CN103616372A - Hepatitis B e-antibody measurement kit and detection method thereof - Google Patents
Hepatitis B e-antibody measurement kit and detection method thereof Download PDFInfo
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- CN103616372A CN103616372A CN201310654654.1A CN201310654654A CN103616372A CN 103616372 A CN103616372 A CN 103616372A CN 201310654654 A CN201310654654 A CN 201310654654A CN 103616372 A CN103616372 A CN 103616372A
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Abstract
The invention provides a hepatitis B e-antibody measurement kit. The kit comprises a reagent R, a reagent N, a reagent M and a reference substance, wherein the reagent R comprises the following components: a biotinylated anti-HBe antibody, an acridinium ester-labeled anti-HBe antibody, an MES buffer solution and a diagnostic reagent preservative; the reagent N comprises the following components: genetic recombined HBeAg, an MES buffer solution and a diagnostic reagent preservative; the reagent M comprises the following components: avidin-coated magnetic particles, wherein the magnetic particles are treated by the diagnostic reagent preservative; the reference substance comprises a human hepatitis B e antibody, new-born calf serum and a diagnostic reagent preservative; the acridinium ester-labeled anti-HBe antibody is formed by coupling acridinium ester to the anti-HBe antibody through a coupling agent, wherein the coupling agent is dimethylimine or glutaraldehyde; the kit also comprises a substrate solution system on the basis of H2O2-NaHO3; the substrate solution system contains triton 100 serving as a luminous enhancer. The kit for measuring the e antibody of hepatitis B provided by the invention has the beneficial effects of high sensitivity, small error and safety and rapidness in detection.
Description
Technical field
The present invention relates to diagnosis of hepatitis b kit, there is the hepatitis B e antibody of design based on neutralization reagent A competitive inhibition method chemiluminescence principle and measure kit and detection method.
Background technology
Hepatitis B is to infect by hepatitis type B virus (HBV) a kind of communicable disease causing.HBV can directly propagate by blood and body fluid, and common circulation way comprises blood transfusion, injection, wound contact, mother-to-baby transmission etc.; Common clinical symptoms has discomfort, heating, jaundice, asymptomatic hepatitis, fulminant hepatitis and chronic hepatitis etc., and chronic infection easily develops into liver cancer, and therefore, the work that detects hepatitis B is most important.
At present, conventional detection method is immunology detection, a kind of means that specific reaction based on antigen and antibody during immunology detection detects, it can utilize isotope, enzyme, chemiluminescent substance etc. that detected signal is amplified and shown, therefore be often used to detect protein, the micro-bioactivator such as hormone.Immunology detection has been handled radio-immunity detection, enzyme linked immunosorbent detection and photobiology Labeled immunoassay technology three phases, makes immunology detection towards the future development of susceptibility, accuracy and property simple to operation.
Chemiluminescence immune assay is worldwide to develop nearly ten years very fast on-radiation immuno analytical method.Detect principle and be and using luminescent substance and directly measure immune combination as signal amplifying system and by its luminous intensity.The method is highly sensitive, sensing range is wide, is the important developing direction of immunology detection.
Summary of the invention
The invention solves deficiency of the prior art, provide a kind of highly sensitive, error is little, the hepatitis B e antibody that detects is safely and fast measured kit and detection method.
Technical scheme of the present invention is: a kind of hepatitis B e antibody is measured kit, it is characterized in that, comprise reagent R, reagent N, reagent M and reference substance, the component in described reagent R comprises anti-HBe antibody, MES damping fluid and the diagnostic reagent antiseptic of biotinylation anti-HBe antibody, acridinium ester mark; Component in described reagent N comprises genetic recombination HBeAg, MES damping fluid and diagnostic reagent antiseptic; In described reagent M, component comprises that Avidin is coated with magnetic particle, and described magnetic particle after diagnosing reagent antiseptic is processed; Described control group comprises humanized's hepatitis B e antibody, NBCS and diagnostic reagent antiseptic; The anti-HBe antibody of described acridinium ester mark is to pass through coupling agent coupling anti-HBe antibody with acridinium ester, and described coupling agent is dimethyl imines or glutaraldehyde; Described kit also comprises with H
2o
2-NaHO
3for basic substrate solution system, in described substrate solution system, containing conduct is the TritonX-100 of luminescence enhancer.
