CN105092855B - A kind of kit detected for liver fibrosis and hepatic sclerosis - Google Patents

Abstract

The invention discloses a kind of kit for being used to detect the liver fibrosis as caused by hepatitis B virus infection or hepatic sclerosis, it determines whether individual has liver fibrosis or hepatic sclerosis situation by detecting the level of CHI3L1 in individual specimen, and the kit is included：(1) CHI3L1 antibody can be combined；(2) when the antibody limited during CHI3L1 is incorporated into (1), CHI3L1 labelled antibody can be incorporated into；And (3), by the standard sample of the solution composition containing known quantity CHI3L1, wherein CHI3L1 is from the liver fibrosis as caused by hepatitis B virus infection or the serum of liver cirrhosis patient.

Description

A kind of kit detected for liver fibrosis and hepatic sclerosis

Technical field

The invention belongs to field of biomedicine technology.Specifically, it is used for liver fibrosis and/or liver the present invention relates to one kind The kit of detection is hardened, more particularly to a kind of kit for being used to detect CHI3L1 levels in sample.

Background technology

Hepatic sclerosis (Cirrhosis) refers to liver fibrosis middle and advanced stage.Its morphological definition is diffusivity hepatic fibrosis-renal tubular ectasia syndrome Formed with abnormal nodule.Only diffusivity liver fibrosis and without tubercle formation (such as congenital hepatic fibrosis), or only nodosity Formed and can not be referred to as hepatic sclerosis without fibrosis (such as nodular regenerative hyperplasia).From the perspective of clinic, hepatic sclerosis is Refer to above-mentioned pathology of hepar and change caused liver failure (seralbumin reduction, choline esterase activity reduction, courage Red pigment rise, prolonged prothrombin etc.) and portal hypertension (esophagus fundus ventricularis varication and its Rupture haemorrhag, ascites, from Hair property bacterial peritonitis and hepatorenal syndrome, hepatic encephalopathy etc.) etc. performance.From pathology, chronic inflammation necrosis is first First cause hepatic fibrosis connective tissue hyperplasia and deposition (fibrosis), then cause the broken ring and pseudolobuli shape of lobuli hepatis structure Into finally developing into hepatic sclerosis.

The methods for clinical diagnosis of hepatic sclerosis includes imaging diagnosis, pathological diagnosis or/and treatment and follow-up of correlation etc.. The serum sample of selection is from patient with liver cirrhosis of clinical definite etc..The diagnostic method of hepatic sclerosis mainly has following 3 class：

1) histopathological examination：Liver biopsy tissue pathological examination is still considered as diagnosing liver fibrosis so far and liver is hard " goldstandard (the Gold standard) " changed.The new classification of international chronic hepatitis in 1994, staging scale suggestion are by hepatic fibrosis The foundation of the state of an illness by stages is turned to, is scored respectively with classification (mainly inflammation, the degree of necrosis).The liver commonly used in the world at present Organize the methods of marking system such as including Knodell, Scheuer, Ishak, Metavir, Chevallier.China 1995 and 2000 Year, virus hepatitis control prece also used corresponding classification, staging scale, and it is fine that professor Wang Tailing has also delivered improved liver The sxemiquantitative integrating system of dimensionization.Using the histochemistry of conventional H E dyeing and various extracellular matrixs, immunohistochemistry very Many information about in terms of fibrosis can be obtained from hepatic tissue sample to hybridization histochemistry technology；Computer image analysis etc. Various technologies can more provide quantitative data to observe the effect of antifibrosis therapy.Used certainly under B ultrasound guiding at present The reliability and safety that dynamic liver wears rifle progress liver biopsy is very high, the painful also very little of patient.But liver biopsy also has limitation, example Such as：Lesion heterogeneity is likely to cause sampling error, it is difficult to which in the repeated multiple times progress of same patient, thus it is fine to be not easy to observation liver The dynamic change or therapeutic effect of dimensionization, thus it is general seldom in the actual use of clinical diagnosis.

2) serodiagnosis of liver fibrosis：In view of the limitation of liver puncture tissue pathology checking, people pass through animal Experiment and clinic-case-control studies are found that many to judging that hepatic fibroplasia has the Serum Indexes of certain values.State planted agent There are serum type III procollagen amino terminal peptide (PIIINP), IV collagens (CIV), laminin P1 (Lam), hyalomitome with more Sour (HA).Generally speaking, in zoopery, these indexs and corresponding extracellular matrix components in liver have good related Property；In clinical studies, these indexs and liver histopathology fibrosis also have preferable correlation, by chronic hepatitis, Liver fibrosis is stepped up to hepatic sclerosis, and Extrahepatic diseases and the movable influence of inflammation except energy, to diagnosing liver fibrosis There is certain help.But have more overlapping between each group, the diagnosis for making affirmative, and state at present are only difficult to once result Interior such kit is badly in need of standardizing and improving its stability.Use in conjunction many index comprehensive descision, Mobile state of going forward side by side is determined It may be more conducive to judge hepatic fibrosis hyperplasia variation tendency and therapeutic effect.

3) imaging diagnosis：Various conventional iconography means such as Type B ultrasound, CT, magnetic resonance imaging (MRI) etc. can be sent out Existing Glisson's capsule is thickened, and liver surface profile is irregular, and the hepatic parenchymal even enhancing of Echo heterogenicity or CT values increase or in nodositas, each leaf Ratio changes, the sign of thickness of spleen increase and portal vein and the hepatic sclerosis such as splenic vein diameter is broadening and portal hypertension.It is colored many General Le ultrasonic examination or radionuclide scanning can determine the CBF and feature Door deformation feelings of liver artery and portal vein Condition.

But the above detection has muting sensitivity and specific defect, cause very high false positive occur in diagnosis And false negative.Therefore, people develop that detection is more convenient, sensitivity and specificity are higher in the urgent need to scientific worker and The high liver cirrhosis diagnosis means of accuracy rate.

It is hepatitis B (HBV) or hepatitis C virus (HCV) infection, the pact of world population sum to cause the most common cause of disease of hepatic sclerosis Once infected or infected hepatitis B (referring to http in 1/3 (2,000,000,000)://www.hepb.org/hepb/ Statistics.htm), wherein there is 400,000,000 people to be lifelong chronic infection.And the HBV infection rate of China is up to 10%.Cause This, hepatic sclerosis increases year by year in the morbidity of China caused by HBV and HCV infection.According to incompletely statistics, China's chronic type b liver Scorching patient is about 20,000,000 people, wherein nearly 25~300,000 chronic hepatitis B patients can develop into hepatic sclerosis, wherein 5~200,000 may hair Open up as liver cancer.And the number that China dies from hepatic sclerosis every year is up to 200,000.If it is possible to find Susceptible population and carry out morning Phase diagnoses and intervened, then can substantially reduce hepatic sclerosis or even the incidence of disease of liver cancer, reduces mortality, reduces medical expense simultaneously Improve the quality of life of patient；Meanwhile, the discovery of hepatic sclerosis Susceptible population is for understanding its pathogenesis and finding intervention means Also there are important scientific meaning and clinical value, it is significant that this will finally capture this persistent ailment to the mankind.Many institute's weeks Know there is the performance of " hepatitis-hepatic sclerosis-liver cancer " trilogy after hepatitis B virus infection, wherein hepatic sclerosis is to invade liver based on virus Cause liver cell long-term chronic inflammation, heptocellular death after dirty, and developed by hepatic fibrosis hyperblastosis.

Inventor is in hepatic sclerosis gene differential expression and the research of the biomarker of correlation, it has unexpectedly been found that in China The high expression of CHI3L1 albumen can occur for the liver fibrosis as caused by hepatitis B and hepatic sclerosis in crowd, and this prompting CHI3L1, which has, to be used Make the possibility of the biomarker of liver fibrosis and hepatic sclerosis.

CHI3L1 be HC gp-39 (HcGP-39) (or for CHI3L1, chitinase 3- Like 1) write a Chinese character in simplified form, YKL-40 is abbreviated as again.People it has been investigated that, CHI3L1 level and many disease conditions in serum Such as osteoarthritis, primary colorectal carcinoma, breast cancer, recurrence oophoroma it is relevant, thus available for some diseases diagnosis, Prognosis evaluation, therapeutic effect monitoring and disease development monitoring.For example, available for oophoroma diagnosis and prognosis (referring to：Cheng Ming Just, etc..Change of serum C HI3L1 is diagnosed and the application in prognosis in ovarian cancer patients.《Guangdong medical science》2 phase 255- of volume 29 in 2008 Page 256).Yale University School of Medicine researcher published on November 15th, 2007《New England Journal of Medicine》(The New England Journal of Medicine) on publish an article title, clinical test results show the molecule may determine it is serious Played a significant role during asthma physiological reaction, compared with ordinary person, the CHI3L1 circulation serum of asthmatics can be more, simultaneously Also it is relevant with the order of severity of asthma.(referring to the logical network address of biology：http://www.ebiotrade.com/newsf/2007- 11/20071116165039.htm).U.S. San Diego University of California Johansen etc. claims, and CHI3L1 is independently of carcinomebryonic antigen (CEA) and lactic dehydrogenase biomarker, the high person of general population's change of serum C HI3L1 levels, its gastroenteric tumor risk 2.7 times of increase, and it is not good enough (referring to the Chinese medicine Tribune network edition to be diagnosed as prognosis after gastroenteric tumor.Network address：http:// www.cmt.com.cn/article/080221/a080221b0301.htm).Change of serum C HI3L1 levels are in alcoholic cirrhosis Raised in patient (referring to Johansen, J.S., Christoffersen, P., Moller, S., Price, P.A., Henriksen,J.H.,Garbarsch,C.,and Bendtsen,F.(2000)Serum CHI3L1is increased in Patients with hepaticfibrosis.Journal of hepatology, 32：911-920.).Kamal's etc. grinds Study carefully the hepatitis C virus chronic infection of CHI3L1 protein levels (Kamal, the et relevant with liver fibrosis shown in blood Al.HEPATOLOGY, 2006；43:771-779.).

