CN106645714A - EV71 virus IgA antibody detection test strip and application thereof - Google Patents
EV71 virus IgA antibody detection test strip and application thereof Download PDFInfo
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- CN106645714A CN106645714A CN201610976069.7A CN201610976069A CN106645714A CN 106645714 A CN106645714 A CN 106645714A CN 201610976069 A CN201610976069 A CN 201610976069A CN 106645714 A CN106645714 A CN 106645714A
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- G—PHYSICS
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G—PHYSICS
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- 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 invention relates to an EV71 virus IgA antibody detection test strip which comprises a sample pad, a label pad, a nitrocellulose membrane, absorbing paper and a back plate. The nitrocellulose membrane is provided with an EV71 virus VP1 protein antigen peptide fragment detection line and a human IgA antibody control line. The label pad is provided with an an-human IgA anti-body-colloidal gold marker. The EV71 virus IgA antibody detection test strip is used for saliva EV71 virus detection; by detecting an IgA anti-body in a saliva sample, EV71 viruses can be quickly screened, reliability of detection results can be improved, and false-negative conditions are reduced. A detection method is simple to operate, the results are quick, the collection of a detected sample has no invasiveness, and discomfort and cross infection caused by conventional blood sampling and collection of throat swab, anus swab samples and cerebrospinal fluid are avoided.
Description
Technical field
The present invention relates to a kind of Viral diagnosis test strips and its application, more particularly to a kind of EV71 virus IgA antibody inspections
Test paper slip and its application.
Background technology
Enterovirns type 71 (abbreviation EV71 is viral) 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
The approach such as the Jing air spittle propagate, have 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
Will be by way of early diagnosis and symptomatic treatment.Below EV71 viruses main infection 6 years old, the particularly children of 2-5 year, the year
Age section children attend 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 kindergarten examination monitoring, reduces propagation of the EV71 viruses in kindergarten.Traditional examination 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' mouth patient's early stage
Without obvious hand, foot, herpes of mouth symptom or fever phenomenon, thus occur in that later EV71 virus early diagnosis side
Method.
The method of early diagnosis of EV71 viruses is more, relates generally to histocyte culture technique, serology, molecular biology
Three arts, the more commonly used has cell separation culture, the detection of RT-PCR nucleic acid molecules and serum IgM antibody ELISA inspections
Three kinds of methods are surveyed, but there is certain defect in these methods：
Cell separation culture is the goldstandard method that EV71 virus infection determines, but because detection time length is (more than 5
My god), experiment condition has high demands, operating personnel's technical requirements limit its clinical diagnostic applications the shortcomings of high.
The detection of RT-PCR nucleic acid molecules is EV71 virus early detection method medium sensitivity highest methods, is suffered from by detecting
The EV71 viruses of person's bottleneck throat swab and anal swab judge infection conditions, need the fluorescence of technical professional and costliness
PCR amplification instrument, primary care department is more difficult to carry out the diagnostic method.In addition, when patient's bottleneck throat cell is gathered, being easier to
Cause patient's discomfort to cause anthostele or vomiting to occur, work more difficult to medical worker, while also easily increasing sampler chamber
The risk of virus pollution.Finally, the EV71 patients with viral infections restrovirus process that circulates in patient's body is more complicated, according to the state of an illness
Development virus can detect that throat swab obtains result in bottleneck throat cell sometimes, sometimes in enteron aisle can by inspection
Survey anus swab and obtain result, sometimes can be while in throat and enteron aisle but sometimes viral not in throat and enteron aisle
In, therefore RT-PCR results often have false-negative result.
EV71 virus IgM antibodies understand patient and feel in the recent period in the main serum by detection of serology IgM antibody ELISA detection
Catch an illness the situation of poison, there is certain help to clinical diagnosis and treatment, but many EV71 Virus patients are less than the children of 6 years old, it is more
The blood vessel of child is thinner, and has fear to blood drawing, therefore brings certain difficulty to clinical staff operation.Have in addition
Substantial portion of children's superinfection EV71 is viral, and the EV71 that the colony produces low concentration when infection EV71 is viral is viral
IgM antibody, therefore detect that serum IgM antibody becomes more readily available false-negative result.
