CN108508217A - A kind of indirect ELISA method kit and its application for detecting people's lower respiratory tract bocavirus - Google Patents

A kind of indirect ELISA method kit and its application for detecting people's lower respiratory tract bocavirus Download PDF

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CN108508217A
CN108508217A CN201810699705.5A CN201810699705A CN108508217A CN 108508217 A CN108508217 A CN 108508217A CN 201810699705 A CN201810699705 A CN 201810699705A CN 108508217 A CN108508217 A CN 108508217A
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bocavirus
respiratory tract
lower respiratory
indirect elisa
elisa method
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赵风强
侯建毅
邬晓乐
邬江
姬志娟
张军龙
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BEIJING BIOSYNTHESIS BIOTECHNOLOGY Co Ltd
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BEIJING BIOSYNTHESIS BIOTECHNOLOGY Co Ltd
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Abstract

The invention discloses a kind of indirect ELISA method kit and its applications for detecting people's lower respiratory tract bocavirus, a kind of indirect ELISA method kit for detecting people's lower respiratory tract bocavirus of the present invention, including coated antibody, antigen, calibration object, quality-control product, sample diluting liquid, the coated antibody is anti-human lower respiratory tract bocavirus VP2 fusion protein monoclonal antibodies；The antigen behaviour lower respiratory tract bocavirus VP2 fusion proteins；The calibration object is with ProSpec companies Procalcitonin Human Recombinant (HOR 304, purity >=97.0%) for HBoV positive serums.The present invention has many advantages, such as, using easy, detection is quick, sensitive, stable, easy to operate, there is higher sensitivity and specificity, strong antijamming capability.

Description

A kind of indirect ELISA for detecting people's lower respiratory tract bocavirus Method kit and its application
Technical field
The present invention relates to molecular immunology technical fields, and in particular to a kind of for detecting people's lower respiratory tract bocavirus Indirect ELISA method kit and its application.
Background technology
Bocavirus (Human Bocavirus) is a kind of new person being separated to from children with lower respiratory tract infections secretion Parvovirus (Parvovirus).It is reported that the Tobias Allander children of Stockholm, SWE Karolinska Institute in Stockholm What the doctor of hospital had found when being treated to 540 children's patients with infection of low respiratory tract.In this batch of child patient, 17 There are this new virus, Sweden doctor " bocavirus " of to be named as in children's body.Children with lower respiratory tract infections can be by not Cause with virus, including respiratory syncytial virus (RSV), influenza virus, adenovirus and coronavirus.So far, about 10- The cause of disease of 39% children with lower respiratory tract infections is unknown.The human bocavirus newly detached is considered at least causing 5% ratio Children with lower respiratory tract infections the cause of disease.According to the statistics of the World Health Organization, lower respiratory tract infection is that five are caused in worldwide Year old or less death of child principal disease, infant often shows the symptom of general asthma.In the U.S., lower respiratory tract infection is small The primary disease of youngster's hospitalization, there are about 2,500,000 patients every year.In China, every year since unknown cause causes children to live The number of institute's treatment also rises year by year.Although the major reason that virus infection is children with lower respiratory tract infections clinically has The children with lower respiratory tract infections etiology unknown of significant proportion.The discovery of bocavirus is undoubtedly to the cause of disease of children with lower respiratory tract infections Research and the diagnosis and treatment of lower respiratory tract infection provide new scientific basis.
It is the key that diagnosing and treating that clinically infectious new hair pathogen, which is fast and accurately detected,.People's Bo Ka diseases Poison is a kind of infected children lower respiratory tract virus found for 2005, therefore, for the virus identification not yet one it is quick, Effective method.Currently, only having Xiaoming Ma etc. to use round pcr the report of the detection and identification of human bocavirus Detect 318 clinical samples, positive rate 5.7%.This seminar carries out successfully table to the VP2 genes in human bocavirus It reaches, and by the antigen binding Western Blot and elisa technique of purifying, establishes the protein level detection body of the virus System, i.e. the molecular immune detection method of human bocavirus.