In a preferred embodiment of the present invention, further comprise, the Avidin of the coated magnetic particle of described Avidin is Streptavidin, neutral Avidin or class Avidin, and the magnetic particle in the coated magnetic particle of described Avidin is to take the polymkeric substance of tri-iron tetroxide as core surface coating active group.
In a preferred embodiment of the present invention, further comprise, described anti-HBe antibody is mouse resource monoclonal anti-HBe antibody.
In a preferred embodiment of the present invention, further comprise, described in reagent R and reagent N, the concentration of MES damping fluid is 0.05M, and pH value is 6.0.
In a preferred embodiment of the present invention, further comprise the Proclin300 antiseptic that described diagnostic reagent antiseptic is 0.1%.
In a preferred embodiment of the present invention, further comprise, the amount of described reagent R and reagent N is 12ml, the amount of described reagent M is 3 milliliters.
In a preferred embodiment of the present invention, further comprise, described reference substance comprises reference substance 1 and 2 liang of groups of reference substance, and the amount of described reference substance 1 is 0.5ml, and concentration is 0.0PEIU/ml, and the amount of described reference substance 2 is 0.5ml, concentration is 2.0PEIU/ml.
In a preferred embodiment of the present invention, further comprise, a kind of detection method of using this hepatitis B e antibody to measure kit, comprises the following steps:
(1) the reagent N of testing sample and the HBeAg that contains genetic recombination is mixed and reacted, will form the reactant liquor 1 of antibody-antigenic complex;
(2) in reactant liquor 1, add the reagent R of the Anti-HBe that contains biotinylated e antibody (Anti-HBe) and acridinium ester mark to react, form the reactant liquor 2 of the quantity minimizing of biotinylated antibody-HBeAg-acridinium ester labelled antibody complex;
(3) in reactant liquor 2, add the reagent M that contains the coated magnetic particle of Avidin, the complex in reactant liquor 2 forms solid phase under the interaction of biotin and Avidin;
(4) reactant liquor is placed on chemical luminescence detector and is detected, the magnetic particle in the detection in magnetic field will be adsorbed, and by cleansing solution, wash, and bond does not rinse and removes; Then, inject chemiluminescence exciting liquid 1 and chemiluminescence exciting liquid 2, detect its chemiluminescence photon intensity; In the light intensity producing and sample, Anti-HBe concentration is inversely proportional to.
In a preferred embodiment of the present invention, further comprise, in described chemiluminescence exciting liquid 1, contain hydrogen peroxide, in described chemiluminescence exciting liquid 2, contain NaOH, described cleansing solution contains phosphate buffer.
The defect that solves prior art of the present invention, has following beneficial effect:
1. the hepatitis B e antibody the present invention relates to is measured kit; contain the coated magnetic particle of Avidin; utilized the chemiluminescence principle of neutralization reagent A competitive inhibition method; magnetic particle in magnetic field in reactant liquor can be adsorbed; by washing, inciting somebody to action not bond rinses and removes; and then inject chemiluminescence exciting liquid and detect chemiluminescence photon intensity; because the anti-HBe antibody concentration in light intensity and sample is inversely proportional to; so be easy to just obtain the anti-HBe antibody concentration in sample, thereby can judge the different phase that HBV infects.
2. use hepatitis B e antibody of the present invention to measure the detection method of kit, only need gather according to the medical technology of routine patient's whole blood sample, make serum separation completely not containing blood cell composition, then use kit provided by the invention, can detect hepatitis B e antibody (Anti-HBe), in conjunction with other HBV, infect serological index, can judge the different phase that HBV infects, the method is simple to operation, and error is little, and accuracy is high.
3. the present invention adds TritonX-100 as luminescence enhancer in the substrate solution system of kit, can improve the luminescence efficiency in testing process.
Embodiment
In order to make those skilled in the art person understand better the present invention, and above-mentioned advantage of the present invention can be become apparent more, below in conjunction with specific embodiment, the present invention is further detailed explanation.
