CN106188248A - A kind of Epstein-Barr virus antigen preparation procedure and utilize the quick detection kit of detection Epstein-Barr virus antibody prepared by this antigen - Google Patents
A kind of Epstein-Barr virus antigen preparation procedure and utilize the quick detection kit of detection Epstein-Barr virus antibody prepared by this antigen Download PDFInfo
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- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- 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
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
- G01N33/56994—Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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Abstract
The present invention relates to a kind of artificial antigen expressed of Epstein-Barr virus genetic engineering；The method preparing this antigen, the method synthetic self merges Epstein-Barr virus capsid protein antigen gene order, build prokaryotic expression carrier, escherichia coli expression Epstein-Barr virus capsid protein antigen, use dialysis, gradient dilution method and gel chromatography renaturation inclusion body, it is thus achieved that there is three dimensional structure and immunocompetent recombined EB virus capsid protein antigen；A kind of method for quick detecting Epstein-Barr virus antibody, the method includes applying Epstein-Barr virus capsid protein antigen；A kind of quick detection kit for Epstein-Barr virus antibody test, this test kit comprises Epstein-Barr virus capsid protein antigen, can be directly used for whole blood test.This test kit comprises rheumatism factor treatment pad, can remove the rheumatism factor in sample, the directly IgM in detection sample.According to the present invention, it is provided that a kind of Epstein-Barr virus antigen, this antigen has the specificity of height.The invention provides the method preparing this antigen, the method quickly measuring Epstein-Barr virus antibody, and for quickly measuring the test kit of Epstein-Barr virus antibody.
Description
Technical field
The invention belongs to field of clinical medical detection, relate to immuno-chromatographic assay technology, be specifically related to a kind of for diagnosing
The Epstein-Barr virus antigen of ebv infection, the method preparing this antigen, by the quick detection kit of this Detection of antigen Epstein-Barr virus.
Background technology
Epstein-Barr virus (Epstein-Barr Virus, EBV) is ebb virus, by Epstein and Barr 1964
Find first during year research burkitt lymphoma, have addicted to human B lymphocyte characteristic.EBV is that a kind of DNA is sick
Poison, spherical in shape, a diameter of 180～200nm, replicate in bone-marrow-derived lymphocyte.People is the host that EBV infects, and virus mainly passes through saliva
Liquid is propagated.Symptomless infection mostly occurs child, and 3～5 years old child of more than 90% once infected EBV.Cellular immunization infects at EBV
In play key effect, this function reduction will cause the activation of EBV.The incubation period that EBV infects is 4～7 weeks, prodrome bag
Including headache, weak etc., the patient of 80% is likely to occur clinical three and levies: pharyngitis, heating and lymphadenopathy.Infection can involve entirely
Each organ of body, typically has heating, loss of appetite, nausea,vomiting,diarrhea, lymph nodes of body as a whole enlargement, hepatosplenomegaly, erythra etc..
Have it may also occur that neurological symptom, typically needs the convalescent period of 2～4 weeks.EB virus actute infection can cause infectiousness monokaryon
The diseases such as cytosis, erythrophage syndrome, the chain lymphoproliferative syndrome of X-.
Upper respiratory tract infection is the general name of a class pathogen infection, and the pathogen more than 80% causing upper sense is virus, Epstein-Barr virus
It it is one of pathogen causing upper sense.Infectious monocytosis (Infectious Mononucleosis, IM) be by
The lymphoproliferative infectious disease that Epstein-Barr virus primary infection causes, clinical manifestation be heating, angina, deradenoncus,
Hepatosplenomegaly etc., similar with upper sense symptom.Mainly by the spittle and saliva through respiratory infectious, thus it is also called " kissing disease ". IM
Most prognosis bonas, minority can cause severe complication, causes the pathological changes of multisystem.So whether being sick by EB for making a definite diagnosis
The upper sense symptom that poison causes has important meaning for prognosis and further medication.
First the IgM for capsid antigen (Capsid Antigen, CA) is produced during constitutional ebv infection
With IgG antibody, actute infection late period, the IgG antibody for early antigen (Early Antigen, EA) occurs；Extensive
In multiple late period phase, produce for nuclear antigen antibody.Anti-EBV-CA-IgG antibody and anti-EBV-NA-IgG antibody are sustainable all the life.In recent years
Coming, antibody affinity detection has been used for judging whether acute HIV infection.Owing to the serological reaction complexity of ebv infection is many
Sample, the anti-EBV-CA-IgM of some cases produces delay, the lasting disappearance that has or exist for a long time, and this brings to making a definite diagnosis of EBV-IM
Certain difficulty.