Currently, shortage is a kind of can quickly, accurately and efficiently to detect the sick for detecting the rich card of people's lower respiratory tract of sample The indirect ELISA method kit of poison and its application.
Invention content
The purpose of the present invention is to provide a kind of one kind that can quickly, accurately and efficiently detect sample for detecting The indirect ELISA method kit of people's lower respiratory tract bocavirus and its application.
In order to achieve the above objectives, present invention employs following technical proposals：One kind of the present invention is breathed for detecting under people The indirect ELISA method kit of road bocavirus, including coated antibody, antigen, calibration object, quality-control product, sample Dilution,
The coated antibody is monoclonal antibody；
The antigen behaviour lower respiratory tract bocavirus VP2 fusion proteins；
The calibration object is with ProSpec companies Procalcitonin Human Recombinant (HOR-304, purity >=97.0%) be reference material prepare 50,100,200,400,800, the HBoV positive serums of 3200pg/ml concentration；
The quality-control product is to be formulated by HBoV clinical detection high level people source sample, and through inactivation treatment, after testing its Anti- TP, HBsAg, anti-HCV and anti-HIV are feminine gender；
The sample diluting liquid is the dilution for detecting sample.
Further, the monoclonal antibody is the mouse source Dan Ke of anti-human lower respiratory tract bocavirus VP2 fusion proteins Grand antibody；
Further, people's lower respiratory tract bocavirus VP2 fusion proteins are the fusion egg of sf9 insect cell expression In vain.
Indirect ELISA method kit of the present invention for detecting people's lower respiratory tract bocavirus Application in it can be detected to the bocavirus antibody corresponding antigens in lower respiratory tract sample using recombinant protein VP2.
Further, applications of the recombination fusion VP2 in the detection of indirect ELISA technology.
Further, recombination fusion VP2 in clinical disease diagnosis, antiviral drugs screening, environmental monitoring and is commented Application in valence, health supervision and port health quarantine, epidemiological survey drug.
Advantageous effect：The present invention has many advantages, such as using easy, detection is quick, sensitive, stable, easy to operate, have compared with High sensitivity and specificity, strong antijamming capability.It is used suitable for vast middle and small hospital.
Compared with prior art, the invention has the advantages that：
(1) anti-using human bocavirus in the indirect ELISA serum of detection people's lower respiratory tract bocavirus Body is horizontal, realizes the high sensitivity (being less than 50pg/ml) and specificity of detection.
(2) one aspect of the present invention can quickly and accurately measure the content of human bocavirus antibody in serum, for inflammation Early diagnosis, early treatment reliable clinical reference value is provided.
(3) on the other hand due to the use of the monoclonal antibody of specificity as coating solid phase, people's Bo Ka diseases are substantially increased The detection sensitivity and specificity of poison, strong antijamming capability.The corresponding kit, can be widely applied to examining for clinical disease Disconnected, antiviral drugs screening, Environmental Monitoring and Assessment, health supervision and food quarantine, epidemiological survey etc..
Description of the drawings
Fig. 1 is that the SDS-PAGE of the present invention analyzes the human bocavirus VP2 albumen of baculovirus expression；The insect of infection Intracellular high efficient expression recombination fusion VP2 albumen, molecular weight of albumen 60kDa, destination protein expression quantity account for insect solubility egg White 80% or more；M：Albumen Marker；1 and 2：HA label VP2 fusion proteins；3 and 4：Express VP2 albumen；5：Insect cell pair According to.
Fig. 2 is the Western blot verifications of the present invention, albumen is expressed with the VP2 of HA mark mirror, through Western blot skills Art demonstrates the specificity of BacHBoVCap expression albumen；M：Albumen Marker；1 and 2：HA label VP2 fusion proteins；3 and 4： Express VP2 albumen；5：Insect cell compares.
