polypeptide antigens encapsulate batch between A value coefficient of variation average all be no more than 10%.
More than the experiment show, batch in interassay coefficient of variation all less than 10%, explain that kit of the present invention has accuracy preferably.
Four, the clinic trial of kit of the present invention
Provide serum sample 200 person-portions by Shanghai Long March Hospital Infectious Disease; Wherein 60 parts of liver cancer patient blood serums, 52 parts of liver cirrhosis patient serum, 49 parts of hepatitis B patient serum, 39 parts of donors with normal serum; Detect with detection kit of the present invention, the positive rate result sees Fig. 2.Visible by Fig. 2: as in liver cancer patient, liver cirrhosis patient and the hepatitis B patient serum, to detect a kind of above positive rate of index and be followed successively by 90.4%, 92.6% and 61.7%, detect 2 kinds of above positive rates of index and be respectively 78.6%, 70.6% and 48.3%.And in 39 parts of donors with normal serum, a kind of above positive rate of index is merely 12.2%, 2 kind of above positive rate of index and is merely 6.3%.The above results shows that kit of the present invention has susceptibility and specificity preferably.
Claims (2)
1. the detection kit of a schistosomiasis japonica blood serum designated object, form as follows:
1) antigen coated ELISA Plate
2) enzyme labelled antibody working fluid: 1 bottle
3) sample dilution: 1 bottle
4) cleansing solution A:1 bottle
5) cleansing solution B:1 pipe
6) positive control serum: 1 pipe
7) negative control sera: 1 pipe
8) colour developing liquid A:1 bottle
9) colour developing liquid B:1 bottle
10) stop buffer: 1 bottle
Said antigen coated ELISA Plate, its envelope antigen are respectively 5 groups of polypeptide antigen: L4-A and L4-B, L7-B, and L11-1, L11-3 and L11-4, L12-A and L12-B, and Sui1-A and Sui1-B, their sequential structure is distinguished as follows:
Except that L7B encapsulated separately, all the other all were polypeptide equal proportion hybrid packet quilt on the same group; The goat-anti people two that said enzyme labelled antibody working fluid is the HRP mark is anti-; The sample dilution is for containing the Tris-HCl damping fluid of 2.5% (W/V) BSA; Cleansing solution A is the PBS of 0.2M; Cleansing solution B is Tween-20; Positive control serum is to take from liver cancer patient and 5 kinds of serum that indicator antibody is all positive; Negative control sera is the serum of all negative donors with normal of 5 kinds of indicator antibodies; Colour developing liquid A is for containing 0.03% (V/V) H
2O
2The phosphate citrate buffer solution; Colour developing liquid B is the citrate buffer solution that contains 1.5mM TMB; Stop buffer is 2M H
2SO
4Solution.
2. by the detection kit of the described schistosomiasis japonica blood serum designated object of claim 1, it is characterized in that forming as follows:
1) antigen coated ELISA Plate is 1, is made up of the enzyme mark bar in 10 1 * 8 holes, and per 2 is 1 group, and being divided into is 5 groups, is labeled as URG4, URG7, URG11, S15a and Sui1 respectively, and the enzyme that is labeled as URG4 is marked bar, and that encapsulate is L4A and L4B; Be labeled as URG7, that encapsulate is L7-B; Be labeled as URG11, that encapsulate is L11-1, L11-2 and L11-3; Be labeled as S15a, that encapsulate is L12-A and L12-B; Be labeled as Sui1, that encapsulate is Sui1-A and Sui1-B;
2) the enzyme labelled antibody working fluid is 1 bottle, contains enzyme labelled antibody working fluid 20ml
3) the sample dilution is 1 bottle, contains sample dilution 20ml
4) cleansing solution A is 1 bottle, contains cleansing solution A 50ml
5) cleansing solution B 1 pipe contains cleansing solution B 1ml
6) positive control serum 1 pipe contains serum 500 μ l
7) negative control sera 1 pipe contains serum 500 μ l
8) colour developing liquid A is 1 bottle, contains colour developing liquid A 10ml
9) colour developing liquid B is 1 bottle, contains colour developing liquid B 10ml
10) stop buffer is 1 bottle, contains stop buffer 10ml.
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| KR101470108B1 (en) * | 2009-07-14 | 2014-12-05 | 도꾸리쯔교세이호진 상교기쥬쯔 소고겡뀨죠 | Method for measurement of glycoprotein, method for detection of hepatic diseases, reagent for quantification of glycoprotein, and sugar chain marker glycoprotein as measure of disease conditions of hepatic diseases |
| CN101643503B (en) * | 2009-09-03 | 2012-04-18 | 广西医科大学 | Neutrophil-activating peptide 2 (72) taken as primary liver cancer markers and application thereof |
| CN102749453B (en) * | 2012-05-09 | 2014-07-23 | 中国人民解放军第二军医大学 | Applications of retinoic acid induced protein 16 (RAI16) as hepatocellular carcinoma marker |
| CN102735831B (en) * | 2012-05-21 | 2014-07-23 | 中国人民解放军第二军医大学 | Uses of truncated retinoic acid induced protein 16 (tRAI16) as hepatocellular carcinoma marker |
| CN106153893B (en) * | 2016-06-14 | 2018-01-16 | 浙江大学 | The enzyme-linked immunologic detecting kit of saliva anti-mitochondrial antibody M2 types |
| CN107942074A (en) * | 2017-11-22 | 2018-04-20 | 南宁科城汇信息科技有限公司 | It is a kind of to establish liver cancer and normal person's diagnostic model and the diagnostic model of liver cancer and chronic liver disease |
| CN107942055A (en) * | 2017-11-22 | 2018-04-20 | 南宁科城汇信息科技有限公司 | The ELISA method of transcript profile and protein science in a kind of liver cancer biological process |
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| CN101393217A (en) | 2009-03-25 |
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