A kind of hepatitis B e antibody of the present invention is measured kit, and group wants constituent as follows:
Reagent R: every bottle of 12ml, contains biotinylation Anti-HBe and acridinium ester mark Anti-HBe, the MES damping fluid of 0.05MPH6.0,0.1%Proclin300 diagnostic reagent antiseptic; Wherein, the anti-HBe antibody of acridinium ester mark is to pass through coupling agent coupling anti-HBe antibody with acridinium ester, and coupling agent is selected conventional coupling agent in the industries such as dimethyl imines or glutaraldehyde.
Reagent M: every bottle of 3.0ml, includes the coated magnetic particle of Avidin, through the preservative treatment of 0.1%Proclin300 diagnostic reagent antiseptic; Wherein, the Avidin of the magnetic particle that Avidin is coated is Streptavidin, neutral Avidin or class Avidin, and the magnetic particle in the coated magnetic particle of described Avidin is to take the polymkeric substance of tri-iron tetroxide as core surface coating active group.
Reagent N: every bottle of 12ml, includes genetic recombination HBeAg, the MES damping fluid of 0.05MPH6.0,0.1%Proclin300 diagnostic reagent antiseptic;
Reference substance (Cal): the reference substance in the embodiment of the present invention is divided into 1 and 2 liang of group, all includes humanized's hepatitis B e antibody, NBCS, 0.1%Proclin300 diagnostic reagent antiseptic; Reference substance concentration is as follows: the approximate concentration of Cal1 is 0.0PEIU/ml, and the approximate concentration of Cal2 is 2.0PEIU/ml.
Kit of the present invention also comprises with H
2o
2-NaHO
3for basic substrate solution system, in described substrate solution system, contain as being the TritonX-100 of luminescence enhancer, add TritonX-100 to make illumination effect better.
The anti-HBe antibody relating in the present invention is mouse resource monoclonal anti-HBe antibody, and it is as follows using the detection principle of this kit:
First step reaction: the HBeAg reaction by sample and gene expression, if contain Anti-HBe in sample, will form antibody-antigenic complex.
Second step reaction: add the Anti-HBe of biotinylated e antibody (Anti-HBe) and acridinium ester mark to react, due to the combination inhibiting effect of Anti-HBe in sample, the quantity that forms biotinylated antibody-HBeAg-acridinium ester labelled antibody complex reduces.
Three-step reaction: add the coated particulate of Avidin, complex forms solid phase under biotin and Avidin interaction.
Reactant liquor is placed in a magnetic field, and the magnetic particle in detection will be adsorbed, and by washing, bond does not rinse and removes; Then, inject chemiluminescence exciting liquid 1 and chemiluminescence exciting liquid 2, detect its chemiluminescence photon intensity; In the light intensity producing and sample, Anti-HBe concentration is inversely proportional to.
Embodiment 1
Specific experiment step in the specific embodiment of the invention is as follows:
First, obtain testing sample, serum, gathers whole blood sample according to conventional application technology, places and makes above aggegation half an hour, centrifugal more than 10 minutes under the rotating speed of 3000rpm, makes serum completely separated, does not contain cell component in serum; Liquaemin, EDTA-K for blood plasma
3, sodium citrate or the anti-freezing of sodium fluoride/potassium oxalate.
Then, to this testing sample, using hepatitis B e antibody to measure kit detects:
(1) the reagent N of testing sample and the HBeAg that contains genetic recombination is mixed and reacted, will form the reactant liquor 1 of antibody-antigenic complex;
(2) in reactant liquor 1, add the reagent R of the Anti-HBe that contains biotinylated e antibody (Anti-HBe) and acridinium ester mark to react, form the reactant liquor 2 of the quantity minimizing of biotinylated antibody-HBeAg-acridinium ester labelled antibody complex;
(3) in reactant liquor 2, add the reagent M that contains the coated magnetic particle of Avidin, the complex in reactant liquor 2 forms solid phase under the interaction of biotin and Avidin; Biotin wherein and Avidin form biotin-avidin system, this biotin-avidin system as the immune response of the process of mensuration after piece-rate system, make magnetic particle there is system.
(4) reactant liquor is placed on chemical luminescence detector and is detected, the magnetic particle in the detection in magnetic field will be adsorbed, and by cleansing solution, wash, and bond does not rinse and removes; Then, inject chemiluminescence exciting liquid 1 and chemiluminescence exciting liquid 2, detect its chemiluminescence photon intensity; In the light intensity producing and sample, Anti-HBe concentration is inversely proportional to.