Fig. 3 detects clinical sample by the bocavirus indirect ELISA of the present invention and is used concentration and absorbance linear relationship； Log (Abs)=- 2.096+0.7521Log (Cone), linear coefficient=0.998.
Specific implementation mode
In order to further illustrate the embodiment party of detection human respiratory bocavirus antibody indirect enzyme-linked immunosorbent assay Formula and purposes, illustrate with reference to following embodiment.Embodiment is in order to explain rather than limit the invention in any way Range, the certain changes and adjustment that those skilled in the art are made within the scope of the claims also are regarded as belonging to the present invention Range.
A kind of indirect ELISA method kit for detecting people's lower respiratory tract bocavirus of the present invention, Including coated antibody, antigen, calibration object, quality-control product, sample diluting liquid, the coated antibody is monoclonal antibody；
The antigen behaviour lower respiratory tract bocavirus VP2 fusion proteins；
The calibration object is with ProSpec companies Procalcitonin Human Recombinant (HOR-304, purity >=97.0%) be reference material prepare 50,100,200,400,800, the HBoV positive serums of 3200pg/ml concentration；
The quality-control product is to be formulated by HBoV clinical detection high level people source sample, and through inactivation treatment, after testing its Anti- TP, HBsAg, anti-HCV and anti-HIV are feminine gender；
The sample diluting liquid is the dilution for detecting sample.
Indirect ELISA method kit of the present invention for detecting people's lower respiratory tract bocavirus Application in it can be detected to the bocavirus antibody corresponding antigens in lower respiratory tract sample using recombinant protein VP2.
Applications of the recombination fusion VP2 in the detection of indirect ELISA technology.
The recombination fusion VP2 is in clinical disease diagnosis, antiviral drugs screening, Environmental Monitoring and Assessment, health prison It superintends and directs and the application in port health quarantine, epidemiological survey drug.
Embodiment 1
The preparation of VP2 recombinant proteins
Human bocavirus coded sequence (NCBI accession number NC007455) is obtained from ncbi database, builds prokaryotic expression Carrier pCMVHBoVi, induced expression recombinates BacHBoVCap and with HA label B acHBoVCapHA recombinant antigen, through Western Blot analysis albumen has immune response, Chelating Sepharose Fast Flow metals and column chromatography largely to prepare BacHBoVCap recombinant proteins.
1 rhabdovirus expression vector is built：Ned I and III double digestions of Hind are cut from plasmid pCMVHBoVi to be marked containing HA The VP2 genetic fragments of note after Klenow enzyme filling-in, are recycled using Glass Milk；After I digestion carrier pAcuW51 filling-in of BamH Recycling, T4 ligases are connected to I downstreams carrier B amH by VP2 segments are recycled, and convert bacillus coli DH 5 alpha.BamH I and Hind III The positive clone of double digestion identification recombination is carried out, DNA purification kits extraction recombinant vector DNA is for transfecting.Build pAcuW51- HBoV-VP2 recombination rhabdovirus expression vectors.
2BacHBoVCap protein expressions：Purify recombinant vector DNA and linear baculovirus DNA cotransfection logarithmic phase insect Cell sf9.Recombination BacHBoVCap and with HA label Bs acHBoVCapHA through prepare and take after purification 2 μ g respectively with 10 μ L lipids Body mixes, and 27 DEG C are cultivated 6 days, and 0.1ml supernatants infected insect cell again is taken, and are collected supernatant cell, are used lysate (50mmol/L Tris-HCl pH8.5,1%NP-40,10% protease inhibitors, 10mmol/l beta -mercaptoethanols, 1mmol/ LPMSF) lytic cell after 12000g centrifuges 10min, takes cracking supernatant to carry out SDS-PAGE electroresis appraisals, is resisted with HA after transferring film Body carries out Western blot identification recombination fusions VP2Protein expression.As a result the high efficient expression recombination in infected insect cell Merge VP2Albumen, molecular weight 60kDa, destination protein expression quantity account for 80% or more insect soluble protein.Mirror VP is marked with HA2Expression Albumen expresses protein-specific through Western blot technical identifications BacHBoVCap.
