CN102901829A - ELISA kit and method for detection of regucalcin level - Google Patents
ELISA kit and method for detection of regucalcin level Download PDFInfo
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Abstract
The invention provides an ELISA kit and a method for detection of the level of regucalcin, which relates to a medical detection reagent and especially to a monoclonal antibody to a regucalcin protein and a hybridoma cell secreting the antibody and having an accession number of CGMCC No. 6509. The ELISA kit and the method for detection of the level of regucalcin are established based on the monoclonal antibody and ELISA reaction principles. The kit provided by the invention can be used for detecting the level of regucalcin in serum and researching clinical significance of regucalcin to liver injury and correlation between regucalcin and other serological indexes of liver injury, which assists in better determination of the degree of inflammatory injury of liver, and therefore, a clear diagnosis of inflammatory injury of a patient with hepatopathy can be realized.
Description
Technical field
The present invention relates to medical science and detect reagent, particularly a kind of ELISA kit and method that detects regucalin level in the patients with liver deficiency serum.
Background technology
Hepatitis is one of main infectious disease of serious threat China broad masses of the people health.Hepatitis is the common consequence of a variety of causes liver injury, and is especially common with factors such as various viral infections, Drug damage, Alcoholic damages.In China, inflammation was as main due to inflammation infected take HBV again.Hepatic failure, cirrhosis and primary hepatoma due to HBV and HCV infect are one of important disease death reasons.
At present, the inflammation serological index of widespread use mainly is glutamic-pyruvic transaminase (ALT) and glutamic-oxalacetic transaminease (AST) clinically.Yet in long-term clinical practice, the doctor finds that serum aminotransferase levels at commencement and inflammation degree are usually inconsistent, has significant limitation.The clinician often need to carry out the liver biopsy pathological tissues to the patient and learn inspection, determines the truth of inflammation.The liver biopsy is the goldstandard of making a definite diagnosis liver inflammatory damage nature and extent, but it is traumatic inspection, and is expensive, and the patient is reluctant to accept, and owing to be difficult to repeat, is not suitable as the conventional means that diagnosis and curative effect are judged.Therefore, carry out auxiliary diagnosis in the urgent need to other more sensitive and accurate serological index clinically, remedy the deficiency of transaminase.
1978, the people such as Yamaguchi found to have a kind of calcium ion-binding protein in liver organization, by regulating intracellular Ca
2+The signal transmitter is regulated hepatocellular function, called after regucalin (regucalcin).1992, when detecting concerning of rats'liver soluble protein and age, the method for the human two dimensional electrophoresises such as Fujita found a kind of new albumen, its expression can independently reduce in the senile cell androgenic synthetic, its molecular weight is about 30KD, so the old and feeble marker protein 30(Senescence of called after Marker Protein-30, SMP30).1993, Shimokawa N and Yamaguchi reported a kind of cDNA of coded protein, confirmed that regucalin and SMP30 are same albumen.
Under physiological conditions, regucalin mRNA is great expression in liver cell and renal cortical cell mainly, in heart, brain, bone and some other histocytes a small amount of expression is arranged also.At liver, regucalin is present in the liver cell endochylema in a large number, accounts for 2% of Soluble Proteins in Liver total amount.Regucalin can be brought into play important physiological action at endochylema and the karyon of Various Tissues cell.Except regulating the intracellular Ca2+ stable state, also have Profilin tyrosine phosphatase, albumen serine and (or) effect of Threonine Phosphatases, thereby affect protein phosphorylation, this important modification of dephosphorylation.In addition, regucalin also has the effect that suppresses cell proliferation.2010, the Chakraborti First Determination crystal structure of regucalin, confirm to only have a metal ion binding site on the one-level crystal structure of human regucalin albumen, can comprise Zn in conjunction with divalent ion
2+, Ca
2+, Mn
2+Or Mg
2+, this crystal structure is more prone in conjunction with Zn itself
2+, strengthen enzymatic activity; Under extraneous action condition, regucalin albumen may be in conjunction with more Ca
2+Thereby promote calcium to assemble.
Regucalin gene and protein expression thereof have participated in the pathogenic process of various diseases unusually, such as high blood pressure, kidney trouble etc.Because regucalin can be released into blood, its serology level may reflect the damage of hepatic tissue to a certain extent.But how to reflect specifically it be unclear that the relation that the variation of regucalin level and liver histological are expressed in the serum also needs further research.