Wherein, in described chemiluminescence exciting liquid 1, contain hydrogen peroxide, in described chemiluminescence exciting liquid 2, contain NaOH, described cleansing solution contains phosphate buffer.
Chemical luminescence detector in the embodiment of the present invention is EVERESYSA1800 I class, when detecting, reagent is loaded on chemical luminescence detector agent bin, and determines the title of loading with regard to reagent on operation system of software; Reagent, before loading, should mix gently, but forbids the opened reagent that turns upside down; Detection sample is loaded in sample storehouse, and determines pattern detection project on operation system of software; Reaction cup is loaded with full, also can loads respective numbers reaction cup according to experiment situation; Each sample size that detects needs 50ul, considers detection system dead volume factor, should guarantee that sample size is more than 200ul, should consider the dead space factor of sample container simultaneously.
Result is calculated
Analyzer calculates Cut-off value automatically, and every part of sample, according to the ratio (S/CO) of the RLU of sample and Cut-off value, calculates the testing result of anti-HBe.The anti-HBe of sample measures S/CO value > 1 and is judged as anti-HBe anergy; The anti-HBe of sample measures S/CO value≤1 and is judged as anti-HBe reactivity.
Sensitivity
Use above-mentioned detection method, the sensitivity Cut-off value that obtains anti-HBe kit of the present invention is≤0.5PEIU/mL; Detect the sensitivity of the national dish of anti-HBe reagent, meet standard; Detect national positive reference material, meet standard.
Precision
Precision is according to the EP5-A of the standardization council of American National clinical labororatory (NCCLS) solution formulation, to 5 parts of anti-HBe quality-control products (three parts of serum wherein, two parts is calibration object), adopt same batch of reagent, each detection of different time every day once, each diplopore, carries out 20 days altogether, and experimental result is as shown in table 1 below.
Table 1 experimental result
This detection method is respectively with haemoglobin (150mg/dL), triglyceride (1200mg/dL), and cholerythrin (30mg/dL), biotin (30ng/ml) is for sample detects, and result is anergy.Illustrate that anti-HBe kit specificity provided by the invention meets national standard.
Hepatitis B e antibody of the present invention is measured kit and be can be used for measuring the antihepatitis b e antibody in human serum (slurry), after individual hepatitis b virus infection (HBV), hepatitis B e antibody (Anti-HBe) in serum, detected, in conjunction with other HBV, infect serological index, can judge the different phase that HBV infects.
When using kit of the present invention to detect the antihepatitis b e antibody in human serum (slurry), should be noted that following some: owing to using the monoclonal antibody of mouse, the patient who the accepts mouse monoclonal antibody diagnosis and treatment process result that can make the mistake, need to diagnose in conjunction with out of Memory; Different heterophil antibody in human serum reacts with reagent immunoglobulin (Ig), interference in vitro immunoassays.Often the patient of contact animal or animal blood serum product is easily interfered, and the result that leads to errors, need to diagnose in conjunction with out of Memory; The testing result of kit only supplies clinical reference, can not be separately as making a definite diagnosis or the foundation of Excluded cases, for reaching diagnostic purpose, this testing result will be combined with clinical examination, medical history and other check result.
The above; be only the specific embodiment of the present invention, protection scope of the present invention is not limited to this, is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; the variation that can expect easily or replacement, within all should being encompassed in protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claim was defined.
Claims (9)
1. a hepatitis B e antibody is measured kit, it is characterized in that, comprise reagent R, reagent N, reagent M and reference substance, the component in described reagent R comprises anti-HBe antibody, MES damping fluid and the diagnostic reagent antiseptic of biotinylation anti-HBe antibody, acridinium ester mark; Component in described reagent N comprises genetic recombination HBeAg, MES damping fluid and diagnostic reagent antiseptic; In described reagent M, component comprises that Avidin is coated with magnetic particle, and described magnetic particle after diagnosing reagent antiseptic is processed; Described control group comprises humanized's hepatitis B e antibody, NBCS and diagnostic reagent antiseptic; The anti-HBe antibody of described acridinium ester mark is to pass through coupling agent coupling anti-HBe antibody with acridinium ester, and described coupling agent is dimethyl imines or glutaraldehyde; Described kit also comprises with H
2o
2-NaHO
3for basic substrate solution system, in described substrate solution system, containing conduct is the TritonX-100 of luminescence enhancer.
