CN106645714B - EV71 virus IgA antibody test strips and its application - Google Patents
EV71 virus IgA antibody test strips and its application Download PDFInfo
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- CN106645714B CN106645714B CN201610976069.7A CN201610976069A CN106645714B CN 106645714 B CN106645714 B CN 106645714B CN 201610976069 A CN201610976069 A CN 201610976069A CN 106645714 B CN106645714 B CN 106645714B
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- G—PHYSICS
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention relates to a kind of EV71 viruses IgA antibody test strips, including sample pad, label pad, nitrocellulose filter, blotting paper and backboard, the nitrocellulose filter is equipped with EV71 virus VP 1s proteantigen polypeptide fragment detection line and people's IgA antibody control line, and the label pad is equipped with anti-human IgA antibody-colloid gold label object.The EV71 virus IgA antibody test strips of the present invention are used for saliva EV71 viral diagnosis, by the IgA antibody in detection saliva sample, the case where capable of realizing the rapid screening of EV71 viruses, and can improve the reliability of testing result, reduce false negative.The detection method is easy to operate, as a result quickly, detects the acquisition of sample without invasive, avoids traditional blood sampling and acquire that throat swab, anus swab, cerebrospinal fluid brings does not accommodate cross-infection.
Description
Technical field
The present invention relates to a kind of viral diagnosis test strips and its applications, are examined more particularly to a kind of EV71 viruses IgA antibody
Test paper slip and its application.
Background technology
Enterovirns type 71 (abbreviation EV71 viruses) belongs to Picornaviridae enterovirus genus, nonencapsulated single-stranded positive RNA
Virus is one of the main pathogens for causing hand-foot-and-mouth disease (hand-foot-mouth disease, HFMD).EV71 viruses can
Through the approach such as airborne droplet propagate, tool it is highly pathogenic, often result in as aseptic meningitis, encephalitis, polio sample paralysis and
The serious central nervous system complication such as neurogenic pulmonary edema, and cause death, it is main currently without effective medicine
It will be by way of early diagnosis and symptomatic treatment.EV71 viruses main infection 6 years old is hereinafter, especially 2-5 Sui children, the year
Age, section children attended school kindergarten mostly, therefore kindergarten is the EV71 virus precaution units of emphasis, in hand-foot-and-mouth disease epidemic season,
It is necessary to carry out screening monitoring to kindergarten, propagation of the EV71 viruses in kindergarten is reduced.Traditional screening hand-foot-and-mouth disease is main
It is the method investigation hand-foot-and-mouth disease by checking herpes of mouth and temperature test, but clinical discovery part brothers mouthful patient's early stage
There is no apparent hand, foot, herpes of mouth symptom or fever phenomenon, therefore the early diagnosis side of later EV71 viruses occurs
Method.
The method of early diagnosis of EV71 viruses is more, relates generally to histocyte culture technique, serology, molecular biology
Three field technologies, it is more commonly used there is cell to be separately cultured, the detection of RT-PCR nucleic acid molecules and serum IgM antibody ELISA inspections
Three kinds of methods are surveyed, but these methods have certain defect：
It is the determining goldstandard method of EV71 viruses infection that cell, which is separately cultured, but since detection time is grown (more than 5
It), experiment condition require high, operating personnel's technology that the shortcomings of high is required to limit its clinical diagnostic applications.
The detection of RT-PCR nucleic acid molecules is the highest method of EV71 virus early detection method medium sensitivities, is suffered from by detecting
The EV71 viruses of person's bottleneck throat swab and anal swab judge infection conditions, need technical professional and expensive fluorescence
PCR amplification instrument, the more difficult development diagnostic method of primary care department.In addition, when acquiring patient's bottleneck throat cell, it is easier to
Cause patient's discomfort that anthostele or vomiting is caused to occur, work to medical worker more difficult, while being also easy to increase sampler chamber
The risk of virus pollution.Finally, EV71 patients with viral infections restrovirus is more complicated in patient's body circulation process, according to the state of an illness
Development virus throat swab can be detected in bottleneck throat cell sometimes obtain as a result, inspection can be passed through in enteron aisle sometimes
Survey anus swab obtain as a result, sometimes can simultaneously in throat and enteron aisle, but sometimes virus not in throat and enteron aisle
In, therefore RT-PCR results often have the result of false negative.