Nuclear antigen, early antigen, capsid antigen, membrane antigen and lymphocyte identification can be produced anti-after human infection Epstein-Barr virus
Former corresponding antibodies antibody such as () IgG, IgA, IgM waiting antigen.Wherein, VCA/IgM antibody is antibody the earliest occur, 90 ~ 94%
Ebv infection person can be detected in initial infection, be Epstein-Barr virus actute infection, be also the important symbol of recurrent infection,
It it is the indispensable important indicator of early diagnosis of ebv infection.Conventional Epstein-Barr virus laboratory diagnostic method mainly has virus
Separate, detect virus protein and the several method such as nucleic acid, EBV Serologic test detection antibody.Isolation of virus typically uses
Pharyngeal aspirate direct inoculation human cord blood lymphocyte, determines the amount of virus according to the efficiency of Transformed Human Lymphocytes.The method consumes
Time and need special conditions of tissue culture, therefore be not suitable as Clinical detection and use；The method that detection viral nucleic acid is conventional
There are nucleic acid hybridization and RT-PCR method, in pathological tissues, detect viral genomic nucleic acid and viral genome transcription product, in inspection
The aspects such as survey condition and technology require higher, therefore should not use in routine clinical detection；Serodiagnosis is mainly exempted from by enzyme connection
Whether the method detection serum such as epidemic disease absorption method exist the specific antibody such as CA-IgM/IgG antibody, anti-nuclear antigen antibody, due to
It has highly sensitive, specificity good and the advantage such as easily operated, is one of the most frequently used way of current laboratory, to disease
Diagnosis has certain reference value.Epstein-Barr virus antibody test reagent quality depends on the performance of antigen, and engineering antigen expressed is system
The best-of-breed technology scheme of standby Epstein-Barr virus antigen.In the antigen of Epstein-Barr virus, capsid antigen is a kind of optimal candidate antigen.
Summary of the invention
The first object of the present invention is to provide a kind of engineering and expresses Epstein-Barr virus self fusion capsid protein specific antigen, should
Antigen has specificity and the immunoreactivity of height.It is high that antigen has immunogenicity, and without specific reaction, epitope collection is medium
Advantage.
The second object of the present invention is to provide a kind of preparation engineering and expresses the method that Epstein-Barr virus self merges capsid antigen, should
Method utilizes synthesis fusion gene pronucleus to express, and by the specific antigen that renaturing inclusion bodies preparation activity is high, prepared by the method
Epstein-Barr virus antigen has specificity and immunoreactivity.
The third object of the present invention is to provide a kind of test kit measuring anti-Epstein-Barr virus antibody, and this test kit is quick " one
Step detection method " test kit, there is highly sensitive, high specificity, detection speed is fast, easy and simple to handle, it is not necessary to Special Equipment, it is adaptable to
The multiple occasions such as Clinical detection, Epidemiological study and place quarantine.
The fourth object of the present invention is to provide a kind of Epstein-Barr virus antibody whole blood device for fast detecting, and it is a kind of equipped with filter membrane
Can filter out erythrocytic Epstein-Barr virus whole blood test device.
The fifth object of the present invention is to provide a kind of direct device for fast detecting of Epstein-Barr virus IgM antibody, it be a kind of equipped with
The Epstein-Barr virus detection device that can remove the rheumatism factor in sample of rheumatism factor treatment pad.
The main contents of the present invention
(1) a kind of Epstein-Barr virus antigen, it is a kind of antigen using technique for gene engineering synthetic.
(2) a kind of method preparing Epstein-Barr virus antigen, merges capsid antigen gene order including synthetic Epstein-Barr virus,
Build prokaryotic expression carrier, escherichia coli expression Epstein-Barr virus fusion capsid protein, use dialysis, gradient dilution method and gel layer
Analysis method renaturation inclusion body, it is thus achieved that there is three dimensional structure and immunocompetent recombined EB virus fusion capsid protein antigen.
(3) a kind of Epstein-Barr virus antibody method for quick, including the Epstein-Barr virus antigen applied above in (1).
