CN104360063A - HCV core antigen detection kit based on magnetic micro-particle chemiluminescence method - Google Patents
HCV core antigen detection kit based on magnetic micro-particle chemiluminescence method Download PDFInfo
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- CN104360063A CN104360063A CN201410740936.8A CN201410740936A CN104360063A CN 104360063 A CN104360063 A CN 104360063A CN 201410740936 A CN201410740936 A CN 201410740936A CN 104360063 A CN104360063 A CN 104360063A
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- hcv
- hcv core
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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
- G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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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
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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Abstract
The invention relates to an HCV core antigen detection kit based on a magnetic micro-particle chemiluminescence method, which belongs to the technical field of clinical in-vitro detection. Total or free HCV core antigens in a serum sample are qualitatively or quantitatively detected by virtue of a double-antibody sandwich method. The HCV core antigen detection kit mainly comprises a reagent R1, a reagent R2, a bottle of positive reference, a bottle of negative reference, a luminous substrate A, a luminous substrate B and a solid washing solution. The kit is high in sensitivity and stability, is simple, convenient and quick to operate, is easy to automatically operate, and can well meet domestic clinical using requirements.
Description
Technical field
The present invention relates to a kind of Magnetism particulate immuno chemistry luminescence method HCV core antigen detection kit, the HCV-cAg total or free in legal or quantitative detection blood serum sample by double-antibody sandwich, belongs to clinical vitro detection technical field.
Background technology
Third type
virus hepatitis, referred to as hepatitis C, the third liver, be that one infects by hepatitis C virus (HCV) virus hepatitis caused, main warp
blood transfusion, acupuncture, the propagation such as drug abuse, according to World Health Organization's statistics, the infection rate of global HCV is about 3%, estimates that about 1.8 hundred million people have infected HCV, newly sends out hepatitis C case about 3.5 ten thousand example every year.Hepatitis C is global prevalence, and can cause the necrosis of liver chronic inflammation and fiberization, some patients can develop into
cirrhosiseven hepatocellular carcinoma (HCC).Mortality ratio relevant to HCV infection in following 20 years (
hepatic failureand the death that causes of hepatocellular carcinoma) continuation is increased, very harmful to the health and lives of patient, become serious society and public health problem.
Hepatitis C virus (HCV) is addicted to liver property slow virus.After HCV infection, onset and the clinical symptoms pole of patient are not true to type, and be common, easily cause and fail to pinpoint a disease in diagnosis with subclinical infection.The chronicity incidence of HCV infection is apparently higher than hepatitis B, and comparatively hepatitis B easily occurs cirrhosis, liver cancer in early days, and mortality ratio is higher.Therefore the detection of HCV has great meaning to the early diagnosis of infection with hepatitis C virus and guiding clinical treatment.
The method detected for HCV at present mainly contains: detection two kinds of methods of hepatitis C virus anti-HCV and hepatitis C virus nucleic acid RNA.Current China many employings third generation anti-HCV ELISA reagent, although be greatly improved in sensitivity and specificity, but still the minority false positive can not got rid of because recombination fusion protein brings and only a few is undetected.Comparatively speaking, the features such as RT-PCR detection HCV RNA has in early days, responsive and special, but the method requires higher on technology and equipment, and time-consuming, recall rate is lower; In addition, the method is also easy there is false positive because of pollution.Therefore RT-PCR method is difficult to promote in routine work or laboratories, limits and generally applies.
Summary of the invention
In order to solve that anti-HCV ELISA reagent in above prior art has that false positive, window phase are long, sensitivity is low, RT-PCR detects HCV RNA and requires higher on technology and equipment, wastes time and energy, is difficult in shortcomings such as routine work or laboratories popularizations.The invention provides a kind of highly sensitive, easy to operate Magnetism particulate immuno chemistry luminescence method HCV core antigen detection kit.
The technical solution adopted in the present invention is:
A kind of Magnetism particulate immuno chemistry luminescence method HCV core antigen detection kit, it is characterized in that, it comprises reagent R1, reagent R2, positive reference material 1 bottle, negative reference product 1 bottle, luminous substrate A, luminous substrate B and solid washing lotion;
Described reagent R1 is the PBS damping fluid being coated with the 0.1M pH=7.6 of HCV-Ab IgG-cAg antibody magnetic bead containing 1g/L;
Described reagent R2 is the PBS damping fluid of the 0.1M pH=7.6 of the HRP mark HCV-Ab IgG-cAg antibody containing 1:500.