But, for CHI3L1 levels and liver in biological specimen such as blood after the common hepatitis B virus infection of Chinese population The relation of fibrosis and hepatic sclerosis, there is presently no report.

Therefore, based on CHI3L1 albumen and liver fibrosis caused by hepatitis B in Chinese population and the correlation of hepatic sclerosis, this Invention proposes by detecting CHI3L1 albumen to judge pathogenesis of cirrhosis, diagnosis and the new application process intervened.

The content of the invention

Based on above-mentioned discovery, primary and foremost purpose of the invention be to provide it is a kind of be used to detecting liver fibrosis and/or hepatic sclerosis, The especially kit of liver fibrosis caused by hepatitis B virus infection and/or hepatic sclerosis, it is by detecting in individual specimen CHI3L1 level come determine individual whether have liver fibrosis and/or hepatic sclerosis situation, the kit is included：

(1) CHI3L1 antibody can be combined；

(2) when the antibody limited during CHI3L1 is incorporated into (1), CHI3L1 labelled antibody can be incorporated into；With

(3) by the standard sample of the solution composition containing known quantity CHI3L1, wherein CHI3L1 is derived from by hepatitis B sense The body fluid such as serum of liver fibrosis and/or liver cirrhosis patient caused by dye.

For convenience, the antibody limited in above-mentioned (1) is referred to as CHI3L1 primary antibodies or CHI3L1 first antibodies； The labelled antibody limited in above-mentioned (2) is referred to as CHI3L1 secondary antibodies.

It is preferred that CHI3L1 first antibodies can be fixed on solid phase carrier, be formed and resisted for " capture " CHI3L1 capture Body.

It is preferred that the label in CHI3L1 secondary antibodies can be catalyzed or activate chemiluminescence compound or fluorescence dye Material, so that colourless substrate rapidly is transformed into coloured product or causes light to change, or the fluorescence of non-fluorescence is contaminated Material is transformed into strong fluorescence-causing substance.

It is preferred that mentioned reagent box is further included：

(4) initiator solution, for triggering the label in CHI3L1 secondary antibodies with reacting object as label Chemical reaction between chemiluminescence compound or chromogenic substrate or fluorescent dye.

It is preferred that being connected with chemical hair on CHI3L1 first antibodies or solid phase carrier for fixing CHI3L1 first antibodies Optical compounds or fluorescent dye.Whereby so that method of immunity is more simplified, it is quantitative more accurate.

Another object of the present invention is to provide it is another be used for detect the liver fibrosis as caused by hepatitis B virus infection and/ Or the kit of hepatic sclerosis, it determines whether individual has liver by detecting the level of CHI3L1 and HBV albumen in individual specimen Fibrosis and/or hepatic sclerosis situation, it is included：

(1 ') can combine CHI3L1 antibody；

(2 ') can be incorporated into CHI3L1 labelled antibody when the antibody limited during CHI3L1 is incorporated into (1 ')；

(3 ') can combine the antibody of HBV albumen；

(4 ') can be incorporated into the labelled antibody of HBV albumen when the antibody that HBV protein bindings are limited in (3 ')；

(5 '), by the standard sample of the solution composition containing known quantity CHI3L1, wherein CHI3L1 is from by hepatitis B sense The body fluid such as serum of liver fibrosis and/or liver cirrhosis patient caused by dye；And by the solution group containing known quantity HBV albumen Into standard sample, wherein HBV albumen sources are in the liver fibrosis as caused by hepatitis B virus infection and/or the body fluid of liver cirrhosis patient Such as serum.

For convenience, the antibody limited in above-mentioned (1 ') is referred to as CHI3L1 primary antibodies or CHI3L1 first is anti- Body；The labelled antibody limited in above-mentioned (2 ') is referred to as CHI3L1 secondary antibodies；The antibody limited in above-mentioned (3 ') is referred to as HBV Primary antibody or HBV first antibodies；The labelled antibody limited in above-mentioned (4 ') is referred to as HBV secondary antibodies；

It is preferred that CHI3L1 first antibodies and HBV first antibodies can be fixed on different solid phase carriers respectively, respectively Form the capture antibody for " capture " CHI3L1 and HBV albumen.

It is preferred that the label in CHI3L1 secondary antibodies and HBV albumen secondary antibodies can be catalyzed or activate chemiluminescence Compound or fluorescent dye, thus rapidly by colourless substrate be transformed into coloured product or cause light change or The fluorescent dye of non-fluorescence is transformed into strong fluorescence-causing substance.

It is preferred that mentioned reagent box is also included：

(6 ') initiator solution, is respectively used to trigger the label in CHI3L1 secondary antibodies and HBV albumen secondary antibodies The chemical reaction between chemiluminescence compound or chromogenic substrate or fluorescent dye with reacting object respectively as label.

It is preferred that CHI3L1 first antibodies and HBV first antibodies or being respectively used to fix CHI3L1 first antibodies and HBV Chemiluminescence compound or fluorescent dye are connected on the solid phase carrier of one antibody.

Above two kit can also include at least one of following articles respectively：(5) instrument is carried, its space is divided For that can house the restriction space of one or more containers, 96 orifice plates or lath, the container is, for example, medicine bottle, test tube and similar Thing, is all individually used for the component of the inventive method per sample container containing one；(6) auxiliary reagent, such as, and developer, enzyme suppression Preparation, buffer solution, stabilizer, diluent, washing reagent and the like；(7) specification, its can write on bottle, test tube and On analog, either write on a single paper or in the outside or inside or multimedia form of container, Such as CD, compact disk, video recording etc..

By means of the kit of the present invention, the level of CHI3L1 in individual specimen can be determined, and then whether determine individual There are liver fibrosis and/or hepatic sclerosis situation.For example, when using serum as sample, if CHI3L1 contents 79ng/ml be set as Boundary's point value is diagnosed, is substantially assured that change of serum C HI3L1 levels exceed the individual of the diagnosis circle point value with liver fibrosis And/or hepatic sclerosis.Moreover, for different sample types, kit of the invention can set corresponding CHI3L1 levels to make For diagnosis circle's point value, boundary's point value can be determined by simple test experiments and conventional statistical means.Therefore, it is of the invention Kit can be used as diagnosis hepatitis B virus infection caused by liver fibrosis and/or hepatic sclerosis supplementary means.

Brief description of the drawings

Fig. 1 is (containing serum of cirrhosis patients sample, 112 caused by 314 hepatitis B virus infections to 1151 serum samples Example Serum of Patients with Hepatitis B sample, 725 normal healthy controls serum samples) carry out the ROC curve interfaces of CHI3L1 detections.

Fig. 2 is that 581 serum samples of Zhejiang University Medical College The First Affiliated Hospital (are drawn containing 162 hepatitis B virus infections Serum of cirrhosis patients sample, 62 Serum of Patients with Hepatitis B samples, the 357 normal healthy controls serum samples risen) carry out CHI3L1 The ROC curve interface of detection.

Fig. 3 is that 297 serum samples of 2nd Affiliated Hospital Zhejiang University School of Medicine (are caused containing 67 hepatitis B virus infections Serum of cirrhosis patients sample, 30 Serum of Patients with Hepatitis B samples, 200 normal healthy controls serum samples) carry out CHI3L1 inspections The ROC curve interface of survey.

Fig. 4 is that 273 serum samples of Shaoyifu Hospital Attached to Zhejiang Univ. Medical College (are drawn containing 85 hepatitis B virus infections Serum of cirrhosis patients sample, 20 Serum of Patients with Hepatitis B samples, the 168 normal healthy controls serum samples risen) carry out CHI3L1 The ROC curve interface of detection.

Embodiment

Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following examples are merely to illustrate this Invention rather than restriction the scope of the present invention.

The present invention provides a kind of for detecting liver caused by liver fibrosis and/or hepatic sclerosis, especially hepatitis B virus infection The method of fibrosis and/or hepatic sclerosis.This method detects the level of CHI3L1 in individual specimen including the use of kit, whereby It is determined that whether individual has liver fibrosis and/or hepatic sclerosis situation.

Sample

Sample used of the invention can include diversified forms, such as whole blood, blood plasma, serum, urine, cerebrospinal fluid, saliva, essence Liquid or tears.Wherein preferred serum.