(4) Epstein-Barr virus antibody method for quick in above-mentioned (3), is fixed to a kind of nitre including the Epstein-Barr virus antigen in (1)
Acid cellulose film, a kind of traget antibody (two resist) is fixed on solid phase carrier, and testing sample contacts with traget antibody (two resist), if
There is Epstein-Barr virus antibody in testing sample, then Epstein-Barr virus antibody and the labelling two anti-complex of anti-formation antibody-marker two, " compound
Thing " move horizontally at nitrocellulose filter, run into the Epstein-Barr virus antigen being fixed on film and produce reaction, carry markd " compound
Thing " just it is fixed to specifically on antigen, the position of immobilized antigen just has chromogenic reaction；If testing sample resists without Epstein-Barr virus
Body, then " complex " can not be fixed on the position of antigen, does not the most just have chromogenic reaction, can fix according to nitrocellulose filter
Whether antigenic site has chromogenic reaction, judges testing result, i.e. whether has Epstein-Barr virus antibody in testing sample.
(5) above-mentioned (4) Epstein-Barr virus antibody detection method, wherein testing sample is the whole blood of people, blood plasma or serum.
(6) above-mentioned (4) or (5) middle method measuring Epstein-Barr virus antibody, traget antibody therein (two resist) is the anti-of labelling
People IgM or anti-human IgG antibodies.
(7) in above-mentioned (4)-(6), traget antibody is colloidal metal particles or the antibody of colored latex particle marker.
(8) test kit of a kind of quick detection Epstein-Barr virus antibody, antigen used by it is the Epstein-Barr virus antigen in above-mentioned (1).
(9) for quickly detecting the test kit of Epstein-Barr virus antibody in above-mentioned (8), traget antibody therein is colloidal metal
Particle marker antibody or colored latex particle marker antibody.
(10) for the test kit of detection in above-mentioned (8), hemofiltration film therein is to be fixed with anti-erythrocyte monoclonal antibody or many
Anti-non-woven fabrics.
(11) for the test kit of detection in above-mentioned (8), rheumatism factor treatment pad therein is to be fixed with the rheumatism factor
Monoclonal antibody or the non-woven fabrics of multi-resistance.
Test kit yin and yang attribute coincidence rate of the present invention, elaboration, sensitivity etc. all meet quality criteria requirements, produce in effect duration
Quality is stable.Rheumatoid factor, hepatitis B virus antibody, treponema pallidum, HIV antibody, giant cell
This test kit testing result will not be interfered by antiviral antibody, herpes simplex virus antibody positive specimen.
The present invention will be described in detail below.
The Epstein-Barr virus antigen of the present invention, mainly by Epstein-Barr virus capsid protein gene is building up to prokaryotic expression carrier
PET30a, pET30a-p18-p23 expression vector converts escherichia coli, the positive recombinant bacterium of screening, and IPTG induction recombinant bacterium expresses EB
Virus self merges capsid antigen protein, through steps such as bacteria lysis, solubilization of inclusion bodies and recombiant protein structure renaturation and purification
Obtain.
Method that the present invention prepare Epstein-Barr virus antigen be will be described below.
Epstein-Barr virus capsid protein gene sequence is obtained, according to respiratory syncystial Epstein-Barr virus capsid protein gene from GenBank
Self fusion capsid protein gene order of P18 and P23 synthetic Epstein-Barr virus.Genes of interest after double digestion with through same pair of enzymes
Cut through to obtain prokaryotic expression carrier connection, transformed competence colibacillus cell (escherichia coli), the positive recombinant bacterium of screening.Positive colony bacterium is extracted
Plasmid, double digestion and order-checking are identified, it was demonstrated that the genes of interest of insertion is correct.Recombinant bacterium through IPTG abduction delivering, centrifugal to obtain restructuring
Bacterium is precipitated, and thalline is resuspended in ultrasonic degradation after buffer, centrifugal obtain inclusion bodies albumen precipitation, scrubbed after be dissolved in
Renaturation in urea liquid.Denatured protein need through structure renaturation and purification could obtain native protein immunoreation originality and
Reach the application standard of Epstein-Barr virus antibody test.
For Epstein-Barr virus capsid antigen recombiant protein renaturing inclusion bodies method, dialysis, gradient dilution method, gel can be applied
The method that chromatography etc., preferably dialysis and gradient dilution method combines, because they are easily controllable, protein renaturation rate is high.
For the purification process of Epstein-Barr virus fusion capsid protein after renaturing inclusion bodies, gel chromatography, affinity chromatograph can be applied
Deng, preferably gel chromatography, because it is easiest to control, yield is high.
The Epstein-Barr virus obtained by gene recombinaton is merged capsid antigen and has higher specificity and sensitivity.
