AKR1B10 albumen and the kit for liver cirrhosis diagnosisTechnical field
The present invention relates to technical field of pharmaceutical biotechnology, specifically, relate to a kind of hepatic sclerosis specific antigen aldehyde ketone alsoProtoenzyme 1B10（Aldo-Keto Reductase 1B10, AKR1B10）, and it is a kind of for the early diagnosis of hepatic sclerosis, diagnosis,Treatment is instructed and the kit of Index for diagnosis..
Background technology
Hepatic sclerosis refers to the chronic liver caused by the various causes of disease, progressive, diffusivity lesion after chronic hepatitis, and it is specialPoint is that fibrosis occurs on the basis of necrosis of liver cells, and the instead abnormal hepatocytes tubercle of fiber wrapping（It is referred to as falseLeaflet）, cause the destruction of normal lobuli hepatis structure and vascular anatomy.The Etiological of China's hepatic sclerosis is virus hepatitis, especiallyIt is chronic, long-term caused by hepatitis type B virus (HBV), HCV (HCV) and hepatitis D (HDV) virusInflammation can cause hepatic sclerosis.
Hepatic sclerosis is a kind of common disease, gradually progress, and liver failure, portal hypertension and a variety of concurrent occurs in late periodDisease, it is serious and irreversible liver diseases.Hepatic sclerosis is at present without effective treatment method.Early stage hepatic sclerosis（It is compensatoryPhase）, timely intervened, allow patient to avoid taskwork momentum from working, be aided with dietary adjustments, such as with high heat, high protein, dimensionBased on the raw abundant and digestible food of element.Forbid to drink, and limit fat, especially the excessive of animal tallow takes the photograph people etc., canAvoid or delay its disease progression, prevent the generation of end-age cirrhosis and various complication.
However, early-phase hepatocirrhosis diagnosis is relatively difficult, many Histopathology turn out to be the patient of hepatic sclerosis, and Chang Kewu appointsOnly there is nonspecific symptom of digestive tract in what symptom.At present, the early diagnosis for hepatic sclerosis essentially consists in：1. to virusThe property tight follow-up observation of hepatitis；2. solid to the hepatomegaly that reason is not clear, particularly liver quality, rough person, there is liverSick face person, there are the sign persons such as spider angioma, liver palms and telangiectasis, lived using including ultrasound, laparoscope and hepatic tissueThe detection methods such as inspection assist in property.First these means lack specificity, second clinical manipulation also has substantive difficulty.It is anxiousNeed more specific diagnosis or follow-up, tracking index, especially non-invasive serum biological indicator.
AKR1B10 albumen is to separate to identify in organizing from human primary tumors.The protein has 316 ammoniaBase acid, the high expression in a variety of mankind tumor tissues, and promote the growth and increment of tumour cell, generation, development in tumourIn play an important role.Before this, the present inventor have developed for mankind's specific antigen aldehyde ketone reductase 1B10 albumen（Hereinafter referred to asFor AKR1B10）Specific detection agents box and its antibody, for cancer examination, diagnosis, curative effect judge, prognosis evaluationOr recurrence monitoring.However, by further research and development, it is found that mankind's specific antigen aldehyde ketone reductase 1B10 albumen is hepatic sclerosisOne it is new, specific serum label, available for the early diagnosis of hepatic sclerosis, diagnosis, treatment is instructed and Index for diagnosis,And the present invention is achieved.
The content of the invention
The high expression in liver cirrhosis with regenerating node of the present inventor's new discovery AKR1B10 albumen, and secreted through cell, into bloodLiquid circulates, and raises the protein level in serum of cirrhosis patients.Therefore AKR1B10 can be as new hepatic sclerosis serum markerThing, available for the examination of hepatic sclerosis, tracking, early diagnosis, treatment guidance and Index for diagnosis, thus as the basis of the present invention.
Thus for mankind's hepatic sclerosis specific antigen aldehyde ketone reductase 1B10 (Aldo-Keto Reductase 1B10,AKR1B10) albumen, develop a kind of new, sensitive, special, efficient diagnostic kit, for the examination of hepatic sclerosis, diagnosis,Curative effect judges and prognosis evaluation.
Specifically, the present invention relates to following content：
1. one kind is based on the diagnostic reagent of the mankind's hepatic sclerosis specific antigen aldehyde ketone reductase 1B10 (AKR1B10) albumenBox.
2. the kit can be used for examination, diagnosis, curative effect judgement and the prognosis evaluation of hepatic sclerosis, it includes specificity and examinedSurvey AKR1B10 reagent.
