CN105092855A - Kit for detecting liver fibration and liver cirrhosis - Google Patents

Kit for detecting liver fibration and liver cirrhosis Download PDF

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Publication number
CN105092855A
CN105092855A CN201410224546.5A CN201410224546A CN105092855A CN 105092855 A CN105092855 A CN 105092855A CN 201410224546 A CN201410224546 A CN 201410224546A CN 105092855 A CN105092855 A CN 105092855A
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antibody
chi3l1
cirrhosis
hbv
kit
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CN105092855B (en
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林标扬
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HANGZHOU PROPRIUM BIOTECH COLTD
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HANGZHOU PROPRIUM BIOTECH COLTD
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Abstract

The invention discloses a kit for detecting liver fibration or liver cirrhosis caused by hepatitis B virus infection. Whether an individual has liver fibration or liver cirrhosis conditions or not is determined through detecting the level of CHI3L1 in an individual sample. The kit includes: (1) an antibody bound to the CHI3L1; (2) a labeled antibody bound to the CHI3L1 when the CHI3L1 is bound to the antibody limited in (1); and (3) a standard sample composed of a solution containing a known amount of the CHI3L1, wherein the CHI3L1 is from serum of a liver fibration or liver cirrhosis patient caused by the hepatitis B virus infection.

Description

A kind of kit detected for liver fibrosis and cirrhosis
Technical field
The invention belongs to field of biomedicine technology.Specifically, the present invention relates to a kind of kit detected for liver fibrosis and/or cirrhosis, particularly relating to a kind of kit for detecting CHI3L1 level in sample.
Background technology
Cirrhosis (Cirrhosis) refers to liver fibrosis middle and advanced stage.Its morphological definition is that diffusivity hepatic fibrosis-renal tubular ectasia syndrome is formed with abnormal nodule.Only have diffusivity liver fibrosis and form (as congenital hepatic fibrosis) without tubercle, or only nodosity is formed and all can not be called cirrhosis without fiberization (as nodular regenerative hyperplasia).From clinical angle, cirrhosis refers to that above-mentioned pathology of hepar changes the performance such as liver failure (seralbumin reduction, choline esterase activity reduction, cholerythrin rising, prolonged prothrombin etc.) and portal hypertension (esophagus fundus ventricularis varication and Rupture haemorrhag, ascites, spontaneous bacterial peritonitis and hepatorenal syndrome, hepatic encephalopathy etc.) caused.From pathology, first chronic inflammation necrosis causes hepatic fibrosis connective tissue proliferation and deposition (fiberization), then causes the broken ring of lobuli hepatis structure and pseudolobuli to be formed, finally develops into cirrhosis.
The methods for clinical diagnosis of cirrhosis comprises relevant imaging diagnosis, pathological diagnosis or/and treat and follow up a case by regular visits to.The serum sample selected derives from the patient with liver cirrhosis etc. of clinical definite.The diagnostic method of cirrhosis mainly contains following 3 classes:
1) histopathological examination: Liver biopsy tissue pathological examination is still considered to " goldstandard (Goldstandard) " of diagnosing liver fibrosis and cirrhosis so far.Hepatic fibrosis is turned to state of an illness foundation by stages by the classification that international chronic hepatitis in 1994 is new, staging scale suggestion, marks respectively with classification (mainly inflammation, downright bad degree).Hepatic tissue methods of marking conventional in the world at present comprises the systems such as Knodell, Scheuer, Ishak, Metavir, Chevallier.China 1995 and virus hepatitis control prece in 2000 also use corresponding classification, staging scale, and professor Wang Tailing has also delivered the sxemiquantitative integrating system of the liver fibrosis improved.Utilize conventional H E to dye and the histochemistry of various extracellular matrix, immunohistochemistry even hybridization histochemistry technology can obtain the much information about fiberization aspect from hepatic tissue sample; The various technology such as computer image analysis more can provide quantitative data so that observe the effect of antifibrosis therapy.Under B ultrasonic guides, adopting at present automatic liver to wear rifle, to carry out the reliability and safety of liver biopsy very high, and the misery of patient is also very little.But liver biopsy also has limitation, such as: pathology heterogeneity likely causes sampling error, be difficult to carry out same patient is repeated multiple times, be thus not easy to dynamic change or the result for the treatment of of observing liver fibrosis, therefore generally little in the actual employing of clinical diagnosis.
2) serodiagnosis of liver fibrosis: in view of the limitation of liver puncture tissue pathology checking, people find many to judging that hepatic fibroplasia has the Serum Indexes of certain values through zoopery and clinical-case-control studies.What domestic application was more has serum type III procollagen amino terminal peptide (PIIINP), IV collagen (CIV), laminin P1 (Lam), hyaluronic acid (HA).Generally speaking, in zoopery, in these indexs and liver, corresponding extracellular matrix components has good correlativity; In clinical studies, these indexs and liver histopathology fibrosis also have good correlativity, progressively raised to cirrhosis by chronic hepatitis, liver fibrosis, and the impact of Extrahepatic diseases and inflammation activity except energy, there is certain help to diagnosing liver fibrosis.But have more overlap between each group, be only difficult to once result the diagnosis making affirmative, and this type of kit domestic is badly in need of standardization and is improved its stability at present.Use in conjunction many index comprehensive descision, Mobile state of going forward side by side measures and may more contribute to judging hepatic fibrosis hyperplasia variation tendency and result for the treatment of.
3) imaging diagnosis: various conventional iconography means are as ultrasonic in Type B, CT, magnetic resonance imaging (MRI) etc. can find that Glisson's capsule thickens, liver surface profile is irregular, the even enhancing of hepatic parenchymal Echo heterogenicity or CT value increase or are nodositas, each leaf ratio changes, and thickness of spleen increases and the sign of portal vein and the cirrhosis such as splenic vein diameter is broadening and portal hypertension.Color Doppler ultrasonography or radionuclide scanning can measure volume of blood flow and the functional door body tapping condition of liver artery and portal vein.
But these detections above have muting sensitivity and specific defect, cause in diagnosis and occur very high false positive and false negative.Therefore, people develop in the urgent need to scientific worker and detect more convenient, sensitivity and specificity is higher and accuracy rate is high liver cirrhosis diagnosis means.
The modal cause of disease of cirrhosis is caused to be that hepatitis B (HBV) or hepatitis C virus (HCV) infect, hepatitis B (see http://www.hepb.org/hepb/statistics.htm) was once infected or infected in about 1/3 (2,000,000,000) of world population sum, wherein has 400,000,000 people to be lifelong chronic infection.And the HBV infection rate of China is up to 10%.Therefore, the cirrhosis caused by HBV and HCV infection increases year by year in the morbidity of China.According to incompletely statistics, China's chronic hepatitis B patients is about 2,000 ten thousand people, and wherein nearly 25 ~ 300,000 chronic hepatitis B patients can develop into cirrhosis, wherein 5 ~ 200,000 may develop into liver cancer.And China dies from the number of cirrhosis every year up to 200,000.Therefore, if can Susceptible population be found and carry out early diagnosis and intervention, then greatly can reduce the incidence of disease of cirrhosis and even liver cancer, reduce mortality, reduce medical expense and improve the quality of life of patient; Meanwhile, the discovery of cirrhosis Susceptible population is for its pathogenesis of understanding and find intervention means and also have important scientific meaning and clinical value, and it is significant that this will finally capture this persistent ailment to the mankind.As everyone knows, have " hepatitis-cirrhosis-liver cancer " trilogy to show after hepatitis B virus infection, wherein cirrhosis causes liver cell long-term chronic inflammation, heptocellular death after invading liver based on virus, and developed by hepatic fibrosis hyperblastosis.
Inventor is in cirrhosis gene differential expression and the research of relevant biomarker, be surprised to find that the high expressed of CHI3L1 albumen can occur for the liver fibrosis that caused by hepatitis B in Chinese population and cirrhosis, this prompting CHI3L1 has the possibility of the biomarker as liver fibrosis and cirrhosis.
CHI3L1 is writing a Chinese character in simplified form of HC gp-39 (HcGP-39) (or being called CHI3L1, chitinase3-like1), is abbreviated as YKL-40 again.People find after deliberation, in serum, the level of CHI3L1 is such as relevant in the oophoroma of osteoarthritis, primary colorectal carcinoma, breast cancer, recurrence with numerous disease situation, thus can be used for the diagnosis of some diseases, prognosis evaluation, result for the treatment of monitoring and PD monitoring.Such as, can be used for oophoroma diagnosis and prognosis (see Cheng Minggang, etc.The application of change of serum C HI3L1 in ovarian cancer patients diagnosis and prognosis." Guangdong medical science " 29 volume 2 phase 255-256 pages in 2008).Yale University School of Medicine researchist publishes an article title on " New England Journal of Medicine " (TheNewEnglandJournalofMedicine) published on November 15th, 2007, clinical test results shows that this molecule may play a significant role when determining severe asthma physiological reaction, compared with ordinary person, the CHI3L1 circulation serum of asthmatics can be more, simultaneously also relevant with the order of severity of asthma.(leading to network address see biology: http://www.ebiotrade.com/newsf/2007-11/20071116165039.htm).The titles such as U.S. San Diego University of California Johansen, CHI3L1 is the biomarker independent of carcinomebryonic antigen (CEA) and lactic dehydrogenase, general population change of serum C HI3L1 level height person, its gastroenteric tumor risk increases by 2.7 times, and after being diagnosed as gastroenteric tumor, prognosis is not good enough (see the Chinese medicine Tribune network edition.Network address: http://www.cmt.com.cn/article/080221/a080221b0301.htm).Change of serum C HI3L1 level raises (see Johansen, J.S., Christoffersen in alcoholic cirrhosis patient, P., Moller, S., Price, P.A., Henriksen, J.H., Garbarsch, C., andBendtsen, F. (2000) SerumCHI3L1isincreasedinpatientswithhepaticfibrosis.Jour nalofhepatology, 32:911-920.).The research of Kamal etc. shows CHI3L1 protein level hepatitis C virus chronic infection (Kamal, etal.HEPATOLOGY, 2006 relevant to liver fibrosis in blood; 43:771-779.).
