CN102955028A - Kit for detecting AMA (anti-mitochondrial antibody)-M2 relative to autoimmune liver diseases, and detection method with kit - Google Patents
Kit for detecting AMA (anti-mitochondrial antibody)-M2 relative to autoimmune liver diseases, and detection method with kit Download PDFInfo
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Abstract
The invention discloses a kit for detecting AMA (anti-mitochondrial antibody)-M2 relative to autoimmune liver diseases, and a quantitative and quantitative detection method for the AMA-M2 with the kit. The detection method comprises the following steps of: combining a sample with IgG or/and anti-human IgM or/and anti-human IgA, which is/are connected with marker(s), and specific antigen corresponding to the detected antibody; adding a substrate of a luminescence system or a developing system or a light exciting system into the sample, and then reading a signal value of the sample to be detected; and calculating a quantitative and quantitative result or reading a quantitative and quantitative result of the sample by naked eyes or instruments according to immunochromatography or immuno-infiltration, thereby accurately diagnosing the type and severity of the autoimmune liver diseases to guide a doctor to dispense medicines. The quantitative and quantitative detection method has the advantages of high sensitivity, strong specificity and high accuracy, and is applicable to interpretation by semi-automatic or full-automatic detection systems or naked eyes.
Description
Technical field
The present invention relates to field of biological detection, especially relate to a kind of kit that detects the anti-AMA-M2 type of patients with autoimmune hepatitis antibody, the invention still further relates to the method for using this kit to detect anti-AMA-M2 type antibody.
Background technology
Autoimmune liver disease (autoimmune liver diseases) mainly comprise three kinds closely-related with autoimmunity, take Bile duct injury as main disease: oneself immunity hepatitis (autoimmune hepatitis, AIH), primary biliary cirrhosis of liver (primary biliary cirrhosis, PBC) and primary sclerotic cholangitis (primary sclerosing cholangitis, PSC).AIH is a kind of liver diseases that circulation autoantibody and hyper immunoglobulinemia, the cause of disease are not bright, be the chronic inflammatory necrosis of accompanying, PBC damages, is later on liver fibrosis, finally causes liver failure as the agnogenic autoimmunity liver diseases of a class of feature take the stones in intrahepatic bile duct that autoimmunity mediates, PSC is a kind of agnogenic chronic syndrome, it is characterized in that liver outer and/or stones in intrahepatic bile duct diffuse inflammation and the caused chronic Intrahepatic Cholestasis of fiberization.
In recent years, in the world the research of autoimmune liver disease Preclinic and clinic is paid much attention to, and progress rapidly, relates generally to pathogenesis, genetic background, related auto-antibodies and the aspects such as target antigen character, clinical epidemiology, clinical diagnosis and treatment and prognosis thereof of autoimmune liver disease.The diagnosis of autoimmune liver disease mainly relies on medical history, clinical manifestation, sign and laboratory examination results.The liver histopathology inspection is considered to AIH, " goldstandard " of PBC diagnosis, but this checks that clinical practice has limitation, and for example, the liver biopsy Pathological specificity of autoimmune liver disease is not strong, and non-self immunity hepatopathy institute is peculiar; The patient is reluctant or discomfort is accepted this traumatic inspection; In addition, present most hospitals adopt the B ultrasonic guided fine needle aspiration to draw materials to carry out the liver histopathology inspection, and this inspection sample of being drawn materials limits to, and whether can obtain pathological tissues, and is very large on the check result impact.However, the liver histopathology inspection is most important to the judgement of autoimmune liver disease diagnosis, antidiastole and lesion degree, is still one of major criterion of diagnosis.AIH, PBC early lesion (cholangitis phase, chronic cholestasis phase) but belong to the reversible refunding of clinical treatment or the Partial Inverse refunding, belong to the irreversible refunding of clinical treatment in later stage or whole latter stage (liver fibrosis/cirrhosis phase, hepatic failure phase), not good after more.Therefore, early diagnosis, early treatment are particularly important.Autoimmune liver disease pathogenesis and immunologic dysfunction are closely related, and autoantibody plays an important role in pathogenesis.Every kind of autoimmune liver disease all has the characteristic autoantibody repertoire, and autoantibody detects significant to diagnosis, somatotype and the antidiastole of autoimmune liver disease.In recent years, abroad to autoimmune liver disease related auto-antibodies spectrum target antigen character, clinical diagnostic applications value (the especially early diagnosis of asymptomatic stage), rapid with the progress such as relation of disease clinical characters and activity, the research of relevant autoantibody repertoire has caused clinical great attention, and autoantibody detects the routine inspection project that has become the autoimmune liver disease clinical diagnosis.