3. the kit described in 2, wherein, the reagent of the specific detection AKR1B10 is specific recognitionThe specific antibody of AKR1B10 albumen；Or the reagent of the specific detection AKR1B10 albumen is specific recognition AKR1B10The primer or probe of protein nucleic acid：5 ' ends：CAGAAAGCAGGACGTGGGACATC, 3 ' ends：GCACGTCGATTCGCGTCTATGAC。
4. the kit any one of a 2-3, wherein the reagent can be used for the detection body fluid such as serum and urine,Or the AKR1B10 albumen in tissue samples.
5. the kit any one of a 2-4, wherein the kit also includes AKR1B10 protein standard substances.
6. the kit any one of a 2-5, wherein the kit is ELISA determining adsorption(ELISA) kit, Time-resolved Fluoroimmunoassay（TRFIA）Kit and chemiluminescence detection kit.
7. the kit described in 6, it includes：
It is coated with the detection plate plate of hepatic sclerosis specific antigen AKR1B10 specificity capture antibody；
The AKR1B10 of another Species origin of biotin labeling specific detection antibody.The capture monoclonal antibody withDetecting antibody can be with AKR1B10 protein binding.
8. the kit described in 7, it also includes：
It is coated with buffer solution (1 × PBS, pH7.3 ± 0.1)；
Sample diluting liquid (1 × PBS, 1%BSA, 0.05%NaN3, pH 7.3 ± 0.1)；
Confining liquid (1 × PBS, 3% BSA)；
Eluent PBST (1 × PBS, 0.05% Tween-20)；
Streptavidin-Eu/HRP。
9. according to the kit of item 7 or 8, wherein capture antibody come total length AKR1B10 mice immunized with antigen of using by oneself, is gone forward side by sideRow cell fusion, monoclonal hybrid cell strain is separated, and prepare, specific recognition AKR1B10 monoclonal antibody；Detection is anti-Body is come the antibody purification for total length AKR1B10 antigen immunes rabbit acquisition of using by oneself.
10. according to the kit any one of item 2-9, wherein the examination, diagnosis, curative effect judgement and prognosis are commentedEstimate including：
A) AKR1B10 in biological sample of the reagent measuring from subject of the specific detection AKR1B10 is utilizedThe level of albumen；
B) by the level measured in a) compared with the respective horizontal in normal specimens.
11. according to the kit of item 10, wherein the sample be serum, urine, etc. body fluid, or tissue sample.
12. the reagent of specific detection AKR1B10 albumen is being prepared for hepatic sclerosis examination, early diagnosis, curative effect judgementWith the purposes in the kit of prognosis evaluation.
13. according to the purposes described in item 12, wherein, the reagent of the specific detection AKR1B10 is specific recognitionThe monoclonal antibody of AKR1B10 albumen, it is preserving number for CGMCC No.9249 or CGMCC No.9250 monoclonal antibodies cell lines instituteCaused monoclonal antibody.
Brief description of the drawings
The specificity of Fig. 1 mouse AKR1B10 monoclonal antibodies.This is to be entered with the AKR1B10 monoclonal antibodies of purifyingRow Western blot are specific to detect its.M, protein markers thing；H460, human lung cancer cell line（ExpressionAKR1B10）；293T, human embryonic kidney cell（AR is expressed, but does not express AKR1B10）；AKR1B10, purifying protein；AR, purifyingAlbumen.Anti- AKR1B10 monoclonal antibodies have high specific, with AR albumen no cross reactions（Note：AR（AldoseReductase）It is highly similar same family protein to AKR1B10）.
Fig. 2：With expression of the anti-AKR1B10 detection of specific antibody AKR1B10 albumen in mankind's cirrhotic tissue.ThisTo carry out Immunohistochemical detection acquired results with the section of mankind's hepatic sclerosis biopsy, show AKR1B10 albumen in the mankindSpecifically expressing in cirrhotic tissue, and can be arrived with AKR1B10 detection of specific antibody.
Fig. 3：Time resolution immunofluorescence assay（TRFIA）The sensitivity of kit.Antibody is wherein captured come total length of using by oneselfMouse monoclonal antibody prepared by AKR1B10 antigens；Detection antibody is come the pure of total length AKR1B10 antigen immunes rabbit acquisition of using by oneselfChange antibody.By the use of purifying, same amount AKR1B10 albumen is not as measuring samples, to determine lowest detection sensitivity.