But, for the relation of CHI3L1 level in the hepatitis B virus infection artifact sample such as blood that Chinese population is common and liver fibrosis and cirrhosis, also do not report at present.
Therefore, the liver fibrosis caused based on hepatitis B in CHI3L1 albumen and Chinese population and the correlativity of cirrhosis, the present invention proposes the new application process judging pathogenesis of cirrhosis, diagnosis and intervention by detecting CHI3L1 albumen.
Summary of the invention
Based on above-mentioned discovery, primary and foremost purpose of the present invention is to provide a kind of kit for detecting liver fibrosis that liver fibrosis and/or cirrhosis, especially hepatitis B virus infection cause and/or cirrhosis, by the level detecting CHI3L1 in individual specimen, it determines whether individuality has liver fibrosis and/or cirrhosis situation, and this kit comprises:
(1) can in conjunction with the antibody of CHI3L1;
(2) when the antibody limited during CHI3L1 is incorporated into (1), the labelled antibody of CHI3L1 can be incorporated into; With
(3) by the standard sample of the solution composition containing known quantity CHI3L1, wherein CHI3L1 derives from the body fluid such as serum of liver fibrosis and/or the liver cirrhosis patient caused by hepatitis B virus infection.
For simplicity, the antibody limited in above-mentioned (1) is called CHI3L1 primary antibody or CHI3L1 first antibody; The labelled antibody limited in above-mentioned (2) is called CHI3L1 second antibody.
Preferred CHI3L1 first antibody can be fixed on solid phase carrier, forms the capture antibody being used for " catching " CHI3L1.
Label in preferred CHI3L1 second antibody can catalysis or activation luminophor or fluorescent dye, thus rapidly colourless substrate be transformed into coloured product or cause light to change, or the fluorescent dye of non-fluorescence is transformed into strong fluorescence-causing substance.
Preferred mentioned reagent box comprises further:
(4) initiator solution, the chemical reaction between the label that causes in CHI3L1 second antibody and the chemiluminescence compound reacting object as label or chromogenic substrate or fluorescent dye.
Preferred CHI3L1 first antibody or be connected with chemiluminescence compound or fluorescent dye on the solid phase carrier of fixation of C HI3L1 first antibody.Whereby, method of immunity is more simplified, quantitatively more accurate.
Another object of the present invention is to provide another kind of for detecting the kit of liver fibrosis and/or the cirrhosis caused by hepatitis B virus infection, by the level detecting CHI3L1 and HBV albumen in individual specimen, it determines whether individuality has liver fibrosis and/or cirrhosis situation, and it comprises:
(1 ') can in conjunction with the antibody of CHI3L1;
(2 '), when the antibody limited during CHI3L1 is incorporated into (1 '), can be incorporated into the labelled antibody of CHI3L1;
(3 ') can in conjunction with the antibody of HBV albumen;
(4 '), when the antibody that HBV protein combination limits in (3 '), can be incorporated into the labelled antibody of HBV albumen;
(5 '), wherein CHI3L1 derived from the body fluid such as serum of liver fibrosis and/or the liver cirrhosis patient caused by hepatitis B virus infection by the standard sample of the solution composition containing known quantity CHI3L1; And by the standard sample of the solution composition containing known quantity HBV albumen, wherein HBV dietary protein origin is in the body fluid such as serum of the liver fibrosis caused by hepatitis B virus infection and/or liver cirrhosis patient.
For simplicity, the antibody limited in above-mentioned (1 ') is called CHI3L1 primary antibody or CHI3L1 first antibody; The labelled antibody limited in above-mentioned (2 ') is called CHI3L1 second antibody; The antibody limited in above-mentioned (3 ') is called HBV primary antibody or HBV first antibody; The labelled antibody limited in above-mentioned (4 ') is called HBV second antibody;
Preferred CHI3L1 first antibody and HBV first antibody can be fixed on different solid phase carriers respectively, form the capture antibody being used for " catching " CHI3L1 and HBV albumen respectively.
Label in preferred CHI3L1 second antibody and HBV albumen second antibody can catalysis or activation luminophor or fluorescent dye, thus rapidly colourless substrate is transformed into coloured product or causes light change or the fluorescent dye of non-fluorescence is transformed into strong fluorescence-causing substance.
Preferred mentioned reagent box also comprises:
(6 ') initiator solution, is respectively used to cause label in CHI3L1 second antibody and HBV albumen second antibody and reacts the chemical reaction between the chemiluminescence compound of object or chromogenic substrate or fluorescent dye respectively as label.
Preferred CHI3L1 first antibody and HBV first antibody or be respectively used to fixation of C HI3L1 first antibody and HBV first antibody solid phase carrier on be connected to chemiluminescence compound or fluorescent dye.
Above-mentioned two kinds of kits also can comprise respectively in following article one of at least: (5) carrying implement, its spatial division is the restriceted envelope can accommodating one or more containers, 96 orifice plates or lath, this container is such as medicine bottle, test tube and analog, and every sample container all contains an independent component for the inventive method; (6) auxiliary reagent, such as, developer, enzyme inhibitor, damping fluid, stabilizing agent, thinning agent, washing reagent and analog; (7) instructions, it can write on bottle, test tube and analog, or writes on an independent paper, or in the outside or inside of container, also can be multimedia form, such as CD, compact disk, video recording etc.
By means of kit of the present invention, the level of CHI3L1 in individual specimen can be measured, and then determine whether individuality has liver fibrosis and/or cirrhosis situation.Such as, when taking serum as sample, if CHI3L1 content 79ng/ml to be set as diagnosis circle point value, substantially just can determine that the individuality that change of serum C HI3L1 level exceedes this diagnosis circle point value suffers from liver fibrosis and/or cirrhosis.And for different sample types, kit of the present invention can arrange corresponding CHI3L1 level as diagnosis circle point value, and this boundary's point value is determined by simple test experiments and conventional statistical means.Therefore, kit of the present invention can as the supplementary means of the cirrhosis diagnosing hepatitis B virus infection cause.
Accompanying drawing explanation
Fig. 1 is the ROC curve interface of 1151 routine serum samples (serum of cirrhosis patients sample, 112 routine Serum of Patients with Hepatitis B samples, 725 routine normal healthy controls serum samples containing 314 routine hepatitis B virus infections cause) being carried out to CHI3L1 detection.
Fig. 2 is the ROC curve interface of the routine serum sample of Zhejiang University Medical College The First Affiliated Hospital 581 (serum of cirrhosis patients sample, 62 routine Serum of Patients with Hepatitis B samples, 357 routine normal healthy controls serum samples containing 162 routine hepatitis B virus infections cause) being carried out to CHI3L1 detection.
Fig. 3 is the ROC curve interface of the routine serum sample of 2nd Affiliated Hospital Zhejiang University School of Medicine 297 (serum of cirrhosis patients sample, 30 routine Serum of Patients with Hepatitis B samples, 200 routine normal healthy controls serum samples containing 67 routine hepatitis B virus infections cause) being carried out to CHI3L1 detection.
Fig. 4 is the ROC curve interface of the routine serum sample of Shaoyifu Hospital Attached to Zhejiang Univ. Medical College 273 (serum of cirrhosis patients sample, 20 routine Serum of Patients with Hepatitis B samples, 168 routine normal healthy controls serum samples containing 85 routine hepatitis B virus infections cause) being carried out to CHI3L1 detection.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples are only not used in for illustration of the present invention and limit scope of the present invention.
The invention provides a kind of method for detecting liver fibrosis that liver fibrosis and/or cirrhosis, especially hepatitis B virus infection cause and/or cirrhosis.The method comprises use kit to detect the level of CHI3L1 in individual specimen, determines whether individuality has liver fibrosis and/or cirrhosis situation whereby.
sample
The present invention's sample used can comprise various ways, such as whole blood, blood plasma, serum, urine, cerebrospinal fluid, saliva, seminal fluid or tears.Wherein preferred serum.