Colloid gold immune analysis (Colloidal gold immunoassay, CGIA) results from the eighties in 20th century, is
The solid phase labelling immune detection skill grown up after fluorescence labeling, emitting isotope mark and the big labelling technique of enzyme mark three
Art, has been applied to the aspects such as Electronic Speculum, light microscopic, agglutination test, Western blotting, spot diafiltration and immunochromatography, colloid therein
Golden immunochromatography technique so that its is simple and quick, it is special it is sensitive, can grow without the need for 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, is particularly well-suited to hospital, basic unit's list
Position, field work and inspection it is pressed for time and large area generaI investigation.
The content of the invention
For the problem for overcoming prior art to exist, the invention provides a kind of EV71 virus IgA antibody test strips
And its application, the rapid screening of EV71 viruses can be realized.
The invention provides a kind of EV71 viruses IgA antibody test strip, including sample pad, label pad, cellulose nitrate
Plain film, blotting paper and backboard, described nitrocellulose filter is provided with EV71 virus VP 1s proteantigen polypeptide fragment detection line and people
IgA antibody control line, described label pad is provided with anti-human IgA antibody-colloid gold label thing.
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 selected from goat-anti people's 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 sticked to successively on the backboard.
Present invention also offers another EV71 virus IgA antibody test strip, including sample pad, label pad, nitre
Acid cellulose film, blotting paper, backboard, described nitrocellulose filter is provided with anti-human IgA antibody detection line and EV71 virus VP 1 eggs
White antibody control line, described label pad is provided with EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label thing.
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, described anti-human IgA antibody resists selected from goat-anti people's IgA polyclonal antibodies, the anti-human IgA monoclonals of mouse
Body, rabbit-anti people IgA monoclonal antibodies or rabbit-anti people's IgA polyclonal antibodies；Described EV71 virus VP 1s protein antibodies are anti-selected from mouse
EV71 virus VP 1 protein monoclonal antibodies, the anti-EV71 virus VP 1s protein polyclone antibody of people, rabbit-anti EV71 virus VP 1 albumen list
Clonal antibody, rabbit-anti EV71 virus VP 1 protein polyclone antibody or goat-anti EV71 virus VP 1 protein polyclone antibodies.
Further, the blotting paper, nitrocellulose filter, label pad, sample pad are sticked 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, in adding sample diluting liquid, described EV71 virus IgA antibody test strips is then placed in and is examined
Survey.
Further, described sample diluting liquid is containing 0.5% bovine serum albumin(BSA), 0.2% casein, 1% pancreas egg
White peptone and the PBS of 0.02% Sodium azide.
EV71 viral capsids are made up of tetra- kinds of protein of VP1, VP2, VP3 and VP4, and wherein VP1 albumen is located at virion
Surface, it is made up of 297 amino acid, is the main neutrality epitope of EV71 viruses, and body can be stimulated to produce high titre
Protectiveness neutralizing antibody.When EV71 viruses invade human body by oral cavity, start to be colonized in people's throat cell and breed,
At this moment human immune system takes the lead in starting mucosal immunity and carries out immune attack to virus, and mucosal immunity produces virus spy by saliva
The IgA antibody opposing virus infection of the opposite sex.
The EV71 virus IgA antibody test strips of the present invention, by the IgA antibody in detection saliva sample, Neng Goushi
The rapid screening of existing EV71 viruses, and the reliability of testing result can be improved, reduce false-negative situation.The detection method operation
Simply, as a result quickly, the collection of detection sample is without invasive, it is to avoid traditional blood sampling and collection 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 representation of the EV71 virus IgA antibody test strips of the present invention.
Fig. 2 salivas acquisition rod discharges 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 the positive findings schematic diagram of the test strip detection EV71 virus IgA antibodies of the present invention.
Fig. 5 is the negative findings schematic diagram of the test strip detection EV71 virus IgA antibodies of the present invention.
Fig. 6 is the null result schematic diagram of the test strip detection EV71 virus IgA antibodies of the present invention.
Specific embodiment
In order to more fully understand and implement, the present invention is described in detail below in conjunction with the accompanying drawings.
【Embodiment 1】EV71 virus IgA antibody test strips are prepared (prize law)
1st, raw material sources：Goat-anti people 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 as shown in Seq ID NO.1,58 amino acid altogether,
Commission Shanghai gill biochemical corp is responsible for synthesis, and purity is more than 98%, freezes and preserves.Colloidal gold solution (40nm) is outstanding purchased from Shanghai
One Bioisystech Co., Ltd.