In recent years, because the accumulation of clinical experience and the progress of laboratory diagnosis technology find that autoimmune liver disease patients increases gradually in the population of China.The article of domestic pathogenesis about autoimmune liver disease, related auto-antibodies target antigen, Case report, clinical case discussion and Clinical and Pathological Analysis increases gradually, and above job description autoimmune liver disease is much in China.
Anti-mitochondrial antibody (AMA) is the general name of one group of autoantibody that can combine with plurality of enzymes compound composition on mitochondria inner membrance or the adventitia.AMA is that a kind of susceptibility in PBC and specificity all are higher than 90%～95% take mitochondria as target antigen, without kind and organ specific autoantibody, has become diagnosis PBC(primary biliary cirrhosis of liver) main inspection item.The early stage AMA of PBC often is low titre, and along with disease progression, AMA can raise gradually, but the high end of its titre and disease severity or prognosis and uncorrelated.If the high titre of AMA is positive, even without PBC symptom and biochemical unusual, also strongly prompt for PBC.Only a few patient clinical, biochemistry and histology all meet the PBC diagnosis but AMA is negative, its natural history and the auto immune conditions of being correlated with and PBC patient's indifference of the AMA positive.
AMA can be divided into M1-M9 totally 9 hypotypes, and closely-related with PBC is M2, M4, M8, M9, and wherein the specificity of M2 diagnosis PBC is the highest.2-ketoacid dehydrogenase complex on the mitochondrial inner membrane is PBC patient's high degree of specificity autoantibody as the AMA-M2 antibody (anti-mitochondrial antibody M2 hypotype) of target antigen, and susceptibility is 95%～98%, and specificity reaches 97%.Have the scholar to think when the high titre of AMA-M2 hypotype antibody, making a definite diagnosis PBC no longer needs the hepatic tissue biopsy pathology to confirm.Indirect immunofluorescence (IIF) commonly used detects the anti-AMA-M2 antibody that occurs among the patient clinically, its experiment matrix is generally liver, stomach, the renal tissue frozen section of different primates, contain complete spectrotype, but the indirect immunofluorescence analysis method dye to such an extent that identify to have certain subjectivity, thereby may be between different time points, the different observers, there are differences between the old and new's histotomy to result's explanation, and need the auxiliary of the instrument such as fluorescent microscope, be difficult to carry out at general middle and small hospital.So the diagnostic method that resists at present AMA-M2 antibody (resist mitochondria M2 type antibody) also needs further to improve.
Summary of the invention
The object of the present invention is to provide a kind of kit that detects the anti-AMA-M2 type of patients with autoimmune hepatitis antibody, use this kit can be quantitatively and the anti-AMA-M2 antibody of qualitative detection, thereby Accurate Diagnosis goes out type and the order of severity of autoimmune liver disease, instructs doctor's medication; The invention also discloses the method for using this kit to detect anti-AMA-M2 type antibody, the method is highly sensitive, high specificity, accuracy are good, is applicable to semi-automatic or automatic detection system or naked eyes interpretation.