The inflammation serological index of widespread use mainly is glutamic-pyruvic transaminase (ALT) and glutamic-oxalacetic transaminease (AST) clinically, and they also are the leading indicators of judging curative effect.In addition, for HBV the infected, ALT and AST also are simultaneously the indications of the most frequently used antiviral therapy.Yet in long-term clinical practice, the doctor finds that serum aminotransferase levels at commencement and inflammation degree are usually inconsistent.In the normal chronic asymptomatic HBV infection person of transaminase, hepatic tissue only accounts for only a few fully normally, and about the infected of about 90% has liver tissues inflammatory and fiberization in various degree.In severe viral hepatitis, " separation of enzyme courage " phenomenon usually appears, and namely total bilirubin is more and more higher, and ALT and AST then are tending towards descending, so transaminase more can not accurately reflect the degree of heavy type hepatitis hepatic injury.In sum, ALT and AST are as present main inflammation index, and its meaning has significant limitation.Based on this point common recognition, the clinician usually needs that the patient is carried out the liver biopsy pathological tissues and learns inspection, determines the truth of inflammation.The liver biopsy is the goldstandard of making a definite diagnosis liver inflammatory damage nature and extent, but it is traumatic inspection, and is expensive, and the patient is reluctant to accept, and owing to be difficult to repeat, is not suitable as the conventional means that diagnosis and curative effect are judged.Therefore, carry out auxiliary diagnosis in the urgent need to other more sensitive and accurate serological index clinically, remedy the deficiency of transaminase.
Regucalin has certain clinical meaning aspect the reaction hepatocellular injury, but owing to still do not detect the kit of serum regucalin both at home and abroad, has greatly limited the research to its clinical meaning.Still lack at present the comparative study of system about the relation between this mark and the hepatocellular injury, the degree of inflammation correlation research of its expression and liver histological is also few, 4 routine serology WB has only been carried out in a research of heavy type hepatitis detected.Obviously, estimate the kit of the clinical meaning urgent need standard criterion of regucalin.For this reason, we attempt to search out a kind of ELISA detection method at this experiment of primary design, and by regucalin level in this method detection serum, explore regucalin for the clinical meaning of hepatic injury, and the correlativity between regucalin and other each serological index of hepatic injury, in the hope of can helping better the degree of our clear and definite inflammation damage, clarify a diagnosis thereby hepatopath's inflammation damnification made.
Summary of the invention
The present invention is based on the demand in above-mentioned field, a kind of method and kit for detection of regucalin level in the serum is provided, it is sensitive and special that the method and kit detect regucalin, the tool and method of the research of regucalin and hepatic injury Relations Among being provided convenience for this area researchist.
Secrete the mouse hybridoma cell DC091 of anti-regucalin protein 18 monoclonal antibody, preserving number is CGMCCNo.6509.
No. 18 monoclonal antibodies of anti-regucalin albumen is characterized in that: secreted by mouse hybridoma cell DC091.
A kind of ELISA kit that detects the regucalin level comprises ELISA Plate, it is characterized in that: described ELISA Plate micropore endoperidium has the coating buffer that contains above-mentioned No. 18 monoclonal antibodies.
The concentration of No. 18 monoclonal antibodies described in the coating buffer is 5ug/ml.
Described ELISA kit also comprises confining liquid, ELIAS secondary antibody, substrate nitrite ion, stop buffer.
A kind of ELISA method that detects the regucalin level is characterized in that, the first antibody in the enzyme linked immunoassay is above-mentioned No. 18 monoclonal antibodies.
In the described ELISA method, the work extension rate of ELIAS secondary antibody is 1:20000.
The preparation of described first antibody becomes in the micropore that coating buffer is coated in ELISA Plate, and the concentration of No. 18 monoclonal antibodies is 5ug/ml in the described coating buffer.
Among the present invention, the inventor has obtained the hybridoma cell strain of the monoclonal antibody of the anti-regucalin albumen of secretion.Based on this monoclonal antibody and DAS-ELISA principle, the inventor further provides a kind of method and kit that detects the regucalin level of patients with liver deficiency serum.Test figure shows that this monoclonal antibody is for detection of regucalin, and sensitivity can reach the level of pg/ml, with other albumen no cross reaction, can be used for the specific detection regucalin.