For the method quickly measuring antibody, enumerate here is preferred method, as applied exempting from of various traget antibodies
Epidemic disease measurement in chromatography, applies the immunity percolation method etc. of various traget antibody.
Immunochromatographic measurement method is explained in detail below.
Anti-Epstein-Barr virus antibody immunochromatographic measurement method comprises the steps: that Epstein-Barr virus self merges capsid antigen and is fixed to
On solid phase carrier 1, traget antibody (two resist) is fixed on solid phase carrier 2, and testing sample contacts, if treating with traget antibody (two resist)
There is Epstein-Barr virus antibody in test sample product, then Epstein-Barr virus antibody and two anti-reflective should form the anti-complex of antibody-marker two, is somebody's turn to do " complex "
Solid phase 1 moves horizontally, runs into the Epstein-Barr virus antigen being fixed on carrier 1 and produce reaction, just carry markd " complex "
Being fixed to specifically on antigen, the position that solid phase carrier is fixed immobilized antigen just has chromogenic reaction；If testing sample is not
Containing Epstein-Barr virus antibody, then " complex " can not be fixed on the position of antigen, does not the most just have chromogenic reaction, can carry according to solid phase
On body, whether immobilized antigen position has chromogenic reaction, judges testing result, i.e. whether has Epstein-Barr virus antibody in testing sample.
For solid phase carrier 1, such as nitrocellulose filter (NC film), pvdf membrane etc. can be applied.Preferably nitrocellulose filter.
For solid phase carrier 2, such as glass fibre element film can be applied, any form such as non-woven fabrics.Preferably non-woven fabrics
Make conjugate release pad, because it is easiest to control.
For the antibody of labelling, refer here to the antibody with different label labellings, refer here to anti-human IgM and resist
Body or anti-human IgG antibodies, suitably can apply according to the kind of detected antibody.
Beneficial effects of the present invention
(1) present invention includes that Epstein-Barr virus self merges the optimization expression of capsid antigen, has synthesized self the fusion capsid egg optimized
White gene order, the expression of this sequence is higher, and specificity is higher.And the present invention has also explored the inclusion body of complete set
Antigen purification and the method for renaturation, the antigen yield prepared is high, and immunogenicity is high.
(2) present invention has also prepared the test kit of a kind of one-step method detection Epstein-Barr virus antibody.Detection EB is sick for this test kit
The IgG of poison or IgM antibody, be beneficial to the stage that confirmation infects, and the treatment suggesting effect for Epstein-Barr virus is obvious.
(3) present invention can also directly detect whole blood sample, has hemofiltration film, can use a small amount of whole blood on detection card
This detection.
(4) present invention can also directly detect the sample without processing the rheumatism factor, has at the rheumatism factor on detection card
Reason pad, can directly detect sample and not disturbing by the rheumatism factor in sample,
Therefore, the present invention can be by the infection of primary sample one-step method whole blood test respiratory tract Epstein-Barr virus.Be suitable to primary care
Mechanism and hospital outpatient.
Accompanying drawing explanation
Fig. 1 shows the surface structure schematic diagram of invention reagent detection plate
1-detects plate 2-well 3-inspection window
Fig. 2 shows the external structure schematic diagram of invention reagent detector bar
4-sample-adding end absorbent paper layer 5-gold labeling antibody layer 6-control line 7-detects line
8-detection layers 9-suction side water accepting layer 10-liner plate
Detailed description of the invention
Embodiment 1 recombined EB virus self fusion capsid protein antigen is recombinant expressed, structure renaturation and purification
Epstein-Barr virus capsid antigen P23-P18 fusion gene, with reference to GenBank sequence V01555.2, chooses P23 encoding gene
BLRF2(amino acid/11 ~ 162) and the C end gene order of P18 encoding gene BFRF3, pass through peptide linker between two fragment genes
(Gly4Ser)3 DNA sequence connects acquisition fusion gene, carries out full genome synthetic, and expression vector is pET30a, during connection
Restriction enzyme site is EcoR I//XhoI, genes of interest 750bp(249 aa altogether), it is transformed into BL21(DE3), the recombiant protein of expression
297aa, molecular weight 30.8 kDa, isoelectric point, IP 11.03 altogether.Recombiant protein is expressed with inclusion bodies, available Ni column purification.