Serum sample preparation can be carried out according to commonsense method is such as centrifugal etc., such as, see such as Publication about Document: Young, D.S. & Bermes, E.W. " Specimencollectionandprocessing " inTietzTextbookofClinicalChemistry2ndEdition " Eds.Burtis; C.A. & Ashwood, E.R., Saunders (1994); MethodsinEnzymology, H.VanVunakisandJ.J.Langone (Eds), 1981,72 (B); PracticeandTheoryofEnzymeImmunoassays, PTijssen, LaboratoryTechniquesinBiochemistryandMolecularBiology, R.J.BurdenandP.H.VanKnippenberg (Eds), Elsevier, 1985; IntroductiontoRadioimmunoassayandRelatedTechniques, T.Chard, ibid, 3rdEdition, 1987; MethodsinEnzymology, H.VanVunakisandJ.J.Langone (Eds) 1981,74 (C).
kit
The first kit of the present invention comprises:
(1) can in conjunction with the antibody of CHI3L1;
(2) when the antibody limited during CHI3L1 is incorporated into (1), the labelled antibody of CHI3L1 can be incorporated into; And
(3) by the standard sample of the solution composition containing known quantity CHI3L1, this standard sample is the CHI3L1 solution of artificial preparation, and wherein CHI3L1 derives from the body fluid such as serum of liver fibrosis and/or the liver cirrhosis patient, especially Chinese population patient caused by hepatitis B virus infection.
For simplicity, the antibody limited in above-mentioned (1) is called CHI3L1 primary antibody or CHI3L1 first antibody; The labelled antibody limited in above-mentioned (2) is called CHI3L1 second antibody.
Wherein, CHI3L1 first antibody comprises any can in conjunction with the antibody of CHI3L1 or antibody fragment, and can be recombinant, chimeric antibody, humanized antibody and murine antibody.Described antibody can be monoclonal antibody or polyclonal antibody, preferred monoclonal antibody.The commercially available CHI3L1 antibody of preferred commodity in use, its example such as comprises: a series of CHI3L1 antibody products of Ai Bokang company (Abcam), such as CHI3L1 antibody [MM0628-9X24], Anti-CHI3L1 antibody (ab77528), Anti-CHI3L1 antibody (ab180569), Anti-CHI3L1 antibody [AT4A3] (ab86428), Anti-CHI3L1 antibody [AT4A3] (ab115328) etc.; Mouse anti human CHI3L1 antibody (ABIN934046); Rabbit Chinese People's Anti-Japanese Military and Political College mouse (Rattus) CHI3L1 antibody (ABIN311477); Rabbit anti-mouse CHI3L1 antibody (ABIN737556), EMDMilliporeclonemAY:MABC196, AvivaSystemsBiology, CHI3L1antibody-middleregion (ARP51929_P050); CHI3L1/YKL-40 mouse monoclonal anti-human (aa22-383) (AT4A3) antibody (LS-B3057-50) of LifeSpanBiosciences company or CHI3L1/YKL-40 mouse monoclonal anti-human (aa22-383) antibody (LS-C125352-50); The HumanChitinase3-like1MAb (Clone384327) of RandDsystems company; The CHI3L1Antibody (4A3) of NovusBiologicals company, CHI3L1Antibody (9X24); The CHI3L1/YKL40 antibody (11227-R101) of Sino Biological Inc. or CHI3L1/YKL40 antibody (AntigenAffinityPurified) (11227-RP02), etc.CHI3L1 first antibody, also by the antibody production techniques of standard, is expressed or synthesis CHI3L1 albumen or polypeptide, screening and preparation for CHI3L1 special monoclonal antibody or resist more.
In an embodiment of the invention, CHI3L1 first antibody can be fixed on solid phase carrier, forms the capture antibody being used for " catching " CHI3L1.This fixing can by covalently bound or undertaken by absorbing process.In embodiments of the present invention can solid phase carrier can be the solid support of various material, various shape and various sizes, comprise: multiple microwell plates more than 96 holes, 384 holes, test tube, sample cup, plastic microsphere, cellulose, papery or plastics testing bar, latex particle, polymer beads, silicon dioxide granule, magnetic particle, especially those mean diameters are the various materials of 0.1-10 μm and nano particle.
It can in conjunction with (with the label) antibody marked of CHI3L1 or antibody fragment, and can be recombinant, chimeric antibody, humanized antibody and murine antibody that CHI3L1 second antibody comprises any.(with the label) monoclonal antibody preferably to have marked or polyclonal antibody, the biotin labeled of such as R & DSystems resists more, CHI3L1-antibody (AlexaFluor488) (No.ABIN890291), the CHI3L1antibody (Cy5.5) of AntibodyOnline company; The CHI3L1/YKL-40 rabbit anti-human polyclonal antibody (LS-B8213-50) of LifeSpanBiosciences company; The anti-CHI3L1 antibody (orb170491) of rabbit polyclonal of Biorbyt company; Goat-anti people CHI3L1 polyclonal antibody (GWB-112941) of alkaline phosphatase monoester enzyme conjugation of GenWayBiotech company or goat-anti people CHI3L1 polyclonal antibody (18-783-77701) of HRP conjugation; The PAb (BAF2599) of the people CHI3L1 biotinylation affinity purification of RandDsystems company; The CHI3L1 antibody (H00001116-D01P) of NovusBiologicals company; The CHI3L1/YKL40 antibody (11227-RP01) of Sino Biological Inc.; The anti-CHI3L1 rabbit polyclonal antibody (TA323195) of OrigeneTechnologies company.CHI3L1 second antibody, also by the antibody production techniques of standard, is expressed or synthesis CHI3L1 albumen or polypeptide, screening and preparation for CHI3L1 special monoclonal antibody or resist more.Can antibody labeling be carried out according to conventional methods or tag, also can the commercially available CHI3L1 antibody of commodity in use.Such as: during using enzyme such as HRP (horseradish peroxidase) as label (label), the associated methods of enzyme and antibody has many kinds, such as glutaraldehyde two step method and Over-voltage protection can be used.
CHI3L1 second antibody can be combined with the label being called activator, activator can catalysis or activation luminophor or fluorescent dye, induced chemical reacts, thus rapidly colourless substrate be transformed into coloured product or cause light to change, or the fluorescent dye of non-fluorescence is transformed into strong fluorescence-causing substance.The compound of label can be used as and comprise transition metal salt and complex compound and enzyme, the peroxidase especially containing transition metal and luciferase, more specifically preferably peroxidase.Transition metal useful in label compound comprises those transition metal in periodic table 3-12 race, especially iron, copper, cobalt, zinc, manganese and chromium.Wherein peroxidase can comprise: the peroxidase of lactoperoxidase, small peroxidase, myeloperoxidase, haloperoxidase such as vanadium bromine peroxide enzyme, horseradish peroxidase, fungi such as lignin peroxidase and the peroxidase of dependence Mn produced in white-rot fungi and soybean peroxidase.The simulated compound of other oxide enzymes is not enzyme, but there is the activity of similar peroxidase, it comprises iron complex such as ferroprotoporphyrin and Mn-TPPS4 (Y.X.Ci etc., Mikrochem.J., 52,257-62 (1995)), this compound known can catalytic substrate chemiluminescence oxidation, this compound is also comprised in the scope of peroxidase implication used in the present invention.Consider the general applicability of ELISA in protein detection, the label in preferred CHI3L1 second antibody is horseradish peroxidase (Horseradishperoxidase is called for short HRP).When CHI3L1 second antibody having had label such as biotin, by adding the Avidin of activator such as HRP mark, compound second antibody such as biotin labelled antibodies-enzyme mark Avidin compound can be formed.
Different labels has respective reaction object, these reactions to as if be applicable to the compound of chemiluminescence detection, color developing detection or fluoroscopic examination.Therefore, in an embodiment of the invention, label in preferred CHI3L1 second antibody can catalysis or activation luminophor or fluorescent dye, thus rapidly colourless substrate be transformed into coloured product or cause light to change, or the fluorescent dye of non-fluorescence is transformed into strong fluorescence-causing substance.Such as, when label is horseradish peroxidase, corresponding color-developing compounds is such as conventional o-phenylenediamine (OPD), tetramethyl benzidine (TMB) such as 3,3 ', 5,5 '-tetramethyl benzidine or 2,2 '-hydrazine-bis-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) diamine salts (ABTS); When label is phosphate (AP), corresponding color-developing compounds is such as conventional p-nitrophenyl phosphate (p-NPP) or corresponding fluorogenic substrate is such as (phosphatase 24-methyl umbrella ketone).Preferred chemiluminescence compound is can be oxidized thus when there is label (namely, activator) and initiator solution time produce chemiluminescence, its exemplary compounds species comprises luminol (luminol), different luminol, the different luminol of aminobutyl ethyl (ABEI), the different luminol of Aminohexyl ethyl (AHEI), 7-dimethylamino naphthalene-l, 2-dicarboxylic acid hydrazides, cyclosubstituted amino phthalyl hydrazine, anthracene-2, 3-dicarboxylic acid hydrazides, luxuriant and rich with fragrance-1, 2-dicarboxylic acid hydrazides, pyrene dicarboxylic acid hydrazides, 5-hydroxyl-phthalylhydrazine, 6-hydroxyl phthalylhydrazine, 2, 3-benzodiazine diketone analog, 9, 10-acridan compounds such as 9, 10-acridan ester, 9, 10-acridan ester, 9, 10-acridan thioesters, 9, 10-acridan sulfonamide, 9, 10-acridan dithio keteal compound.In general, anyly knownly can produce chemiluminescent compound under the effect of hydrogen peroxide and peroxidase and all can be used as chemiluminescence compound in the present invention to produce chemiluminescence, such compound comprises and accounts for pungent dyestuff, aromatic amine and heterocyclic amine.The fluorescent dye that can use in the present invention comprises the compound of the fluorescence immunoassay that can be used for protein, and it can conjugate to protein such as antibody.Preferred fluorescent dye comprises firefly luciferin compound.Fluorescein is the substrate of luciferase, and it is oxidized thus produce oxyluciferin and luminous under luciferase existent condition.
