CN103616513A - Hepatitis B virus e-antigen measurement kit and detection method thereof - Google Patents
Hepatitis B virus e-antigen measurement kit and detection method thereof Download PDFInfo
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- CN103616513A CN103616513A CN201310653455.9A CN201310653455A CN103616513A CN 103616513 A CN103616513 A CN 103616513A CN 201310653455 A CN201310653455 A CN 201310653455A CN 103616513 A CN103616513 A CN 103616513A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5762—Hepatitis B core antigen
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
- G01N33/5761—Hepatitis B
- G01N33/5764—Hepatitis B surface antigen
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Abstract
The invention relates to a hepatitis B virus e-antigen measurement kit which comprises a reagent M, a reagent R and a reference substance, wherein the components in the reagent M include streptavidin coated magnetic particles treated by a diagnostic reagent preservative; the streptavidin coated magnetic particles are universal magnetic particles; 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 acridinium ester-labeled anti-HBe antibody is formed by coupling acridinium ester to the anti-HBe antibody through dimethylimine; the control group comprises a human hepatitis B virus e-antigen, newborn calf serum and a diagnostic reagent preservative. The hepatitis B virus e-antigen measurement kit 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 virus e antigen of design based on double antibody 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 virus e antigen that detects is safely and fast measured kit and detection method.
Technical scheme of the present invention is: a kind of hepatitis B virus e antigen is measured kit, it is characterized in that, comprise reagent M, reagent R and reference substance, in described reagent M, component comprises the coated magnetic particle of Avidin that reagent antiseptic is processed after diagnosing, and the coated magnetic particle of described Avidin is universal magnetic particle; 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, and the anti-HBe antibody of described acridinium ester mark is by dimethyl imines coupling anti-HBe antibody with acridinium ester; Described control group comprises humanized's hepatitis B virus e antigen, NBCS and diagnostic reagent antiseptic.
In a preferred embodiment of the present invention, further comprise, 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.
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, the concentration of MES damping fluid described in reagent R 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, described reagent R is 23ml, and the amount of described reagent M is 3ml.
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, and concentration is 1.5PEIU/ml.
In a preferred embodiment of the present invention, further comprise, a kind of detection method of using this hepatitis B virus e antigen to measure kit, comprises the following steps:
(1) testing sample is reacted with the reagent R of the Anti-HBe that contains biotinylated e antibody (Anti-HBe) and acridinium ester mark, form the reactant liquor 1 that contains antibody-Ag-Ab sandwich complex;
(2) in reactant liquor 1, add the reagent M that contains the coated magnetic particle of Avidin, the complex in reactant liquor 1 forms solid phase under the interaction of biotin and Avidin;
(3) reactant liquor 1 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; The light intensity producing is directly proportional to hepatitis B virus e antigen concentration in sample.
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 virus e antigen the present invention relates to is measured kit; contain the coated magnetic particle of Avidin; utilized double antibody chemiluminescence principle (sandwich 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 light intensity is directly proportional to hepatitis B virus e antigen (HBeAg) concentration in sample; so be easy to just obtain the HBeAg concentration in sample, thus can auxiliary diagnosis acute and chronic HBV infection.
2. use hepatitis B virus e antigen 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 virus e antigen (HBeAg), in conjunction with several marks of other HBV, can judge the clinical state that HBV infects, the method is simple to operation, and error is little, and accuracy is high.
3. the biotin in kit of the present invention 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.
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 virus e antigen of the present invention is measured kit, and group wants constituent as follows:
Reagent R: every bottle of 23ml, contain 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 by dimethyl imines coupling anti-HBe antibody with acridinium ester.
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, Avidin in the coated magnetic particle of Avidin is Streptavidin, neutral Avidin or class Avidin, magnetic particle in the coated magnetic particle of described Avidin is to take the polymkeric substance that tri-iron tetroxide is core surface coating active group, and the magnetic particle in the present invention is universal magnetic particle.
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 virus e antigen, 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 1.5PEIU/ml.
Hepatitis B virus e antigen of the present invention is measured kit and is used for measuring human serum (blood plasma) hepatitis B virus e antigen.Hepatitis B virus e antigen HBeAg is the front C/C gene coded protein of hepatitis B (HBV) product, can appear in acute hepatitis b virus infections person's serum, with other several the mark combinations of HBV, the clinical state that diagnosable HBV infects, can be used for the acute and chronic HBV infection of auxiliary diagnosis.
The anti-HBe antibody relating in the present invention is mouse resource monoclonal anti-HBe antibody, and the detection principle of using this kit is to adopt double antibody chemiluminescence principle, and step is as follows:
First step reaction: the Anti-HBe of sample and biotinylated antihepatitis b e antibody (Anti-HBe) and acridinium ester mark is reacted, if contain HBeAg in sample, will form antibody-Ag-Ab sandwich complex.
Second step reaction: add the coated particulate of Avidin, complex forms solid phase under the interaction of biotin and Avidin.
Reactant liquor is placed in a magnetic field, and the magnetic particle in detection will be held, 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; The light intensity producing is directly proportional to HBeAg concentration in sample.
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-K3, sodium citrate or the anti-freezing of sodium fluoride/potassium oxalate for blood plasma.