The present invention is based on same principle and the monoclonal antibody of acquisition, the kit that detects the regucalin level also is provided, the chief component assembly of kit is ELISA Plate, is coated with No. 18 monoclonal antibodies that the present invention obtains on the ELISA Plate.
Biological preservation information
Preserving number: CGMCC No.6509
Biomaterial title: mouse hybridoma cell DC091
Preservation date: on September 6th, 2012
Preservation mechanism: China Committee for Culture Collection of Microorganisms common micro-organisms center
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Description of drawings
Fig. 1. different regucalin concentration and OD value correlativity.
Ordinate is the OD value, and horizontal ordinate is regucalin concentration, the pg/ml of unit.
Embodiment
Below by concrete test figure technical scheme of the present invention is described.
Preparation and the checking of embodiment 1 regucalin antibody
1.1 the preparation of monoclonal antibody
Mouse hybridoma cell DC091 be through selectivity cultivate, screening and identify after the hybridoma cell strain that can secreting specificity antibody resists natural regucalin albumen.
Select mice celiac inoculation, be about to hybridoma and be seeded to mouse peritoneal, in Growth of Hybridoma Cell, ooze out a large amount of ascites, just contain antibody in the ascites.
Specific operation process is as follows: choose the BALb-c mouse and carry out the lumbar injection paraffin oil and induce, 0.5ml/ only; After 10-14 days, with Hybridoma Cell Culture to 5 * 10
5～10
6Individual cells/ml is carried out the hybridoma inoculation at mouse peritoneal, and 0.5ml/ only; Mouse peritoneal has ascites to ooze out after 10-14 days, can once kill mouse according to the health status of mouse and get ascites, or the interval a few days is extracted ascites several times.Adopt the water yield what and adopt the water time and should determine according to the size of mouse and health status.With the centrifugal removal precipitation of the ascites that extracts, leave and take supernatant, contain a large amount of monoclonal antibodies in the gained supernatant.
1.2 the preparation of polyclonal antibody
Regucalin albumen: this institute gets according to disclosed prokaryotic protein expression method preparation in " molecular cloning experiment guide (third edition) ".
Carry out immunity with injecting New Zealand's large ear rabbit after the emulsification of regucalin albumen (Beijing's hepatopathy research institute) process Fu Shi antigen adjuvant, thereby how anti-ly obtain.
Concrete operations are as follows: will inject New Zealand's large ear rabbit after the emulsification of regucalin albumen (Beijing's hepatopathy research institute) process Fu Shi antigen adjuvant, 1mg/ only, again injection after 4 weeks, and the 7-10 after injection days collection blood, with blood transfer to plastic centrifuge tube, 4 ° of C are centrifugal, collect supernatant and are antiserum, obtain the anti-human polyclonal antibody of rabbit.Detect antibody titer by the ELISA method.
1.3 the Western Blot of regucalin monoclonal antibody and polyclonal antibody checking
The present invention passes through behind hepatic tissue or the hepatoma cell line extraction albumen, use again antibody of the present invention and commercially available antibody (Rabbit-anti-Rat Regucalcin antibody originates from Cosmo Bio company) to carry out Western Blot experiment as primary antibodie respectively.
Whether observations can detect respectively same albumen is verified;
Prepare simultaneously the hepatic tissue paraffin section and carry out immunohistochemical staining, and utilize hepatoma carcinoma cell HepG2 cell to make cell climbing sheet and carry out immunocytochemical stain further to monoclonal antibody and many anti-checkings.
The result shows:
(1 part of tissue specimen is patients with viral hepatitis gravis by choosing 2 parts of hepatic tissue samples, in addition 1 part is liver tissues of cirrhosis) and the albumen that extracts of human hepatoma cell line HepG2's clone carry out the Westernblot experiment, confirm that self-control monoclonal antibody and many anti-and commercially available resist all detect positive bands more near 34KD.
Adopt immunocytochemical stain to detect the expression of regucalin albumen in human hepatoma cell line HepG2's cell, the result shows that regucalin all has expression in liver cell endochylema and karyon, particularly is in the nucleus of division stage, and expression is the highest.