The reaction object of these labels can separately as the ingredient of kit, that is, reaction substrate solution.
In another preferred embodiment of the present invention, can be connected on CHI3L1 first antibody or the solid phase carrier be connected to for fixation of C HI3L1 first antibody as the label reaction chemical luminous substrate of object or chromogenic substrate or fluorescent dye.Whereby, when being tested by double antibody sandwich method, must rinsing remove compared with the method for immunity of second antibody with traditional before detecting step, because eliminate separation (second antibody) and washing step and make assay method simplify.In traditional method of immunity such as ELISA, after antigen is combined with first antibody, add second antibody; Through incubation after a while, rinsing must remove the excessive second antibody be not combined with antigen, and then add zymolyte incubation, form color products.By the optimization to traditional immunization assay method, the feature of above-mentioned detection method of the present invention is, use the label in the chemiluminescence compound that is fixed on solid phase carrier or fluorescent dye and second antibody, for induced chemical luminescence-producing reaction or fluorescence radiation reaction.The common location of the chemiluminescence compound that CHI3L1 mediates or fluorescent dye and second antibody cause chemiluminescence subsequently or fluorescence radiation reaction only to occur in the site of CHI3L1 molecule.The existence of excessive second antibody does not participate in or does not hinder chemiluminescence reaction or fluorescence radiation reaction.Consequently, the light intensity of transmitting is directly proportional to the quantity of CHI3L1.Therefore more simplify compared with the method for immunity of prior art, quantitatively more accurate.In another preferred embodiment of the present invention, can be used in antibody has been connected with chemical luminous substrate or chromogenic substrate or fluorescent dye antibody as CHI3L1 first antibody, such as can use CHI3L1 antibody such as BYK-1093R-FITC, bs-10215R-FITC etc. of combined with fluorescent element on it.(with the label) monoclonal antibody of mark or polyclonal antibody comprise the biotin labeled of R & DSystems and resist more, CHI3L1-antibody (AlexaFluor488) (No.ABIN890291), the CHI3L1antibody (Cy5.5) of AntibodyOnline company.
In embodiments of the present invention, immunoassay employs, and " CHI3L1 standard sample " is for referencial use, for Criterion control curve or mathematical model.CHI3L1 standard sample is by the solution composition containing known quantity CHI3L1, and this standard sample is the CHI3L1 solution of artificial preparation.Theoretically, the CHI3L1 in standard sample can derive from any biosome comprising or produce CHI3L1, such as derives from the body fluid of genetic engineering bacterium expression or animal.But when the CHI3L1 of separate sources is as antigen, its epitope specificity may be different, thus also may be variant with the combination of same antibody, and then have influence on immune detection.As antigen, there is higher epitope specificity for making CHI3L1, preferred CHI3L1 standard sample derives from the body fluid corresponding to animal, such as derive from animal body fluid such as whole blood, blood plasma, serum, urine, cerebrospinal fluid, saliva, seminal fluid or the tears corresponding to examined biological specimen type, wherein more preferably derive from serum.Research finds, when CHI3L1 standard sample takes from the serum of liver fibrosis that hepatitis B virus infection causes and/or liver cirrhosis patient, this standard sample has wide spectrum applicability, detect the result obtained especially accurate, even if examined biological specimen type is not serum but whole blood, blood plasma, urine, cerebrospinal fluid, saliva, seminal fluid or tears.Therefore preferably CHI3L1 standard sample derives from the body fluid such as serum of the liver fibrosis that caused by hepatitis B virus infection and/or liver cirrhosis patient, the liver cirrhosis patient preferably caused by hepatitis B virus infection, especially Chinese population patient.Certainly, also can derive from genetic engineering bacterium and express or the body fluid of animal, prerequisite is that the epitope specificity of the change of serum C HI3L1 of the liver fibrosis that causes of epitope specificity and the hepatitis B virus infection of its CHI3L1 and/or liver cirrhosis patient is roughly equal to.Material as standard sample and control sample can adopt various ways, and suitable material can be in aqueous solution state.Typically, a kind of standard sample being suitable for immunoassays of the present invention can comprise the solution of known CHI3L1 content, and it can carry out gradient dilution.
Homemade CHI3L1 standard items can be used in the present invention, such as, by the protein separation means such as affine chromatography of routine, CHI3L1 is isolated from the serum of the liver cirrhosis patient caused by hepatitis B virus infection, be dissolved in damping fluid (the 20mM phosphate buffer such as containing 2M urea, citric acid-citrate damping fluid or Tris damping fluid, pH7.4), in, can dilute with identical damping fluid during use.Also some commercial CHI3L1 standard items can be used, the active people CHI3L1 full-length proteins (ab182706) of such as Abcam company in the present invention; The recombined human CHI3L1 of CreativeBiomart company, His-tagged (CHI3L1-7262H); Recombined human CHI3L1 albumen (cartilageglycoprotein-39) (CHI3L1) (TP303769) of OriGeneTechnologies company; People CHI3L1 total length ORF (AAH08568.1,1a.a.-383a.a.) of AbnovaCorporation company, has the recombinant protein of GST-tag at N-end; The CHI3L11-383aaHumanHistagCHO (IBATGP1962) of IBL-America (Immuno-BiologicalLaboratories) company; (CartilageGlycoprotein-39) (CHI3L1) albumen (Histag) (ABIN1098537) of Antibodies-online company; The recombined human CHI3L1 of R & DSystems company, CF (2599-CH-050).The dilution point of standard items can be respectively 1200pg/ml, 600pg/ml, 300pg/ml, 150pg/ml, 75pg/ml, 0pg/ml.
In a preferred embodiment of the present invention, in order to realize chemical reaction between label in CHI3L1 second antibody and chemiluminescence compound or chromogenic substrate or fluorescent dye or enzymic catalytic reaction, also can comprise in kit of the present invention:
(4) initiator solution.Initiator solution provides as producing for the reactant required for excited state compound needed for chemiluminescence.This reactant can be that one is reacted for by direct and chemiluminescence compound and carried out the necessary reactant of chemiluminescence reaction.Such as, when activator is peroxidase, will this thing happens.In a preferred embodiment, initiator solution comprises peroxide compound.This peroxide ingredient be any can with the superoxide of peroxidase reaction or alkyl hydroperoxide.Preferred superoxide comprises hydrogen peroxide (hydrogen peroxide), urea peroxide and perborate.This superoxide and peroxidase reaction, estimate it may is, at the active site of enzyme, the oxidation state of iron is become different oxidation state.Described initiator solution can also comprise the peroxidase enhancer being selected from lower group: oxybenzene compound, aromatic amine, arylboronic acid compound, aryl boric acid ester compounds, aryl boric acid anhydride compound.
In embodiments of the present invention, mentioned reagent box also comprise in following article one of at least: (5) carrying implement, its spatial division is the restriceted envelope can accommodating one or more containers, 96 orifice plates or lath, this container is such as medicine bottle, test tube and analog, and every sample container all contains an independent component for the inventive method; (6) auxiliary reagent, such as, developer, enzyme inhibitor, damping fluid, stabilizing agent, thinning agent, washing reagent and analog; (7) instructions, it can write on bottle, test tube and analog, or writes on an independent paper, or in the outside or inside of container, also can be multimedia form, such as CD, compact disk, video recording etc.
The research of the present inventor finds, the biological marker that the cirrhosis that the expression of hepatitis B viruses (HBV) also can cause as hepatitis B virus infection detects, it combines with the expression of CHI3L1, carries out the success ratio that joint-detection can significantly improve liver fibrosis that HBV infection causes and/or liver cirrhosis diagnosis.Therefore, the detection of hepatitis B in sample, the detection of CHI3L1 are incorporated in a kit, the accuracy of liver fibrosis and/or cirrhosis detection will be improved further, and improve the accuracy determining liver fibrosis and/or the cirrhosis cause of disease further.