2nd, the pretreatment of sample pad and label pad
Sample pad and label pad are polyester film material, and immersion treatment need to be carried out with pretreatment fluid using before.Pretreatment fluid
It is phosphate buffer (pH7.0～7.5), the non-ionic surface of 3g/100ml～6g/100ml by 0.01～0.02mol/L
It is mixed that the Macrogol 6000 of activating agent, the tween of 1ml/100ml～4ml/100ml and 5g/100ml～20g/100ml is constituted
Close liquid.Polyester film material is soaked into 5～24h in pretreatment fluid, is taken out after 37～45 DEG C of exhausting drying, sealed after drying standby
With.Pretreated sample pad can be directly used for the assembling of test strips, and label pad need to be used for after the coating of label is processed
The assembling of test strips.
3rd, the coating of goat-anti people IgA antibody-colloid gold label pad is processed
(1) preparation of goat-anti people IgA antibody-colloid gold label thing 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 in the ratio of 1mg antibody/ml colloidal gold solutions goat-anti people's IgA antibody is added,
0.9% sodium chloride solution isopyknic with reaction system is subsequently adding, stirring reaction 2h is mixed, 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 sediment borate buffer solution (boric acid of the 0.02M pH9.0 of 1/20 initial colloid gold solution volume
0.1237g, PEG 20000 1g, with ultra-pure water 1L is settled to, and adjusts pH to being centrifuged again after 9.0) resuspended, such repeated washing
3 times, thoroughly to remove unconjugated protein, goat-anti people IgA antibody-colloid gold label thing solution is finally given, in 4 DEG C of preservations
It is standby.
(2) coating of goat-anti people IgA antibody-colloid gold label pad is processed：With Isoflow film instrument is sprayed by the goat-anti for preparing
People's IgA antibody-colloid gold label thing solution even application sprays 0.03ml in the label pad pre-processed through step 2, per 1cm
Goat-anti people IgA antibody-colloid gold label thing solution, dries under the conditions of the mark for having sprayed is padded on into humidity 15%, 30 DEG C of temperature
Process more than 12 hours, seal after drying standby.
4th, the coating of nitrocellulose filter is processed
Take people's IgA antibody coating buffer (10mM, pH7.4 phosphate buffer containing 1% sucrose) and be diluted to 1.5mg/ml,
Then the control line (C lines) being coated in Isoflow point film instruments on nitrocellulose filter；Take EV71 virus VP 1s albumen to resist
Former polypeptide fragment coating buffer (10mM, pH7.4 phosphate buffer containing 1% sucrose) is diluted to 1.0mg/ml, Ran Houyong
Isoflow point film instruments are coated in the detection line on nitrocellulose filter (T lines).The nitrocellulose filter being coated with is in humidity
Drying and processing more than 12 hours under the conditions of 10-30%, temperature 20-35 DEG C, seal standby after drying.
5th, assembling, slitting
Accompanying drawing 1 is referred to, accompanying drawing 1 is the structural representation of the EV71 virus IgA antibody test strips of the present invention.To inhale
Water paper 4, nitrocellulose filter 3, label pad 2, sample pad 1 are sticked to successively on PVC backboards 5, are then adjusted cutting machine parameter and are entered
Row slitting, is cut into the test strips of 4mm width.
【Embodiment 2】EV71 virus IgA antibody test strips are prepared (indirect method)
1st, raw material sources：Goat-anti people IgA antibody is purchased from Sigma companies, and EV71 virus VP 1s 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, altogether 58 ammonia
Base acid, commission Shanghai gill biochemical corp is responsible for synthesis, and purity is more than 98%, freezes and preserves.Colloidal gold solution (40nm) is purchased from
Shanghai one Bioisystech Co., Ltd of outstanding person.
2nd, the pretreatment of sample pad and label pad
Sample pad and label pad are polyester film material, and immersion treatment need to be carried out with pretreatment fluid using before.Pretreatment fluid
It is phosphate buffer (pH7.0～7.5), the non-ionic surface of 3g/100ml～6g/100ml by 0.01～0.02mol/L
It is mixed that the Macrogol 6000 of activating agent, the tween of 1ml/100ml～4ml/100ml and 5g/100ml～20g/100ml is constituted
Close liquid.Polyester film material is soaked into 5～24h in pretreatment fluid, is taken out after 37～45 DEG C of exhausting drying, sealed after drying standby
With.Pretreated sample pad can be directly used for the assembling of test strips, and label pad need to be used for after the coating of label is processed
The assembling of test strips.