The foundation of embodiment 2 serum regucalin double-antibody sandwich elisa detection methods
2.1 the ELISA of self-control regucalin monoclonal antibody, polyclonal antibody and ELIAS secondary antibody identifies
2.1.1 measure the regucalin protein concentration
To storE the regucalin albumen taking-up of-20 ℃ of refrigerators and treat that it melts, mixing dilutes with the PBS dilution, gets cuvette and numbers respectively mark 0,1(0 is contrast, 1 by being surveyed albumen), number 0 glass and add PBS dilution 3ml, number 1 glass of adding the albumen 0.5ml that surveys and 2.5ml PBS dilution, spectrophotometer transfers to wavelength 280nm place and detects, record OD, the calculating concentration formula: concentration=0.625 * OD * extension rate, the mg/ml of unit.According to the previous experiments method, use spectrophotometer, under wavelength 280nm condition, detect, respectively regucalin albumen, monoclonal antibody and polyclonal antibody are diluted 50 times, 6 times and 6 times, calculate by formula: the regucalin protein concentration is 2.0625mg/ml; No. 18 monoclonal antibodies (being that hybridoma DC091 is secreted) concentration is 26mg/ml; The concentration of polyclonal antibody is 15.446mg/ml.
2.1.296 orifice plate is coated with regucalin
(1) coated: with regucalin albumen coated elisa plate, coated concentration is 1 μ g/ml, 100 μ l/ holes, and 4 ℃ are spent the night or 37 ℃ after 2 hours, and cleansing solution is washed plate 3 times.
(2) sealing: add 150 μ l/ holes or fill it up with/the hole confining liquid, 4 ℃ are spent the night or 37 ℃ after 2 hours, and cleansing solution is washed plate 3 times, pats dry.It is for subsequent use to put 4 ℃ of Refrigerator stores.
2.1.3 the ELISA of monoclonal antibody identifies and titration
(1) add testing sample: the monoclonal antibody behind the doubling dilution, 100 μ l/ holes are loaded onto in the antigen coated good ELISA Plate, simultaneously, hatch 1h for 37 ℃, wash plate 6 times, pat dry.
(2) adding two resists: add the goat-anti rabbit HRP ELIAS secondary antibody (available from ancient cooking vessel state prosperous biotechnology Ltd) of dilution, its original liquid concentration is 1.0mg/ml, and 30min is hatched for 37 ℃ in 100 μ l/ holes, and cleansing solution is washed plate 6 times, pats dry.
(3) colour developing: add substrate nitrite ion A, each 80 μ l/ hole of B liquid (available from ancient cooking vessel state prosperous biotechnology Ltd), 37 ℃ were developed the color 15 minutes.
(4) stop: add stop buffer (available from ancient cooking vessel state prosperous biotechnology Ltd) 80 μ l/ holes, measure the OD value with microplate reader 450nm wavelength.
(5) reading: measure each hole OD value with the single wavelength of 450nm.
To be limited as positive or determine the critical points of tiring greater than 2.1 with the ratio (P/N) of negative control hole OD value.The ELISA of No. 18 monoclonal antibodies of regucalin tires to reach and is 1:409.6 ten thousand, and the result is as shown in table 1:
Table 1ELISA method is measured No. 18 monoclonal antibodies and is tired
Monoclonal antibody detects OD value-blank value 〉=2.1 * (negative control OD value-blank value) can be judged to be positive findings.
2.1.4 many anti-ELISA identify and titration
How anti-the evaluation of regucalin be similar with monoclonal antibody, identifies by doubling dilution, and how anti-tiring reaches 1:102 more than ten thousand, result such as table 2 to identify regucalin:
Table 2ELISA method is measured many anti-the tiring of regucalin
Many anti-detection OD value-blank values 〉=2.1 * (negative control OD value-blank value) can be judged to be positive findings.
2.1.5 goat-anti rabbit HRP ELIAS secondary antibody best effort concentration determination
Be diluted to 1mg/ml with regucalin is how anti-, goat-anti rabbit HRP ELIAS secondary antibody concentration is diluted in proportion, chooses normal human serum as negative control.
The result shows, is in the situation of 1:2 ten thousand in goat-anti rabbit HRP ELIAS secondary antibody concentration, and yin and yang attribute comparison values gap is obvious, so the best effort concentration of goat-anti rabbit HRP ELIAS secondary antibody is 1:2 ten thousand, the result is as shown in table 3:
The mensuration of ELIAS secondary antibody best effort concentration when the how anti-concentration of table 3 is 1mg/ml
2.2 the foundation of double-antibody sandwich elisa detection method
(1) coated:
The coating buffer prescription: No. 18 antibody mab concentration are 5 μ g/ml
In the little reacting hole of ELISA Plate, every hole adds 60 μ l coating buffers, and 4 ℃ are spent the night, PBST cleansing solution washing 5 times.