Therefore, the second kit of the present invention, except above-mentioned CHI3L1 first antibody and CHI3L1 second antibody, also comprises:
(3 ') can in conjunction with the antibody of HBV albumen;
(4 '), when the antibody that HBV protein combination limits in (3 '), can be incorporated into the labelled antibody of HBV albumen; And
(5 ') is by the standard sample of the solution composition containing known quantity CHI3L1, this standard sample is the CHI3L1 solution of artificial preparation, and wherein CHI3L1 derives from the body fluid such as serum of liver fibrosis and/or the liver cirrhosis patient, especially Chinese population patient caused by hepatitis B virus infection; And by the standard sample of the solution composition containing known quantity HBV albumen, wherein HBV albumen can derive from the body fluid such as serum of liver fibrosis and/or the liver cirrhosis patient, especially Chinese population patient caused by hepatitis B virus infection.
For simplicity, the antibody limited in above-mentioned (3 ') is called HBV primary antibody or HBV first antibody; The labelled antibody limited in above-mentioned (4 ') is called HBV second antibody.
As for HBV albumen, it can be the protein of any kind comprised in HBV, is such as HBx, HBs, HBe, HBc etc.
It can in conjunction with the antibody of HBV albumen or antibody fragment, and can be recombinant, chimeric antibody, humanized antibody and murine antibody that HBV first antibody comprises any.Described antibody can be monoclonal antibody or polyclonal antibody, preferred monoclonal antibody, the commercially available HBV antibody of preferred commodity in use, its example such as comprises: the HBVPreS2antibody [H.11] of ABCAM company, the HBVPres1antibody of Biorbyt company, the HBV-PreS1 (CaptureAb) of Abbexa company and HepatitisBvirus (coreantigen) monoclonalantibody, the cloneLF161 of Abnova company.
Similar with the situation of above-mentioned CHI3L1 first antibody and CHI3L1 second antibody, preferred CHI3L1 first antibody and HBV first antibody can be fixed on different solid phase carriers respectively, form the capture antibody being used for " catching " CHI3L1 and HBV albumen respectively.
It can in conjunction with (with the label) antibody marked of HBV albumen or antibody fragment, and can be recombinant, chimeric antibody, humanized antibody and murine antibody that HBV second antibody comprises any.(with the label) monoclonal antibody preferably to have marked.Can antibody labeling be carried out according to conventional methods or tag, also can the commercially available HBV antibody of commodity in use.Such as: during using enzyme such as HRP (horseradish peroxidase) as label (label), the associated methods of enzyme and antibody has many kinds, such as glutaraldehyde two step method and Over-voltage protection can be used.The antibody commodity of specifiable (with label) HBV marked such as have the conjugation of Prosci company to have the goat anti-hepatitis B surface antigen polyclonal antibody of biotin; The conjugation of CreativeDiagnostics company has rabbit anti-hepatitis B surface antigen polyclonal antibody (Cat.No.DPATB-H81654) of FITC; The conjugation of ThermoScientificPierce company has the mouse anti-hepatitis B cAg monoclonal antibody Clone4H5 of HRP.
Label in HBV albumen second antibody can catalysis or activation luminophor or fluorescent dye, thus rapidly colourless substrate is transformed into coloured product or causes light change or the fluorescent dye of non-fluorescence is transformed into strong fluorescence-causing substance.The label comprised in above-mentioned CHI3L1 second antibody can be used as the label in HBV albumen second antibody equally.
Similarly, the reaction object of the label comprised in above-mentioned CHI3L1 second antibody can be used as the reaction object of the label in HBV albumen second antibody equally.
Similarly, for inducing, the initiator solution of the label in CHI3L1 second antibody and the chemical reaction between chemiluminescence compound or chromogenic substrate or fluorescent dye or enzymic catalytic reaction is same may be used for inducing chemical reaction between label in HBV albumen second antibody and chemiluminescence compound or chromogenic substrate or fluorescent dye or enzymic catalytic reaction.Therefore, in embodiments of the present invention, preferred mentioned reagent box comprises further:
(6 ') initiator solution, is respectively used to cause label in CHI3L1 second antibody and HBV albumen second antibody and reacts the chemical reaction between the chemiluminescence compound of object or chromogenic substrate or fluorescent dye respectively as label.Such as, when label is peroxidase, initiator solution comprises peroxide compound.This peroxide ingredient be any can with the superoxide of peroxidase reaction or alkyl hydroperoxide.Preferred superoxide comprises hydrogen peroxide (hydrogen peroxide), urea peroxide and perborate.
In embodiments of the present invention, HBV protein standard sample is by the solution composition containing known quantity HBV albumen, and this standard sample is the HBV protein solution of artificial preparation.Theoretically, standard sample can derive from any biosome comprising or produce HBV albumen, such as derives from the body fluid of genetic engineering bacterium expression or animal.But when the HBV albumen of separate sources is as antigen, its epitope specificity may be different, thus also may be variant and then have influence on immune detection with the combination of same antibody.As antigen, there is higher epitope specificity for making HBV albumen, preferred HBV protein standard sample derives from the body fluid corresponding to animal, such as HBV dietary protein origin, in the animal body fluid such as whole blood, blood plasma, serum, urine, cerebrospinal fluid, saliva, seminal fluid or the tears that correspond to examined biological specimen type, wherein more preferably derives from serum.Research finds, when the serum of liver fibrosis that hepatitis B virus infection causes and/or liver cirrhosis patient taken from by HBV protein standard sample, this standard sample has wide spectrum applicability, detect the result obtained especially accurate, even if examined biological specimen type is not serum but whole blood, blood plasma, urine, cerebrospinal fluid, saliva, seminal fluid or tears.Therefore preferably HBV dietary protein origin in the body fluid such as serum of the liver fibrosis caused by hepatitis B virus infection and/or liver cirrhosis patient, the liver cirrhosis patient preferably caused by hepatitis B virus infection, especially Chinese population patient.Certainly, also can derive from genetic engineering bacterium and express or the body fluid of animal, prerequisite is that the epitope specificity of the serum HBV albumen of the liver fibrosis that causes of epitope specificity and the hepatitis B virus infection of its HBV albumen and/or liver cirrhosis patient is roughly equal to.Material as standard sample and control sample can adopt various ways, and suitable material can be in aqueous solution state.Typically, the standard sample being suitable for immunoassays of the present invention can comprise the solution of known CHI3L1 content and comprise the solution of known HBV protein content, and they can carry out gradient dilution.When not causing the content detection of CHI3L1 and HBV albumen to produce error because of cross reaction, the standard sample of immunoassays of the present invention can comprise the solution of known CHI3L1 content and known HBV protein content simultaneously.
In embodiments of the present invention, preferred CHI3L1 first antibody and HBV first antibody or be respectively used to fixation of C HI3L1 first antibody and HBV first antibody solid phase carrier on be connected to chemiluminescence compound or fluorescent dye.Whereby, when testing respectively by double antibody sandwich method, must rinsing remove compared with the method for immunity of second antibody with traditional before detecting step, because eliminate separation (second antibody) and washing step and make assay method simplify, quantitatively more accurate.
Similarly, the second kit of the present invention also can comprise in following article one of at least: (5) carrying implement, its spatial division is the restriceted envelope can accommodating one or more containers, 96 orifice plates or lath, this container is such as medicine bottle, test tube and analog, and every sample container all contains an independent component for the inventive method; (6) auxiliary reagent, such as, developer, enzyme inhibitor, damping fluid, stabilizing agent, thinning agent, washing reagent and analog; (7) instructions, it can write on bottle, test tube and analog, or writes on an independent paper, or in the outside or inside of container, also can be multimedia form, such as CD, compact disk, video recording etc.
ELISA
Kit of the present invention is preferred for ELISA (enzyme linked immunosorbent assay (ELISA)), detects the sample of individuality.
Enzyme-linked immunosorbent assay (enzymelinkedimmunosorbentassay, ELISA) is the protein content analyzing method that biology field is commonly used, and it can be used for measuring antigen, also can be used for measuring antibody.According to the source of reagent, the satisfying the requirements of the proterties of sample and detection, the present invention can adopt number of different types, such as: double antibody sandwich method, dibit point single stage method, indirect method are surveyed antibody, competition law, prize law survey IgM antibody and applied the ELISA etc. of Avidin and biotin.The antibody coupling matter detecting instrument of preferred CHI3L1 antibody and HBV antibody detects, and such as, microplate reader carries out absorbance measurement.