Fig. 4：AKR1B10 concentration in normal population and serum of cirrhosis patients.Detected with above-mentioned TRFIA.
Embodiment
The present invention relates to a kind of new discovery, i.e. AKR1B10 albumen high expression in liver cirrhosis with regenerating node, and divide through cellSecrete, into blood circulation, the protein level in serum of cirrhosis patients is raised (referring to Fig. 2 and Fig. 4).Therefore AKR1B10 canAs new hepatic sclerosis serum markers, to be instructed available for the examination of hepatic sclerosis, tracking, early diagnosis, treatment and prognosis sentencedIt is disconnected.
The kit of detection AKR1B10 albumen provided by the invention, include the monoclonal of specific recognition AKR1B10 albumenAntibody, the antibody are coated on a solid support.
The present invention is based on anti-AKR1B10 monoclonal antibody specifics, newly developed to go out to utilize sandwich time resolution immunofluorescenceAnalysis（TRFIA）The kit of technology for detection serum AK R1B10 antigens, this kit are applied to the examination, tracking, morning of hepatic sclerosisPhase diagnoses and guiding treatment and judging prognosis.
The sandwich time-resolved fluorescent immunoassay of detection AKR1B10 antigens provided by the invention（TRFIA）Kit is specialSign is to mainly include：Be coated with capture AKR1B10 albumen mouse monoclonal antibody specific time resolution be immunized it is glimmeringLight measurement（TRFIA）Plate；The detection antibody (2 μ g/ml rabbit-anti AKR1B10 antibody) of biotin labeling.It is anti-with detection to capture antibodyBody can be with AKR1B10 antigen bindings.
The sandwich time resolution immunofluorescence assay of the detection AKR1B10 antigens（TRFIA）Kit also includes：BagBy buffer solution (1 × PBS, pH7.3 ± 0.1)；Sample dilution/antibody diluent (1 × PBS, 1%BSA, 0.05%NaN3, pH7.3±0.1)；Confining liquid (1 × PBS, 3% BSA)；Eluent PBST (1 × PBS, 0.05% Tween-20)；Horseradish peroxidatingThe Streptavidin of thing enzyme (HRP) or Eu marks；Standard items (the pure antigens of various concentrations AKR1B10：0、0.3125、0.625、1.25、2.5、5、10、20 ng/ml)。
The capture antibody is come the mouse monoclonal antibody for total length AKR1B10 antigens preparation of using by oneself；Detection antibody comes fromThe antibody purification obtained with total length AKR1B10 antigen immunes rabbit.
It is a further object to provide the preparation method of mentioned reagent box.
A kind of sandwich time-resolved fluorescent immunoassay for preparing above-mentioned detection AKR1B10 antigens（TRFIA）KitMethod, comprise the following steps：
7-10 μ g/ml are diluted to by antibody is captured with coating buffer solution, in time-resolved fluorescent immunoassay（TRFIA）The 100 above-mentioned antibody diluents of μ l are added in every hole of plate, 4oC is overnight；Take out, be placed in 37oC 30 minutes；Liquid is got rid of, is usedPBST is washed 5 times；Sky is dry, and 100 μ l confining liquids, 37oC 2 hours are added per hole；Liquid is got rid of, is washed 2 times with PBS；In room temperatureUnder, natural air drying 2 hours；With masking foil vacuum sealing, 4oC is put in, it is standby.
In use, by serum sample 1:10 dilutions, added per hole 100 μ l dilute serums and standard items (0,0.3125,0.625th, 1.25,2.5,5,10,20ng/ml AKR1B10 pure proteins dilution product), 37 oC are incubated 1 hour.Sample liquid is removed, is usedPBST is washed 5 times, and the rabbit-anti AKR1B10 antibody (1 of biomarker is diluted with Antibody/Conjugate buffer:500), so100 μ l antibody diluents are added per hole afterwards, 37 oC are incubated 1 hour.Antibody liquid is removed, is washed 5 times with PBST, uses Antibody/Conjugate buffer dilute Streptavidin-EU (1:5000) 100 μ l dilutions, are then added per hole, 37oC is incubated 1Hour.Streptavidin-Eu dilutions are removed, are washed 5 times with PBST, 100 μ l Stop solution are added per hole and are terminated insteadShould, read OD values.Standard curve is made with standard items, the concentration of AKR1B10 in dilute serum is calculated, is then multiplied by concentration10, obtain the final concentration in serum sample.
