CN102955030A - Kit for detecting autoimmune liver disease related antibody autoantibody to nuclear antigen (ANA) and detection method thereof - Google Patents
Kit for detecting autoimmune liver disease related antibody autoantibody to nuclear antigen (ANA) and detection method thereof Download PDFInfo
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Abstract
The invention discloses a kit for detecting an autoimmune liver disease related antibody autoantibody to nuclear antigen (ANA). The invention also discloses a method for quantitatively and qualitatively detecting ANA by using the kit. The method comprises the following steps of: combining a detection sample with a specific antigen which corresponds to a marker-linked anti-human IgG or/and anti-human IgM or/and anti-human IgA and a detection antibody, adding substrates of a luminous system or a developing system or a light exciting system, reading a signal value of a sample to be detected, calculating a quantitative and qualitative result, and reading the sample through immunochromatography or immuno-infiltration naked eyes or instruments to obtain a quantitative and qualitative result, thus accurately diagnosing the type of severity of the autoimmune liver disease, and guiding the medication of a doctor. The kit and the detection method have high sensitivity, high specificity and high accuracy and are suitable for semi-automatic or full-automatic detection systems or naked eye interpretation.
Description
Technical field
The present invention relates to field of biological detection, especially relate to a kind of kit that detects patients with autoimmune hepatitis ANA, the invention still further relates to the method for using this kit to detect ANA.
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 outside the liver 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; And 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 healing.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.
The ANA(antinuclear antibodies) be one of modal autoantibody of AIH, approximately have 75% I type AIH patient ANA positive, and 10% AIH patient, ANA are arranged is unique autoantibody that detects in its serum.But ANA does not have specificity, not only is found in AIH, is found in multiple connective tissue disease (CTD) yet.The titre of ANA in AIH is generally higher, usually surpasses 1:160.Its ANA of typical case's I type AIH is positive, and titre obviously raise (adult 〉=1:80, children 〉=1:40).Indirect immunofluorescence (IIF) commonly used detects the ANA 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, at present the diagnostic method of antinuclear antibodies (ANA) also needed further to improve.
Summary of the invention
The object of the present invention is to provide a kind of kit that can accurately detect patients with autoimmune hepatitis ANA, another purpose of the present invention is to improve the method for using this kit to detect antinuclear antibodies.
For achieving the above object, the present invention can take following technical proposals:
The kit of detection patients with autoimmune hepatitis ANA of the present invention, comprise solid phase carrier, the ELIAS secondary antibody working fluid, positive reference substance, the negative control product, calibration object, sample diluting liquid, substrate and cleansing solution, described solid phase carrier are that microwell plate, magnetic particle or magnetic ball etc. can carry out physisorption with the antinuclear antibodies target antigen or carry out the carrier that chemistry is connected; 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 10% sodium chloride damping fluid with less than 1% protected protein 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; Luminous substrate is luminous substrate A and luminous substrate B, and 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 is as follows:
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.
The invention has the advantages that can be quantitatively and qualitative detection antinuclear antibodies (ANA) by chemoluminescence method or enzyme linked immunosorbent assay or golden mark method or time resolution method or fluorescence method, when ANA concentration during more than or equal to 1AU/ml, can be judged as the AIH 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) of detection patients with autoimmune hepatitis ANA of the present invention 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 unnecessary damping fluid afterwards, place dry environment to dry, 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 forms) and luminous substrate B(0.85mM amino acid oxidase, 0.8%Tween 20,0.5mM DTPA, 0.12mM the vitamin C composition) 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 respectively to add afterwards 100 μ l variable concentrations;
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 ELIAS secondary antibody working fluid 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 AIH 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 AIH is larger.
Embodiment 2:
The kit (magnetic particle) of detection patients with autoimmune hepatitis ANA of the present invention comprising:
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 AIH 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 AIH is larger.
Claims (7)
1. kit that detects patients with autoimmune hepatitis ANA, 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 the antinuclear antibodies 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 detection patients with autoimmune hepatitis ANA according to claim 1 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 detection patients with autoimmune hepatitis ANA according to claim 2, it is characterized in that: described protected protein is casein, albumin, peptone, gelatin, animal blood serum or human serum.
4. the kit of detection patients with autoimmune hepatitis ANA according to claim 1, 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 detection patients with autoimmune hepatitis ANA according to claim 1, it is characterized in that: described substrate is luminous substrate or chromogenic substrate.
6. the kit of detection patients with autoimmune hepatitis ANA according to claim 5, 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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Cited By (5)
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| CN106248943A (en) * | 2016-06-30 | 2016-12-21 | 深圳市亚辉龙生物科技股份有限公司 | Antinuclear antibody chemiluminescence immune detection reagent kit and preparation method thereof |
| CN108267579A (en) * | 2016-12-30 | 2018-07-10 | 深圳市新产业生物医学工程股份有限公司 | Stablize and preserve dilution, antinuclear antibody detection reagent and its preparation method and application |
| CN108333347A (en) * | 2017-01-19 | 2018-07-27 | 深圳市新产业生物医学工程股份有限公司 | Antinuclear antibodies target antigen conjugate reagent, preparation method, the kit comprising it and application |
| CN108431818A (en) * | 2015-12-31 | 2018-08-21 | 豪夫迈·罗氏有限公司 | Analyte in detection flash of light and aura reaction |
| CN109270272A (en) * | 2018-10-18 | 2019-01-25 | 郑州安图生物工程股份有限公司 | A kind of kit and preparation method thereof detecting the citrullinated vimentin antibodies of anti-saltant type |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108431818A (en) * | 2015-12-31 | 2018-08-21 | 豪夫迈·罗氏有限公司 | Analyte in detection flash of light and aura reaction |
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| CN108267579A (en) * | 2016-12-30 | 2018-07-10 | 深圳市新产业生物医学工程股份有限公司 | Stablize and preserve dilution, antinuclear antibody detection reagent and its preparation method and application |
| CN108333347A (en) * | 2017-01-19 | 2018-07-27 | 深圳市新产业生物医学工程股份有限公司 | Antinuclear antibodies target antigen conjugate reagent, preparation method, the kit comprising it and application |
| CN109270272A (en) * | 2018-10-18 | 2019-01-25 | 郑州安图生物工程股份有限公司 | A kind of kit and preparation method thereof detecting the citrullinated vimentin antibodies of anti-saltant type |
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Application publication date: 20130306 |