Serology IgM antibody ELISA detections are mainly felt by detecting EV71 virus IgM antibodies understanding patient in serum in the recent period
It catches an illness the situation of poison, there is certain help to clinical diagnosis and treatment, but many EV71 Virus patients are less than 6 years old children, it is more
The blood vessel of child is thinner, and has fear to blood drawing, therefore brings certain difficulty to clinical staff operation.In addition have
Substantial portion of children's superinfection EV71 viruses, the group generate the EV71 viruses of low concentration when infecting EV71 viruses
IgM antibody, therefore detect the result that serum IgM antibody becomes more readily available false negative.
Colloid gold immune analysis (Colloidal gold immunoassay, CGIA) results from the 1980s, being
The solid phase labelling immune detection skill to grow up after fluorescent marker, emitting isotope label and three big labelling technique of enzyme label
Art has been applied to Electronic Speculum, light microscopic, agglutination test, immunoblotting, spot diafiltration and immunochromatography etc., colloid therein
Golden immunochromatography technique is simple and quick with its, it is special it is sensitive, can be grown without auxiliary reagent and instrument, visual results, result
The features such as phase preserves becomes one of convenient immunology detection technology of current most rapid sensitive, single especially suitable for hospital, base
Position, field work and inspection it is pressed for time and large area generaI investigation.
Invention content
Of the existing technology in order to overcome the problems, such as, the present invention provides a kind of EV71 viruses IgA antibody test strips
And its application, it can realize the rapid screening of EV71 viruses.
The present invention provides a kind of EV71 viruses IgA antibody test strips, including sample pad, label pad, cellulose nitrate
Plain film, blotting paper and backboard, the nitrocellulose filter are equipped with EV71 virus VP 1s proteantigen polypeptide fragment detection line and people
IgA antibody control line, the label pad are equipped with anti-human IgA antibody-colloid gold label object.
Further, the amino acid sequence such as sequence table Seq ID of the EV71 virus VP 1s proteantigen polypeptide fragment
Shown in NO.1.
Further, the anti-human IgA antibody be selected from goat-anti people IgA polyclonal antibodies, the anti-human IgA monoclonal antibodies of mouse,
Rabbit-anti people IgA monoclonal antibodies or rabbit-anti people's IgA polyclonal antibodies.
Further, the blotting paper, nitrocellulose filter, label pad, sample pad are adhered to successively on the backboard.
The present invention also provides another EV71 virus IgA antibody test strips, including sample pad, label pad, nitre
Acid cellulose film, blotting paper, backboard, the nitrocellulose filter are equipped with anti-human IgA antibody detection line and EV71 virus VP 1 eggs
White antibody control line, the label pad are equipped with EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label object.
Further, the amino acid sequence such as sequence table Seq ID of the EV71 virus VP 1s proteantigen polypeptide fragment
Shown in NO.1.
Further, it is anti-to be selected from goat-anti people IgA polyclonal antibodies, the anti-human IgA monoclonals of mouse for the anti-human IgA antibody
Body, rabbit-anti people IgA monoclonal antibodies or rabbit-anti people's IgA polyclonal antibodies；The EV71 virus VP 1s protein antibodies are anti-selected from mouse
EV71 virus VP 1s protein monoclonal antibody, the anti-EV71 virus VP 1s protein polyclone antibody of people, rabbit-anti EV71 virus VP 1 albumen lists
Clonal antibody, rabbit-anti EV71 virus VP 1s protein polyclone antibody or goat-anti EV71 virus VP 1 protein polyclone antibodies.
Further, the blotting paper, nitrocellulose filter, label pad, sample pad are adhered to successively on the backboard.
The EV71 virus IgA antibody test strips of the present invention can be used for the EV71 viral diagnosis in saliva, detecting step
For：Saliva sample is taken, is added in sample diluting liquid, the EV71 virus IgA antibody test strips is then placed in and is examined
It surveys.
Further, the sample diluting liquid is to contain 0.5% bovine serum albumin(BSA), 0.2% casein, 1% pancreas egg
The PBS buffer solution of white peptone and 0.02% Sodium azide.
