CN109765384A - A kind of canine coronavirus antibody fluorescence test strip and its preparation method and application - Google Patents
A kind of canine coronavirus antibody fluorescence test strip and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of canine coronavirus antibody fluorescence test strips and its preparation method and application.Test strips provided by the present invention include sample absorption pad, conjugate release pad a, conjugate release pad b, nitrocellulose filter, water absorption pad and bottom plate, the anti-dog antiantibody-biotin conjugates of chicken are coated on the conjugate release pad a, Streptavidin-Fluorescent microsphere marker is coated on the conjugate release pad b, there are detection line and nature controlling line on the nitrocellulose filter, it is coated with canine coronavirus N expression albumen in the detection line, is coated with goat-anti chicken antiantibody on the nature controlling line.Test strips of the invention have many advantages, such as high sensitivity, high specificity, easy to operate, economical and practical, it can be achieved that canine coronavirus antibody scene rapid quantitative detection.
Description
Technical field
The present invention relates to the detections of canine coronavirus antibody, and in particular to a kind of canine coronavirus antibody fluorescence Test paper
Item and its preparation method and application.
Technical background
Canine Coronavirus Infection is a kind of with dog caused by canine coronavirus (Canine coronavirus, CCV)
Acute gastroenteritis is the highly contagious disease of cardinal symptom, frequently to vomit, characterized by diarrhea, spiritual depressed and anorexia.
Two genotype, i.e. CCV-I and CCV- II has been determined in CCV at present.Under normal circumstances, CCV will lead to the abdomen that dog differs in weight
It rushes down, the other pathogens of concurrent infection dog will cause mortality final result.The disease is in worldwide distribution, China Yi You army police dog and the people
The report fallen ill with dog.With the pernicious infection disease vaccine of the dogs such as canine distemper, canine parvoviral enteritis succeeding in developing at home
And extensive use, these diseases gradually decrease the harm of dog, and canine Coronavirus Infection is not due to having a kind of effective control
Method endangers canine farming serious, the more and more attention by numerous animal doctors circle personage.CCV has multiform state property, and size is not
One, it is most rounded or oval, there is one layer of approximate double-deck cyst membrane outside, petal sample protrusion is distributed on cyst membrane, is about 20nm.
CCV is made of 4 kinds of structural proteins: fibre is dashed forward as a kind of glycoprotein (S), molecular weight 220kD；Memebrane protein (M), molecular weight 32kD；It is small
Memebrane protein (SM), molecular weight 912kD；Nucleoprotein (N), molecular weight 50kD combine closely with the underlying stock RNA of virus.1992,
Horsburgh measures the gene order that 916kb is held in CCV-Insavc-1 strain 3 ', and it comprises in addition to polymerase area for analytical proof
Encoded information.Share 10 Open reading frames (ORF) from 5 ' ends to 3 ' ends: 1b, 2S, 3a, 3x, 3b, 4SM, 5M, 6N, 7a,
7b, wherein S, SM, M and the ORF of N protein are the encoding gene of virus structural protein, remaining is non-structural protein encoding gene, mesh
The function of albumen coded by preceding nonstructural protein gene is not fully understood.
Canine coronavirus disease mainly passes through vaccine prevention, and knot will be generated in vaccine immunity animal and infection animal body
Structure protein antibodies, therefore the diagnostic method of detection structure protein antibodies can determine that animal to the resistance of Strain.It is daily simultaneously
It is that the daily monitoring to group selects vaccine, assessment immune programme reasonability, grasps health status that monitoring vaccine, which generates antibody,
Important means, while can also reflect from side and manage main foundation that is whether reasonable, and vaccinating in due course.
The serological method for detecting CCV antibody established at present mainly have hemagglutination-inhibition test, neutralization test,
ELISA method and Colloidal Gold etc..Hemagglutination-inhibition test needs fresh and high sensibility red blood cell, and detection time needs 2
~3h；Neutralization test is cumbersome, needs higher experimental condition and operation horizontal, and entire test needs 1~2 week time could
Result out；ELISA method high operation requirements needs microplate reader measurement result, and is not suitable for doing the detection of single sample；Immune glue
Body gold method is although easy to detect, is not required to professional technician and working environment, but the sensitivity of its detection is low, and can only be qualitative,
Detection range is narrow.These all limit their applications in the detection of canine coronavirus antibody clinical, therefore, the invention patent
Purpose is exactly that it is anti-to establish a kind of canine coronavirus easy to operate, sensitive, special, quick the shortcomings that overcoming the above several method
Body quantitative detecting method, applied to the monitoring of canine coronavirus immune effect, to limit the prevalence of canine coronavirus.
