CN108169475A - A kind of Respiratory Syncytial Virus(RSV) IgM antibody detection kit - Google Patents
A kind of Respiratory Syncytial Virus(RSV) IgM antibody detection kit Download PDFInfo
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- CN108169475A CN108169475A CN201711367577.6A CN201711367577A CN108169475A CN 108169475 A CN108169475 A CN 108169475A CN 201711367577 A CN201711367577 A CN 201711367577A CN 108169475 A CN108169475 A CN 108169475A
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- 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
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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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Abstract
The invention discloses a kind of Respiratory Syncytial Virus(RSV) IgM antibody detection kits, include the magnetic particle of anti-human IgM antibodies' coupling, respiratory tract closes packet viral antigen solution, the anti respiratory syncitial virus antibodies of horseradish peroxidase-labeled, Sample dilution and Chemoluminescent substrate.Prize law is used in kit technical principle of the present invention, sensitivity and specificity are better than the kit using indirect method principle, in addition prize law is by the use of natural Respiratory Syncytial Virus(RSV) as bridging, by the use of the monoclonal antibody of Respiratory Syncytial Virus(RSV) as tracer, its principle is different from using prize law kit of the antigen as tracer, when avoiding using antigen as tracer because the purity of antigen not enough or site not comprehensively caused by specific the problem of being affected with sensitivity.The instruments such as kit of the present invention and the Full-automatic chemiluminescence apparatus AutoLumo A2000 of applicant company are used cooperatively, it can be achieved that being detected to the stochastic automation of sample, have many advantages, such as it is easy to operate, fast and automatically change.
Description
Technical field
The present invention relates to vitro diagnostic techniques fields, detect and try more particularly, to a kind of Respiratory Syncytial Virus(RSV) IgM antibody
Agent box.
Background technology
Respiratory Syncytial Virus(RSV)（Respiratory syncytial virus, RSV）Belong to paramyxovirus section Pneumovirinae
Belong to, be sub-thread minus-stranded rna virus, two hypotypes of A, B are divided into according to memebrane protein F and G antigenicity differences.
Respiratory Syncytial Virus(RSV) generally in winter-spring season prevalence, passes through air（The spittle, dust）It propagates, it also can be by polluting
Hand or article be in direct contact eye or schneiderian membrance infection.Incubation period 2-8 days after infection, the sustainable row of infant of hypoimmunity
Malicious 1-3 weeks.
Respiratory Syncytial Virus(RSV) is to cause the important virus causing disease of infant's lower respiratory tract infection, and infection for the first time mostly occurs in
Within 1 year old, almost all people has infected at 2-4 Sui.Clinical symptoms are mainly bronchiolitis, pneumonia, rami tracheales gas
Guan Yan cannot generate lasting immunocompetence after infection, can be with repeated infection, congenital heart disease, lung's disease less than 2 years old
Sick infant, the children of hypoimmunity, can lead to serious disease.Recurrent infection rate is relatively low within 2 years old or more, usual asymptomatic or disease
Shape is lighter.
The detection method of respiratory syncytial virus infection has：Detection of nucleic acids, antigen detection, the inspection of serological specificity antibody
It surveys, virus purification culture, wherein the serum specificity IgM antibody positive has important references meaning to etiological diagnosis.
Virus purification culture is to diagnose the goldstandard of respiratory syncytial virus infection, but since technology requires height, goes out knot
The time of fruit is long, seldom in clinical practice.The technology of detection of nucleic acids also requires that height, it is also necessary to which specific equipment and place are received
It is also higher to take price.Antigen detection sensitivity it is relatively low, testing result is affected by sample quality, simultaneously because nucleic acid and
Antigen detection acquisition is secretion sample, and sample collection difficulty is also larger, limits it in clinical extensive use.Serum is special
The detection of different in nature IgM antibody has easy to operate, and sample easily acquires, and disturbing factor is few, the advantages that having reasonable fee, at present in clinic
On be widely used.
At present the Respiratory Syncytial Virus(RSV) IgM antibody detection method that has listed have indirect immunofluorescence, enzyme-linked immunization,
Colloidal gold chromatography.Indirect immunofluorescence is that antigen is fixed on glass slide, using fluorescein FITC as marker, is passed through
Fluorescence microscope is as a result, result interpretation basic need visually observes, and easily there are errors.Enzyme-linked immunization be by antigen or
Antibody is fixed on polystyrene micropore plate, using horseradish peroxidase or alkaline phosphatase as marker, is inhaled by developing the color
Shading value judging result, the reaction time of experiment is long, and the degree of automation is low.Colloidal gold chromatography is to be fixed on antigen or antibody
On nitrocellulose filter, using colloidal gold as marker, developed the color by liquid capillarity in detection line, as a result interpretation basic need
It visually observes, although quick accuracy is low.