Described luminous substrate A is the luminol of 1 mol/L and the mixed liquor to iodophenol of 0.5 mol/L; Described luminous substrate B is the H of 1%
2o
2solution.
Described solid washing lotion is the PBS damping fluid of the 0.1M pH=7.6 containing 0.5% polysorbas20.
Described positive reference material is the PBS damping fluid containing HCV-cAg, and concentration is 200ng/ml;
Described negative reference product are the PBS damping fluid containing BSA, and concentration is 200ng/ml.
The using method of kit of the present invention is as follows:
Tube-type chemical luminometer detects by following program:
(1) in instrument reaction small test tube, add reagent R1 300 μ l, then add yin and yang attribute reference material or blood serum sample 100 μ l, then add reagent R2 50 μ l, 37 DEG C of incubations 1 hour;
(2) in magnetic field, use solid wash liquid magnetic bead 5 times, wash away in conjunction with material;
(3) add each 50 μ l of luminous substrate liquid A, B, incubated at room temperature detected luminous intensity after 5 minutes.
The determination of critical value (Cut off value): equal RLU value × 2.1 of negative reference product examine lining.If during equal RLU value≤60 of negative reference product examine lining, calculate by 60; During RLU>60, calculate by average RLU value × 2.1 of practical measurement negative control).
The invention has the beneficial effects as follows:
(1) with HCV-cAg total or free in the legal or quantitative detection blood serum sample of double-antibody sandwich, expand sensing range, shorten window phase.
(2) magnetic microparticle chemiluminescence technology is adopted immunomagnetic beads system, chemical luminous system to be combined with immune response system, for detecting micro-HCV antigen.It had both had the high sensitivity of RT-PCR, had again easy, the quick feature of enzyme linked immunological operation, was easy to automation mechanized operation.
Accompanying drawing explanation
Fig. 1 is embodiment 1 kit range of linearity figure;
Fig. 2 is embodiment 1 kit and real-time fluorescence quantitative PCR correlativity figure;
Fig. 3 is embodiment 1 reagent thermal stability results figure.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in further detail.
Embodiment 1
The composition of kit and preparation method
1. reagent R1: the magnetic bead 0.1g being coated with HCV-Ab IgG-cAg antibody got purchased from dynal company joins in the PBS damping fluid of 100ml 0.1M pH=7.6 and mixes.
2. in reagent R2, the preparation method of HRP mark HCV-Ab IgG-cAg antibody is as follows:
(1) taking HRP25mg is dissolved in 1.25% glutaraldehyde solution, in room temperature hold over night.
(2) reacted enzyme solutions is through Sephadex G-25 chromatographic column, uses physiological saline wash-out.Flow control, at 1ml/min, collects brown efflux.As volume is greater than 5ml, be then concentrated into 5ml with PEG.Place in 25ml small beaker, slowly stir.
(3) by antibody 12.5mg normal saline dilution to be marked to 5ml, dropwise add in enzyme solutions under stirring.
(4) with 1M PH9.5 carbonic acid buffer 0.25ml, stirring 3 hours is continued.
(5) add 0.2M lysine 0.25ml, after mixing, put room temperature 2 hours.
(6) under agitation dropwise add equal-volume saturated ammonium sulfate, put 4 DEG C 1 hour.
(7) 3000rpm centrifugal half an hour, supernatant is abandoned.Sediment semi-saturation ammonium sulfate washes secondary, and last sediment is dissolved in the PBS of a small amount of 0.15M PH7.4.
(8) above-mentioned solution is loaded in bag filter, the PB buffer saline of 0.15M PH7.4 is dialysed, (detect with Nai Shi reagent) after removing ammonium ion, 10000rpm removes precipitation in centrifugal 30 minutes, supernatant is enzyme mark bond, after packing, and-20 DEG C of stored frozen.
The PBS damping fluid of above-mentioned obtained enzyme mark bond and 0.1M pH=7.6 is obtained R1 reagent according to volume ratio 1:500 mixed preparing.
3. positive reference material: 1 bottle, 0.5ml/ bottle, containing the PBS damping fluid of HCV-cAg antigen, concentration is 200ng/ml.
4. negative reference product: 1 bottle, 0.5ml/ bottle, containing the PBS damping fluid of BSA, concentration is 200ng/ml.
5. luminous substrate A: add in 100ml distilled water after 12.11g Tris dissolves and add appropriate HCl solution adjustment pH, be finally mixed with the Tris-HCl damping fluid of 0.1M pH 7.6.Add in this damping fluid the luminol of 17.72g and 11g to iodophenol, mix.