EV71 viral capsids are made of tetra- kinds of protein of VP1, VP2, VP3 and VP4, and wherein VP1 albumen is located at virion
Surface is made of 297 amino acid, is the main neutrality epitope of EV71 viruses, body can be stimulated to generate high titre
Protectiveness neutralizing antibody.When EV71 viruses by oral cavity invade human body, start can in people's throat cell be colonized and breed,
At this moment human immune system takes the lead in starting mucosal immunity to virus progress immune attack, and mucosal immunity is generated viral special by saliva
Anisotropic IgA antibody resists virus infection.
The EV71 virus IgA antibody test strips of the present invention, by detecting the IgA antibody in saliva sample, Neng Goushi
The case where showing the rapid screening of EV71 viruses, and the reliability of testing result can be improved, reduce false negative.The detection method operates
Simply, as a result quickly, the acquisition of sample is detected without invasive, avoids traditional blood sampling and acquisition throat swab, anus swab, brain ridge
What liquid band was come does not accommodate cross-infection.
Description of the drawings
Fig. 1 is the structural schematic diagram of the EV71 virus IgA antibody test strips of the present invention.
Fig. 2 saliva acquisition rods discharge the method schematic diagram of saliva sample.
Fig. 3 is the detection method schematic diagram of the EV71 virus IgA antibody test strips of the present invention.
Fig. 4 is that the test strip of the present invention detects the positive findings schematic diagram of EV71 virus IgA antibodies.
Fig. 5 is that the test strip of the present invention detects the negative findings schematic diagram of EV71 virus IgA antibodies.
Fig. 6 is that the test strip of the present invention detects the null result schematic diagram of EV71 virus IgA antibodies.
Specific implementation mode
In order to better understand and implement, the invention will now be described in detail with reference to the accompanying drawings.
【Embodiment 1】EV71 virus IgA antibody test strips are prepared (prize law)
1, raw material sources：Goat-anti people's IgA antibody is purchased from Sigma companies, and people's IgA antibody thinks biotechnology purchased from Guangzhou promise
Co., Ltd.The sequence of EV71 virus amalgamation protein antigenic polypeptide fragments is as shown in Seq ID NO.1,58 amino acid in total,
Commission Shanghai gill biochemical corp is responsible for synthesis, and purity is more than 98%, and freeze-drying preserves.Colloidal gold solution (40nm) is outstanding purchased from Shanghai
One Bioisystech Co., Ltd.
2, the pretreatment of sample pad and label pad
Sample pad and label pad are polyester film material, need to carry out immersion treatment with pretreatment fluid before use.Pretreatment fluid
For by the non-ionic surface of the phosphate buffer of 0.01~0.02mol/L (pH7.0~7.5), 3g/100ml~6g/100ml
The Macrogol 6000 composition of activating agent, the tween of 1ml/100ml~4ml/100ml and 5g/100ml~20g/100ml mixes
Close liquid.Polyester film material impregnated in pretreatment fluid to 5~for 24 hours, it dries, is sealed after drying standby in 37~45 DEG C of exhausting after taking-up
With.Pretreated sample pad can be directly used for the assembling of test strips, and label pad need to be used for after the processing of the coating of marker
The assembling of test strips.
3, the coating processing of goat-anti people IgA antibody-colloid gold label pad
(1) preparation of goat-anti people IgA antibody-colloid gold label object solution：Under magnetic agitation, with 0.1M potassium carbonate or hydrochloric acid
Solution adjusts the pH value of colloidal gold solution to 7.4, and goat-anti people's IgA antibody is added in the ratio of 1mg antibody/ml colloidal gold solutions,
Then it is added and 0.9% sodium chloride solution isometric with reaction system is then added, mixing is stirred to react 2h, is added final concentration of
0.3% bovine serum albumin(BSA) adjusts pH value of solution to 8.5, stands 30min.Above-mentioned solution is centrifuged at 14000rpm, 4 DEG C
15min abandons supernatant, the borate buffer solution (boric acid of the sediment 0.02M pH9.0 of 1/20 initial colloid gold solution volume
0.1237g, PEG 20000 1g are settled to 1L with ultra-pure water, adjust pH to be centrifuged again to after 9.0) being resuspended, so wash repeatedly
3 times, unbonded protein is removed with thorough, finally obtains goat-anti people IgA antibody-colloid gold label object solution, in 4 DEG C of preservations
It is spare.