3 recombinant VP 2 protein purifications：Recombinant virus infects a large amount of cells (2x107 cells/bottle) by best MOI, is received after 72h Collect cell, washing, lytic cell, 12000g centrifuges 30min, collects supernatant and filters, measures albumen concentration.It prepares Chelating Sepharose Fast Flow metal affinity chromatography columns.With sample-loading buffer (50mmol/l NaH2PO4, 300mmol/lNaCl, 10% glycerine, pH8.6) balance chromatographic column, with washing buffer (50mmol/l NaH2PO4, 300mmol/lNaCL, 10% glycerine, pH7.6) column is washed until protein peaks drop to baseline, add elution buffer (50mmol/l NaH2PO4,300mmol/lNaCL, 10% glycerine, pH4.5) elution, eluent is collected, spectrophotometer determines protein content, SDS-PAGE electrophoresis determines purity of protein, and being stored in -20 DEG C after freeze-drying saves backup.The result shows that VP2 purity of protein reaches 95% or more.
4.Western blottings are identified
Human bocavirus antigen after purification takes out offset plate through SDS-PAGE electrophoresis, and glue is dipped in transfering buffering liquid and is put down Weigh 10min；Pvdf membrane need to be impregnated into saturation 3-5 seconds with pure methanol.Assembly transfer sandwich：2 layers of filter paper-film--2 layers of glue filter Paper.24V, transfer time 30min is arranged in transfer, voltage.4 DEG C are set in film 15ml Block buffers (1g/100ml BSA), overnight. TBS/T is washed 3 times (5min/T).
BacHBoVCap monoclonal antibodies are added, are incubated at room temperature 2h.TBS/T is washed 3 times (5min/T).1ml horseradish mistakes are added The secondary antibody of the sheep anti mouse of oxidizing ferment (HRP) label, is incubated at room temperature 2h.TBS/T is washed 3 times (5min/T).15ml TBS are washed 2 times.Add Enter 0.5ml chemoluminescence method liquid, is placed in gel imaging system and develops the color.As a result it shows：Anti-human bocavirus monoclonal antibody can To be reacted with human bocavirus antigen binding.
5.PCR detects the specificity of lower respiratory tract bocavirus primer
Primer specificity is most important factor in the detection method that this experiment is established.According to human bocavirus in GenBank Sequence chooses NS1 specific gene design primers,
PCT-F:AGC TTT TGT TGA TTC AAG GCT ATA ATC(SEQ IDNo.1)；
PCT-R:TGT TTC CCG AAT TGT TTG TTC A(SEQ IDNo.2)。
PCR amplification human bocavirus gene, PCR system：
2xbuffer 20ul
PCT-F 2ul
PCT-R 2ul
Template 1ul
H2O 15ul
PCR programs：
94 DEG C of 10min, 1 cycle, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 72 DEG C of cycle cycles of 10min 1.
Using being connect with carrier T after the recycling of Ago-Gel QIAquick Gel Extraction Kit, 16 DEG C of connection 1h are obtained PCR product PCMVHBoVi converts bacillus coli DH 5 alpha.PCMVHBoVi carriers send company to be sequenced after digestion is identified.
Correct pCMVHBoVi carriers are sequenced with BacHBoVCapHA expression vectors after BanmH I and Xho I digestions 3h, Connection product is added in 100 μ l bacillus coli DH 5 alpha competent cells, ice bath 20min, the thermal shock 90s in 42 DEG C of water-baths, so After be immediately placed in 2~3min on ice, after the fresh LB culture medium mixings of 900ul are added, 37 DEG C of shaking table culture 1h make DH5 α restore Normal growth state.Transformed cells solution coating is contained into kanamycins LB culture mediums；37 DEG C of constant temperature incubations are stayed overnight.It reflects through digestion It is fixed.It is compared by BLAST, as a result shows that design primer sequence all has very high specificity, detection people Bo Ka diseases are fully achieved The requirement of poison.