For DAS-ELISA, the inspection principle that CHI3L1 kit detects CHI3L1 content in sample is: with anti-human CHI3L1 antibody (first antibody) bag by microwell plate, make insolubilized antibody; In the microwell plate of coated antibody, add sample to be checked, if containing measured object in sample, just can be incorporated on insolubilized antibody, thus form antigen-antibody complex; Add the anti-human CHI3L1 antibody (second antibody) that horseradish peroxidase (HRP) marks again, form antibody-antigene-HRP labelled antibody compound (biotin labeled anti-human CHI3L1 antibody also can be used as second antibody, form antibody-antigene-biotin labelled antibodies compound; Add the Avidin that horseradish peroxidase (HRP) marks again, form antibody-antigene-biotin labelled antibodies-enzyme mark Avidin compound); Finally add TMB (3,3 ', 5,5 '-TMB) substrate system (comprising hydrogen peroxide), chromogenic reaction occurs.Chromogenic reaction terminates rear microplate reader and such as detects at 450nm.Make typical curve according to the concentration of CHI3L1 in calibration object and the OD value of its correspondence, calculate CHI3L1 concentration in sample by typical curve.
rOC curve
ROC curve full name is Receiver operating curve (receiveroperatorcharacteristiccurve), is also called recipient's operating characteristic curve, is mainly used in clinical biochemical diagnostic test.ROC curve is the (sensitivity of reflection True Positive Rate, also known as susceptibility, sensitivity) and the overall target of false positive rate (1-specificity, specificity) continuous variable, be disclose sensitivity and specific mutual relationship by composition method.It is by setting a series of different cut off value (threshold value or critical value, cutoff value (cut-offvalue), divide the normal and abnormal dividing value of diagnostic test result) as continuous variable, thus calculate a series of sensitivity and specificity, be ordinate again with sensitivity, the curve drawn for horizontal ordinate of 1-specificity, area under curve (AUC) is larger, and diagnostic accuracy is higher.On ROC curve, be the critical value that sensitivity and specificity are all higher near the upper left point of coordinate diagram.ROC curve A UC value is between 1.0 and 0.5.When AUC > 0.5, AUC, more close to 1, illustrates that diagnosis effect is better.AUC has lower accuracy 0.5 ~ 0.7 time, and AUC has certain accuracy 0.7 ~ 0.9 time, has high accuracy when AUC is more than 0.9.
The evaluation method of ROC curve is different from traditional evaluation method, according to actual conditions, has allowed intermediateness, test findings can be divided into multiple ordered categorization, such as: normally, roughly normal, suspicious, roughly abnormal and abnormal five grades.
Above-mentioned ordered categorization, for the diagnosis of disease, can be divided into: negative, uncertain, positive.Further, for liver cirrhosis diagnosis, can be divided into: have cirrhosis, health.
Therefore, to detect PROTEIN C HI3L1 in sample, the present invention detects the method for cirrhosis, can comprise the steps:
(1) content of PROTEIN C HI3L1 in sample is measured respectively;
(2) with CHI3L1 protein content for variable, according to different threshold values, ROC curve is drawn out to the sensitivity of liver cirrhosis diagnosis and specificity, and area AUC under calculated curve; And
(3) sensitivity desirably and specificity, classifies (cirrhosis or health) to mensuration sample.
The drafting of ROC curve can use software or the system in prior art field, such as: MedCalc9.2.0.1 medical statistics software, SPSS9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER_POWER.SAS, CREATE_ROC.SAS, GBSTATV10.0 (DynamicMicrosystems, Inc.SilverSpring, MD, USA) etc.
the diagnosis of cirrhosis, prognosis evaluation, result for the treatment of are monitored or course of disease monitoring
The present invention, at the embody rule of medical domain, is mainly reflected in the diagnosis of cirrhosis, prognosis evaluation, result for the treatment of monitoring or course of disease monitoring aspect, comprises method of operating and realize the utensil-kit of the method.As everyone knows, the course of disease of hepatitis and cirrhosis transform closely related, and the typical course of disease is: hepatitis (such as hepatitis B, the third liver) → cirrhosis.The research of the present inventor finds, the expression of hepatitis virus also can as the biological marker of cirrhosis detection, it combines with the expression of CHI3L1, carries out the success ratio that joint-detection can significantly improve liver fibrosis and/or the liver cirrhosis diagnosis caused by hepatitis B virus infection.Therefore, present invention also offers the method for the diagnosis of a kind of liver fibrosis for being caused by HBV infection and/or cirrhosis, prognosis evaluation, result for the treatment of monitoring or course of disease monitoring, it comprises: the expression detecting HBV in individual blood sample; Detect the expression of CHI3L1 in individual blood sample; Mathematical analysis is carried out to the expression of the HBV expression recorded and CHI3L1; And according to mathematical analysis result, mensuration blood sample is classified, makes the judged result of cirrhosis.This result can be divided into: cirrhosis early stage, cirrhosis mid-term, cirrhosis late period etc.
Wherein, the expression of HBV can adopt the method for the art standard to carry out, the blood examination test paper method of such as standard, blood examination RNA isolation kit, and the blood sample test method of routine, etc.
Carry out liver cirrhosis diagnosis for the serum of individuality for sample below, the present invention is described in further detail.
Embodiment
1. clinical trial
1.1 sample sources: that collects in Zhejiang University Medical College The First Affiliated Hospital, 2nd Affiliated Hospital Zhejiang University School of Medicine and Shaoyifu Hospital Attached to Zhejiang Univ. Medical College is diagnosed as the serum sample of the cirrhosis caused by HBV infection and the serum sample of normal control, sample is the serum that in 1 year, less than-80 DEG C are preserved or the serum that in 1 month, less than-20 DEG C are preserved.Examination reagent is used to carry out the quantitative measurement of CHI3L1; With the cirrhosis of clinical unit, clinical or pathological diagnosis result compares, and utilizes ROC curve to determine suitable positive reference value, evaluates the specificity of this detection kit, susceptibility, total coincidence rate and correlation analysis.
1.2 samples selection foundations, inclusion criteria, exclusion standard and rejecting standard
Inclusion criteria: the residue serum sample after routine clinical detection or stock's serum sample; The acquisition and processing of sample meets the requirement of standard laboratory procedure code and product description; The original relevant information of sample at least comprises for the sex of sample person, sample collection date, sample type.
Exclusion standard: the sample not meeting sample collection and processing requirements; The sample of microbial contamination; Significant hemolysis, muddy sample (except interference sample group); The sample do not conformed to clinical setting.
Rejecting standard:
The sample causing obtaining a result because sample size is not enough;
The sample gone wrong in testing process.
1.3 sample collections, preservation and grouping
1) require according to the inclusion criteria of sample the blood serum sample collecting Normal group, non-cirrhosis disease control group, liver cirrhosis group.
2) sample is the serum of separating out after venous blood collection, and every part of serum volume is not less than 200uL.
3) after blood specimen collection, should not add anti-coagulants, and be placed on clean, dry in vitro standing 1 hour, centrifugal (2000rpm × 5min) gets upper serum, to be measured.
4) haemolysis is not suitable for, the detection of the sample of piarhemia or jaundice.
5) adopt fresh serum to detect, the sample of fresh collection as far as possible, if be temporarily not used in detection, must by Sample preservation at-80 DEG C.After sample freeze thawing, should detect immediately, unsuitable multigelation.As must be freeze thawing then number of freezing and thawing controlled within 3 times.Detect before sample should with equilibrium at room temperature.Freezing sample slowly need rise to room temperature, and the sample after thawing should mix.
6) if sample contains fibrin, particle or erythrocyte, reply sample carries out aggegation and centrifuging.
Blood sample is by following grouping:
Normal group
The serum sample selected is from healthy individuals person, and this group crowd is without clear and definite tumour medical history and other common chronic disease histories, and related neoplasms serological index testing result is in normal range.This group also can be described as Apparently healthy individuals group.
Namely non-cirrhosis disease control group disturbs sample group
The serum sample selected derives from the patient of the other diseases suffered from except cirrhosis.These non-cirrhosis diseases comprise hepatitis, liver cancer etc.These serum samples are easily obscured with cirrhosis in blood serum designated object context of detection maybe may occur that single testing result has positive findings to intersect and shows.
Liver cirrhosis group
The methods for clinical diagnosis of cirrhosis comprises relevant imaging diagnosis, and pathological diagnosis is or/and treat and follow up a case by regular visits to.The serum sample selected derives from the patient with liver cirrhosis etc. of clinical definite.
The clinical diagnosis of 1.4 cirrhosis
Adopt the methods for clinical diagnosis (comprising goldstandard) of prior art, complete the detection of tested crowd's cirrhosis.
1.5CHI3L1 detection kit
The plurality of reagents box that Hangzhou Pu Wang Bioisystech Co., Ltd can be used to produce carries out CHI3L1 horizontal detection to biological specimen such as serum, such as:
1.5.1CHI3L1 detection kit (euzymelinked immunosorbent assay (ELISA)) 1, Hangzhou Pu Wang Bioisystech Co., Ltd produces.It mainly comprises:
CHI3L1 first antibody is CHI3L1/YKL-40 mouse monoclonal anti-human (aa22-383) antibody (LS-C125352-50) of LifeSpanBiosciences company.The original concentration of antibody is 1mg/ml, and the dilutability of antibody can be 10-1000 doubly.With first antibody bag by 96 hole microwell plates, make insolubilized antibody, i.e. capture antibody.
CHI3L1 second antibody is the anti-human CHI3L1 polyclonal antibody PAb (BAF2599) of the biotinylation affinity purification of R & Dsystems company, it is biotin labeled anti-CHI3L1 polyclonal antibody, leaves in and adds in the damping fluid of protein stabilizing agent.The original concentration of antibody is 1mg/ml, and the dilutability of antibody can be 10-5000 doubly, and every hole addition can be 100 μ l.