Optionally, the kit can also include following：To normal or disease sample caused by the related information of signal, markStandardization material, such as be used for compare from normal and/or disease sample AKR1B10 protein standard substances, appropriate enzyme, buffer solutionAnd solution.Optionally, the kit can also include any specification, optionally provided for describing how to implement the inventive methodStandard drawing, data and the software of the result obtained when implementing the inventive method for parsing.
The sandwich time-resolved fluorescent immunoassay for the detection AKR1B10 that we establish（TRFIA）Kit and chemistry hairLight detection kit, it is applicable to early diagnosis of liver cirrhosis and Mass screening, therapeutic effect judgement and prognosis evaluation etc., toolThere is quick, objective and accuracy.
In early days（The compensatory phase）Diagnosis：There is without any symptom or only nonspecific symptom of digestive tract, but pathological tissueLearn the patient for turning out to be hepatic sclerosis.By detecting the change of AKR1B10 albumen in serum, diagnosed.The early stage of hepatic sclerosis examinesDisconnected is to avoid developing into end-age cirrhosis, causes the key of death.
Guiding treatment：The change of AKR1B10 albumen, can determine whether curative effect in the patient with liver cirrhosis serum of detection receiving treatment.Reduction is then effective, and then sb.'s illness took a turn for the worse for indication for rise.
Judging prognosis：In patient with liver cirrhosis serum AKR1B10 albumen it is horizontal often with AKR1B10 in cirrhotic tissueExpression is relevant, both at the sign of prognosis mala.There is the change of AKR1B10 in the patient with liver cirrhosis serum for receiving to treat againChange, also indicate the effect for the treatment of and the prognosis of patient.
Embodiment
With reference to specific embodiment, the invention will be further described, but the present invention is not limited to following examples.UnderState in embodiment, be conventional method unless otherwise specified.The reagent used in experiment is public purchased from Sigma-Aldrich ChinaDepartment（Sigma-Aldrich Co. LLC）.
Embodiment 1:The preparation of the anti-AKR1B10 protein monoclonal antibodies hybridoma cell strain of mouse
With the AKR1B10 full-length proteins antigen 1 .0mg of purifying, isometric Freund's adjuvant is added, so as to the spy of enhancement antigenSpecific immunological reacts, and aspirates said mixture repeatedly with 5ml syringes and is emulsified, reaches the state for having Water-In-Oil.From 2The health male of 6-8 week old, is immunized in three times, and the AKR1B10 specific antigens emulsified only, are expelled to by 300 μ L/ every timeMouse subcutaneously and abdominal cavity, every 2 weeks carries out primary immune response, and immunizing dose and approach are constant, 3 days before cell fusion, by antigenDirectly mixed with PBS and do not add any adjuvant, be injected directly into mouse peritoneal and carry out booster immunization.Then, under aseptic condition, takeGo out to have taken the mouse spleen of blood, be ground into cell suspension, be placed in 50 mL centrifuge tubes, the r/min of room temperature 1,400 centrifugations 3min.Supernatant is abandoned, splenocyte is resuspended with 10 mL erythrocyte cracked liquids, is mixed after hooking, stands l0min, then be directly added into 15 mLMD6 serum free mediums terminate erythrocyte splitting, room temperature, 1,400 r/min, centrifuge 3 min.Abandon supernatant, with 10 mL MD6 withoutSplenocyte is resuspended blood serum medium, is drawn after mixing and uses Trypan Blue a little, cell counting count board is added, in inverted microscopeUpper counting, 1 is pressed with melanoma cells:1~5:1 ratio mixes, room temperature, 1,400/min, centrifuges 3 min.Supernatant is abandoned, gentlyAttack centrifuge tube bottom loosens two kinds of cell precipitations, and 45s interior edges centrifugation inside pipe wall adds 1.2 mL PEG cell fusion agent, sideEdged rotates centrifuge tube, makes the abundant exposing cells of PEG.The MD6 serum free mediums of 45 mL, 37 °C of preheatings are added, terminate PEGEffect, slowly stand 5 min at reverse centrifuge tube 2 ~ 3 times, 37 DEG C.Room temperature, 1,400 r/min, centrifuge 3 min.Abandon supernatant,37 DEG C in 96 porocyte culture plates, 5%C02 cultures.The 10th day i.e. growth conditions of observable hybridoma, if it was observed thatObvious individual cells colony, you can take Hybridoma Cell Culture supernatant, anti-AKR1B10 is carried out using indirect ELISA methodAntibody test, the hybridoma cell strain of screening choosing energy secreting specificity antibody.Further secretion pin is obtained using limiting dilution assayTo the hybridoma cell strain of the monoclonal antibody of single epitope.The present invention obtains two plants of mouse monoclonal antibody cell lines, i.e. 8-DC-1And 9-DC-2, preserving number are respectively CGMCC No.9249 and CGMCC No.9250, it is micro- that China is preserved on June 11st, 2014Biological inoculum preservation administration committee common micro-organisms center（CGMCC）（Preservation address is：BeiChen West Road, Chaoyang District, BeiJing City 1Number institute 3）, survive after testing.Then, with protein g affinity chromatography method monoclonal antibody purification is used, dialysis, quantify.