Embodiment 2
The mass production of monoclonal antibody
Take 1x107Cell concentration is injected in Balb/c mouse peritoneals, and ascites is collected after 10 days.It follows these steps to carry out：
1. collecting gained ascites to centrifuge with 2500rpm, supernatant is taken.Isometric PBS (pH7.4) is added to be saturated sulphur with 1/2 volume Sour ammonium, 4 DEG C, standing 30min；
2. with 3000rpm, 4 DEG C, 20min is centrifuged；
3. going to precipitate, above reset and add isometric saturated ammonium sulfate, stands 30min；
4. with 3000rpm, 4 DEG C, 20min is centrifuged；
5. taking precipitation, 5ml physiological saline is added to stand 30min with 5ml saturated ammonium sulfates；
6. with 3000rpm, 4 DEG C, 20min is centrifuged；
7. the PB buffer solutions (PH7.4) of precipitation plus 5ml physiological saline and 5ml0.02M；
8. adding 8 times of column volume PB buffer solutions balance Protein G gel columns (being purchased from GE companies)；
9. the mixed liquor in the 7th step is added in gel column；
10. the PB buffer solutions using 10 times of column volumes elute foreign protein；
11. with 0.2M pH2.8 glycine buffers antibody elutions and collecting；
12. equilibrium liquid is added to balance pillar.
Embodiment 3
The preparation of the ELISA detection kit of human bocavirus
1. preparing the porous plate for being coated with HBoV monoclonal antibodies：
A) it is coated with：Human bocavirus list is prepared using 9.6 carbonate buffer solution of 0.05M, pH and debita spissitudo embodiment 2 Clonal antibody is mixed and made into coating mixed liquor, and is loaded on microwell plate, 4 DEG C of coating 12h；
Carbonate buffer solution standard recipe：
B) board-washing：20 times of concentration washing lotions of dilution use 1 times of concentration washing lotion board-washing 2 times to 1 times of concentration.20 times of concentration washing lotions Standard recipe：
C) it closes：By confining liquid (disodium hydrogen phosphate 5.8g, sodium dihydrogen phosphate 0.593g, sodium chloride 8.0g, lowlenthal serum 100ml, Proclin-3000.5ml, purified water are settled to 1000ml) it loads on microwell plate, room temperature 2h, it dries, it is dried Night.
2. Sample dilution is prepared (10x)：1000ml Sample dilutions include 22.4gTris alkali, 2.9g anhydrous calcium chlorides, 81.8g sodium chloride, 5mlProclin-300.In use, diluting 10 times with deionized water.
3. prepared by substrate solution：Horseradish peroxidase substrate：Substrate solution A is to contain 0.6 ‰ urea peroxide 10mmol/L lemons Lemon acid buffer is protected from light storage；Substrate solution B is the 5mmol/L citrate buffer solutions containing 0.4 ‰ TMB, is protected from light storage.
4.HBoV calibration objects are prepared：The very high positive serum of HBoV detected values is collected, with 1x sample diluting liquids by positive serum Dilution so that calibration object HBoV concentration gradients be 50,100,200,400,800,3200pg/ml.Calibration object can trace to the source to ProSpec companies reference material Procalcitonin Human Recombinant (HOR-304, purity >=97.0%).
5.HBoV quality-control products are prepared：Quality-control product is collected from HBoV clinical detection high level people source sample, through inactivation treatment, through inspection It is feminine gender to survey its anti-TP, HBsAg, anti-HCV and anti-HIV.It is diluted within the scope of normal concentration with Sample dilution.High level Quality Control：800pg/ml-1200pg/ml；Low value Quality Control：40pg/ml-60pg/ml.2-8 DEG C saves backup.