(2) the coating processing of goat-anti people IgA antibody-colloid gold label pad：The goat-anti that will be prepared with Isoflow spray film instrument
People's IgA antibody-colloid gold label object solution even application is by the pretreated label pad of step 2,0.03ml is sprayed per 1cm
The label sprayed is padded under the conditions of humidity 15%, 30 DEG C of temperature and dries by goat-anti people IgA antibody-colloid gold label object solution
Handle 12 hours or more, it is sealed after drying spare.
4, the coating processing of nitrocellulose filter
People's IgA antibody is taken to be diluted to 1.5mg/ml with coating buffer (10mM, pH7.4 phosphate buffer for containing 1% sucrose),
Then the control line (C lines) being coated in Isoflow point film instruments on nitrocellulose filter；Take EV71 virus VP 1 albumen anti-
Former polypeptide fragment is diluted to 1.0mg/ml with coating buffer (10mM, pH7.4 phosphate buffer for containing 1% sucrose), then uses
Isoflow point film instruments are coated in the detection line (T lines) on nitrocellulose filter.The nitrocellulose filter being coated with is in humidity
Drying and processing 12 hours or more, seals spare after drying under the conditions of 10-30%, 20-35 DEG C of temperature.
5, it assembles, cut
Attached drawing 1 is please referred to, attached drawing 1 is the structural schematic diagram of the EV71 virus IgA antibody test strips of the present invention.It will inhale
Water paper 4, nitrocellulose filter 3, label pad 2, sample pad 1 are adhered to successively on PVC backboards 5, then adjust cutting machine parameter into
Row slitting, is cut into the test strips of 4mm width.
【Embodiment 2】EV71 virus IgA antibody test strips are prepared (indirect method)
1, raw material sources：Goat-anti people's IgA antibody is purchased from Sigma companies, and EV71 virus VP 1 protein antibodies are purchased from Guangzhou promise
Think bio tech ltd.The sequence of EV71 virus amalgamation protein antigenic polypeptide fragments such as Seq ID NO.1, in total 58 ammonia
Base acid, commission Shanghai gill biochemical corp are responsible for synthesis, and purity is more than 98%, and freeze-drying preserves.Colloidal gold solution (40nm) is purchased from
Shanghai one Bioisystech Co., Ltd of outstanding person.
2, the pretreatment of sample pad and label pad
Sample pad and label pad are polyester film material, need to carry out immersion treatment with pretreatment fluid before use.Pretreatment fluid
For by the non-ionic surface of the phosphate buffer of 0.01~0.02mol/L (pH7.0~7.5), 3g/100ml~6g/100ml
The Macrogol 6000 composition of activating agent, the tween of 1ml/100ml~4ml/100ml and 5g/100ml~20g/100ml mixes
Close liquid.Polyester film material impregnated in pretreatment fluid to 5~for 24 hours, it dries, is sealed after drying standby in 37~45 DEG C of exhausting after taking-up
With.Pretreated sample pad can be directly used for the assembling of test strips, and label pad need to be used for after the processing of the coating of marker
The assembling of test strips.
3, the coating processing of EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label pad
(1) preparation of EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label object solution：Under magnetic agitation, use
The pH value that 0.1M potassium carbonate or hydrochloric acid solution adjust colloidal gold solution is added to 7.4 in the ratio of 1mg antibody/ml colloidal gold solutions
Enter EV71 virus VP 1 proteantigen polypeptide fragments, 0.9% sodium chloride solution isometric with reaction system, mixing is then added
It is stirred to react 2h, final concentration of 0.3% bovine serum albumin(BSA) is added, pH value of solution is adjusted to 8.5, stands 30min.It will be above-mentioned molten
Liquid centrifuges 15min at 14000rpm, 4 DEG C, abandons supernatant, the 0.02M of 1/20 initial colloid gold solution volume of sediment
(boric acid 0.1237g, PEG 20000 1g are settled to 1L with ultra-pure water, adjust pH to 9.0) borate buffer solution of pH9.0
It is centrifuged again after resuspension, so repeated washing 3 times, removes unbonded protein with thorough, finally obtain EV71 virus VP 1 albumen
Antigenic polypeptide fragments-colloid gold label object solution, save backup in 4 DEG C.