Serum sample is prepared such as be centrifuged according to commonsense method etc. and carried out, for example, with reference to following document：Young, D.S.&Bermes,E.W."Specimen collection and processing"in Tietz Textbook of Clinical Chemistry2nd Edition"Eds.Burtis,C.A.&Ashwood,E.R.,Saunders(1994)； Methods in Enzymology,H.Van Vunakis and J.J.Langone(Eds),1981,72(B)；Practice and Theory of Enzyme Immunoassays,P Tijssen,Laboratory Techniques in Biochemistry and Molecular Biology,R.J.Burden and P.H.Van Knippenberg(Eds), Elsevier,1985；Introduction to Radioimmunoassay and Related Techniques, T.Chard,ibid,3rd Edition,1987；Methods in Enzymology,H.Van Vunakis and J.J.Langone(Eds)1981,74(C)。

Kit

The first kit of the present invention is included：

(1) CHI3L1 antibody can be combined；

(2) when the antibody limited during CHI3L1 is incorporated into (1), CHI3L1 labelled antibody can be incorporated into；And

(3) by the standard sample of the solution composition containing known quantity CHI3L1, the standard sample is that the CHI3L1 manually prepared is molten Liquid, wherein CHI3L1 derive from the liver fibrosis as caused by hepatitis B virus infection and/or liver cirrhosis patient, especially Chinese population The body fluid of patient such as serum.

For convenience, the antibody limited in above-mentioned (1) is referred to as CHI3L1 primary antibodies or CHI3L1 first antibodies； The labelled antibody limited in above-mentioned (2) is referred to as CHI3L1 secondary antibodies.

Wherein, CHI3L1 first antibodies can combine CHI3L1 antibody or antibody fragment including any, and can be Recombinant, chimeric antibody, humanized antibody and murine antibody.The antibody can be monoclonal antibody or polyclonal antibody, It is preferred that monoclonal antibody.The commercially available CHI3L1 antibody of commercialization is preferably used, its example such as includes：Ai Bokang companies (Abcam) a series of CHI3L1 antibody products, 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-anti rat (Rattus) CHI3L1 antibody (ABIN311477)；Rabbit anti-mouse CHI3L1 antibody (ABIN737556), EMD Millipore clone mAY:MABC196,Aviva Systems Biology,CHI3L1antibody-middle region (ARP51929_P050)；CHI3L1/YKL-40 mouse monoclonal anti-humans (the aa22- of LifeSpan Biosciences companies 383) (AT4A3) antibody (LS-B3057-50) or CHI3L1/YKL-40 mouse monoclonal anti-humans (aa22-383) antibody (LS- C125352-50)；The Human Chitinase 3-like 1MAb (Clone 384327) of R and D systems companies； CHI3L1Antibody (4A3), the CHI3L1Antibody (9X24) of Novus Biologicals companies；Yi Qiao Divine Land, Beijing The CHI3L1/YKL40 antibody (11227-R101) or CHI3L1/YKL40 antibody (Antigen of Bioisystech Co., Ltd Affinity Purified) (11227-RP02), etc..CHI3L1 first antibodies also can by the antibody production techniques of standard, Expression or synthesis CHI3L1 albumen or polypeptide, screening and preparation are directed to CHI3L1 special monoclonal antibody or resisted more.

In an embodiment of the invention, CHI3L1 first antibodies can be fixed on solid phase carrier, are formed and used In " capture " CHI3L1 capture antibody.This fixation can be by being covalently attached or being carried out by absorbing process.In this hair Available solid phase carrier can be various materials, variously-shaped and various sizes solid supports, bag in bright embodiment Include：A variety of microwell plates more than 96 holes, 384 holes, test tube, specimen cup, plastic microsphere, cellulose, papery or plastics testing bar, glue Milk particle, polymer beads, silicon dioxide granule, magnetic particle, especially those average diameters be 0.1-10 μm and nanometer The various materials of particle.

CHI3L1 secondary antibodies can combine CHI3L1 marked (carrying label) antibody or antibody including any Fragment, and can be recombinant, chimeric antibody, humanized antibody and murine antibody.It is preferred that marked (carries label ) monoclonal antibody or polyclonal antibody, such as R＆D Systems biotin labeling resists more, Antibody Online CHI3L1-antibody (Alexa Fluor 488) (No.ABIN890291), the CHI3L1antibody (Cy5.5) of company； The CHI3L1/YKL-40 rabbits anti-human polyclonal antibody (LS-B8213-50) of LifeSpan Biosciences companies；Biorbyt The anti-CHI3L1 antibody (orb170491) of rabbit polyclonal of company；The alkaline phosphatase monoester enzyme conjugation of GenWay Biotech companies Goat-anti people CHI3L1 polyclonal antibodies (GWB-112941) or HRP conjugation goat-anti people's CHI3L1 polyclonal antibodies (18- 783-77701)；The PAb (BAF2599) of people's CHI3L1 biotinylation affinity purifications of R and D systems companies；Novus The CHI3L1 antibody (H00001116-D01P) of Biologicals companies；Sino Biological Inc. CHI3L1/YKL40 antibody (11227-RP01)；The anti-CHI3L1 rabbit polyclonal antibodies of Origene Technologies companies (TA323195).CHI3L1 secondary antibodies also can be by the antibody production techniques of standard, expression or synthesis CHI3L1 albumen or many The special monoclonal antibody or more anti-of peptide, screening and preparation for CHI3L1.Antibody labeling or mark-on can be carried out according to conventional methods Label, it is possible to use the commercially available CHI3L1 antibody of commercialization.Such as：Label is used as using enzyme such as HRP (horseradish peroxidase) When (label), the associated methods of enzyme and antibody have many kinds, for example, can use glutaraldehyde two step method and Over-voltage protection.

The label of referred to as activator is may incorporate in CHI3L1 secondary antibodies, activator can be catalyzed or activate chemistry Luminophor or fluorescent dye, induced chemical reaction so that rapidly by colourless substrate be transformed into coloured product or Person causes light to change, or the fluorescent dye of non-fluorescence is transformed into strong fluorescence-causing substance.It can act as the chemical combination of label Thing is including transition metal salt and complex compound and enzyme, peroxidase and luciferase especially containing transition metal, more especially It is preferably peroxidase.Those mistakes during useful transition metal includes periodic table 3-12 races in mark compounds Cross metal, especially iron, copper, cobalt, zinc, manganese and chromium.Wherein peroxidase can include：Lactoperoxidase, small peroxide Compound enzyme, myeloperoxidase, haloperoxidase such as vanadium bromine peroxide enzyme, horseradish peroxidase, the mistake of fungi The peroxidase and soybean mistake of oxide enzyme such as lignin peroxidase and the dependence Mn produced in white-rot fungi Oxide enzyme.The simulated compound of other oxide enzymes is not enzyme, but the activity with similar peroxidase, and it includes Iron complex such as ferroprotoporphyrin and Mn-TPPS4 (Y.X.Ci etc., Mikrochem.J., 52,257-62 (1995)), it is known that The compound can be catalyzed the chemiluminescence oxidation of substrate, and this compound is also contained in peroxide used in the present invention In the range of enzyme implication.In view of general applicability of the ELISA in protein detection, the preferably mark in CHI3L1 secondary antibodies Remember that thing is horseradish peroxidase (Horseradish peroxidase, abbreviation HRP).Have when in CHI3L1 secondary antibodies During label such as biotin, by adding the Avidin of activator such as HRP marks, compound secondary antibody can be formed such as Biotin labelled antibodies-enzyme mark Avidin compound.

Different labels has respective reaction object, and these reaction objects apply to chemiluminescence detection, colour developing Detection or the compound of fluoroscopic examination.Therefore, in an embodiment of the invention, preferably in CHI3L1 secondary antibodies Label can be catalyzed or activate chemiluminescence compound or fluorescent dye, so as to rapidly be transformed into colourless substrate Coloured product or cause light to change, or the fluorescent dye of non-fluorescence is transformed into strong fluorescence-causing substance.Such as, when When label is horseradish peroxidase, corresponding color-developing compounds are, for example, conventional o-phenylenediamine (OPD), tetramethyl biphenyl Amine (TMB) such as 3,3 ', 5,5 '-tetramethyl benzidine or 2,2 '-hydrazine-bis- (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid) diamines Salt (ABTS)；When label is phosphate (AP), corresponding color-developing compounds are, for example, conventional p-nitrophenyl phosphate (p-NPP) or accordingly fluorogenic substrate is, for example, (phosphatase 24-methyl umbrella ketone).It is preferred that chemiluminescence compound be can be by Oxidation when there is label (that is, activator) and initiator solution so that produce chemiluminescence, its exemplary compounds species Including luminol (luminol), different luminol, the different luminol of aminobutyl ethyl (ABEI), the different luminol of Aminohexyl ethyl (AHEI), 7- dimethylamino naphthalene-l, 2- dicarboxylic acids hydrazides, ring take The amino phthalyl hydrazine in generation, anthracene -2,3- dicarboxylic acids hydrazides, phenanthrene -1,2- dicarboxylic acids hydrazides, pyrene dicarboxylic acids hydrazides, 5- hydroxyls - Phthalylhydrazine, 6- hydroxyls phthalylhydrazine, 2,3- benzodiazine diketone analog, acridan class chemical combination Thing such as acridan ester, acridan ester, acridan thioesters, acridan Sulfonamide, acridan dithio keteal compound.In general, it is any it is known can be in hydrogen peroxide and peroxide Chemiluminescent compound is produced in the presence of compound enzyme can all be used as chemiluminescence compound in the present invention come generation Learn luminous, such compound includes accounting for pungent dyestuff, aromatic amine and heterocyclic amine.The fluorescent dye that can be used in the present invention includes Available for the compound of the fluorescence immunoassay of protein, it can conjugate to protein such as antibody.It is preferred that fluorescent dye bag Include firefly luciferin compound.Fluorescein is the substrate of luciferase, its luciferase presence under conditions of be oxidized from And produce oxyluciferin and light.