For achieving the above object, the present invention can take following technical proposals:
The kit of the anti-AMA-M2 type of detection patients with autoimmune hepatitis of the present invention antibody, comprise solid phase carrier, the ELIAS secondary antibody working fluid, positive reference substance, negative control product, calibration object, sample diluting liquid, substrate and cleansing solution, described solid phase carrier are microwell plate, magnetic particle or the magnetic ball that is fixed with anti-AMA-M2 type antibody target antigen, the target antigen that is about to AMA-M2 antibody specific binding is attached on the identical solid phase carrier as capture antibody, forms antigen-carrier conjugates; In the described ELIAS secondary antibody working fluid used two anti-for mouse or sheep or rabbit anti-human igg or/and anti-human IgM or/and anti-human IgA, used two anti-labels are horseradish peroxidase, alkaline phosphatase, acridinium ester, different luminol or rare earth element.
Described sample diluting liquid is comprised of less than 1% protected protein less than 10% sodium chloride damping fluid and concentration concentration.Described protected protein is casein, albumin, peptone, gelatin, animal blood serum or human serum.
Described target antigen is native antigen, recombinant antigen, synthetic peptide, HepG2 cell, Hep2 cell, Hela cell or lysis antigen.
Described substrate is luminous substrate or chromogenic substrate.Described luminous substrate is luminous substrate A and luminous substrate B, and described luminous substrate A is comprised of 0.2M Tris-HCl, 0.15mM Luminol, 0.59mM Hydroxycoumarin, 0.35mM gallic acid; Described luminous substrate B is comprised of 0.85mM amino acid oxidase, 0.8%Tween 20,0.5mM DTPA, 0.12mM vitamin C.
The detection method of kit of the present invention comprises the steps:
The first step, serum to be detected is diluted to serum sample with sample diluting liquid, respectively positive reference substance, negative control product, calibration object and serum sample are joined in the solid phase carrier and hatch, the test serum sample is contacted with antigen-carrier conjugates, so that the AMA-M2 antibody (if present) in the serum sample is hunted down, form antibody-antigen-carrier conjugates; When if sample is blood plasma, detect after needs are centrifugal;
Second step after the sample reaction finishes, discards reactant liquor, washes plate with cleansing solution;
The 3rd step added the ELIAS secondary antibody working fluid in each reacting hole of solid phase carrier after second step is washed plate, hatch; This step can make ELIAS secondary antibody contact reaction with above-mentioned antibody-antigen-carrier conjugates;
In the 4th step, reaction discards reactant liquor after finishing, and washes plate with cleansing solution;
The 5th step added corresponding substrate (such as luminous substrate or chromogenic substrate) and detects, and the instrument detected signal value is come measured antibody in the qualitative or quantitative judgement sample, or read the qualitative or quantitative result of sample by immunochromatography or immunity percolation method naked eyes or instrument.
The invention has the advantages that can be quantitatively and the anti-AMA-M2 antibody of qualitative detection by chemoluminescence method or enzyme linked immunosorbent assay or golden mark method or time resolution method or fluorescence method, when AMA-M2 antibody concentration during more than or equal to 1AU/ml, can be judged as the PBC positive, severity and the positive correlation of detection concentration of specimens value, be applicable to semi-automatic or automatic detection system or naked eyes sentence read result, thereby Accurate Diagnosis goes out type and the order of severity of autoimmune liver disease, instruct doctor's medication, for patient's early diagnosis, early treatment provide safeguard.
Embodiment
The below enumerates respectively plank frame kit and magnetic particle structure kit, and illustrates that it detects using method.It should be understood that following embodiment only is used for the purpose of illustration, never only limits the scope of the invention.