Invention content
It is an object of the invention in view of the defects existing in the prior art, provide a kind of new Respiratory Syncytial Virus(RSV) IgM
Antibody assay kit, the product which uses more at present have more technical advantage.
To achieve the above object, the present invention can take following technical proposals：
Respiratory Syncytial Virus(RSV) IgM antibody detection kit of the present invention, the magnetism including anti-human IgM antibodies' coupling are micro-
Grain, respiratory tract close packet viral antigen solution, the anti respiratory syncitial virus antibodies of horseradish peroxidase-labeled, Sample dilution
And Chemoluminescent substrate.
The anti-human IgM antibodies are the anti-human IgM monoclonal antibody of mouse or goat-anti people IgM polyclonal antibodies or rabbit-anti people IgM
Polyclonal antibody.
A diameter of 0.2 ~ 4um of the magnetic particle, the method that the anti-human IgM antibodies pass through chemical reagent covalent cross-linking
It is coupled on the magnetic particle, working concentration is 10 ~ 250ug/ml.
The antigen that the respiratory tract closes in packet viral antigen solution is the Respiratory Syncytial Virus(RSV) with cell culture, used
Cell is Vero cells, BSC-1 cells, Hep-2 cells or FRhK cells, and virus used is Long plants of standard strain（A is sub-
Type）, can also be clinical separation strain（A or subtype B）.
The anti respiratory syncitial virus antibodies are monoclonal antibody, and the albumen that wherein antibody is directed to is respiratory syncystial disease
Malicious F protein.
The Chemoluminescent substrate include substrate A liquid and substrate B liquid, wherein substrate A liquid be different luminol derivative, bottom
Object B liquid is hydrogen peroxide.
The Sample dilution is the diluted bovine serum albumin(BSA) of PBS buffer solution, during with its diluted sample, ratio for 10 ~
100 times.
The kit further includes negative control and positive control, and wherein positive control participates in the calculating of critical value.
The advantage of the invention is that using solid phase carrier of the magnetic particle as antibody, chemiluminescence immune assay skill is utilized
Single part, random, fast and automatically change detection can be achieved in art on Full-automatic chemiluminescence analyzer.Since the present invention is in technology
Using prize law in principle, sensitivity and specificity are better than the kit using indirect method principle, and the present invention is caught
Method is obtained by the use of natural Respiratory Syncytial Virus(RSV) as bridging, by the use of the monoclonal antibody of Respiratory Syncytial Virus(RSV) as tracer,
Principle is different from using prize law kit of the antigen as tracer, because of antigen when avoiding using antigen as tracer
The problem of purity is not enough or not comprehensively caused specificity and the sensitivity in site are affected.
Specific embodiment
The present invention is illustrated in more detail with reference to specific embodiment.Reagent used in the present invention and detection
It is commercial product if instrument is without specified otherwise.
First, the preparation of Respiratory Syncytial Virus(RSV) IgM antibody detection kit
1st, the preparation of anti-human IgM antibodies' conjugated magnetic particle
1.1 magnetic particles wash
The carboxylic magnetic particle in 200ul surfaces is taken to be placed in vial, magnetic particle is adsorbed in vial bottom with magnet, is removed
Supernatant；Add in 2ml 0.02M PBS（pH 8.0）, repeat above operation 3 times.
1.2 magnetic particles activate
EDC and NHS are dissolved in 0.1M MES respectively（pH 5.0）In buffer solution, concentration is in 10 ~ 70mg/ml, then respectively takes 1ml
It is added in magnetic particle；Ambient temperature with gentle concussion reaction 30 ~ 60 minutes；Magnetic particle is adsorbed in bottom with magnet, removes supernatant；Again
Add in 2ml 0.1M MES（pH 5.0）Magnetic particle is resuspended in buffer solution；It repeats above operation 2 times.
1.3 coupling anti-human IgM antibodies
It takes in the magnetic particle that the anti-human IgM monoclonal antibody 0.2mg ~ 1mg of mouse is added to after activating；Add 0.1M MES（pH
5.0）Buffer solution, until final volume 1ml；Ambient temperature with gentle concussion reaction 30 ~ 60 minutes；Magnetic particle is adsorbed in bottom with magnet, is removed
Supernatant is removed, with 2ml 0.02M PBS（pH 8.0）Washing 3 times；Add in PBS bufferings of the 20ml containing 1% BSA and 0.02% Sodium azide
Liquid（pH 8.0）, 2~8 DEG C of preservations.
2nd, respiratory tract closes the preparation of packet viral antigen solution
2.1 cell culture
By Hep-2 cell inoculations in the MEM culture mediums containing 2% fetal calf serum, it is placed in 35 ~ 37 DEG C of CO2It is cultivated in incubator, until
Exponential phase.