3rd, the coating of EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label pad is processed
(1) preparation of EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label thing solution：Under magnetic agitation, use
0.1M potassium carbonate or hydrochloric acid solution adjust the pH value of colloidal gold solution to 7.4, add in the ratio of 1mg antibody/ml colloidal gold solutions
Enter EV71 virus VP 1 proteantigen polypeptide fragments, be subsequently adding 0.9% sodium chloride solution isopyknic with reaction system, mix
Stirring reaction 2h, adds final concentration of 0.3% bovine serum albumin(BSA), adjusts pH value of solution to 8.5, stands 30min.Will be above-mentioned molten
Liquid is centrifuged 15min at 14000rpm, 4 DEG C, abandons supernatant, the sediment 0.02M of 1/20 initial colloid gold solution volume
(boric acid 0.1237g, PEG 20000 1g, with ultra-pure water 1L is settled to the borate buffer solution of pH9.0, adjusts pH to 9.0)
It is centrifuged again after resuspended, such repeated washing 3 times, thoroughly to remove unconjugated protein, finally gives EV71 virus VP 1 albumen
Antigenic polypeptide fragments-colloid gold label thing solution, save backup in 4 DEG C.
(2) coating of EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label pad is processed：Film instrument is sprayed with Isoflow
The EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label thing solution even application for preparing is being located in advance through step 2
In the label pad of reason, 0.03ml EV71 virus VP 1s proteantigen polypeptide fragments-colloid gold label thing solution is sprayed per 1cm, will
The mark for having sprayed is padded on drying and processing more than 12 hours under the conditions of humidity 15%, 30 DEG C of temperature, seals after drying standby.
4th, the coating of nitrocellulose filter is processed
Take EV71 virus VP 1 protein antibodies coating buffers (10mM, pH7.4 phosphate buffer containing 1% sucrose) dilution
To 1.5mg/ml, the control line (C lines) being then coated in Isoflow point film instruments on nitrocellulose filter；Take goat-anti people
IgA antibody coating buffer (10mM, pH7.4 phosphate buffer containing 1% sucrose) is diluted to 1.0mg/ml, Ran Houyong
Isoflow point film instruments are coated in the detection line on nitrocellulose filter (T lines).The nitrocellulose filter being coated with is in humidity
Drying and processing more than 12 hours under the conditions of 10-30%, temperature 20-35 DEG C, seal standby after drying.
5th, assembling, slitting
Accompanying drawing 1 is referred to, accompanying drawing 1 is the structural representation of the EV71 virus IgA antibody test strips of the present invention.To inhale
Water paper 4, nitrocellulose filter 3, pad 2, sample pad 1 are sticked to successively on PVC backboards 5, are then adjusted cutting machine parameter and are entered
Row slitting, is cut into the test strips of 4mm width.
【Embodiment 3】The detection (prize law) of EV71 virus IgA antibodies
Accompanying drawing 2 is referred to, the saliva acquisition rod of accompanying drawing 2 discharges the method schematic diagram of saliva sample.The present invention is adopted by saliva
Collection rod 22 adsorbs saliva sample in patient oral cavity, aforementioned acquisition rod is placed into and (is contained equipped with 1-2ml sample diluting liquids 23
The PBS 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 for, the cotton swab head liquid of acquisition rod is extruded, discard acquisition rod.
Accompanying drawing 3 is referred to, Fig. 3 is the detection method schematic diagram of the EV71 virus IgA antibody test strips of the present invention.Take
【Embodiment 1】The EV71 virus IgA antibody test strips of preparation are put in the above-mentioned sample cell 21 equipped with saliva sample, sample
Pad end in contact saliva sample, places 15-30 minutes, then visual results.