(2) sealing: add 100 μ l/ hole confining liquids, 4 ℃ are spent the night, cleansing solution washing 5 times.Put 4 ℃ of Refrigerator stores.
(3) antigen: measure the regucalin protein concentration, in proportion dilution is arranged according to concentration after the dilution and is added respectively in the different holes.Adopt normal human serum as negative control, do simultaneously blank.
(4) two is anti-: add regucalin and resist more, concentration is 1mg/ml, and hatched 40 minutes for 37 ℃ in 60 μ l/ holes.
(5) ELIAS secondary antibody: add goat-anti rabbit HRP ELIAS secondary antibody, 1:2 ten thousand dilutions, 100 μ l/ holes.
(6) colour developing: add respectively substrate nitrite ion A liquid and B liquid, each 50 μ l/ hole, 37 ℃ were developed the color 10 minutes.
(7) stop: add stop buffer 50 μ l/ holes, microplate reader is inserted in cotton ball soaked in alcohol wiping ELISA Plate bottom, adjusts wavelength to 450nm, measures the OD value.
(8) reading: measure each hole OD value with the single wavelength of 450nm, with the ratio (P/N) of negative control hole OD value
2.2.1 choose best value drawing standard curve
By revision test repeatedly, it is linear with the OD value in regucalin protein concentration 50pg/ml ~ 300ng/ml scope that we draw this detection method, the typical curve equation is y=0.0002x+0.3821, wherein the x value is the concentration of regucalin, the y value is for recording the OD value, related coefficient reaches 0.9931, shows that correlativity is fine, result such as table 4 and shown in Figure 1:
The different regucalin concentration of table 4 and OD value correlativity
2.3 the checking of double-antibody sandwich elisa detection method
The regucalin double-antibodies sandwich ELISA need to identify that to its specificity and sensitivity the inventor has designed following experiment and verified after setting up.
2.3.1 specificity
Choose 7 routine liver function damage patients serums, 7 routine Healthy Human Serums, totally 14 examples, every part of serum is divided into two parts, and a copy of it sample adds the grand antibody 50 μ l of monoclonal antibody of the concentration of surveying among this present embodiment 2.1.1, makes in the serum regucalin by the decline of " neutralization " concentration; Second part of sample adds isopyknic PBS contrast liquid, measures the absorbance of two parts of samples and observes the degree that absorbance is suppressed.
The result shows, 7 routine patients serums in antibody of the present invention and after, absorbance all obviously reduces, inhibiting rate is 72.6%-96.8%, and the serum of Healthy People light absorption value after neutralization reaction does not have difference substantially with contrast, albumen in the monoclonal anti physical efficiency specific binding patients with liver deficiency serum that the present invention obtains is described, but for the albumen in the healthy serum neutralization reaction does not occur, the result is as shown in table 5:
Table 5 is used in the monoclonal antibody that the present invention obtains and the detection of absorbance before and after hepatopath's serum and the healthy serum
2.3.2 sensitivity
In the varying number level scope of ug/ml, ng/ml and pg/ml, exchange calcium fibroin doubling dilution, how anti-ELISA method (how anti-monoclonal antibody and concentration is ditto described) is carried out repeated experiment according to No. 18 monoclonal antibody+regucalin antigen+regucalins, obtains the minimum number magnitude unit of positive reaction.
The result shows that in ug/ml, ng/ml and pg/ml level, regucalin protein positive group and normal human serum contrast feminine gender are organized, and its OD ratio confirms that all greater than 2.1 the method is highly sensitive, can reach and detect regucalin pg/ml level, and the result is shown in subordinate list 6,7,8:
(μ g/ml) OD value detected when table 6 regucalin concentration was diluted in proportion.
(ng/ml) OD value detected when table 7 regucalin concentration was diluted in proportion
(pg/ml) OD value detected when table 8 regucalin protein concentration diluted in proportion
The preparation of embodiment 3, kit
Kit 1:
Comprising:
Container 1, and be arranged in No. 18 regucalin monoclonal antibodies that container 1 concentration is 5 μ g/ml;
Container 2, and to be arranged in container 2 concentration be that the regucalin rabbit of 1mg/ml is how anti-;
Working volume is the 96 hole ELISA Plate in 100 μ l/ holes.