The structure of recombinant expression carrier and qualification: respectively with EcoRI and XhoI to Epstein-Barr virus fusion capsid protein purpose base
Because fragment and pET30a plasmid carry out double digestion, after digestion products purification reclaims, 16 DEG C connect overnight, then proceed toE.coli
DH5 α competent cell, picking converts bacterium colony extraction plasmid and carries out PCR, enzyme action and order-checking qualification.Identify through PCR and double digestion,
All confirm the genetic fragment insertion vector having size about 750bp, consistent with expected result.Sequencing result shows to insert purpose base
Because fragment is 750bp, nucleotide sequence is identical with the Epstein-Barr virus p18-p23 fusion gene sequence submitting synthesis to, the table of structure
Reach carrier open reading frame correct, recombiant protein can be expressed.
The abduction delivering of recombiant protein: take 50 L pET30a-p18-p23 BL21 (DE3) bacterium solution and join 5mL containing 100
In the LB culture medium of g/mL kanamycin, 37 DEG C, 200 rpm overnight incubation, it is inoculated in containing kanamycin by 1:50 next day
In LB culture medium, when cultivation to light absorption value OD600 is about 0.6, adding IPTG is 1mmol/L to concentration, carries out abduction delivering 4
Hour, 5000rpm is centrifuged 30 minutes, collects thalline, suspends with TE buffer, fully mixes, and-80 DEG C of multigelations 3 times are super
Sound breaks bacterium, 4 DEG C, 12000 rpm be centrifuged 15 min, collect supernatant, precipitate and wash 2 times with 2 M carbamide, 4 DEG C, 12000 rpm from
The heart 15 min, residue precipitation 8 M carbamide dissolvings, 4 DEG C of preservations.Precipitation SDS-PAGE analyzing proteins expression.Electrophoresis strip
Part, concentrating gum concentration is 5%, and resolving gel concentration is 12%.Protein staining is dying method with coomassie brilliant blue, coomassie brilliant blue R_250
Dyeing 3h, acetic acid-ethanol decolorization loss of thick fluid color to background is colourless.
The structure renaturation of recombiant protein and purification: sick to EB with the PBS of 10mmol/L, pH7.2 under room temperature condition
The lysate of self fusion capsid protein p18-p23 recombinant expression protein 8M carbamide of poison carries out gradient dialysis dilution, by solution
Urea concentration is respectively 6mol/L, 4mol/L, 3 mol/L control dilution gradient, and the time of each gradient dilution is respectively 3-4h.
The lysate 4 DEG C containing 3mol/L carbamide completing dilution refolding is placed 24h.The solution 10000r/min of dilution refolding is centrifugal to be removed
Removing deposit, supernatant is purified by the gel chromatography partition method with S-300 as medium, thus obtains recombined EB virus
Self merges capsid antigen.Gained recombined EB virus capsid antigen component carries out SDS-PAGE, to determine the molecular weight of protein
And purity of protein.Deposition condition, concentrating gum concentration is 5%, and resolving gel concentration is 12%.Protein staining is coomassie brilliant blue staining
Method, coomassie brilliant blue R_250 dyeing liquor dyeing 3h, acetic acid-ethanol decolorization loss of thick fluid color to background is colourless.Main albumen one is taken out of
At present 30.3 kDa, purity of protein reaches more than 95%.
The preparation of 2 one kinds of Epstein-Barr virus IgG antibody of embodiment/IgM gold labeled quick detection reagent box
Obtained by above-mentioned, self fusion capsid protein p18-p23 of gene recombinaton Epstein-Barr virus makees detection antigen, at nitrocellulose filter
It is coated detection line；With reference to Fig. 2, preparing Epstein-Barr virus antibody gold label quick detection reagent, its constituent includes: set on liner plate 10
There are sample-adding end water accepting layer 4, detection layers 8 and water accepting layer 9, between detection layers and sample-adding end water accepting layer 4, are provided with gold mark anti EB virus
Antibody layer 5, is coated with detection line 7 and nature controlling line 6 in detection layers 8.Wherein sample-adding end water accepting layer 4 and suction side water accepting layer 9 by
Multi-layer filter paper is made: detection layers 8 is nitrocellulose filter；Gold labeling antibody layer is glass fibre or non-woven fabrics leaching colloid gold label
Antibody.