2.2 virus inoculations and culture
Respiratory Syncytial Virus Strain Long is inoculated on culture cell, is placed in 35 ~ 37 DEG C of CO2It is cultivated in incubator, after 48h
Observe cytopathy.
2.3 collection virus and inactivation
Low-speed centrifugal abandons supernatant, collects the cell of infection, adds in a small amount of MEM culture solutions, ultrasonic lytic cell；Use gamma ray
Inactivation of viruses puts -20 DEG C of preservations.
It is prepared by 2.4 antigenic solutions
By the viral antigen of preparation according to 1:10~1:100 dilution proportion is in the buffer solution containing 1%BSA and preservative, and 2~8
DEG C preserve.
3. preventing respiratory closes the preparation of packet viral monoclonal antibodies enzyme conjugates
Horseradish peroxidase（HRP）It is activated with conventional improvement Over-voltage protection, adds in anti respiratory syncytial virus F protein Dan Ke
Grand antibody, 2~8 DEG C of reactions overnight, add sodium borohydride reduction enzyme conjugates, and dialysis removes unreacted reagent, adds 50% volume sweet
Oil puts -20 DEG C of preservations.According to 1 during use:1000~1:5000 dilution proportion is prevented containing 20% calf serum and 0.1% P300
In the buffer solution of rotten agent, 2~8 DEG C of preservations.
4th, the preparation of Sample dilution
Prepare 1000ml Sample dilutions：Disodium hydrogen phosphate 5.80g, sodium dihydrogen phosphate dihydrate 0.59g, sodium chloride
9.00g, BSA 10g, Tween-20 1ml are settled to 1000ml with deionized water, add in 0.02% Sodium azide preservative.
5. prepared by negative control and positive control
Negative control is matrix liquid, prepares 1000ml negative controls：Disodium hydrogen phosphate 5.80g, sodium dihydrogen phosphate dihydrate
0.59g, sodium chloride 9.00g, BSA 10g are settled to 1000ml with deionized water, add in 0.02% Sodium azide preservative.
Positive control is that the respiratory tract of the addition 1/100 ~ 1/1000 in matrix liquid closes packet virus IgM antibody positive material system
It is standby to form.
6th, Chemoluminescent substrate：Substrate A liquid uses different luminol derivative, and substrate B liquid uses hydrogen peroxide.
2nd, detection example
The kit prepared using embodiment 1 is sent out with the full-automatic chemical that Zhengzhou Autobio Engineering Co., Ltd. produces
Light instrument AutoLumo A2000 or AutoLumo A2000 Plus are used cooperatively, and are resisted to detect Respiratory Syncytial Virus(RSV) IgM
Body：
In reaction vessel（Hereinafter referred to as " hole "）In sequentially add 3 hole of positive control（For determining Cutoff values）, negative control 2
Hole, 100 μ L/ holes；In remaining hole 10 μ L samples and 100 μ L sample dilutions are separately added into per hole；
20 μ L of magnetic particle suspension are separately added into per hole；After mixing, 37 DEG C incubate 15 minutes；Then it is washed 5 times with cleaning solution；
50 μ L of enzyme conjugates, 50 μ L of antigenic solution are added respectively per hole；It is incubated 17 minutes for 37 DEG C after mixing；Cleaning solution washing 5
It is secondary；
It is last to add substrate A liquid and each 50 μ L of substrate B liquid per hole；1~5 minute detection luminous intensity after mixing；
As a result it calculates：Cutoff values=Positive control wells average irradiance × 0.4；
Positive cutoff value：S/CO=sample to be tested luminous value/Cutoff values；
During S/CO >=1.00, it is as a result judged to the positive；During S/CO ＜ 1.00, it is as a result judged to feminine gender.
3rd, the performance evaluation of kit of the present invention
1st, it is repeated
Repeatability refers to the degree of closeness to same a sample replication, each measurement result and mean value, with coefficient of variation CV
It represents.
3 parts of samples of various concentration are selected, detect 4 repetitions, continuous detection 5 days, coefficient of variation CV daily<8%, as a result
It is shown in Table 1：
Table 1
Result can be seen that from table 1：The coefficient of variation of sample 1 is 3.95%, and the coefficient of variation of sample 2 is 4.25%, sample 3
The coefficient of variation is 6.72%, respectively less than the 10% of industry requirement, it was demonstrated that its repeatability is good.
2. specificity
Detect seven kinds of pathogen：Mycoplasma pneumoniae, chlamydia pneumoniae, adenovirus, legionella pneumophilia, enterovirns type 71, Ke Sa
Strange B groups, parainfluenza virus；Each 10 parts of IgM antibody positive samples of each pathogen, no cross reaction.