Kit 2:
Comprising:
Working volume is the 96 hole ELISA Plate that are coated with No. 18 regucalin monoclonal antibodies in 100 μ l/ holes,
Container 1, and to be arranged in container 1 concentration be that the regucalin rabbit of 1mg/ml is how anti-.
Claims (9)
1. secrete the mouse hybridoma cell DC091 of anti-regucalin protein 18 monoclonal antibody, preserving number is CGMCC No.6509.
2. anti-regucalin albumen No. 18 monoclonal antibodies is characterized in that: by the described hybridoma secretion of claim 1.
3. the solution that contains No. 18 monoclonal antibodies claimed in claim 2.
4. an ELISA kit that detects the regucalin level comprises ELISA Plate, it is characterized in that: described ELISA Plate micropore endoperidium requirement 2 described No. 18 monoclonal antibodies of having the right.
5. ELISA kit according to claim 4 is characterized in that: coated described No. 18 monoclonal antibodies are dissolved in the coating buffer, and concentration is 5ug/ml.
6. arbitrary described ELISA kit is characterized in that: also comprise confining liquid, ELIAS secondary antibody, substrate nitrite ion, stop buffer according to claim 4～5.
7. ELISA method that detects the regucalin level, it is characterized in that: the first antibody in the enzyme linked immunoassay is No. 18 monoclonal antibodies claimed in claim 2.
8. ELISA method according to claim 7, it is characterized in that: the work extension rate of goat-anti rabbit HRP ELIAS secondary antibody is 1:20000.
9. ELISA method according to claim 7 is characterized in that: described first antibody preparation becomes in the micropore that coating buffer is coated in ELISA Plate, and the concentration of No. 18 monoclonal antibodies described in the described coating buffer is 5ug/ml.
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| CN103592438A (en) * | 2013-05-29 | 2014-02-19 | 首都医科大学附属北京佑安医院 | ELISA detection kit of new diagnosis marker regucalcin for liver damage |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2007039550A2 (en) * | 2005-09-29 | 2007-04-12 | Proyecto De Biomedicina Cima, S.L. | Molecular markers of hepatocellular carcinoma and their applications |
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| WO2007039550A2 (en) * | 2005-09-29 | 2007-04-12 | Proyecto De Biomedicina Cima, S.L. | Molecular markers of hepatocellular carcinoma and their applications |
Non-Patent Citations (5)
| Title |
|---|
| MASAMI OMURA: "Effect of anti-regucalcin antibody on neutral phosphatase activity in rat liver cytosol: Involvement of endogenous regucalcin", 《MOLECULAR AND CELLULAR BIOCHEMISTRY》, vol. 197, 31 July 1999 (1999-07-31), pages 25 - 29 * |
| MASAYOSHI YAMAGUCHI: "Potential sensitivity of hepatic specific protein regucalcin as a marker of chronic liver injury", 《MOLECULAR AND CELLULAR BIOCHEMISTRY 》, vol. 167, 28 February 1997 (1997-02-28), pages 187 - 190 * |
| MASAYOSHI YAMAGUCHI: "Tissue concentration of calcium-binding protein regucalcin in rats by enzyme-linked immunoadsorbent assay", 《MOLECULAR AND CELLULAR BIOCHEMISTRY》, vol. 122, 12 May 1993 (1993-05-12), pages 65 - 68 * |
| SU-FANG ZHOU: "Serum immunoreactivity of SMP30 and its tissues expression in hepatocellular carcinoma", 《CLINICAL BIOCHEMISTRY》, vol. 44, 1 November 2010 (2010-11-01), pages 331 - 336, XP028361601, DOI: doi:10.1016/j.clinbiochem.2010.10.008 * |
| 莫发荣: "衰老标记蛋白-30 的表达与肝疾病的关系", 《解剖学报》, vol. 38, no. 5, 17 October 2007 (2007-10-17), pages 589 - 592 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103592438A (en) * | 2013-05-29 | 2014-02-19 | 首都医科大学附属北京佑安医院 | ELISA detection kit of new diagnosis marker regucalcin for liver damage |
| CN103592438B (en) * | 2013-05-29 | 2015-06-03 | 首都医科大学附属北京佑安医院 | ELISA detection kit of new diagnosis marker regucalcin for liver damage |
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