The preparation of 6.20 times of concentrated cleaning solutions：20 times of concentrated cleaning solutions of 1000ml include：90g sodium chloride, 12 water of 29g Close disodium hydrogen phosphate, bis- hypophosphite monohydrate sodium dihydrogens of 2.97g, 10ml polysorbas20s, pH value 6.20-6.60.In use, using deionized water 20 times of dilution.
Test case
1. human bocavirus ELISA kit application method of test case
1. preparing before experiment
1. taking out kit from 4 DEG C of refrigerators, room temperature (18-25 DEG C) should be equilibrated to, it is proposed that no less than 30min.
2. 20 times of concentrated cleaning solution purified waters or distilled water use after diluting 20 times.
3. 10 times of Sample dilution purified waters or distilled water use after diluting 10 times.
2. test method
1. adding calibration object and sample to be tested：Sufficient amount porous plate is taken, is fixed on frame.Be respectively set calibration sample wells, Sample to be tested hole and blank control wells record each hole site.In addition to blank control wells, calibrated after dilution is added in calibrating sample wells HBoV Sample dilutions 100ul is added in product 100ul, calibration object zero hole (0pg/ml calibration objects)；It is added and waits in sample to be tested hole This 100ul of test sample, is placed at room temperature for 120min.
2. adding horseradish enzyme label：The horseradish enzyme being added per hole after dilution marks 100ul, (blank control wells are not added with) room temperature to put Set 30min.
3. board-washing：Liquid is discarded, is patted dry on blotting paper, 5-10s is stood, gets rid of cleaning solution, patted dry on blotting paper, washed 5 times.
4. colour developing：50ul substrate solution A and 50ul substrate solution B are added per hole, mix well, 37 DEG C of incubation 15min.
5. terminating：After colour developing, 50ul terminate liquids are added per hole, mix well.
6. measuring：After terminating reaction, microplate reader 450nm wavelength (returning to zero with blank well) or dual wavelength 450nm/ should be set OD values are measured under 630nm.
7. calculating：According to calibration object concentration and corresponding OD values, use double-log linear fit mode (log (X)-log (Y)) As a result result of calculation is multiplied by extension rate, as sample ultimate density.
Test case 2
The methodology index of the ELISA kit of HBoV
1. accuracy：The rate of recovery should be between 95%~105%；
2. blank detection limit：50pg/ml should be not higher than；
3. measuring system is linear：Linear correlation coefficient r >=0.9900 within the scope of 50pg/ml~3200pg/ml.Accurately Property, minimum detectability, system linear testing result are as shown in table 1：
Table 1
4 batches of internal stability experiments：4 parts of positive serum samples are taken, carry out being repeated 9 times ELISA measurement, meter in same block ELISA Plate Stablize experimental variations coefficient in calculating batch between 5.6%-8.1%, average 6.87%, with good stability and repetition Property.
Stability test coefficient of variation result of calculation is as shown in table 2 in batch：
Table 2
Repetitive test between 5 batches：5 parts of positive serum samples are taken, equal quality difference ELISA Plate is carried out and is repeated 3 times ELISA Measuring calculates and repeats experimental variations coefficient between criticizing between 0.74%-8.12%, and average 4.67%, have good Stability and repeatability.
It is as shown in table 3 that experiment coefficient of variation result of calculation is repeated between batch：
Table 3
6 specificabsorptions inhibit experiment：With special VP2Antigen is with after serum effect, and OD is significantly reduced, and negative serum inhibits Rate 6.67 levels off to 0,8 parts of positive serum inhibiting rates between 50.44-56.94, and it is the positive that specificabsorption, which inhibits experiment, Illustrate that this method has preferable specificity.Specificabsorption inhibits test result as shown in table 4：
Table 4
The sensitivity testing of 7 antibody：6 parts of anti-established indirect elisa methods of HBoV Positive Seras measure antibody effect The equal ＞ of valence 1:800, it is 1 that tube agglutination test, which measures anti-HBoV antibody titers,:160-1:640, show that indirect ELISA method has There is good susceptibility.ELISA measures HBoV antibody sensitivities and compares that the results are shown in Table 5：
Table 5
8 distinct methods detection clinical samples compare：In 40 samples, indirect elisa method detects HBoV antibody positives 2 Example, positive rate 5.0%；Fluorescence quantitative PCR detection goes out the HBoV positives 2, positive rate 5.0%, and two methods detect together A sample；Occurs band at 60KD through 2 samples of Western blot technical identifications；Two methods detect HBoV sun Property coincidence rate 100%.