6. luminous substrate B: the H getting 3.3ml commercially available 30%
2o
2join in the Tris-HCl damping fluid of 0.1M pH 7.6 and mix, make the H of 1%
2o
2.
7. solid washing lotion: get 0.5g polysorbas20 and join in the PBS damping fluid of 0.1M pH 7.6 and mix.
Embodiment 2
Sensitivity and linear measurement range is tested
Detect analysis by experiment, the determination of this reagent critical value (Cut off value): if during equal RLU value≤60 of negative reference product examine lining equal RLU value × 2.1(negative reference product examine lining, calculate by 60; During RLU>60, calculate by average RLU value × 2.1 of practical measurement negative control).Low value sample is detected, contrasts the luminous value of negative reference product and normal human serum simultaneously, as shown in table 1.By comparison, when low value concentration of specimens is 0.5ng/mL, still test positive, illustrates that the sensitivity of embodiment 1 reagent can reach 0.5ng/mL, has good sensitivity.
Table 1 sensitivity determination
Test item | Luminous value (RLU) | Result judges |
HCV-cAg 0.5ng/ mL | 331 | + |
20 portions of normal human serums | 92 | - |
Negative reference product | 64 |
High level HCV-cAg positive serum is diluted to different concentration measure, with the theoretical concentration of dilution for horizontal ordinate, the corresponding luminous value detected is ordinate, as shown in Figure 1, recording the typical curve range of linearity is 0.5 ~ 200ng/mL, its r > 0.99, illustrates that kit of the present invention has good linear dependence.
Embodiment 3
Magnetism particulate immuno chemistry luminescence method HCV core antigen detection kit of the present invention and real-time fluorescence quantitative PCR acquired results have good correlativity.
Concrete grammar is as follows: detect 40 routine clinical samples by the hepatitis C virus nucleic acid detection kit (PCR-fluorescence probe method) that embodiment 1 gained kit and Da'an Gene Company, Zhongshan University produce simultaneously, result has good correlativity, and concrete outcome as shown in Figure 2.
Embodiment 4
Thermal stability determination
Embodiment 1 kit is put the luminous intensity of the positive reference material measuring the dilution of each concentration gradient under 37 DEG C of conditions after 7d, and with compared with batch placement 4 DEG C with observation caliber curve with or without significant change, the luminous value change that Fig. 3 shows each reference material concentration value corresponding is little, background is not high, illustrates that this stabilization of kit is good.
By checking, it is good that this reagent and RT-PCR reagent contrast correlativity, and highly sensitive, good stability, and fast easy and simple to handle, easily realizes automation mechanized operation, can meet domestic Clinical practice demand better.
Claims (3)
1. a Magnetism particulate immuno chemistry luminescence method HCV core antigen detection kit, is characterized in that, it comprises reagent R1, reagent R2, positive reference material 1 bottle, negative reference product 1 bottle, luminous substrate A, luminous substrate B and solid washing lotion; Described reagent R1 is the PBS damping fluid being coated with the 0.1M pH=7.6 of HCV-Ab IgG-cAg antibody magnetic bead containing 1g/L; Described reagent R2 is the PBS damping fluid of the 0.1M pH=7.6 of the HRP mark HCV-Ab IgG-cAg antibody containing 1:500.
2. Magnetism particulate immuno chemistry luminescence method HCV core antigen detection kit according to claim 1, is characterized in that, described luminous substrate A is the luminol of 1 mol/L and the mixed liquor to iodophenol of 0.5 mol/L; Described luminous substrate B is the H of 1%
2o
2solution.
3. Magnetism particulate immuno chemistry luminescence method HCV core antigen detection kit according to claim 1, is characterized in that, described solid washing lotion is the PBS damping fluid of the 0.1M pH=7.6 containing 0.5% polysorbas20.
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105486856A (en) * | 2015-12-24 | 2016-04-13 | 泰州泽成生物技术有限公司 | Qualitative detection kit for antibody to hepatitis C (anti-HCV) and preparing method and application thereof |
Citations (7)
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WO2003002749A2 (en) * | 2001-06-26 | 2003-01-09 | Abbott Laboratories | Methods for the simultaneous detection of hcv antigens and hcv antibodies |
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2014
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Non-Patent Citations (2)
Title |
---|
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105486856A (en) * | 2015-12-24 | 2016-04-13 | 泰州泽成生物技术有限公司 | Qualitative detection kit for antibody to hepatitis C (anti-HCV) and preparing method and application thereof |
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Application publication date: 20150218 |