Embodiment 1:
The kit (board-like) that the present invention detects the anti-AMA-M2 type of patients with autoimmune hepatitis antibody (anti-mitochondrial antibody M2 hypotype) comprising:
1, the preparation of target antigen-carrier conjugates: target antigen (native antigen, recombinant antigen, synthetic peptide, HepG2 cell, Hep2 cell, Hela cell or lysis antigen) the 100 μ l that will not be higher than 10ug/ml join in 96 orifice plates, 4 ℃ of coated 16-24h or 37 ℃ of coated 2h are afterwards with twice of (PBST) cleansing solution washing for preparing;
2, sealing: with the 0.05-0.5%Tween20 that contains of 50-200 μ l, 1-5%BSA, 1-5%Casein, the 1-5% peptone, the phosphate buffer of the 0.01-0.1M of the sucrose of 5-10%, 4 ℃ of sealing 16-24h or 37 ℃ of sealing 2h, get rid of afterwards unnecessary damping fluid, place dry environment to dry, after drying, be sealed in 2-8 ℃ of preservation;
3, the preparation of calibration object, positive reference substance and negative control product: get the clinically high value sample of definite value, doubly dilute by being not less than 1:50 with calibration object dilution (PBS+3%BSA), line taking preferably 4～8 points as calibration object, selection is not less than the calibration object of 2AU/ml concentration as positive reference substance, selects the calibration object of 0AU/ml concentration as the negative control product;
4, the preparation of ELIAS secondary antibody working fluid: in the described ELIAS secondary antibody working fluid used two anti-for anti-human IgG or/and anti-human IgM or/and anti-human IgA, doubly dilute and prepare the ELIAS secondary antibody working fluid by not being higher than 1:200 with enzyme dilution (PBS+1%Casein); Used two anti-labels are horseradish peroxidase, alkaline phosphatase, acridinium ester, different luminol or rare earth element.
5, assembling: with one of above-mentioned 96 hole antigen coated microplate, calibration object 6 * 1.0ml, negative control 1 * 1.0ml, positive control 1 * 1.0ml, ELIAS secondary antibody working fluid 1 * 12ml, sample diluting liquid (PBS+3%BSA) 1 * 100ml, concentrated washing lotion 20 * 50ml(PBST), luminous substrate A(0.2M Tris-HCl, 0.15mM Luminol, 0.59mM Hydroxycoumarin, 0.35mM gallic acid) and luminous substrate B(0.85mM amino acid oxidase, 0.8%Tween 20,0.5mM DTPA, 0.12mM vitamin C) each 1 * 7ml fits together and forms complete kit.
Detection method:
The processing of test sample book:
Serum: use not contain pyrogen and endotoxic test tube extracting vein blood 2-5ml, avoid any cytositimulation in the operating process, after collecting blood, 3000 turn/the centrifugal 10min of min, with serum and rapidly careful separating of red blood cell, serum specimen is stored in 2-8 ℃, and the sample that can not test in 24h should be placed in-20 ℃ of refrigerators and preserve, and significant hemolysis, has the serum specimen of floccus can affect test result.
Blood plasma: use EDTA, citrate or anticoagulant heparin, 3000 turn/min is centrifugal, and 30min gets supernatant, the same serum of store method.
1, application of sample dilutes sample to be checked with sample diluting liquid (the Tris-NaCl damping fluid that contains BSA) according to 1:100, (sample to be checked after 1～100AU/ml) calibration object and the 100 μ l dilution is put into 37 ℃ of constant temperature ovens and is hatched 30min in target antigen-carrier conjugates fully respectively to add 100 μ l variable concentrations behind the mixing;
2, wash after plate sample reaction finishes, discard reactant liquor, manual or at automatic washer washing 6 * 1min with the washing lotion for preparing;
3, the mouse-anti human IgG antibody 1:500 that adds the ELIAS secondary antibody horseradish peroxidase-labeled doubly dilutes, and adds 100 μ l in each reacting hole, puts into 37 ℃ of constant temperature ovens and hatches 30min;
4, wash after plate sample reaction finishes, discard reactant liquor, manual or at automatic washer washing 6 * 1min with the washing lotion for preparing;
5, add the substrate detection and in each reacting hole, add luminous substrate A and each the 50 μ l of B that prepare, behind the concussion mixing, room temperature 5min light-emitting appearance testing result, take calibration object concentration as horizontal ordinate, corresponding luminous value is ordinate, draw out calibration object linear regression curve, press the concentration value that curvilinear equation calculates sample to be tested.