Cleaning Principle：Due to capillarity, saliva sample flows 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 thing, the colloid gold label thing is redissolved into into completely free state.If saliva sample
Containing EV71 virus IgA antibodies in product, can be combined with free goat-anti people IgA antibody-colloid gold label thing, formation collaurum-
Goat-anti people IgA antibody-IgA antibody compound, the compound is up to detection line (T lines) place of nitrocellulose filter, then with its
Upper coated EV71 virus VP 1s proteantigen polypeptide fragment specific bond, is presented red stripes (accompanying drawing 4), unnecessary free glue
Body gold-goat-anti people's IgA antibody continues control line (C lines) place for being up to nitrocellulose filter, with coated people's IgA antibody thereon
Specific binding, forms collaurum-goat-anti people IgA antibody-people's IgA antibody compound, and red stripes (accompanying drawing 4) are presented.If
EV71 virus IgA antibodies are not contained in sample, free goat-anti people IgA antibody-colloid gold label thing can cross detection line (T
Line), continue control line (C lines) place for being up to nitrocellulose filter, with coated people's IgA antibody specific binding thereon, shape
Into collaurum-goat-anti people IgA antibody-people's IgA antibody compound, red stripes (accompanying drawing 5) are presented.If nitrocellulose filter
Control line (C lines) place does not form red stripes, illustrates that test strips fail, as a result unreliable (accompanying drawing 6).
Testing result judges：If detection line (T lines) and control line (C lines) occurs in test strip simultaneously, sample is illustrated
It is positive, containing IgA antibody, that is, indicates EV71 viruses infection (accompanying drawing 4)；If only there is control line (C lines), sample is illustrated
Be negative (accompanying drawing 5), without IgA antibody, that is, indicates without EV71 virus infection；If there is not any line or only occurring
Detection line (T lines), illustrates this test strips invalid (accompanying drawing 6).
【Embodiment 4】The detection (indirect method) of EV71 virus IgA antibodies
Accompanying drawing 2 is referred to, the saliva acquisition rod of accompanying drawing 2 discharges the method schematic diagram of saliva sample.The present invention is adopted by saliva
Collection rod 22 adsorbs saliva sample in patient oral cavity, aforementioned acquisition rod is placed into and (is contained equipped with 1-2ml sample diluting liquids 23
The PBS 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 for, the cotton swab head liquid of acquisition rod is extruded, discard acquisition rod.
Accompanying drawing 3 is referred to, Fig. 3 is the detection method schematic diagram of the EV71 virus IgA antibody test strips of the present invention.Take
【Embodiment 1】The EV71 virus IgA antibody test strips of preparation are put in the above-mentioned sample cell 21 equipped with saliva sample, sample
Pad end in contact saliva sample, places 15-30 minutes, then visual results.
Cleaning Principle：Due to capillarity, saliva sample flows 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 thing, the colloid gold label thing is redissolved into completely free
State.If containing EV71 virus IgA antibodies in saliva sample, could be with free EV71 virus VP 1s proteantigen polypeptide fragment-glue
Body gold label is combined, and forms collaurum-EV71 virus VP 1s proteantigen polypeptide fragment-IgA antibody compound, the compound
Be up to detection line (T lines) place of nitrocellulose filter, then with coated goat-anti people IgA antibody specific bond thereon, form glue
Body gold-EV71 virus VP 1s proteantigen polypeptide fragment-IgA antibody-goat-anti people's IgA antibody compound, is presented red stripes (attached
Fig. 4), unnecessary free collaurum-EV71 virus VP 1s proteantigen polypeptide fragment continues to be up to the control of nitrocellulose filter
Line (C lines), with coated EV71 virus VP 1s protein antibodies specific binding thereon, forms collaurum-EV71 virus VP 1 albumen
Antigenic polypeptide fragments-EV71 virus VP 1 protein antibodies compounds, are presented red stripes (accompanying drawing 4).If do not contained in sample
EV71 virus IgA antibodies, free EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label thing 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
With reference to, collaurum-EV71 virus VP 1 proteantigen polypeptide fragment-EV71 virus VP 1 protein antibodies compounds are formed, present red
Vitta band (accompanying drawing 5).If the control line (C lines) of nitrocellulose filter does not form red stripes, illustrate that test strips fail, knot
Really unreliable (accompanying drawing 6).
Testing result judges：If detection line (T lines) and control line (C lines) occurs in test strip simultaneously, sample is illustrated
It is positive, containing IgA antibody, that is, indicates EV71 viruses infection (accompanying drawing 4)；If only there is control line (C lines), sample is illustrated
It is negative, without IgA antibody, that is, indicates without EV71 viruses infection (accompanying drawing 5)；If there is not any line or only occurring
Detection line (T lines), illustrates this test strips invalid (accompanying drawing 6).