(2) the coating processing of EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label pad：Film instrument is sprayed with Isoflow
The EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label object solution even application prepared is being located in advance by step 2
In the label pad of reason, spraying 0.03ml EV71 virus VP 1s proteantigens polypeptide fragment-colloid gold label object solution per 1cm will
The label sprayed is padded under the conditions of humidity 15%, 30 DEG C of temperature drying and processing 12 hours or more, is sealed after drying spare.
4, the coating processing of nitrocellulose filter
Take EV71 virus VP 1 protein antibodies coating buffer (10mM, pH7.4 phosphate buffer for containing 1% sucrose) dilution
To 1.5mg/ml, the control line (C lines) that is then coated in Isoflow point film instruments on nitrocellulose filter；Take goat-anti people
IgA antibody is diluted to 1.0mg/ml with coating buffer (10mM, pH7.4 phosphate buffer for containing 1% sucrose), then uses
Isoflow point film instruments are coated in the detection line (T lines) on nitrocellulose filter.The nitrocellulose filter being coated with is in humidity
Drying and processing 12 hours or more, seals spare after drying under the conditions of 10-30%, 20-35 DEG C of temperature.
5, it assembles, cut
Attached drawing 1 is please referred to, attached drawing 1 is the structural schematic diagram of the EV71 virus IgA antibody test strips of the present invention.It will inhale
Water paper 4, nitrocellulose filter 3, bonding pad 2, sample pad 1 are adhered to successively on PVC backboards 5, then adjust cutting machine parameter into
Row slitting, is cut into the test strips of 4mm width.
【Embodiment 3】The detection (prize law) of EV71 virus IgA antibodies
Attached drawing 2 is please referred to, 2 saliva acquisition rod of attached drawing discharges the method schematic diagram of saliva sample.The present invention is adopted by saliva
Collection stick 22 adsorbs saliva sample in patient oral cavity, and aforementioned acquisition rod is placed into and (is contained equipped with 1-2ml sample diluting liquids 23
The PBS buffer solution of 0.5% bovine serum albumin(BSA), 0.2% casein, 1% tryptone and 0.02% Sodium azide) sample cell 21
In, it is sufficiently stirred, squeezes the cotton swab head liquid of acquisition rod, discard acquisition rod.
Attached drawing 3 is please referred to, Fig. 3 is the detection method schematic diagram of the EV71 virus IgA antibody test strips of the present invention.It takes
【Embodiment 1】The EV71 virus IgA antibody test strips of preparation are put into the above-mentioned sample cell 21 equipped with saliva sample, sample
End in contact saliva sample is padded, is placed 15-30 minutes, then visual results.
Testing principle：Due to capillarity, saliva sample is flowed from sample pad end to blotting paper end, by way of being loaded with goat-anti
After the label pad of people's IgA antibody-colloid gold label object, which is redissolved into free state completely.If saliva sample
Contain EV71 virus IgA antibodies in product, can be combined with free goat-anti people IgA antibody-colloid gold label object, forms colloidal gold-
Goat-anti people IgA antibody-IgA antibody compound, the compound are up at the detection line (T lines) of nitrocellulose filter, then with its
Upper coated EV71 virus VP 1s proteantigen polypeptide fragment specific bond, is presented red stripes (attached drawing 4), extra free glue
Body gold-goat-anti people's IgA antibody continues to be up at the control line (C lines) of nitrocellulose filter, with coated people's IgA antibody thereon
Specific binding forms colloidal gold-goat-anti people IgA antibody-people's IgA antibody compound, and red stripes (attached drawing 4) are presented.If
EV71 virus IgA antibodies are not contained in sample, free goat-anti people IgA antibody-colloid gold label object can cross detection line (T
Line), continue to be up at the control line (C lines) of nitrocellulose filter, be specifically bound with coated people's IgA antibody thereon, shape
At colloidal gold-goat-anti people IgA antibody-people's IgA antibody compound, red stripes (attached drawing 5) are presented.If nitrocellulose filter
Red stripes are not formed at control line (C lines), illustrate that test strips fail, it is as a result unreliable (attached drawing 6).
Testing result judges：If detection line (T lines) and control line (C lines) occurs simultaneously in test strip, illustrate sample
It is positive, containing IgA antibody, that is, indicates EV71 viruses infection (attached drawing 4)；If only there is control line (C lines), illustrate sample
Be negative (attached drawing 5), is free of IgA antibody, that is, indicates no EV71 viruses infection；If there is not any line or only occurring
Detection line (T lines) illustrates that this test strips is invalid (attached drawing 6).