When the concentration value that calculates during less than 1AU/ml, sample is negative, and illustrates that the possibility of diagnosis PBC is smaller.
When the concentration value that calculates during more than or equal to 1AU/ml, sample is positive, and illustrates that the possibility of diagnosis PBC is larger.
Embodiment 2:
The kit (magnetic particle) of the anti-AMA-M2 type of detection patients with autoimmune hepatitis of the present invention (anti-mitochondrial antibody M2 hypotype) antibody comprises:
1, the preparation of target antigen-carrier conjugates: get the 10mg magnetic particle, with phosphate buffer washing 5 times, add be not less than 0.15mlEDC or (with) NHS or glutaraldehyde (with) behind the activator activation 2h, wash 3 times, the target antigen that adds afterwards 0.1mg, the coated 2h of room temperature concussion;
2, sealing: with the 0.05-0.5%Tween20 that contains of 1ml, 1-5%BSA, 1-5%Casein, the 1-5% peptone, the phosphate buffer of the 0.01-0.1M of the sucrose of 5-10%, room temperature vibration sealing 10min, sealing is four times continuously, adds afterwards the 2ml confining liquid in 2-8 ℃ of preservation;
3, the preparation of calibration object, positive reference substance and negative control product: get the clinically high value sample of definite value, doubly dilute by being not less than 1:50 with calibration object dilution (PBS+3%BSA), line taking preferably 4～8 points as calibration object, selection is not less than the calibration object of 2AU/ml concentration as positive reference substance, selects the calibration object of 0AU/ml concentration as the negative control product;
4, the preparation of ELIAS secondary antibody working fluid: in the described ELIAS secondary antibody working fluid used two anti-for anti-human IgG or/and anti-human IgM or/and anti-human IgA, doubly dilute and prepare the ELIAS secondary antibody working fluid by not being higher than 1:200 with enzyme dilution (PBS+1%Casein); Used two anti-labels are horseradish peroxidase, alkaline phosphatase, acridinium ester, different luminol or rare earth element.
5, the assembling of kit: with 2ml target antigen-magnetic particle bond, calibration object 6 * 1.0ml, negative control 1 * 1.0ml, positive control 1 * 1.0ml, sample diluting liquid 1 * 100ml, concentrated washing lotion 20 * 50ml(PBST), luminous substrate A(0.2M Tris-HCl, 0.15mM Luminol, 0.59mM Hydroxycoumarin, 0.35mM gallic acid) and luminous substrate B(0.85mM amino acid oxidase, 0.8%Tween 20,0.5mM DTPA, 0.12mM vitamin C) respectively 1 * 7ml fit together and get final product.
Detection method:
The processing of test sample book: with embodiment 1.
1, application of sample dilutes sample to be checked with sample diluting liquid (the Tris-NaCl damping fluid that contains BSA) according to 1:100, (sample to be checked after 1～100AU/ml) calibration object and the 100 μ l dilution is in reacting hole respectively to add afterwards 100 μ l variable concentrations, respectively add afterwards 20 μ l target antigen-magnetic particle bonds in each reacting hole, put into 37 ℃ of constant temperature ovens and hatch 15min;
2, wash after plate sample reaction finishes, discard reactant liquor, manual or at automatic washer washing 6 * 1min with the washing lotion for preparing;
3, the mouse-anti human IgG antibody 1:500 that adds the ELIAS secondary antibody horseradish peroxidase-labeled doubly dilutes, and adds 100 μ l in each reacting hole, puts into 37 ℃ of constant temperature ovens and hatches 15min;
4, wash after plate sample reaction finishes, discard reactant liquor, manual or at automatic washer washing 6 * 1min with the washing lotion for preparing;
5, add the substrate detection and in each reacting hole, add luminous substrate A and each the 50 μ l of B that prepare, behind the concussion mixing, room temperature 5min light-emitting appearance testing result, take calibration object concentration as horizontal ordinate, corresponding luminous value is ordinate, draw out calibration object linear regression curve, press the concentration value that curvilinear equation calculates sample to be tested.