Detectable preparation procedure includes: prepares gold labeling antibody layer 5 and the nature controlling line 6 of detection layers 8, detect being coated of line 7,
On liner plate 10, combine gold label test strip the most again and detection blocks 1. and pastes sample-adding end water suction at the two ends of plastics lining board 10 respectively
Ply of paper 4 and suction side water accepting layer 9；Section is pasted and is coated detection line and the cellulose layer 8 of nature controlling line wherein, in sample-adding end absorbent paper
Layer 4 and the handing-over position of cellulose membrane layer 8, press from both sides the patch glass fibre containing gold labeling antibody layer 5, and 4/5 part of glass fibre is adding
In the middle of sample end absorbent paper layer 4,1/5 part is on cellulose membrane layer 8.Then according to 4 mm wides, 7 centimeter length specification cuttings.Join again
According to Fig. 2, in detector bar is assembled in plastic casing, form detection card 1.Well 2 just sample-adding end absorbent paper to test strip on lid
Layer 4, observation port 3 just detection layers 8 to cellulose membrane.
The preparation method of above-mentioned golden labeling antibody layer comprises the steps: the preparation of gold colloidal, takes 100mg gold chloride molten
In 1000mL tri-distilled water, adding 15mL concentration is the citric acid three sodium solution of 1%, boils 15 minutes, available a diameter of 15-
The colloid gold particle solution of 50 nanometers；Colloid gold label anti-human igg or IgM monoclonal antibody, take 100ml colloidal gold solution,
Adjust pH to be 8.4 with 0.2M solution of potassium carbonate, add 1mg anti-human igg/IgM monoclonal antibody, stir 20 minutes, add 240mg
Bovine serum albumin (BSA), continues stirring 5 minutes, and 4 DEG C stand 2-4 hour；By above-mentioned colloidal gold solution through 2000 revs/min
Zhongli's heart 10-15 minute, goes precipitation, obtains supernatant；Supernatant is centrifuged through 10000 revs/min and within 60 minutes, is precipitated；
The Tris-Hcl buffer that precipitation is dissolved in 4mL0.02M pH7.4 obtains colloidal gold solution, containing 0.25%BSA in this solution
With 0.02% sodium azide；Being immersed by colloidal gold solution glass fibre or non-woven fabrics to liquid starts to ooze out, 37 DEG C are dried
Within 2 hours, form gold labeling antibody layer 5.
Described detection layers 8 is to be provided with recombined EB virus on nitrocellulose filter to merge the detection line that capsid antigen is constituted
7 and the nature controlling line 6 that formed by sheep or rabbit anti-Mus IgM/IgG antibody.Its preparation method is to take the restructuring EB disease prepared by above-mentioned (1)
Poison capsid antigen, degree of thickening is 1.5mg/mL, adds the methanol of 2%, with Membrane jetter at cellulose membrane stage casing spray detection line 7；Take again
Sheep or rabbit anti-Mus IgM/IgG antibody degree of thickening are 1.5mg/mL, with Membrane jetter in cellulose membrane stage casing, at detection line 0.5cm,
Spraying nature controlling line 6, by 2uL/cm arrange spray film amount, spray film be placed on 37 DEG C be dried 2 hours, then with 0.01mLpH7.0 containing 10%
The PBS of calf serum closes 30 minutes at 37 DEG C, and the PBS rinsing of 0.01mLpH7.0,45 DEG C are dried.
The mensuration of embodiment 3 Epstein-Barr virus antibody
Take serum or plasma sample 10 μ l drips in the sample well 2 of detection plate 1, then drip 100 μ l sample diluting liquids in sample
In hole 2, observing testing result at observation window 3, observed result is effective in 20 minutes.If containing anti-Epstein-Barr virus antibody in sample, then
Seeing that in observation window two red lines occur in detection line and nature controlling line, testing result is judged to the positive；If serum does not contains
Anti-Epstein-Barr virus antibody, then see a red line in observation window nature controlling line position, and testing result is judged to feminine gender；If in observation window
Article one, redness all be can't see, then testing result is invalid.
The detection of embodiment 4 whole blood sample
Anticoagulant whole blood sample can directly be detected by the Epstein-Barr virus immunochromatography quick detection kit that the present invention realizes, it is not necessary to blood
Slurry or serum separate, its realize principle be, increase by a layers of blood separating film in sample pad, hemofiltration film is solidified with anti-human blood red carefully
Born of the same parents' antibody (monoclonal antibody or polyclonal antibody), during detection, after anticoagulation whole blood enters hemofiltration film, in blood, erythrocyte is tied by antibody
Closing on hemofiltration film, blood plasma penetrates into sample pad, completes detection.