【Embodiment 4】The detection (indirect method) of EV71 virus IgA antibodies
Attached drawing 2 is please referred to, 2 saliva acquisition rod of attached drawing discharges the method schematic diagram of saliva sample.The present invention is adopted by saliva
Collection stick 22 adsorbs saliva sample in patient oral cavity, and aforementioned acquisition rod is placed into and (is contained equipped with 1-2ml sample diluting liquids 23
The PBS buffer solution of 0.5% bovine serum albumin(BSA), 0.2% casein, 1% tryptone and 0.02% Sodium azide) sample cell 21
In, it is sufficiently stirred, squeezes the cotton swab head liquid of acquisition rod, discard acquisition rod.
Attached drawing 3 is please referred to, Fig. 3 is the detection method schematic diagram of the EV71 virus IgA antibody test strips of the present invention.It takes
【Embodiment 1】The EV71 virus IgA antibody test strips of preparation are put into the above-mentioned sample cell 21 equipped with saliva sample, sample
End in contact saliva sample is padded, is placed 15-30 minutes, then visual results.
Testing principle：Due to capillarity, saliva sample is flowed from sample pad end to blotting paper end, by way of being loaded with EV71
After the label pad of virus VP 1 proteantigen polypeptide fragment-colloid gold label object, which is redissolved completely at free
State.It, can be with free EV71 virus VP 1s proteantigen polypeptide fragment-glue if containing EV71 virus IgA antibodies in saliva sample
Body gold marker combines, and forms colloidal gold-EV71 virus VP 1s proteantigen polypeptide fragment-IgA antibody compound, the compound
Be up at the detection line (T lines) of nitrocellulose filter, then with coated goat-anti people IgA antibody specific bond thereon, formed glue
It is (attached that red stripes are presented in body gold-EV71 virus VP 1s proteantigen polypeptide fragment-IgA antibody-goat-anti people's IgA antibody compound
Fig. 4), extra free colloidal gold-EV71 virus VP 1s proteantigen polypeptide fragment continues the control for being up to nitrocellulose filter
Line (C lines) specifically binds with coated EV71 virus VP 1s protein antibodies thereon, forms colloidal gold-EV71 virus VP 1 albumen
Red stripes (attached drawing 4) are presented in antigenic polypeptide fragments-EV71 virus VP 1 protein antibodies compounds.If do not contained in sample
EV71 virus IgA antibodies, free EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label object can cross detection line (T
Line), continue the control line (C lines) for being up to nitrocellulose filter, with coated EV71 virus VP 1s protein antibodies specificity thereon
In conjunction with formation colloidal gold-EV71 virus VP 1 proteantigen polypeptide fragment-EV71 virus VP 1 protein antibodies compounds are presented red
Vitta band (attached drawing 5).If the control line (C lines) of nitrocellulose filter does not form red stripes, illustrate that test strips fail, knot
Fruit is unreliable (attached drawing 6).
Testing result judges：If detection line (T lines) and control line (C lines) occurs simultaneously in test strip, illustrate sample
It is positive, containing IgA antibody, that is, indicates EV71 viruses infection (attached drawing 4)；If only there is control line (C lines), illustrate sample
It is negative, is free of IgA antibody, that is, indicate no EV71 viruses infection (attached drawing 5)；If there is not any line or only occurring
Detection line (T lines) illustrates that this test strips is invalid (attached drawing 6).
【Embodiment 5】EV71 viruses IgA antibody test strip method of the present invention and fluorescence PCR method Comparative result
Laboratory sample 88 is chosen, acquires the saliva sample and Pharyngeal swab samples of patient respectively, uses EV71 viruses IgA respectively
Antibody test test strips method is tested with fluorescence PCR method, as a result such as table 1：
The EV71 viruses IgA antibody test strip method and fluorescence PCR method comparison result of 1 present invention of table
Compared with fluorescence PCR method, sensitivity reaches EV71 viruses IgA antibody test strip method of the present invention
97.50%, specificity 87.50% has actual application value to EV71 virus screenings.