When the concentration value that calculates during less than 1AU/ml, sample is negative, and illustrates that the possibility of diagnosis PBC is smaller.
When the concentration value that calculates during more than or equal to 1AU/ml, sample is positive, and illustrates that the possibility of diagnosis PBC is larger.
Claims (7)
1. kit that detects the anti-AMA-M2 type of patients with autoimmune hepatitis antibody, comprise solid phase carrier, the ELIAS secondary antibody working fluid, positive reference substance, the negative control product, sample diluting liquid, substrate and cleansing solution is characterized in that: described solid phase carrier is microwell plate, magnetic particle or the magnetic ball that is fixed with anti-AMA-M2 type antibody target antigen; In the described ELIAS secondary antibody working fluid used two anti-for mouse or rabbit or goat anti-human igg or/and anti-human IgM or/and anti-human IgA, used two anti-labels are horseradish peroxidase, alkaline phosphatase, acridinium ester, different luminol or rare earth element.
2. the kit of the anti-AMA-M2 type of detection patients with autoimmune hepatitis according to claim 1 antibody is characterized in that: described sample diluting liquid is comprised of less than 10% sodium chloride damping fluid with less than 1% protected protein concentration.
3. the kit of the anti-AMA-M2 type of detection patients with autoimmune hepatitis according to claim 2 antibody, it is characterized in that: described protected protein is casein, albumin, peptone, gelatin, animal blood serum or human serum.
4. the kit of the anti-AMA-M2 type of detection patients with autoimmune hepatitis according to claim 1 antibody, it is characterized in that: described target antigen is native antigen, recombinant antigen, synthetic peptide, HepG2 cell, Hep2 cell, Hela cell or lysis antigen.
5. the kit of the anti-AMA-M2 type of detection patients with autoimmune hepatitis according to claim 1 antibody, it is characterized in that: described substrate is luminous substrate or chromogenic substrate.
6. the kit of the anti-AMA-M2 type of detection patients with autoimmune hepatitis according to claim 5 antibody, it is characterized in that: described luminous substrate is luminous substrate A and luminous substrate B, and described luminous substrate A is comprised of 0.2M Tris-HCl, 0.15mM Luminol, 0.59mM Hydroxycoumarin, 0.35mM gallic acid; Described luminous substrate B is comprised of 0.85mM amino acid oxidase, 0.8%Tween 20,0.5mM DTPA, 0.12mM vitamin C.
7. the detection method of kit as claimed in claim 1 is characterized in that: comprise the steps:
The first step is diluted to serum sample with serum to be detected with sample diluting liquid, positive reference substance, negative control product, calibration object and serum sample is joined in the solid phase carrier hatch respectively;
Second step after the sample reaction finishes, discards reactant liquor, washes plate with cleansing solution;
The 3rd step added the ELIAS secondary antibody working fluid in each reacting hole, hatched;
In the 4th step, reaction discards reactant liquor after finishing, and washes plate with cleansing solution;
The 5th step added substrate and detects, and the instrument detected signal value is come measured antibody in the qualitative or quantitative judgement sample, or read the qualitative or quantitative result of sample by immunochromatography or immunity percolation method naked eyes or instrument.
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| CN111505268A (en) * | 2020-04-29 | 2020-08-07 | 四川携光生物技术有限公司 | Autoimmune antibody detection method |
| CN113009149A (en) * | 2021-02-10 | 2021-06-22 | 中国医学科学院北京协和医院 | Sugar chain marker for diagnosing PBC patients positive and negative to AMA-M2 antibody and application thereof |
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