The determination of the wet factor treatment pad of embodiment 5 points
The Epstein-Barr virus immunochromatography quick detection kit that the present invention realizes is provided with a point wet factor treatment pad on detection card, can
To get rid of point wet factor in sample, it is not necessary to remove point wet factor in blood sample in advance.Its principle realized is,
Sample pad increases by one layer of anti-point of wet factor treatment pad, processes and is solidified with anti-human point of wet factor antibody (monoclonal antibody or many on pad
Clonal antibody), during detection, after blood sample enters sample pad, point wet factor in blood is selectively bound by the antibody on process pad,
Remaining sample penetrates into sample pad, completes detection.
The embodiment 6 Sensitivity and Specificity to clinical sample
Epstein-Barr virus antigen and detection method by the present invention prepare Epstein-Barr virus antibody (IgM) gold mark detectable, detect 58 parts of EB
Virus antibody IgM positive clinical blood serum sample and 92 parts of Epstein-Barr virus antibody IgM feminine gender clinical serum samples carry out specificity and
Sensitivity tests.Meanwhile, in order to verify the effect of the present invention, to the gold mark detectable carried out with the Epstein-Barr virus antigen of the present invention
The test kit produced with Ou Meng company carries out coincidence rate analysis, the results are shown in Table 1, table 2.
Table 1: the specificity of the Epstein-Barr virus antigen of the present invention and sensitivity Detection result
The result of table 1 shows, the sensitivity of antigen of the present invention reaches 93.10%, and specificity reaches 95.65%.The present invention's is anti-
Former can be as further research and development Epstein-Barr virus antibody assay kit.
Table 2: the detectable carried out with antigen of the present invention and the coincidence rate analysis of Ou Meng company (EUROIMMUN) reagent
Table 2 result shows, detection kit of the present invention is 94. 83% with the positive coincidence rate of Ou Meng company reagent, and feminine gender meets
Rate is 97.83%, and total coincidence rate is 96.67%, and testing result and the Ou Meng company Epstein-Barr virus antibody of test kit of the present invention are described
Detection kit has preferable coincidence rate.Therefore, the present invention has preferable clinical value.
It is pointed out that and the foregoing is only presently preferred embodiments of the present invention, be not limiting as the present invention, all at this
Any amendment, equivalent and the improvement etc. made within the spirit of invention and principle, should be included in the protection model of the present invention
In enclosing.
<110>Lanzhou Yahua Biotech. Co., Ltd.
<120>a kind of Epstein-Barr virus antigen preparation procedure and utilize detection Epstein-Barr virus antibody prepared by this antigen quickly detect examination
Agent box
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 749
<212> DNA
<213>artificial sequence
<400> 1
atgtcagctc cacgcaaagt cagattgcct tctgttaagg ctgttgacat gagcatggaa 60
gacatggccg cccgcctggc tcgcctggag tctgagaata aggctctgaa gcaacaggtc 120
ctcagagggg gtgcctgtgc ctcgtctacc tctgttcctt ctgctccagt gcctccgcct 180
gagccgctta cagctcgaca gcgagaggta atgattacgc aggccacggg ccgtttggcg 240
tctcaggcta tgaagaagat tgaagacaag gttcggaaat ctgttgacgg tgtaactacc 300
cgcaatgaaa tggaaaatat attgcaaaat ctgaccctcc gcattcaagt atctatgttg 360
ggtgcaaaag gccaacccag ccctggtgag ggaacacgac cacgagaatc aaacgacccc 420
aacgccaccc gacgtgcccg ctcccgctcc cggggacgtg aagcaaagaa agtgcaaatt 480
tctgatggtg gcggtggaag cggcggtggc ggaagcggcg gtggcggcag cgccgcatcc 540
gctgggaccg gggccttggc atcatcagcg ccgtccacgg ccgtagccca gtccgcgacc 600
ccctctgttt cttcatctat tagcagcctc cgggccgcga cttcgggggc gactgccgcc 660
gcctccgccg ccgcagccgt cgataccggg tcaggtggcg ggggacaacc ccacgacacc 720
gccccacgcg gggcacgtaa gaaacagtag 750
Claims (6)
1. Epstein-Barr virus (EBV) antigen, it is a kind of employing technique for gene engineering prokaryotic expression, dialysis, gradient dilution method
The recombinant antigen that size is 30.8 kDa obtained with gel chromatography renaturation.
Antigen the most according to claim 1, it is that the p23-p18 section partial sequence of Epstein-Barr virus capsid antigen is manually closed
The gene prokaryotic become, described antigen has expression stable, and expression is high, immunogenicity advantages of higher.