Detect the rheumatoid factor RF positives and antinuclear antibodies（ANA）Each 12 parts of positive sample, it is noiseless, it the results are shown in Table 2：
Table 2
As can be seen from Table 2：Seven kinds of respiratory pathogen IgM antibodies do not generate cross reaction, rheumatoid with this kit
Factor R F and autoantibody ANA does not generate interference to this kit.
3rd, sensitivity
The child patient that acute respiratory infection is selected to be hospitalized, the same day of being admitted to hospital acquire throat swab and serum sample, pharynx are wiped respectively
Daughter nucleus acid is detected as the patient of the Respiratory Syncytial Virus(RSV) positive, before discharge（5 ~ 14 days）Serum sample is acquired again to observe antibody
Variation, qualified patient totally 21.The serum sample of 2 acquisitions is detected with kit prepared by the present invention, wherein having
The serum sample that 11 patients acquire twice is the IgM antibody positive, and the sample IgM antibody of 10 patients acquisition for the first time is the moon
Property, the sample IgM antibody of second of acquisition is the positive, IgM antibody seropositive conversion occurs, the results are shown in Table 3：
As can be seen from Table 3：21 Respiratory Syncytial Virus(RSV) detection of nucleic acids have detected IgM antibody sun for positive patient
Property or antibody serum conversion, it was demonstrated that this kit have very high sensitivity.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, it is impossible to assert
The specific implementation of the present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, several equivalent substitute or obvious modifications are made, and performance or use is identical, all should
It is considered as belonging to protection scope of the present invention.
Claims (8)
1. a kind of Respiratory Syncytial Virus(RSV) IgM antibody detection kit, it is characterised in that：Include the magnetic of anti-human IgM antibodies' coupling
Property particle, respiratory tract closes packet viral antigen solution, and the anti respiratory syncitial virus antibodies of horseradish peroxidase-labeled, sample is dilute
Release liquid and Chemoluminescent substrate.
2. Respiratory Syncytial Virus(RSV) IgM antibody detection kit according to claim 1, it is characterised in that：It is described anti-human
IgM antibody is the anti-human IgM monoclonal antibody of mouse or goat-anti people IgM polyclonal antibodies or rabbit-anti people's IgM polyclonal antibodies.
3. Respiratory Syncytial Virus(RSV) IgM antibody detection kit according to claim 1 or 2, it is characterised in that：The magnetic
A diameter of 0.2 ~ 4um of property particle, the anti-human IgM antibodies are coupled at the magnetism by the method for chemical reagent covalent cross-linking
On particle, working concentration is 10 ~ 250ug/ml.
4. Respiratory Syncytial Virus(RSV) IgM antibody detection kit according to claim 1 or 2, it is characterised in that：It is described to exhale
It is the Respiratory Syncytial Virus(RSV) with cell culture to inhale the antigen closed in packet viral antigen solution in road, and cell used is thin for Vero
Born of the same parents, BSC-1 cells, Hep-2 cells or FRhK cells, virus used are Long plants of standard strain or clinical separation strain.
5. Respiratory Syncytial Virus(RSV) IgM antibody detection kit according to claim 1 or 2, it is characterised in that：It is described anti-
Respiratory Syncytial Virus(RSV) antibody is monoclonal antibody, and the albumen that wherein antibody is directed to is respiratory syncystial virus F protein.
6. Respiratory Syncytial Virus(RSV) IgM antibody detection kit according to claim 1 or 2, it is characterised in that：Describedization
It learns luminous substrate liquid and includes substrate A liquid and substrate B liquid, wherein substrate A liquid is different luminol derivative, and substrate B liquid is peroxidating
Hydrogen.
7. Respiratory Syncytial Virus(RSV) IgM antibody detection kit according to claim 1 or 2, it is characterised in that：The sample
This dilution is the diluted bovine serum albumin(BSA) of PBS buffer solution.
8. Respiratory Syncytial Virus(RSV) IgM antibody detection kit according to claim 1 or 2, it is characterised in that：The examination
Agent box further includes negative control and positive control, and wherein positive control participates in the calculating of critical value.
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Cited By (3)
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| CN110501493A (en) * | 2019-08-28 | 2019-11-26 | 郑州安图生物工程股份有限公司 | A kind of detection kit of hemadsorption virus type 1's IgM antibody |
| CN111999494A (en) * | 2020-08-18 | 2020-11-27 | 北京贝尔生物工程股份有限公司 | Kit for detecting adenovirus IgM antibody by magnetic particle chemiluminescence method and preparation method thereof |
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| CN111999494A (en) * | 2020-08-18 | 2020-11-27 | 北京贝尔生物工程股份有限公司 | Kit for detecting adenovirus IgM antibody by magnetic particle chemiluminescence method and preparation method thereof |
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Application publication date: 20180615 |