The reaction object of these labels can separately as kit part, i.e. reaction substrate solution.

In another preferred embodiment of the present invention, chemical luminous substrate or the colour developing of object are reacted as label Substrate or fluorescent dye can be connected on CHI3L1 first antibodies or are connected to for fixing CHI3L1 first antibodies Solid phase carrier on.Whereby, when being tested by double antibody sandwich method, removing must be rinsed before detecting step with traditional The method of immunity of secondary antibody is compared, and makes assay method simple because separation (secondary antibody) and washing step is eliminated Change.In traditional method of immunity such as ELISA, after antigen is combined with first antibody, secondary antibody is added；By The incubation of a period of time, it is necessary to which rinsing removes excessive secondary antibody not with antigen binding, then adds zymolyte incubation, Form color products.By the optimization to traditional immunization assay method, above-mentioned detection method of the invention is characterised by, is used The label on chemiluminescence compound or fluorescent dye and secondary antibody being fixed on solid phase carrier, for inductionization Learn luminescence-producing reaction or fluorescence radiation reaction.The chemiluminescence compound of CHI3L1 mediations or the common location of fluorescent dye and the Two antibody cause subsequent chemiluminescence or fluorescence radiation the reaction generation only at the site of CHI3L1 molecules.Excessive second resists The presence of body is not involved in or without prejudice to chemiluminescence reaction or fluorescence radiation reaction.As a result, transmitting luminous intensity with CHI3L1 quantity is directly proportional.Therefore more simplify compared with the method for immunity of prior art, it is quantitative more accurate.In this hair In another bright preferred embodiment, can use be connected with antibody chemical luminous substrate or chromogenic substrate or The antibody of fluorescent dye can for example use as CHI3L1 first antibodies and combine the CHI3L1 antibody of fluorescein such as thereon BYK-1093R-FITC, bs-10215R-FITC etc..(the carrying label) monoclonal antibody of mark or polyclonal antibody bag Include more anti-, the CHI3L1-antibody (Alexa of Antibody Online companies of R＆D Systems biotin labeling Fluor 488)(No.ABIN890291)、CHI3L1antibody(Cy5.5)。

In embodiments of the present invention, immunoassay has used " CHI3L1 standard samples " to refer to, for setting up standard Control curve or mathematical modeling.CHI3L1 standard samples are artificial by the solution composition containing known quantity CHI3L1, the standard sample The CHI3L1 solution of preparation.Theoretically, the CHI3L1 in standard sample can include or produce CHI3L1's from any Organism, such as from genetic engineering bacterium expression or the body fluid of animal.However, when the CHI3L1 of separate sources is as antigen, Its epitope specificity may be different so that and same antibody combination may also can be variant, and then have influence on immune Detection.To make CHI3L1 have higher epitope specificity as antigen, preferably CHI3L1 standard samples, which are derived from, corresponds to animal Body fluid, such as from the animal body fluid corresponding to examined biological specimen type such as whole blood, blood plasma, serum, urine, brain Spinal fluid, saliva, seminal fluid or tears, wherein more preferably deriving from serum.Research is found, when CHI3L1 standard samples are derived from B-type hepatitis Caused by poison infection during the serum of liver fibrosis and/or liver cirrhosis patient, the standard sample has wide spectrum applicability, what detection was obtained As a result it is especially accurate, even if examined biological specimen type is not serum but whole blood, blood plasma, urine, cerebrospinal fluid, saliva, essence Liquid or tears.Therefore preferably CHI3L1 standard samples are suffered from from the liver fibrosis as caused by hepatitis B virus infection and/or hepatic sclerosis The body fluid such as serum of person, the preferably liver cirrhosis patient as caused by hepatitis B virus infection, especially Chinese population patient.Certainly, Can also be from genetic engineering bacterium expression or the body fluid of animal, on condition that its CHI3L1 epitope specificity and hepatitis B The change of serum C HI3L1 of liver fibrosis and/or liver cirrhosis patient epitope specificity is substantially equivalent caused by infection.As standard sample It can be taken various forms with the material of control sample, it is in aqueous solution state that suitable material, which can be,.Typically, one kind is suitable for The standard sample of the immunoassays of the present invention can include the solution of known CHI3L1 contents, and it can carry out gradient dilution.

Homemade CHI3L1 standard items can be used in the present invention, for example, passing through conventional protein separation means Such as affine chromatography, CHI3L1 is isolated from the serum of the liver cirrhosis patient as caused by hepatitis B virus infection, is dissolved in buffering Liquid (20mM phosphate buffers, citric acid-citrate buffer solution or Tris buffer solutions such as containing 2M ureas, pH 7.4) in, it can be diluted when using with identical buffer solution.The CHI3L1 of some commercializations can also be used to mark in the present invention The active people CHI3L1 full-length proteins (ab182706) of quasi- product, such as Abcam companies；The restructuring of Creative Biomart companies People CHI3L1, His-tagged (CHI3L1-7262H)；The recombined human CHI3L1 albumen of OriGene Technologies companies (cartilage glycoprotein-39)(CHI3L1)(TP303769)；The people of Abnova Corporation companies CHI3L1 total lengths ORF (AAH08568.1,1a.a.-383a.a.), has GST-tag recombinant protein at N- ends；IBL- CHI3L1,1-383aa Human, the His tag, CHO of America (Immuno-Biological Laboratories) company (IBATGP1962)；(Cartilage Glycoprotein-39) (CHI3L1) albumen of Antibodies-online companies (His tag)(ABIN1098537)；The recombined human CHI3L1, CF (2599-CH-050) of R＆D Systems companies.Standard items Dilution point 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 the label in CHI3L1 secondary antibodies with chemistry send out In chemical reaction or enzymic catalytic reaction between optical compounds or chromogenic substrate or fluorescent dye, kit of the invention also It can include：

(4) initiator solution.Initiator solution is provided needed for the excitation state compound for needed for producing for chemiluminescence The reactant wanted.The reactant can be a kind of anti-for carrying out chemiluminescence by directly being reacted with chemiluminescence compound Should necessary reactant.For example, when activator is peroxidase, it will this thing happens.It is preferable to carry out in one kind In mode, initiator solution includes peroxide compound.The peroxide ingredient be it is any can be with peroxidase reaction Peroxide or alkyl hydroperoxide.It is preferred that peroxide include hydrogen peroxide (hydrogen peroxide), urea peroxide and mistake Borate.The peroxide and peroxidase reaction, estimation are probably to become the oxidation state of iron in the active site of enzyme Different oxidation state.The initiator solution can also include the peroxidase enhancer being selected from the group：Oxybenzene compound, virtue Fragrant amine, arylboronic acid compound, aryl boric acid ester compounds, aryl boric acid anhydride compound.

In embodiments of the present invention, mentioned reagent box is also comprising at least one of following articles：(5) work is carried Tool, its space, which is divided into, can house the restriction space of one or more containers, 96 orifice plates or lath, and the container is, for example, medicine Bottle, test tube and analog, are all individually used for the component of the inventive method per sample container containing one；(6) auxiliary reagent, than Such as, developer, enzyme inhibitor, buffer solution, stabilizer, diluent, washing reagent and the like；(7) specification, it can write On bottle, test tube and analog, either write on a single paper or container outside or inside or Multimedia form, such as CD, compact disk, video recording etc..

The research of the present inventor finds that the expression of hepatitis B viruses (HBV) can also be hard as liver caused by hepatitis B virus infection Change the biological marker of detection, it combines with CHI3L1 expression, progress joint-detection can significantly improve HBV infection and cause Liver fibrosis and/or liver cirrhosis diagnosis success rate.Therefore, it is the detection of hepatitis B, CHI3L1 detection in sample is whole Close in a kit, it will further improve the accuracy of liver fibrosis and/or hepatic sclerosis detection, and further improve Determine the accuracy of liver fibrosis and/or the hepatic sclerosis cause of disease.