Embodiment 2:Prepare the sandwich time-resolved fluorescent immunoassay of detection AKR1B10 antigens（TRFIA）Kit
Capture antibody and detection antibody used in embodiment 1 are that mountain is immunized in wild type full-length AKR1B10 albumen respectivelySerum is obtained after sheep and rabbit, and obtained after purification with the affinity column of AKR1B10 antigens preparation.Rabbit-anti AKR1B10 antibody quiltsBiotin labeling.The capture antibody and the sensitivity and specificity of detection antibody that the present invention obtains are all very strong, time-resolved fluorescenceImmunoassays（TRFIA）Detectable 0.03125ng/ml AKR1B10 albumen.Wherein biotin labeling reagent box is purchased fromPierce companies.Streptavidin-Eu is purchased from Perkin Elmer companies.Detection AKR1B10 time-resolved fluorescence is exempted fromEpidemic disease determines（TRFIA）Plate is prepared by oneself；Agents useful for same autogamy：It is coated with buffer solution (1 × PBS, pH7.3 ± 0.1)；Sample DilutionLiquid/antibody diluent (1 × PBS, 1%BSA, 0.05%NaN3, pH 7.3 ± 0.1)；Confining liquid (1 × PBS, 3% BSA)；ElutionLiquid PBST (1 × PBS, 0.05% Tween-20)；Detect antibody (1 μ g/ml rabbit-anti AKR1B10 antibody)；Standard items are (different denseSpend AKR1B10 antigens：0、0.3125、0.625、1.25、2.5、5、10、20 ng/ml).
Embodiment 3：AKR1B10 concentration in normal population and hepatic sclerosis person's serum
The preparation of time-resolved fluorescent immunoassay plate：7-10 μ g/ml are diluted to by antibody is captured with coating buffer solution,In time-resolved fluorescent immunoassay（TRFIA）The 100 above-mentioned antibody diluents of μ l are added in every hole of plate, 4oC is overnight；Take out,It is placed in 37oC 30 minutes；Liquid is got rid of, is washed 5 times with PBST；Sky is dry, and 100 μ l confining liquids, 37oC 2 hours are added per hole；Liquid is got rid of, is washed 2 times with PBS；At room temperature, natural air drying 2 hours；With masking foil vacuum sealing, 4oC is put in, it is standby.
The detection of serum sample：Serum sample is with PBS with 1:10 dilutions, 100 μ l sample diluting liquids or standard are added per holeProduct (various concentrations AKR1B10 antigens：0th, 0.3125,0.625,1.25,2.5,5,10,20 ng/ml), 37oC, 60 minutes；Get rid ofLiquid is removed, is washed 5 times with lavation buffer solution, sky is dry；Antibody antibody diluent is detected with 1:500 dilutions, 100 μ are added per holeL detection antibody diluents, 37oC, 60 minutes；Liquid is got rid of, is washed 5 times with lavation buffer solution, sky is dry；Streptavidin-EuWith antibody diluent with 1:80000 dilutions, the 100 μ l Streptavidin-Eu dilutions of addition per hole, 37oC, 60 minutes；Get rid ofLiquid is removed, is washed 5 times with lavation buffer solution, sky is dry；100 μ l add reaction solution；With ELIASA OD is determined with 450 nm wavelengthValue.Standard curve is drawn, according to standard curve, the concentration of AKR1B10 in serum sample, and × 10 is calculated, obtains serumAKR1B10 final concentrations.
Analysis of test results
100 Healthy Peoples, 40 liver cirrhosis patients progress serum sample detections are chosen, are as a result counted with Graphpad 4.0Software is learned to be analyzed.T assays show that average AKR1B10 antigen levels have conspicuousness statistics poor in two groups of crowdsDifferent (p< 0.001).In this 100 normal populations, serum AK R1B10 concentration average out to 138pg/ml, liver cirrhosis patientSerum AK R1B10 concentration is 562pg/ml.