2. hepatitis B e antibody according to claim 1 is measured kit, it is characterized in that, Avidin in the coated magnetic particle of described Avidin is Streptavidin, neutral Avidin or class Avidin, and the magnetic particle in the coated magnetic particle of described Avidin is to take the polymkeric substance of tri-iron tetroxide as core surface coating active group.
3. hepatitis B e antibody according to claim 1 is measured kit, it is characterized in that, described anti-HBe antibody is mouse resource monoclonal anti-HBe antibody.
4. hepatitis B e antibody according to claim 1 is measured kit, it is characterized in that, described in reagent R and reagent N, the concentration of MES damping fluid is 0.05M, and pH value is 6.0.
5. hepatitis B e antibody according to claim 1 is measured kit, it is characterized in that the Proclin300 antiseptic that described diagnostic reagent antiseptic is 0.1%.
6. hepatitis B e antibody according to claim 1 is measured kit, it is characterized in that, the amount of described reagent R and reagent N is 12ml, and the amount of described reagent M is 3 ml.
7. hepatitis B e antibody according to claim 1 is measured kit, it is characterized in that, described reference substance comprises reference substance 1 and 2 liang of groups of reference substance, the amount of described reference substance 1 is 0.5ml, concentration is 0.0 PEI U/ml, and the amount of described reference substance 2 is 0.5ml, and concentration is 2.0 PEI U/ml.
8. the hepatitis B e antibody described in right to use requirement 1-7 is measured a detection method for kit, it is characterized in that, comprises the following steps:
(1) the reagent N of testing sample and the HBeAg that contains genetic recombination is mixed and reacted, will form the reactant liquor 1 of antibody-antigenic complex;
(2) in reactant liquor 1, add the reagent R of the Anti-HBe that contains biotinylated e antibody (Anti-HBe) and acridinium ester mark to react, form the reactant liquor 2 of the quantity minimizing of biotinylated antibody-HBeAg-acridinium ester labelled antibody complex;
(3) in reactant liquor 2, add the reagent M that contains the coated magnetic particle of Avidin, the complex in reactant liquor 2 forms solid phase under the interaction of biotin and Avidin;
(4) reactant liquor is placed on chemical luminescence detector and is detected, the magnetic particle in the detection in magnetic field will be adsorbed, and by cleansing solution, wash, and bond does not rinse and removes; Then, inject chemiluminescence exciting liquid 1 and chemiluminescence exciting liquid 2, detect its chemiluminescence photon intensity; In the light intensity producing and sample, Anti-HBe concentration is inversely proportional to.
9. hepatitis B e antibody according to claim 8 is measured the detection method of kit, it is characterized in that, in described chemiluminescence exciting liquid 1, contain hydrogen peroxide, in described chemiluminescence exciting liquid 2, contain NaOH, described cleansing solution contains phosphate buffer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109633168A (en) * | 2018-12-29 | 2019-04-16 | 郑州安图生物工程股份有限公司 | The antibody combined detection kit of hepatitis B virus e antigen, e |
| CN113252901A (en) * | 2021-06-02 | 2021-08-13 | 中山生物工程有限公司 | Hepatitis B virus e antibody detection kit |
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| CN101620229A (en) * | 2008-07-02 | 2010-01-06 | 博阳生物科技（上海）有限公司 | Hepatits B virus e antibody assay kit and assay method thereof |
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| JPH08150000A (en) * | 1994-11-28 | 1996-06-11 | Immuno Japan:Kk | Oligonucleotide and detection of mutant hepatitis b virus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109633168A (en) * | 2018-12-29 | 2019-04-16 | 郑州安图生物工程股份有限公司 | The antibody combined detection kit of hepatitis B virus e antigen, e |
| CN113252901A (en) * | 2021-06-02 | 2021-08-13 | 中山生物工程有限公司 | Hepatitis B virus e antibody detection kit |
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