The results are shown in Table 6 with fluorescence quantitative PCR detection clinical samples for ELISA detections：
Table 6
9 samples screen：Acquisition First People's Hospital of Wenling, Zhejiang province city acute respiratory infection patient's pharynx nasalis extraction object, There are unexplained fever, the symptoms such as cough in throat swab and each 520 samples of serum, patient age 2M-6Y, clinic.With people Bocavirus VP2 recombinant proteins carry out protein immunoblot detection as specific antigen, to the antibody in serum specimen, two kinds Method confirms 13 human bocavirus infection positive patients altogether.
13 infection human bocavirus positive patient particulars are as shown in table 7：
Table 7
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, the present invention Claimed range is delineated by the appended claims, the specification and equivalents thereof from the appended claims.

Claims (5)

1. a kind of indirect ELISA method kit for detecting people's lower respiratory tract bocavirus, including coating are anti- Body, antigen, calibration object, quality-control product, sample diluting liquid, it is characterised in that：
The coated antibody is monoclonal antibody；
The antigen behaviour lower respiratory tract bocavirus VP2 fusion proteins；
The calibration object be with ProSpec companies Procalcitonin Human Recombinant (HOR-304, purity >= 97.0%) be reference material prepare 50,100,200,400,800, the HBoV positive serums of 3200pg/ml concentration；
The quality-control product is to be formulated by HBoV clinical detection high level people source sample, and through inactivation treatment, its is anti-after testing TP, HBsAg, anti-HCV and anti-HIV are feminine gender；The sample diluting liquid is the dilution for detecting sample.
2. the indirect ELISA method examination according to claim 1 for detecting people's lower respiratory tract bocavirus Agent box, it is characterised in that：Gone out using Bac-to-Bac recombinant baculovirus expression system expressions in the sf9 insect cell of infection VP2 albumen in HBoV, molecular weight of albumen 60kDa, expressing quantity account for 80% or more insect soluble protein, and VP2 albumen obtains It to purifying, is purified through Chelating Sepharose Fast Flow metal affinity chromatographies, purity reaches 95% or more, as VP2 antigens；The antibody of anti-bocavirus non-structural protein is had detected using Western blot technologies, determines virus infection feelings Condition, the method that indirect ELISA detection HBoV antibody is established using this VP2 fusion protein as antigen.
3. the indirect ELISA method kit for detecting people's lower respiratory tract bocavirus described in claim 2 Application in it can be detected to the bocavirus antibody corresponding antigens in lower respiratory tract sample using recombinant protein VP2.
4. the indirect ELISA method examination according to claim 3 for detecting people's lower respiratory tract bocavirus The purposes of agent box, it is characterised in that：Applications of the recombination fusion VP2 in the detection of indirect ELISA technology.
5. the indirect ELISA method examination according to claim 4 for detecting people's lower respiratory tract bocavirus The purposes of agent box, it is characterised in that：The recombination fusion VP2 is in clinical disease diagnosis, antiviral drugs screening, environmental monitoring With the application in evaluation, health supervision and port health quarantine, epidemiological survey drug.
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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN112362717A (en) * 2020-11-25 2021-02-12 武汉生物制品研究所有限责任公司 Purity analysis method for SARS-CoV-2 inactivated vaccine
CN117126270A (en) * 2023-10-25 2023-11-28 首都儿科研究所 Type 2 human bocavirus type specific antibody and application thereof

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