For the Avidin marked for horseradish peroxidase (HRP) with the label (i.e. activator) of CHI3L1 second antibody conjugation.Can use by after 1:99 dilution; After dilution, every hole addition can be 100 μ l;
CHI3L1 standard items, by the protein separation means such as affine chromatography of routine, CHI3L1 is isolated the serum of the liver cirrhosis patient caused from the hepatitis B virus infection provided by Zhejiang University Medical College The First Affiliated Hospital, be dissolved in the 20mM phosphate buffer pH7.4 containing 2M urea, can dilute with identical damping fluid during use.The dilution point of standard items can be respectively CHI3L1 concentration 1200pg/ml, 600pg/ml, 300pg/ml, 150pg/ml, 75pg/ml, 0pg/ml.
The chromogenic reaction substrate (developer B) of label is containing the damping fluid of 3,3', 5,5'-tetramethyl benzidine, and every hole adds and can be 50 μ l.
Initiator solution (developer A), the damping fluid containing hydrogen peroxide.To press 1:1 used in combination with developer B, and every hole addition can be 50 μ l.
CHI3L1 Sample dilution, the damping fluid containing protein stabilizing agent, for detecting the front dilution to sample.
1.5.2CHI3L1 detection kit (euzymelinked immunosorbent assay (ELISA)) 2, Hangzhou Pu Wang Bioisystech Co., Ltd produces.It mainly comprises:
CHI3L1 first antibody is CHI3L1/YKL-40 mouse monoclonal anti-human (aa22-383) (AT4A3) antibody (LS-B3057-50) of LifeSpanBiosciences company.The original concentration of antibody is 1mg/ml, and the dilutability of antibody can be 10-1000 doubly.With first antibody bag by 96 hole microwell plates, make insolubilized antibody, i.e. capture antibody.
CHI3L1 second antibody is goat-anti people CHI3L1 polyclonal antibody (18-783-77701) of the HRP conjugation of GenWayBiotech company, leaves in and adds in the damping fluid of protein stabilizing agent.The original concentration of antibody is 1mg/ml, and the dilutability of antibody can be 10-5000 doubly, and every hole addition can be 100 μ l.
CHI3L1 standard items, the active people CHI3L1 full-length proteins (ab182706) of Abcam company.The dilution point of standard items is respectively CHI3L1 concentration 1200pg/ml, 600pg/ml, 300pg/ml, 150pg/ml, 75pg/ml, 0pg/ml.
The chromogenic reaction substrate (developer B) of label HRP, be the damping fluid containing 3,3', 5,5'-tetramethyl benzidine, every hole addition can be 50 μ l.
Initiator solution (developer A), the damping fluid containing hydrogen peroxide.To press 1:1 used in combination with developer B, and every hole addition amount can be 50 μ l.
CHI3L1 Sample dilution, the damping fluid containing protein stabilizing agent, for detecting the front dilution to sample.
Can also comprise other reagent that ELISA is conventional in these kits, such as cleansing solution, stop buffer etc., this is well-known to those skilled in the art.
Hereinafter list the experimental result using CHI3L1 detection kit (euzymelinked immunosorbent assay (ELISA)) 1 to detect CHI3L1 level in serum.
1.6 clinical laboratory evaluations indexs
1.6.1 clinical specificity
Be detected as negative blood sample number of cases with kit and account for ratio in the number of cases that clinical diagnosis is " non-cirrhosis ", investigate the ability that kit correctly gets rid of patients's cirrhosis.
1.6.2 clinical sensibilisin
Detecting blood sample by kit is the ratio that positive number of cases accounts in the case that clinical diagnosis is " cirrhosis ", investigates the ability that kit correctly judges patient's whether suffering from liver cirrhosis.
1.6.3 overall coincidence rate
The sensitivity of COMPREHENSIVE CALCULATING product and specific index, the accordance of reflection kit testing result and patients's cirrhosis.And carry out consistency analysis by Kappa value.
1.7 clinical testing data process and statistical analysis techniques
1.7.1 for statistics, the description such as (mean ± sd) difference partially of average ± standard is adopted; To enumeration data, number of cases and number percent is adopted to describe.
1.7.2 according to data type, the statistical analysis technique that Group Design is corresponding is selected respectively, as methods such as t inspection/rank test/Chi-square Test/exact probability.By SAS software statistics kit measurement clinical sample CHI3L1 concentration value, the correlativity of examination CHI3L1 and cirrhosis and linear regression analysis.
1.7.3 or pathological diagnosis standard such as " goldstandard " clinical with the cirrhosis of prior art compares, and carrys out the clinical analysis performance of kits for evaluation by detection sensitivity, the specificity, always coincidence rate calculating kit:
According to professional knowledge, each sample measurement result is analyzed, determine the bound of measured value, group distance and truncation points (cut-offpoint), group selectively lists cumulative frequency distribution table apart from interval, calculates the sensitivity (True Positive Rate) of all truncation points, specificity and false positive rate (1-specificity) respectively.Take True Positive Rate as ordinate, false positive rate is horizontal ordinate, draws ROC curve, calculates ROC area under curve (AUC) and 95%CI, to evaluate the value of diagnosis, and determines to block reference value.
With above-mentioned determine block reference value for basis for estimation, feminine gender, positive judgement are carried out to all detection samples, and testing result and clinical (or pathology) diagnostic result paired data are arranged as shown in table 1, calculate the indexs such as susceptibility, specificity, total coincidence rate, and by the consistance that testing result and clinical (or pathology) of Kappa test evaluation detection kit are diagnosed.If Kappa value >=0.75, consistance is better; If 0.75 > Kappa value >=0.40, consistance is medium; If Kappa value < 0.40, consistance is undesirable.
Table 1 qualitative detection result paired data statistical form
Wherein A is true positives; B is false positive; C is false negative; D is true negative.
(1) sensitivity, actual diseased and be diagnosed as positive probability, also referred to as True Positive Rate, is:
Sen(sensitivity)＝TPR＝A/(A+C)*100％
(2) specificity, actual not ill and be diagnosed as negative probability, be:
Spe(specificity)＝D/(B+D)*100％
(3) total coincidence rate (consistent percent)=(A+D)/(A+B+C+D) * 100%
(4) positive predictive value: refer in the patient of positive findings, the possibility that cirrhosis exists.Computing formula is:
Positive predictive value=true-positive results number/positive findings sum * 100%
Wherein: positive findings sum=(true-positive results number+false positive results number)
(5) negative predictive value: refer in the patient of negative findings, the possibility that non-cirrhosis exists.Computing formula is:
Negative predictive value=true negative number of results/negative findings sum * 100%
Wherein: negative findings sum=(true negative number of results+false negative result number)
(6) misdiagnosis rate: i.e. false positive rate, computing formula is:
ɑ＝FPR＝1-Spe
(7) rate of missed diagnosis: i.e. false negative rate, computing formula is:
β＝1-Sen
(8) positive likelihood ratio (LR +): the ratio of True Positive Rate and false positive rate, be the ratio of sensitivity and misdiagnosis rate, computing formula is: LR +=TPR/FPR
Wherein, the span of LR+ is (0, ∞), and its value is larger, and detection method confirms that the ability of disease is stronger.
(9) negative likelihood (LR -): the ratio of false negative rate and true negative rate, the i.e. ratio of rate of missed diagnosis and specificity, computing formula is: LR -=(1-TPR)/(1-FPR)
Wherein, the span of LR-is (0, ∞), and its value is less, and the ability that detection method gets rid of disease is better.
2 clinical study results and analysis
2.1 sample essential informations
This clinical testing is completed jointly by Zhejiang University Medical College The First Affiliated Hospital's (being called for short center 01 or Zhejiang one), 2nd Affiliated Hospital Zhejiang University School of Medicine (being called for short center 02 or Zhejiang two), Shaoyifu Hospital Attached to Zhejiang Univ. Medical College's (being called for short center 03 or Shao Yifu), selected 1151 examples altogether, samples sources is in clinical detection residue (namely remaining) sample or stock's serum sample.Each center is selected in sample situation see table 2:
Table 2 each center case distribution table
1151 routine subjects ages be 18-88 year (42.8 ± 13.7 years old), the male sex 601 example, women 550 example.Wherein healthy individuals 725 example, man 310 example, female 415 example, the age be 19-88 year (37.5 ± 11.2 years old); 314 Cases of Hepatocirrhosis, man 223 example, female 91 example, the age be 23-82 year (55.0 ± 11.1 years old); Hepatitis B 112 example, man 68 example, female 44 example, the age be 18-78 year (43.0 ± 13.6 years old).The age characteristics of samples sources refers to table 3, and Sex distribution is in table 4.
Table 3 sample object physical data table
Table 4 sample object Sex distribution situation
2.2 clinical evaluation index analysis
2.2.1 the correlation analysis of change of serum C HI3L1 level and cirrhosis
The serum sample of healthy individuals and liver cirrhosis patient is carried out to the quantitative measurement of CHI3L1, concrete measurement result refers to following table.
Compare with healthy individuals control group, CHI3L1 content obviously raises (P<0.0001), in table 5 in the expression of liver cirrhosis group.