Therefore, second of kit of the invention be in addition to above-mentioned CHI3L1 first antibodies and CHI3L1 secondary antibodies, Also include：

(3 ') can combine the antibody of HBV albumen；

(4 ') can be incorporated into the labelled antibody of HBV albumen when the antibody that HBV protein bindings are limited in (3 ')；With And

(5 '), by the standard sample of the solution composition containing known quantity CHI3L1, the standard sample is that the CHI3L1 manually prepared is molten Liquid, wherein CHI3L1 derive from the liver fibrosis as caused by hepatitis B virus infection and/or liver cirrhosis patient, especially Chinese population The body fluid of patient such as serum；And by the standard sample of the solution composition containing known quantity HBV albumen, wherein HBV albumen can be with From the liver fibrosis as caused by hepatitis B virus infection and/or the body fluid ratio of liver cirrhosis patient, especially Chinese population patient Such as serum.

For convenience, the antibody limited in above-mentioned (3 ') is referred to as HBV primary antibodies or HBV first antibodies；Will be upper State the labelled antibody limited in (4 ') and be referred to as HBV secondary antibodies.

As for HBV albumen, it can be any kind of protein included in HBV, such as is HBx, HBs, HBe, HBc Deng.

HBV first antibodies include it is any can combine HBV albumen antibody or antibody fragment, and can be recombinant, Chimeric antibody, humanized antibody and murine antibody.The antibody can be monoclonal antibody or polyclonal antibody, preferably Dan Ke Grand antibody, preferably uses the commercially available HBV antibody of commercialization, and its example such as includes：The HBV of ABCAM companies PreS2antibody [H.11], the HBV-PreS1 of HBV Pres1antibody, the Abbexa companies of Biorbyt companies Hepatitis B virus (core antigen) monoclonal antibody of (Capture Ab) and Abnova companies, clone LF161。

It is similar with the situation of above-mentioned CHI3L1 first antibodies and CHI3L1 secondary antibodies, preferably CHI3L1 first antibodies and HBV first antibodies can be fixed on different solid phase carriers respectively, be formed respectively for " capture " CHI3L1 and HBV albumen Capture antibody.

HBV secondary antibodies can combine marked (the carrying label) antibody or antibody piece of HBV albumen including any Section, and can be recombinant, chimeric antibody, humanized antibody and murine antibody.It is preferred that marked (with label) Monoclonal antibody.Antibody labeling can be carried out according to conventional methods or tagged, it is possible to use the commercially available HBV antibody of commercialization. Such as：During using enzyme such as HRP (horseradish peroxidase) as label (label), the associated methods of enzyme and antibody have many Kind, it can for example use glutaraldehyde two step method and Over-voltage protection.Specifiable marked (carrying label) HBV antibody business Product for example have the conjugation of Prosci companies to have the goat anti-hepatitis B surface antigen polyclonal antibody of biotin；Creative The conjugation of Diagnostics companies has FITC rabbit-anti hepatitis B surface antigen polyclonal antibody (Cat.No.DPATB-H81654)； The conjugation of Thermo Scientific Pierce companies has HRP mouse anti-hepatitis B cAg monoclonal antibody Clone 4H5。

Label in HBV albumen secondary antibodies can be catalyzed or activate chemiluminescence compound or fluorescent dye, So as to which colourless substrate rapidly is transformed into coloured product or causes light to change or by the fluorescent dye of non-fluorescence It is transformed into strong fluorescence-causing substance.The label included in above-mentioned CHI3L1 secondary antibodies may also serve as HBV albumen second Label on antibody.

Similarly, the reaction object of the label included in above-mentioned CHI3L1 secondary antibodies may also serve as HBV albumen The reaction object of label in secondary antibody.

Similarly, for induce label in CHI3L1 secondary antibodies and chemiluminescence compound or chromogenic substrate or The initiator solution of chemical reaction or enzymic catalytic reaction between fluorescent dye can be equally used for induction HBV albumen second and resist Chemical reaction between label and chemiluminescence compound or chromogenic substrate or fluorescent dye or enzymatic on body is anti- Should.Therefore, in embodiments of the present invention, preferably mentioned reagent box is further included：

(6 ') initiator solution, is respectively used to trigger the label in CHI3L1 secondary antibodies and HBV albumen secondary antibodies The chemical reaction between chemiluminescence compound or chromogenic substrate or fluorescent dye with reacting object respectively as label. For example, when label is peroxidase, initiator solution includes peroxide compound.The peroxide ingredient is any Peroxide or alkyl hydroperoxide that can be with peroxidase reaction.It is preferred that peroxide include hydrogen peroxide (hydrogen peroxide), urea peroxide and perborate.

In embodiments of the present invention, HBV protein standards sample is by the solution composition containing known quantity HBV albumen, the mark Quasi- sample is the HBV protein solutions manually prepared.Theoretically, standard sample can include or produce HBV albumen from any Organism, such as from genetic engineering bacterium express or animal body fluid.However, the HBV albumen of separate sources is used as antigen When, its epitope specificity may be different, so as to can be variant and then may also have influence on and exempt from the combination of same antibody Epidemic disease is detected.To make HBV albumen have higher epitope specificity as antigen, preferably HBV protein standards sample is derived from and corresponded to The body fluid of animal, such as HBV albumen sources in the animal body fluid corresponding to examined biological specimen type such as whole blood, blood plasma, Serum, urine, cerebrospinal fluid, saliva, seminal fluid or tears, wherein more preferably deriving from serum.Research is found, when HBV protein standards When sample is derived from the serum of liver fibrosis caused by hepatitis B virus infection and/or liver cirrhosis patient, there is the standard sample wide spectrum to be applicable Property, detect that obtained result is 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 albumen sources in the liver fibrosis as caused by hepatitis B virus infection and/ Or liver cirrhosis patient, preferably the liver cirrhosis patient as caused by hepatitis B virus infection, especially Chinese population patient body fluid such as Serum.It is of course also possible to from genetic engineering bacterium expression or the body fluid of animal, on condition that the epitope specificity of its HBV albumen Substantially it is equal with the epitope specificity of liver fibrosis caused by hepatitis B virus infection and/or the serum HBV albumen of liver cirrhosis patient. Material as standard sample and control sample can take various forms, and it is in aqueous solution state that suitable material, which can be,.Typically, The solution of known CHI3L1 contents can be included and comprising known HBV albumen by being suitable for the standard sample of the immunoassays of the present invention The solution of content, they can carry out gradient dilution.Do not causing the content detection of CHI3L1 and HBV albumen because of cross reaction In the case of producing error, the standard sample of immunoassays of the invention can include known CHI3L1 contents simultaneously and known The solution of HBV protein contents.

In embodiments of the present invention, preferably CHI3L1 first antibodies and HBV first antibodies or be respectively used to fix Chemiluminescence compound or fluorescent dye are connected on the solid phase carrier of CHI3L1 first antibodies and HBV first antibodies.Borrow This, when being tested respectively by double antibody sandwich method, secondary antibody is removed with traditional must be rinsed before detecting step Method of immunity is compared, and assay method is simplified because separation (secondary antibody) and washing step is eliminated, and is quantified more Accurately.

Similarly, second of kit of the invention may also include at least one of following articles：(5) instrument is carried, Its space, which is divided into, can house the restriction space of one or more containers, 96 orifice plates or lath, and the container is, for example, medicine bottle, examination Pipe and analog, are all individually used for the component of the inventive method per sample container containing one；(6) auxiliary reagent, such as, show Toner, enzyme inhibitor, buffer solution, stabilizer, diluent, washing reagent and the like；(7) specification, it can write on bottle On son, test tube and analog, either write on a single paper or the outside or inside in container or many matchmakers The form of body, such as CD, compact disk, video recording etc..

ELISA

The kit of the present invention is preferred for ELISA (enzyme linked immunosorbent assay (ELISA)), and the sample of individual is detected.

Enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) is molecular biosciences The conventional protein content analyzing method in field, it can be used for determining antigen, it can also be used to determine antibody.According to reagent come Source, the character of sample and detection satisfy the requirements, and the present invention can use a variety of different types, such as：Double antibody sandwich method, Double site one-step method, indirect method survey antibody, competition law, prize law and survey IgM antibody and application Avidin and biotin ELISA etc..It is preferred that the antibody coupling matter of CHI3L1 antibody and HBV antibody is detected with detecting instrument, such as, ELIASA carries out extinction Degree is determined.

By taking DAS-ELISA as an example, the inspection principle of CHI3L1 contents is in CHI3L1 kits detection sample： Microwell plate is coated with anti-human CHI3L1 antibody (first antibody), insolubilized antibody is made；Add and treat in the microwell plate of coated antibody Sample sheet, if containing measured object in sample, just can be combined on insolubilized antibody, so as to form antigen-antibody complex；Again plus Enter the anti-human CHI3L1 antibody (secondary antibody) of horseradish peroxidase (HRP) mark, form antibody-antigene-HRP marks anti- Nanocrystal composition (can also use the anti-human CHI3L1 antibody of biotin labeling as secondary antibody, form antibody-antigene-life Thing element labelled antibody compound；The Avidin of horseradish peroxidase (HRP) mark is added, antibody-antigene-biotin is formed Labelled antibody-enzyme mark Avidin compound)；It is eventually adding 3,3 ', 5,5 '-tetramethyl benzidine (3,3 ', 5,5 '-TMB) substrate System (including hydrogen peroxide), occurs chromogenic reaction.Chromogenic reaction is such as detected after terminating with ELIASA in 450nm.According to CHI3L1 concentration OD values corresponding with its make standard curve in calibration object, and it is dense to calculate CHI3L1 in sample by standard curve Degree.