【Embodiment 5】EV71 virus IgA antibody test strip methods of the present invention and fluorescence PCR method Comparative result
Laboratory sample 88 is chosen, the saliva sample and Pharyngeal swab samples of patient is gathered respectively, respectively with EV71 virus IgA
Antibody test test strips method is tested with fluorescence PCR method, as a result such as table 1：
EV71 virus IgA antibody test strip methods and the fluorescence PCR method comparative result of the present invention of table 1
EV71 viruses IgA antibody test strip method of the present invention compares with fluorescence PCR method, and sensitivity reaches
97.50%, specificity 87.50% possesses actual application value to EV71 virus screenings.
【Embodiment 6】EV71 virus IgA antibody test strip methods of the present invention and serum IgM antibody detection method result
Contrast
88, sample is chosen, the saliva sample and blood sample of patient are gathered respectively, respectively with EV71 virus IgA antibody inspections
Test paper slip method is tested with serum IgM antibody detection method, as a result such as table 2：
EV71 virus IgA antibody test strip methods and the fluorescence PCR method comparative result of the present invention of table 2
EV71 virus IgA antibody test strip methods of the present invention and serum IgM antibody Comparison between detecting methods, sensitivity reaches
To 95.00%, specificity 89.58% possesses actual application value to EV71 virus screenings.
Although above with a general description of the specific embodiments the present invention is described in detail,
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, without departing from theon the basis of the spirit of the present invention these modifications or improvements, belong to the scope of protection of 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 (10)
1. a kind of EV71 virus IgA antibody test strip, it is characterised in that including sample pad, label pad, celluloid
Film, blotting paper and backboard, described nitrocellulose filter is provided with EV71 virus VP 1s proteantigen polypeptide fragment detection line and people
IgA antibody control line, described label pad is provided with anti-human IgA antibody-colloid gold label thing.
2. EV71 according to claim 1 virus IgA antibody test strip, it is characterised in that the EV71 virus VP 1s
The amino acid sequence of proteantigen polypeptide fragment is as shown in sequence table Seq ID NO.1.
3. EV71 according to claim 1 virus IgA antibody test strip, it is characterised in that the anti-human IgA antibody
It is many selected from goat-anti people's IgA polyclonal antibodies, the anti-human IgA monoclonal antibodies of mouse, rabbit-anti people IgA monoclonal antibodies or rabbit-anti people IgA
Clonal antibody.
4. EV71 according to claim 1 virus IgA antibody test strip, it is characterised in that the blotting paper, nitric acid
Cellulose membrane, label pad, sample pad are sticked to successively on the backboard.
5. a kind of EV71 virus IgA antibody test strip, it is characterised in that including sample pad, label pad, celluloid
Film, blotting paper and backboard, described nitrocellulose filter is provided with anti-human IgA antibody detection line and EV71 virus VP 1 protein antibodies
Control line, described label pad is provided with EV71 virus VP 1s proteantigen polypeptide fragment-colloid gold label thing.
6. EV71 according to claim 5 virus IgA antibody test strip, it is characterised in that the EV71 virus VP 1s
The amino acid sequence of proteantigen polypeptide fragment is as shown in sequence table Seq ID NO.1.
7. EV71 according to claim 5 virus IgA antibody test strip, it is characterised in that described anti-human IgA resists
Body is selected from goat-anti people's IgA polyclonal antibodies, the anti-human IgA monoclonal antibodies of mouse, rabbit-anti people IgA monoclonal antibodies or rabbit-anti people IgA
Polyclonal antibody；Described EV71 virus VP 1s protein antibodies resist selected from the anti-EV71 virus VP 1s protein monoclonal antibody of mouse, people
EV71 virus VP 1 protein polyclone antibodies, rabbit-anti EV71 virus VP 1 protein monoclonal antibody, rabbit-anti EV71 virus VP 1 albumen are more
Clonal antibody or goat-anti EV71 virus VP 1 protein polyclone antibodies.
8. EV71 according to claim 5 virus IgA antibody test strip, it is characterised in that the blotting paper, nitric acid
Cellulose membrane, label pad, sample pad are sticked to successively on backboard.
9. the EV71 virus IgA antibody test strips described in one of claim 1-8 are in saliva EV71 Viral diagnosis
In application.
10. application according to claim 9, it is characterised in that：Saliva sample is taken, in adding sample diluting liquid, Ran Houfang
Enter described EV71 virus IgA antibody test strips to be detected；Described sample diluting liquid is pure containing 0.5% ox blood
The PBS of albumen, 0.2% casein, 1% tryptone and 0.02% Sodium azide.
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