Table 5 change of serum C HI3L1 detection level (ng/mL, mean value ± standard deviation)
Carry out Spearman rank correlation analysis to the correlativity of CHI3L1 and cirrhosis, result shows correlation coefficient r=0.751, p<0.0001, and correlation between CHI3L1 and cirrhosis is described, the CHI3L1 of liver cirrhosis group is higher than healthy group.
2.2.2ROC curve
Sample measurement result is analyzed, sets up CHI3L1 and see Fig. 1 for the ROC curve of liver cirrhosis diagnosis.The ROC curve at all the other each centers is shown in Fig. 2 to Fig. 4.The method calculating Youden index is adopted to confirm excellent diagnostics critical value.Youden index is larger, and the diagnostic value of test is higher, and Youden index is 0.870 to the maximum, and corresponding CHI3L1 diagnoses boundary point value 74ng/mL, and namely can be used as diagnosis cutoff value, its diagnostic sensitivity and specificity are respectively 92.4% and 94.6%, in detail in table 6.
Table 6 change of serum C HI3L1 content is ROC area under the curve and susceptibility, specificity in liver cirrhosis diagnosis
2.2.3 susceptibility, specificity, total coincidence rate evaluation
ROC curve adopts the method calculating Youden index to confirm that excellent diagnostics critical value is 79ng/mL, feminine gender, positive judgement are carried out to all detection samples at this center, and testing result and clinical (or pathology) diagnostic result paired data are arranged as shown in table 7.
Table 7 testing result side shows
According to above-mentioned statistics table data, the indexs such as meter sensitivity, specificity, overall coincidence rate, sum up in table 8:
Table 8CHI3L1 is used for each index of the clinical efficiency evaluation of liver cirrhosis diagnosis
Adopt the consistance that the testing result of Kappa test evaluation detection kit and clinical (or pathology) are diagnosed.If Kappa value >=0.75, consistance is better; If 0.75 > Kappa value >=0.40, consistance is medium; If Kappa value < 0.40, consistance is undesirable.The Kappa value passed judgment on healthy individuals crowd cirrhosis is 0.8586,95% fiducial interval (0.950,0.991), and P < 0.0001 can think two kinds of assay method result high consistencies.
2.2.4 the evaluation analysis of sample is disturbed
In 112 routine hepatitis interference samples, the CHI3L1/MASP2 ratio of totally 51 examples (45.5%) does not exceed diagnosis dividing value 79ng/mL.Therefore, hepatitis cases may detect this reagent interference.
3 results and discussions
This clinical testing is that the CHI3L1 kit adopting Hangzhou Pu Wang Bioisystech Co., Ltd to produce detects cirrhosis, hepatitis B, healthy individuals crowd serum sample, clinical or pathological diagnosis result compares with the cirrhosis of each clinical unit, utilize ROC curve to determine suitable positive reference value, evaluate the specificity of this detection kit, susceptibility, total coincidence rate and correlation analysis.
Test is completed jointly by Zhejiang University Medical College The First Affiliated Hospital, 2nd Affiliated Hospital Zhejiang University School of Medicine and Shaoyifu Hospital Attached to Zhejiang Univ. Medical College (center 03), have collected 1151 routine samples altogether, wherein cirrhosis sample 314 example, healthy individuals sample 725 example, hepatitis B sample 112 example.Samples sources age 18-88 year, 42.8 years old mean age, the male sex 601 example, women 550 example.
The serum sample CHI3L1 quantified results of healthier individuality and liver cirrhosis patient, healthy individuals control group change of serum C HI3L1 horizontal is 47.4 ± 23.7 (5-395) ng/ml, and liver cirrhosis group change of serum C HI3L1 level obviously raises, average out to 338.7 ± 328.1 (20-2520) ng/ml, both comparing differences have significant (P<0.0001).Carry out Spearman rank correlation analysis to the correlativity of CHI3L1 and cirrhosis further, result shows correlation coefficient r=0.751, p<0.0001, and correlation between CHI3L1 and cirrhosis is described, liver cirrhosis group CHI3L1 is higher than healthy group.
The serum sample CHI3L1 quantified results of healthy individuals and liver cirrhosis patient is analyzed, sets up the ROC curve of CHI3L1 for liver cirrhosis diagnosis.ROC area under curve is 0.972,95% credibility interval is (0.0.960-0.981).Larger according to Youden index, the diagnostic value of test is higher, and Youden index is 0.850 to the maximum, and corresponding CHI3L1 diagnoses boundary point value 79ng/ml, and namely can be used as diagnosis cutoff value, now diagnostic sensitivity and specificity are respectively 92.4% and 94.6%.Selected CHI3L1 is 79ng/ml for diagnosing the diagnosis dividing value of cirrhosis, and the sample (containing hepatitis sample) selected to 1151 examples is investigated and examined the diagnostic sensitivity of reagent and specificity to be respectively 92.4% and 89.3%, and total coincidence rate is 90.1%.The consistance that the testing result of further employing Kappa test evaluation detection kit and clinical (or pathology) are diagnosed, result shows that this test Kappa value is 0.766, P < 0.0001, can think that two kinds of diagnostic method results have consistance.
In sum, the CHI3L1 kit adopting Hangzhou Pu Wang Bioisystech Co., Ltd to produce for detecting cirrhosis, hepatitis B, healthy individuals person change of serum C HI3L1 level be respectively 92.4% and 89.3% to the sensitivity and specificity of diagnosing cirrhosis, total coincidence rate is 90.1%, diagnosis dividing value is 79ng/ml, illustrates that the mensuration of examination kit can as the supplementary means of diagnosis cirrhosis.

Claims (10)

1., for detecting a kit for liver fibrosis or the cirrhosis caused by hepatitis B virus infection, by the level detecting CHI3L1 in individual specimen, it determines whether individuality has liver fibrosis or cirrhosis situation, and this kit comprises:
(1) can in conjunction with the antibody of CHI3L1;
(2) when the antibody limited during CHI3L1 is incorporated into (1), the labelled antibody of CHI3L1 can be incorporated into; And
(3) by the standard sample of the solution composition containing known quantity CHI3L1, wherein CHI3L1 derives from the serum of the liver cirrhosis patient caused by hepatitis B virus infection.
2., for detecting a kit for liver fibrosis or the cirrhosis caused by hepatitis B virus infection, by the level detecting CHI3L1 and HBV albumen in individual specimen, it determines whether individuality has liver fibrosis or cirrhosis situation, and it comprises:
(1 ') can in conjunction with the antibody of CHI3L1;
(2 '), when the antibody limited during CHI3L1 is incorporated into (1 '), can be incorporated into the labelled antibody of CHI3L1;
(3 ') can in conjunction with the antibody of HBV albumen;
(4 '), when the antibody that HBV protein combination limits in (3 '), can be incorporated into the labelled antibody of HBV albumen; And
(5 '), wherein CHI3L1 derived from the serum of liver fibrosis or the liver cirrhosis patient caused by hepatitis B virus infection by the standard sample of the solution composition containing known quantity CHI3L1; And by the standard sample of the solution composition containing known quantity HBV albumen, wherein HBV dietary protein origin is in the serum of the liver fibrosis caused by hepatitis B virus infection or liver cirrhosis patient.
3. kit as claimed in claim 1 or 2, it is characterized in that, described sample is whole blood, blood plasma, serum, urine, cerebrospinal fluid, saliva, seminal fluid or tears.
4. kit as claimed in claim 1 or 2, it is characterized in that, also comprise initiator solution, if the antibody limited in (1) of claim 1 is called CHI3L1 first antibody, the labelled antibody limited in (2) in claim 1 is called CHI3L1 second antibody, the antibody limited in (3 ') of claim 2 is called HBV first antibody, the labelled antibody limited in (4 ') of claim 2 is called HBV second antibody, described initiator solution is for the chemical reaction between the label that causes in CHI3L1 second antibody and HBV albumen second antibody and the chemiluminescence compound reacting object respectively as label or chromogenic substrate or fluorescent dye.
5. kit as claimed in claim 4, is characterized in that, the label in described CHI3L1 second antibody and HBV albumen second antibody is peroxidase, phosphate or luciferase.
6. kit as claimed in claim 5, it is characterized in that, described peroxidase is horseradish peroxidase.
7. kit as claimed in claim 4, it is characterized in that, CHI3L1 first antibody and HBV first antibody are fixed on different solid phase carriers respectively, and described solid phase carrier is selected from the solid support of lower group: microwell plate, test tube, sample cup, plastic microsphere, cellulose, papery or plastics testing bar, latex particle, silicon dioxide granule, magnetic particle.
8. kit as claimed in claim 7, it is characterized in that, CHI3L1 first antibody and HBV first antibody or be respectively used to fixation of C HI3L1 first antibody and HBV first antibody solid phase carrier on be connected to as the chemiluminescence compound of label reaction object or chromogenic substrate or fluorescent dye.
9. kit as claimed in claim 1 or 2, it is characterized in that, described antibody is monoclonal antibody.
10. kit as claimed in claim 1 or 2, it is characterized in that, in individual specimen, the horizontal detection of CHI3L1 is implemented by ELISA.
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