ROC curve

ROC curve full name is Receiver operating curve (receiver operator characteristic Curve), also known as recipient's operating characteristic curve, is mainly used in clinical biochemical diagnostic test.ROC curve is reflection true positives Rate (sensitivity, also known as sensitiveness, sensitivity) and false positive rate (1- specificity, specificity) continuous variable it is comprehensive Index is closed, is the correlation that sensitivity and specificity are disclosed with composition method.It is by setting a series of different cut off value (thresholds Value or critical value, cutoff value (cut-off value), are the dividing values for dividing diagnostic test result normally with exception) as continuous Variable, so as to calculate a series of sensitivity and specificity, then by ordinate of sensitivity, 1- specificity be what abscissa was drawn Curve, TG-AUC (AUC) is bigger, and diagnostic accuracy is higher.On ROC curve, it is near the upper left point of coordinate diagram Sensitivity and specificity higher critical value.ROC curve AUC is between 1.0 and 0.5.In the case of AUC ＞ 0.5, AUC illustrates that diagnosis effect is better closer to 1.AUC has relatively low accuracy at 0.5~0.7, and AUC has one at 0.7~0.9 Determine accuracy, there is 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, it is allowed to have intermediateness, can So that result of the test is divided into multiple ordered categorizations, such as：Normally, substantially normal, suspicious, substantially abnormal and abnormal five grades.

Above-mentioned ordered categorization, for the diagnosis of disease, can be divided into：Negative, the uncertain, positive.Further, for For liver cirrhosis diagnosis, it can be divided into：There are hepatic sclerosis, health.

Therefore, to detect in sample exemplified by PROTEIN C HI3L1, the method for present invention detection hepatic sclerosis can include following step Suddenly：

(1) content of PROTEIN C HI3L1 in sample is determined respectively；

(2) using CHI3L1 protein contents as variable, according to sensitivity and specificity of the different threshold values to liver cirrhosis diagnosis Draw out area AUC under ROC curve, and calculated curve；And

(3) sensitivity and specificity desirably, are classified (hepatic sclerosis or health) to determining sample.

The drafting of ROC curve can use the software or system of prior art, such as：MedCalc 9.2.0.1 are cured Learn statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER_POWER.SAS, CREATE_ ROC.SAS, GB STAT V10.0 (Dynamic Microsystems, Inc.Silver Spring, MD, USA) etc..

Diagnosis, prognosis evaluation, therapeutic effect monitoring or the course of disease monitoring of hepatic sclerosis

The present invention is mainly reflected in the diagnosis of hepatic sclerosis, prognosis evaluation, therapeutic effect prison in the concrete application of medical domain Survey or course of disease monitoring aspect, including operating method and the utensil-kit for realizing this method.It is well known that the course of disease of hepatitis Closely related with hepatic sclerosis conversion, the typical course of disease is：Hepatitis (such as hepatitis B, hepatitis) → hepatic sclerosis.The research of the present inventor It was found that, the biological marker that the expression of hepatitis viruse can also be detected as hepatic sclerosis, it combines with CHI3L1 expression, entered Row joint-detection can significantly improve the success rate of the liver fibrosis as caused by hepatitis B virus infection and/or liver cirrhosis diagnosis.Cause This, present invention also offers it is a kind of be used for the liver fibrosis as caused by HBV infection and/or the diagnosis of hepatic sclerosis, prognosis evaluation, control The method that therapeutic effect is monitored or the course of disease is monitored, it includes：Detect the expression of HBV in individual blood sample；Detect individual blood sample CHI3L1 expression in this；Mathematical analysis is carried out to the HBV expressions and CHI3L1 expression measured；And according to number Analysis result is learned, classifies to determining blood sample, makes the judged result of hepatic sclerosis.This result can be divided into：Hepatic sclerosis Early stage, hepatic sclerosis mid-term, hepatic sclerosis late period etc..

Wherein, HBV expression can be carried out using the method for the art standard, such as the blood examination test paper of standard Method, blood examination RNA isolation kit, and conventional blood sample test method, etc..

Below so that the serum of individual carries out liver cirrhosis diagnosis for sample as an example, the present invention is described in further detail.

Embodiment

1. clinical trial

1.1 sample source：Zhejiang University Medical College The First Affiliated Hospital, 2nd Affiliated Hospital Zhejiang University School of Medicine and What Shaoyifu Hospital Attached to Zhejiang Univ. Medical College collected has been diagnosed as the serum sample and just of the hepatic sclerosis as caused by HBV infection The serum sample often compareed, the blood that sample is the serum that less than -80 DEG C preserve in 1 year or less than -20 DEG C preserve in 1 month Clearly.CHI3L1 quantitative determination is carried out using examination reagent；Carried out with the hepatic sclerosis clinic or pathological diagnosis result of clinical unit Compare, suitable positive reference value is determined using ROC curve, evaluate the specificity, sensitiveness, total coincidence rate of the detection kit And correlation analysis.

1.2 samples selection foundations, inclusion criteria, exclusion standard and rejecting standard

Inclusion criteria：Remaining serum sample or stock's serum sample after routine clinical detection；The collection and processing of sample Meet the requirement of standard laboratory procedure code and product description；The original relevant information of sample is at least included for sample person's Sex, sample collection date, sample type.

Exclusion standard：The sample of sample collection and processing requirement is not met；The sample of microorganism pollution；It is significant hemolysis, mixed Turbid sample (except interference sample group)；The sample not being consistent with clinical setting.

Rejecting standard：

Because sample size deficiency leads to not the sample obtained a result；

The sample gone wrong in detection process.

1.3 sample collections, preservation and packet

1) required to collect Normal group, non-hepatic sclerosis disease control group, liver cirrhosis group according to the inclusion criteria of sample Blood serum sample.

2) sample is the serum separated out after venous blood collection, and every part of serum volume is not less than 200uL.

3) after blood specimen collection, anti-coagulants should be not added with, and be placed on standing 1 hour, centrifugation in test tube that is clean, drying (2000rpm × 5min) takes upper serum, to be measured.

4) haemolysis it is not suitable for, the detection of the sample of piarhemia or jaundice.

5) detected as far as possible using fresh serum, the sample of fresh collection, if being temporarily not used in detection, it is necessary to by sample Originally -80 DEG C are stored in.After sample freeze thawing, it should detect immediately, unsuitable multigelation.By number of freezing and thawing control if necessary freeze thawing Within 3 times.Sample should be with equilibrium at room temperature before detection.The sample of freezing need to be slowly increased to room temperature, and the sample after defrosting should be mixed.

If 6) sample contains fibrin, particulate matter or erythrocyte, aggegation and centrifugation should be carried out to sample.

Blood sample presses following packet：

Normal group

The serum sample of selection comes from healthy individuals person, and this group of crowd is without clear and definite tumour medical history and other common chronic diseases Medical history, related neoplasms serological index testing result is in normal range (NR).The group is alternatively referred to as Apparently healthy individuals group.

Non- hepatic sclerosis disease control group is interference sample group

The serum sample of selection derives from the patient with the other diseases in addition to hepatic sclerosis.These non-hepatic sclerosis disease bags Include hepatitis, liver cancer etc..These serum samples are easily obscured or it is possible that single in blood serum designated object context of detection with hepatic sclerosis Secondary testing result has positive findings to intersect performance.

Liver cirrhosis group

The methods for clinical diagnosis of hepatic sclerosis includes related imaging diagnosis, pathological diagnosis or/and treatment and follow-up etc.. The serum sample of selection is from patient with liver cirrhosis of clinical definite etc..

The clinical diagnosis of 1.4 hepatic sclerosis

Using the methods for clinical diagnosis (including goldstandard) of prior art, the detection of tested crowd's hepatic sclerosis is completed.

1.5CHI3L1 detection kit

The plurality of reagents box of Hangzhou Pu Wang Bioisystech Co., Ltd production can be used to carry out biological specimen such as serum The detection of CHI3L1 levels, such as：

1.5.1CHI3L1 detection kit (ELISA) 1, the production of Hangzhou Pu Wang Bioisystech Co., Ltd.It is led Comprising：

CHI3L1 first antibodies are the CHI3L1/YKL-40 mouse monoclonal anti-humans of LifeSpan Biosciences companies (aa22-383) antibody (LS-C125352-50).The original concentration of antibody is 1mg/ml, and the dilution factor of antibody can be 10-1000 Times.96 hole microwell plates are coated with first antibody, insolubilized antibody is made, that is, captures antibody.

CHI3L1 secondary antibodies are the anti-human CHI3L1 Anti-TNF-αs of the biotinylation affinity purification of R＆D systems companies Body PAb (BAF2599), it is the anti-CHI3L1 polyclonal antibodies of biotin labeling, is stored in and adds the slow of protein stabilizing agent In fliud flushing.The original concentration of antibody is 1mg/ml, and the dilution factor of antibody can be 10-5000 times, can be 100 μ l per hole addition.