Then, to this testing sample, using hepatitis B virus e antigen to measure kit detects:
(1) testing sample is reacted with the reagent R of the Anti-HBe that contains biotinylated e antibody (Anti-HBe) and acridinium ester mark, form the reactant liquor 1 that contains antibody-Ag-Ab sandwich complex;
(2) in reactant liquor 1, add the reagent M that contains the coated magnetic particle of Avidin, the complex in reactant liquor 1 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.
(3) reactant liquor 1 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; The light intensity producing is directly proportional to hepatitis B virus e antigen concentration in sample.
Wherein, in 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-HBeAg.The HBeAg of sample measures S/CO value > 1 and is judged as anti-HBeAg anergy; The HBeAg of sample measures S/CO value≤1 and is judged as HBeAg reactivity.
Sensitivity
Use above-mentioned detection method, the sensitivity Cut-off value that obtains HBeAg kit of the present invention is≤0.5PEIU/mL; Detect the dish sensitivity of HBeAg reagent country, 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 HBeAg 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.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 virus e antigen is measured kit, it is characterized in that, comprise reagent M, reagent R and reference substance, in described reagent M, component comprises the coated magnetic particle of Avidin that reagent antiseptic is processed after diagnosing, and the coated magnetic particle of described Avidin is universal magnetic particle; 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, and the anti-HBe antibody of described acridinium ester mark is by dimethyl imines coupling anti-HBe antibody with acridinium ester; Described control group comprises humanized's hepatitis B virus e antigen, NBCS and diagnostic reagent antiseptic.
2. hepatitis B virus e antigen 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 virus e antigen 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 virus e antigen according to claim 1 is measured kit, it is characterized in that, the concentration of MES damping fluid is 0.05M described in reagent R, and pH value is 6.0.
5. hepatitis B virus e antigen 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 virus e antigen according to claim 1 is measured kit, it is characterized in that, described reagent R is 23ml, and the amount of described reagent M is 3ml.
7. hepatitis B virus e antigen 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, the amount of described reference substance 2 is 0.5ml, and concentration is 1.5 PEI U/ml.
8. the hepatitis B virus e antigen 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) testing sample is reacted with the reagent R of the Anti-HBe that contains biotinylated e antibody (Anti-HBe) and acridinium ester mark, form the reactant liquor 1 that contains antibody-Ag-Ab sandwich complex;
(2) in reactant liquor 1, add the reagent M that contains the coated magnetic particle of Avidin, the complex in reactant liquor 1 forms solid phase under the interaction of biotin and Avidin;
(3) reactant liquor 1 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; The light intensity producing is directly proportional to hepatitis B virus e antigen concentration in sample.
9. hepatitis B virus e antigen 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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| CN201310653455.9A CN103616513A (en) | 2013-12-06 | 2013-12-06 | Hepatitis B virus e-antigen measurement kit and detection method thereof |
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| CN201310653455.9A CN103616513A (en) | 2013-12-06 | 2013-12-06 | Hepatitis B virus e-antigen measurement kit and detection method thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107632163A (en) * | 2017-10-16 | 2018-01-26 | 苏州长光华医生物医学工程有限公司 | A kind of detection method of hs-CRP |
| 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 |
| CN112526136A (en) * | 2020-11-03 | 2021-03-19 | 广州市达瑞生物技术股份有限公司 | Sample pretreatment liquid and kit for combined detection of hepatitis B virus core-associated antigen |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0411938A2 (en) * | 1989-08-03 | 1991-02-06 | Merck & Co. Inc. | Radio-immuno assay for hepatitis B virus pres2 antibodies |
| WO2005071401A2 (en) * | 2004-01-15 | 2005-08-04 | Chiron Corporation | Homogeneous multiplex assay for nucleic acid targets |
| CN101592663A (en) * | 2008-05-30 | 2009-12-02 | 北京科美东雅生物技术有限公司 | Hepatitis B e antigen magnetic particle chemiluminescent immunoassay kit and preparation method thereof |
| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
-
2013
- 2013-12-06 CN CN201310653455.9A patent/CN103616513A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0411938A2 (en) * | 1989-08-03 | 1991-02-06 | Merck & Co. Inc. | Radio-immuno assay for hepatitis B virus pres2 antibodies |
| WO2005071401A2 (en) * | 2004-01-15 | 2005-08-04 | Chiron Corporation | Homogeneous multiplex assay for nucleic acid targets |
| CN101592663A (en) * | 2008-05-30 | 2009-12-02 | 北京科美东雅生物技术有限公司 | Hepatitis B e antigen magnetic particle chemiluminescent immunoassay kit and preparation method thereof |
| CN102565405A (en) * | 2011-08-24 | 2012-07-11 | 苏州长光华医生物试剂有限公司 | Method for immunological detection by combining acridinium ester labeling technology with general magnetic particles |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107632163A (en) * | 2017-10-16 | 2018-01-26 | 苏州长光华医生物医学工程有限公司 | A kind of detection method of hs-CRP |
| 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 |
| CN112526136A (en) * | 2020-11-03 | 2021-03-19 | 广州市达瑞生物技术股份有限公司 | Sample pretreatment liquid and kit for combined detection of hepatitis B virus core-associated antigen |
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Application publication date: 20140305 |