3. an Epstein-Barr virus antibody method for quick, fixes including the Epstein-Barr virus antigen of arbitrary claim in application claim 1
To nitrocellulose filter, colloidal metal particles or colored latex particle marker antibody (two resist) contact, if testing sample exists
Epstein-Barr virus antibody, then Epstein-Barr virus antibody and labelling two anti-reflective should form the anti-complex of antibody-marker two, and " complex " is fine at nitric acid
Dimension element film moves horizontally, and runs into the Epstein-Barr virus antigen being fixed on nitrocellulose filter and produces reaction, carries markd " compound
Thing " just it is fixed to specifically on antigen, the position of immobilized antigen just has chromogenic reaction, if testing sample closes without respiratory tract
Born of the same parents' Epstein-Barr virus antibody, then " complex " can not be fixed on the position of antigen, does not the most just have chromogenic reaction, can be fine according to nitric acid
On dimension element film, whether immobilized antigen position has chromogenic reaction, whether to have in judgement sample Epstein-Barr virus antibody；Its detection used is anti-
Body is the IgM/IgG of serum or whole blood, and sampling is convenient.
4. a test kit for quick detection Epstein-Barr virus antibody, antigen used by it is that the P23-P18 described in claim 2 self melts
Hop protein, exists with inclusion bodies, the expressing protein obtained by purification and renaturation；Antigen used by it is claim 1
In respiratory syncystial Epstein-Barr virus antigen, method therefor is method described in claim 2.
5. a hemofiltration device for quick detection Epstein-Barr virus antibody, described hemofiltration device is to detecting device described in claim 4
Transforming, it is combined by glass fibre or non-woven fabrics and hemofiltration sample pad and constitutes；Described hemofiltration sample pad is by through anti-human blood red
Cell monoclonal antibodies or the glass fibre of many anti antibodys cured or non-woven fabrics are constituted.
6. a rheumatism factor treatment pad, described rheumatism factor treatment pad is to carry out the detection device described in claim 4
Transformation and come；It is made up of the non-woven fabrics being coated rheumatism factor antibody or nitrocellulose filter；The described rheumatism factor
Process pad by through anti-human rheumatism factor monoclonal antibodies or the non-woven fabrics of polyclonal antibody cured or nitrocellulose filter structure
Become.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679975A (en) * | 2019-01-23 | 2019-04-26 | 济宁医学院 | A kind of fusion and its application of transformation BZLF1 and transformation LMP1 |
| CN111474352A (en) * | 2020-04-29 | 2020-07-31 | 北京乐普医疗科技有限责任公司 | Novel coronavirus COVID-2019 detection card and preparation method thereof |
| CN111574621A (en) * | 2020-04-30 | 2020-08-25 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Monoclonal antibody for neutralizing EB virus and application thereof |
| CN112305218A (en) * | 2020-08-18 | 2021-02-02 | 上海纳米技术及应用国家工程研究中心有限公司 | Novel coronavirus antibody colloidal gold immune lateral chromatography detection method and application thereof |
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2016
- 2016-07-26 CN CN201610590575.2A patent/CN106188248A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109679975A (en) * | 2019-01-23 | 2019-04-26 | 济宁医学院 | A kind of fusion and its application of transformation BZLF1 and transformation LMP1 |
| CN109679975B (en) * | 2019-01-23 | 2020-12-08 | 济宁医学院 | Fusion gene for modifying BZLF1 and LMP1 and application thereof |
| CN111474352A (en) * | 2020-04-29 | 2020-07-31 | 北京乐普医疗科技有限责任公司 | Novel coronavirus COVID-2019 detection card and preparation method thereof |
| CN111474352B (en) * | 2020-04-29 | 2023-12-22 | 北京乐普诊断科技股份有限公司 | Novel coronavirus COVID-2019 detection card and preparation method thereof |
| CN111574621A (en) * | 2020-04-30 | 2020-08-25 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Monoclonal antibody for neutralizing EB virus and application thereof |
| CN111574621B (en) * | 2020-04-30 | 2022-03-29 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | Monoclonal antibody for neutralizing EB virus and application thereof |
| CN112305218A (en) * | 2020-08-18 | 2021-02-02 | 上海纳米技术及应用国家工程研究中心有限公司 | Novel coronavirus antibody colloidal gold immune lateral chromatography detection method and application thereof |
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