What the label (i.e. activator) for being conjugated with CHI3L1 secondary antibodies marked for horseradish peroxidase (HRP) Avidin.Can be by 1：Used after 99 dilutions；Every hole addition can be 100 μ l after dilution；

CHI3L1 standard items, by the conventional such as affine chromatography of protein separation means, from by Zhejiang University CHI3L1 is isolated in the serum of liver cirrhosis patient caused by the hepatitis B virus infection that the attached First Hospital of medical college is provided, is dissolved in In 20mM phosphate buffers pH 7.4 containing 2M ureas, it can be diluted when using with identical buffer solution.Standard items it is dilute It can be respectively a little CHI3L1 concentration 1200pg/ml, 600pg/ml, 300pg/ml, 150pg/ml, 75pg/ml, 0pg/ml to release.

The chromogenic reaction substrate (developer B) of label, to contain the buffer solution of 3,3', 5,5'- tetramethyl benzidines, often It can be 50 μ l that hole, which is added,.

Initiator solution (developer A), the buffer solution containing hydrogen peroxide.1 is pressed with developer B：1 is used in mixed way, and adds per hole It can be 50 μ l to enter amount.

CHI3L1 Sample dilutions, the buffer solution containing protein stabilizing agent, for detecting the preceding dilution to sample.

1.5.2CHI3L1 detection kit (ELISA) 2, the production of Hangzhou Pu Wang Bioisystech Co., Ltd.It is led Comprising：

CHI3L1 first antibodies are the CHI3L1/YKL-40 mouse monoclonal anti-humans of LifeSpan Biosciences companies (aa22-383) (AT4A3) antibody (LS-B3057-50).The original concentration of antibody is 1mg/ml, and the dilution factor of antibody can be 10- 1000 times.96 hole microwell plates are coated with first antibody, insolubilized antibody is made, that is, captures antibody.

Goat-anti people's CHI3L1 polyclonal antibodies that CHI3L1 secondary antibodies are conjugated for the HRP of GenWay Biotech companies (18-783-77701), is stored in and adds in the buffer solution of protein stabilizing agent.The original concentration of antibody is 1mg/ml, antibody Dilution factor can be 10-5000 times, can be 100 μ l per hole addition.

CHI3L1 standard items, the active people CHI3L1 full-length proteins (ab182706) of Abcam companies.The dilution point of standard items Respectively CHI3L1 concentration 1200pg/ml, 600pg/ml, 300pg/ml, 150pg/ml, 75pg/ml, 0pg/ml.

Label HRP chromogenic reaction substrate (developer B), to contain the buffering of 3,3', 5,5'- tetramethyl benzidines Liquid, can be 50 μ l per hole addition.

Initiator solution (developer A), the buffer solution containing hydrogen peroxide.1 is pressed with developer B：1 is used in mixed way, and adds per hole It can be 50 μ l to enter amount amount.

CHI3L1 Sample dilutions, the buffer solution containing protein stabilizing agent, for detecting the preceding dilution to sample.

Other reagents that can also be commonly used in these kits comprising ELISA, such as cleaning solution, terminate liquid etc., this is this Known to art personnel.

Hereinafter list the reality using CHI3L1 levels in the detection serum of CHI3L1 detection kits (ELISA) 1 Test result.

1.6 clinical laboratory evaluations indexs

1.6.1 clinical specificity

The ratio that the blood sample number of cases of feminine gender is accounted in the number of cases that clinical diagnosis is " non-hepatic sclerosis " is detected as with kit, is investigated Kit correctly excludes the ability of patients' hepatic sclerosis.

1.6.2 clinical sensibilisin

Detect that blood sample is the ratio that positive number of cases is accounted in the case that clinical diagnosis is " hepatic sclerosis " by kit, investigate Kit correctly judge patient whether the ability of suffering from liver cirrhosis.

1.6.3 overall coincidence rate

The index of COMPREHENSIVE CALCULATING product sensitivity and specificity, reflection kit testing result and patients' hepatic sclerosis Accordance.And carry out consistency analysis with Kappa values.

The processing of 1.7 clinical testing datas and statistical analysis technique

1.7.1 for statistics, described using average ± standard deviation (mean ± sd) difference etc.；To enumeration data, adopt Described with number of cases and percentage.

1.7.2 according to data type, the corresponding statistical analysis technique of Group Design, such as t inspections/sum of ranks inspection are selected respectively Test/the method such as Chi-square Test/exact probability.Pass through SAS software statistics kit measurement clinical sample CHI3L1 concentration values, examination CHI3L1 and hepatic sclerosis correlation and linear regression analysis.

1.7.3 compared with the hepatic sclerosis clinic or pathological diagnosis standard such as " goldstandard " of prior art, pass through to calculate and try The detection sensitivity of agent box, specificity, total coincidence rate carry out the clinical analysis performance of kits for evaluation：

According to professional knowledge, each sample measurement result is analyzed, determine the bound of measured value, group away from and block Point (cut-off point), lists cumulative frequency distribution table away from interval by the group of selection, the spirit of all point of cut-offs is calculated respectively Sensitivity (True Positive Rate), specificity and false positive rate (1- specificity).Using True Positive Rate as ordinate, false positive rate is sat to be horizontal Mark, draws ROC curve, calculates area (AUC) and 95%CI under ROC curve, to evaluate the value of diagnosis, and determines to block reference Value.

Reference value is blocked as basis for estimation using above-mentioned determination, negative, positive judgement is carried out to all detection samples, and will Testing result and clinical (or pathology) diagnostic result paired data arrange as shown in table 1, calculate susceptibility, specificity, always meet The indexs such as rate, and testing result and the uniformity of clinical (or pathology) diagnosis with Kappa test evaluation detection kits.If Kappa value >=0.75, uniformity is preferable；If 0.75 ＞ Kappa value >=0.40, uniformity is medium；If Kappa values ＜ 0.40, one Cause property is undesirable.

The qualitative detection result paired data statistical form of table 1

Wherein A is true positives；B is false positive；C is false negative；D is true negative.

(1) sensitivity, actual diseased and the probability for being diagnosed as the positive, also referred to as True Positive Rate, be：

Sen (sensitivity)=TPR=A/ (A+C) * 100%

(2) specificity, probability that is actual ill and being diagnosed as feminine gender, as：

Spe (specificity)=D/ (B+D) * 100%

(3) total coincidence rate (consistent percentage)=(A+D)/(A+B+C+D) * 100%

(4) positive predictive value：Refer in the patient of positive findings, the possibility that hepatic sclerosis is present.Calculation 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-hepatic sclerosis is present.Calculation 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：That is false positive rate, calculation formula is：

ɑ=FPR=1-Spe

(7) rate of missed diagnosis：That is false negative rate, calculation formula is：

β=1-Sen

(8) positive likelihood ratio (LR⁺)：The ratio between the ratio between True Positive Rate and false positive rate, as sensitivity and misdiagnosis rate, are calculated Formula is：LR⁺=TPR/FPR

Wherein, LR+ span is (0, ∞), and its value is bigger, and detection method confirms that the ability of disease is stronger.

(9) negative likelihood (LR^-)：The ratio between the ratio between false negative rate and true negative rate, i.e. rate of missed diagnosis and specificity, calculate public Formula is：LR^-=(1-TPR)/(1-FPR)

Wherein, LR- span is (0, ∞), and its value is smaller, and the ability that detection method excludes disease is better.

2 clinical study results and analysis

2.1 sample essential informations

This clinical test is big by Zhejiang University Medical College The First Affiliated Hospital (abbreviation center 01 or Zhejiang one), Zhejiang Attached second hospital of institute (abbreviation center 02 or Zhejiang two), Shaoyifu Hospital Attached to Zhejiang Univ. Medical College study medicine (in abbreviation The heart 03 or Shao Yifu) common completion, 1151 are selected in altogether, samples sources are in remaining (remaining) sample of clinical detection or storehouse Deposit serum sample.Each center is selected in sample situation referring to table 2：

Each center case distribution table of table 2

1151 subjects ages are 18-88 Sui (42.8 ± 13.7 years old), male 601, women 550.Wherein it is good for Health individual 725, man 310, female 415, the age is 19-88 Sui (37.5 ± 11.2 years old)；314 Cases of Hepatocirrhosis, man 223, Female 91, the age is 23-82 Sui (55.0 ± 11.1 years old)；Hepatitis B 112, man 68, female 44, the age is 18-78 Sui (43.0 ± 13.6 years old).The age characteristics of samples sources refers to table 3, and Sex distribution is shown in Table 4.

The sample object general information table of table 3

The sample object Sex distribution situation of table 4

2.2 clinical evaluation index analysis

2.2.1 the correlation analysis of change of serum C HI3L1 levels and hepatic sclerosis

CHI3L1 quantitative determination is carried out to the serum sample of healthy individuals and liver cirrhosis patient, specific measurement result is referred to Following table.

Compared with healthy individuals control group, expression significantly raised (P of the CHI3L1 contents in liver cirrhosis group<0.0001), see Table 5.