【Embodiment 6】EV71 viruses IgA antibody test strip method of the present invention and serum IgM antibody detection method result
Comparison
88, sample is chosen, acquires the saliva sample and blood sample of patient respectively, uses EV71 virus IgA antibodies to examine respectively
Test paper slip method is tested with serum IgM antibody detection method, as a result such as table 2：
The EV71 viruses IgA antibody test strip method and fluorescence PCR method comparison result of 2 present invention of table
EV71 viruses IgA antibody test strip method of the present invention and serum IgM antibody Comparison between detecting methods, sensitivity reach
To 95.00%, specificity 89.58% has actual application value to EV71 virus screenings.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Sequence table
<110>Guangzhou Ruihui Biomedical Medical Technology Co., Ltd.
<120>EV71 virus IgA antibody test strips and its application
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 58
<212> PRT
<213>Amino acid sequence
<400> 1
Ser Arg Glu Ser Leu Ala Trp Gln Thr Ala Thr Asn Pro Ser Val
5 10 15
Phe Val Lys Leu Ser Asp Pro Pro Ala Gln Val Ser Val Pro Phe
20 25 30
Met Ser Pro Ala Ser Ala Tyr Gln Trp Phe Tyr Asp Gly Tyr Pro
35 40 45
Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu Tyr
50 55
Claims (6)
1. a kind of EV71 viruses IgA antibody test strip, which is characterized in that including sample pad, label pad, nitrocellulose
Film, blotting paper and backboard, the nitrocellulose filter are equipped with EV71 virus VP 1s proteantigen polypeptide fragment detection line and people
IgA antibody control line, the label pad are equipped with anti-human IgA antibody-colloid gold label object；The EV71 virus VP 1s albumen is anti-
The amino acid sequence of former polypeptide fragment is as shown in sequence table Seq ID NO.1；The EV71 virus IgA antibody test strips
It is detection sample with the saliva of people.
2. EV71 viruses IgA antibody test strip according to claim 1, which is characterized in that the anti-human IgA antibody
It is more selected from goat-anti people IgA polyclonal antibodies, the anti-human IgA monoclonal antibodies of mouse, rabbit-anti people IgA monoclonal antibodies or rabbit-anti people IgA
Clonal antibody.
3. EV71 viruses IgA antibody test strip according to claim 1, which is characterized in that the blotting paper, nitric acid
Cellulose membrane, label pad, sample pad are adhered to successively on the backboard.
4. a kind of EV71 viruses IgA antibody test strip, which is characterized in that including sample pad, label pad, nitrocellulose
Film, blotting paper and backboard, the nitrocellulose filter are equipped with anti-human IgA antibody detection line and EV71 virus VP 1 protein antibodies
Control line, the label pad are equipped with EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label object；The EV71 viruses
The amino acid sequence of VP1 proteantigen polypeptide fragments is as shown in sequence table Seq ID NO.1；The EV71 virus IgA antibodies
Test strip is detection sample with the saliva of people.
5. EV71 viruses IgA antibody test strip according to claim 4, which is characterized in that the anti-human IgA is anti-
Body is selected from goat-anti people IgA polyclonal antibodies, the anti-human IgA monoclonal antibodies of mouse, rabbit-anti people IgA monoclonal antibodies or rabbit-anti people IgA
Polyclonal antibody；The EV71 virus VP 1 protein antibodies are selected from the anti-EV71 virus VP 1s protein monoclonal antibody of mouse, people resists
EV71 virus VP 1s protein polyclone antibody, rabbit-anti EV71 virus VP 1s protein monoclonal antibody, rabbit-anti EV71 virus VP 1 albumen are more
Clonal antibody or goat-anti EV71 virus VP 1 protein polyclone antibodies.
6. EV71 viruses IgA antibody test strip according to claim 4, which is characterized in that the blotting paper, nitric acid
Cellulose membrane, label pad, sample pad are adhered on backboard successively.
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| CN108254554B (en) * | 2017-11-23 | 2020-04-24 | 广州瑞辉生物科技股份有限公司 | Influenza A virus IgA antibody immunofluorescence detection test strip and preparation method, detection method and application thereof |
| CN108152506B (en) * | 2017-11-23 | 2020-04-24 | 广州瑞辉生物科技股份有限公司 | Influenza B virus IgA antibody immunofluorescence detection test strip and preparation method, detection method and application thereof |
| CN108267577B (en) * | 2018-01-15 | 2020-10-09 | 广州市妇女儿童医疗中心 | EV71 virus IgA antibody detection test strip |
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