The change of serum C HI3L1 detection levels of table 5 (ng/mL, average value ± standard deviation)

Correlation to CHI3L1 and hepatic sclerosis carries out Spearman rank correlation analysises, as a result show correlation coefficient r= 0.751, p<0.0001, illustrate correlation between CHI3L1 and hepatic sclerosis, the CHI3L1 of liver cirrhosis group is higher than healthy group.

2.2.2ROC curve

Sample measures result is analyzed, the ROC curve for setting up CHI3L1 for liver cirrhosis diagnosis is shown in Fig. 1.Remaining is each The ROC curve at center is shown in Fig. 2 to Fig. 4.Excellent diagnostics critical value is confirmed using the method for calculating Youden indexes.Youden indexes Bigger, the diagnostic value of experiment is higher, and Youden indexes are 0.870, correspondence CHI3L1 diagnosis circle point value 74ng/mL to the maximum, i.e., Can be as diagnosis cutoff value, its diagnostic sensitivity and specificity are respectively 92.4% and 94.6%, and 6 are shown in Table in detail.

The change of serum C HI3L1 contents of table 6 ROC curve area and sensitiveness, specificity in liver cirrhosis diagnosis

2.2.3 susceptibility, specificity, total coincidence rate evaluation

ROC curve uses the method for calculating Youden indexes to confirm excellent diagnostics critical value for 79ng/mL, to this center All detection samples carry out negative, positive judgement, and testing result and clinical (or pathology) diagnostic result paired data are arranged As shown in table 7.

The testing result side's table of table 7

According to above-mentioned statistics table data, the index such as meter sensitivity, specificity, overall coincidence rate, summary is shown in Table 8：

Table 8CHI3L1 is used for each index of the clinical efficiency evaluation of liver cirrhosis diagnosis

Using the testing result and the uniformity of clinical (or pathology) diagnosis of Kappa test evaluation detection kits.If Kappa value >=0.75, uniformity is preferable；If 0.75 ＞ Kappa value >=0.40, uniformity is medium；If Kappa values ＜ 0.40, one Cause property is undesirable.It is 0.8586 to the Kappa values that healthy individuals crowd hepatic sclerosis is judged, 95% confidential interval (0.950, 0.991), P ＜ 0.0001, it will be recognized that two kinds of assay method result high consistencies.

2.2.4 the evaluation analysis of sample is disturbed

In 112 hepatitis interference sample the CHI3L1 values of totally 51 (45.5%) not less than diagnosing dividing value 79ng/mL.Cause This, hepatitis cases may have interference to the detection of this reagent.

3 results and discussion

This clinical test be using Hangzhou Pu Wang Bioisystech Co., Ltd produce CHI3L1 kits to hepatic sclerosis, Hepatitis B, healthy individuals crowd's serum sample are detected, are carried out with the hepatic sclerosis clinic or pathological diagnosis result of each clinical unit Compare, suitable positive reference value is determined using ROC curve, evaluate the specificity, sensitiveness, total coincidence rate of the detection kit And correlation analysis.

Experiment is big by Zhejiang University Medical College The First Affiliated Hospital, 2nd Affiliated Hospital Zhejiang University School of Medicine and Zhejiang The attached Shao Yi husbands hospital (center 03) of institute of studying medicine completion jointly, have collected 1151 samples, wherein hepatic sclerosis sample 314 altogether Example, 725, healthy individuals sample, 112, hepatitis B sample.Samples sources age 18-88 Sui, average age 42.8 years old, male 601 Example, women 550.

The serum sample CHI3L1 quantified results of healthier individual and liver cirrhosis patient, healthy individuals control group blood Horizontal average out to 47.4 ± 23.7 (5-395) ng/ml of clear CHI3L1, and liver cirrhosis group change of serum C HI3L1 levels are significantly raised, put down It is 338.7 ± 328.1 (20-2520) ng/ml, both comparing differences have significant (P<0.0001).It is further right CHI3L1 and hepatic sclerosis correlation carry out Spearman rank correlation analysises, as a result show correlation coefficient r=0.751, p<0.0001, Illustrate correlation between CHI3L1 and hepatic sclerosis, liver cirrhosis group CHI3L1 is higher than healthy group.

The serum sample CHI3L1 quantified results of healthy individuals and liver cirrhosis patient are analyzed, CHI3L1 is set up ROC curve for liver cirrhosis diagnosis.Area is 0.972 under ROC curve, and 95% credibility interval is (0.0.960-0.981).Root Bigger according to Youden indexes, the diagnostic value of experiment is higher, and Youden indexes are 0.850, correspondence CHI3L1 diagnosis circle's points to the maximum Value 79ng/ml, you can as diagnosis cutoff value, now diagnostic sensitivity and specificity are respectively 92.4% and 94.6%.It is selected The diagnosis dividing value that CHI3L1 is used to diagnose hepatic sclerosis is 79ng/ml, and the sample (containing hepatitis sample) selected to 1151 is examined The diagnostic sensitivity and specificity of core reagent are respectively 92.4% and 89.3%, and total coincidence rate is 90.1%.Further use The testing result of Kappa test evaluation detection kits and the uniformity of clinical (or pathology) diagnosis, as a result show this experiment Kappa It is worth for 0.766, P ＜ 0.0001, it is believed that two kinds of diagnostic method results have uniformity.

In summary, the CHI3L1 kits produced using Hangzhou Pu Wang Bioisystech Co., Ltd are used to detect that liver is hard Change, hepatitis B, healthy individuals person's change of serum C HI3L1 levels come diagnose hepatic sclerosis sensitivity and specificity be respectively 92.4% and 89.3%, total coincidence rate is 90.1%, and diagnosis dividing value is 79ng/ml, illustrates to examine the measure of kit can be as diagnosis liver The supplementary means of hardening.

Claims (10)

1. a kind of kit for being used to detect the hepatic sclerosis as caused by hepatitis B virus infection, it is by detecting in individual specimen CHI3L1 level come determine individual whether have hepatic sclerosis situation, the kit is included：

(1) CHI3L1 antibody can be combined；

(2) when the antibody limited during CHI3L1 is incorporated into (1), CHI3L1 labelled antibody can be incorporated into；And

(3) by the standard sample of the solution composition containing known quantity CHI3L1, wherein CHI3L1 is derived to be drawn by hepatitis B virus infection The serum of the liver cirrhosis patient risen.

2. a kind of kit for being used to detect the hepatic sclerosis as caused by hepatitis B virus infection, it is by detecting in individual specimen The level of CHI3L1 and HBV albumen come determine individual whether have hepatic sclerosis situation, it is included：

(1 ') can combine CHI3L1 antibody；

(2 ') can be incorporated into CHI3L1 labelled antibody when the antibody limited during CHI3L1 is incorporated into (1 ')；

(3 ') can combine the antibody of HBV albumen；

(4 ') can be incorporated into the labelled antibody of HBV albumen when the antibody that HBV protein bindings are limited in (3 ')；And

(5 '), by the standard sample of the solution composition containing known quantity CHI3L1, wherein CHI3L1 is derived to be drawn by hepatitis B virus infection The serum of the liver cirrhosis patient risen；And by the standard sample of the solution composition containing known quantity HBV albumen, wherein HBV albumen comes Come from the serum of the liver cirrhosis patient as caused by hepatitis B virus infection.

3. kit as claimed in claim 1 or 2, it is characterised in that the sample is whole blood, blood plasma, serum, urine, brain Spinal fluid, saliva, seminal fluid or tears.

4. kit as claimed in claim 1 or 2, it is characterised in that also comprising initiator solution, if by claim 1 (1) in antibody for limiting be referred to as CHI3L1 first antibodies, the labelled antibody limited in (2) of claim 1 is referred to as CHI3L1 secondary antibodies, are referred to as HBV first antibodies, by (4 ') of claim 2 by the antibody limited in (3 ') of claim 2 The labelled antibody of middle restriction is referred to as HBV secondary antibodies, and the initiator solution is used to trigger CHI3L1 secondary antibodies and HBV albumen Chemiluminescence compound or chromogenic substrate or fluorescence of the label with reacting object respectively as label in secondary antibody Chemical reaction between dyestuff.

5. kit as claimed in claim 4, it is characterised in that the CHI3L1 secondary antibodies and HBV albumen secondary antibodies On label be peroxidase, phosphate or luciferase.

6. kit as claimed in claim 5, it is characterised in that the peroxidase is horseradish peroxidase.

7. kit as claimed in claim 4, it is characterised in that CHI3L1 first antibodies and HBV first antibodies are consolidated respectively It is scheduled on different solid phase carriers, the solid phase carrier is the solid support being selected from the group：Microwell plate, test tube, specimen cup, modeling Expect microballoon, cellulose, papery or plastics testing bar, latex particle, silicon dioxide granule, magnetic particle.

8. kit as claimed in claim 7, it is characterised in that CHI3L1 first antibodies and HBV first antibodies, or respectively It is connected on solid phase carrier for fixing CHI3L1 first antibodies and HBV first antibodies and reacts object as label Chemiluminescence compound or chromogenic substrate or fluorescent dye.

9. kit as claimed in claim 1 or 2, it is characterised in that the antibody is monoclonal antibody.

10. kit as claimed in claim 1 or 2, it is characterised in that CHI3L1 level detection passes through in individual specimen ELISA is implemented.