Summary of the invention
It is an object of the present invention to provide a kind of canine coronavirus antibody fluorescence test strips.
Canine coronavirus antibody fluorescence test strip provided by the present invention, including the release of sample absorption pad, conjugate
Pad a, conjugate release pad b, nitrocellulose filter, water absorption pad and bottom plate；It is anti-that the anti-dog of chicken is coated on the conjugate release pad a
Antibody-biotin conjugate；Streptavidin-Fluorescent microsphere marker is coated on the conjugate release pad b；The nitric acid
There are detection line and nature controlling line on cellulose membrane, canine coronavirus N expression albumen is coated in the detection line, on the nature controlling line
It is coated with goat-anti chicken antiantibody.
The anti-dog antiantibody of chicken used on the conjugate release pad a is the IgY of the anti-dog IgG of chicken, the nitrocellulose
The goat-anti chicken antiantibody that nature controlling line uses on film is the IgG of goat-anti chicken IgY.
The sample absorption pad, conjugate release pad a, conjugate release pad b, nitrocellulose filter, water absorption pad are sequentially solid
It is scheduled on the bottom plate.
It is a further object to provide a kind of preparation sides of above-mentioned canine coronavirus antibody fluorescence test strip
Method comprising step:
1) Quality Control that preparation has the detection line for being coated with canine coronavirus N expression albumen and is coated with goat-anti chicken antiantibody
The nitrocellulose filter of line；
2) preparation is coated with the conjugate release pad a of the anti-dog antiantibody-biotin conjugates of chicken and to be coated with strepto- affine
Element-Fluorescent microsphere marker conjugate release pad b；
3) sample absorption pad, conjugate release pad a, conjugate release pad b, nitrocellulose filter, water absorption pad is sequentially solid
It is scheduled on bottom plate.
Specifically, step includes:
1) expression and purifying of canine coronavirus N protein；
2) 1) the canine coronavirus N protein of expression is coated on composition detection line (T) on nitrocellulose filter, it will be commercially available
Goat-anti chicken antiantibody is coated on composition nature controlling line (C) on nitrocellulose filter；
3) the anti-dog antiantibody-biotin conjugates of commercially available chicken are sprayed in conjugate release pad, after 37 DEG C of drying 2h
It takes out, as conjugate release pad a is placed in dry environment and saves backup；
4) Streptavidin is added in the fluorescent microsphere solution activated, prepares Streptavidin-fluorescent microsphere mark
Remember object；
5) Streptavidin-Fluorescent microsphere marker by 4) preparation is sprayed in conjugate release pad, 37 DEG C of drying 2h
After take out, as conjugate release pad b is placed in dry environment and saves backup；
6) sample absorption pad is used to the 0.02mol/L phosphate-buffered containing 0.5% bovine serum albumin(BSA) (BSA), pH 7.2
Liquid impregnates 2h, and 37 DEG C of drying 2h are spare；
7) sample absorption pad, conjugate release pad a, conjugate release pad b, nitrocellulose filter, water absorption pad are successively pressed
Sequence is pasted on bottom plate, and the small item of 3.95mm wide is cut into machine, close with aluminium foil bag in special plastics fabrication shell
It seals, can be reserved for 12 months under the conditions of 2~30 DEG C.
Application of the above-mentioned canine coronavirus antibody fluorescence test strip in detection canine coronavirus antibody is also this hair
The range of bright protection.
Canine coronavirus antibody fluorescence test strip of the invention is reacted and is exempted from using the antibody antigen of high degree of specificity
Epidemic disease Chromatographic techniques fix the anti-dog antiantibody-biotin conjugates of chicken and Streptavidin-Fluorescent microsphere marker respectively
In in two conjugate release pads, the canine coronavirus antibody in sample is in flow process, first and on conjugate release pad a
The anti-dog antiantibody of chicken-biotin conjugates combine, then by biotin-Streptavidin system and conjugate release pad b
Streptavidin-Fluorescent microsphere marker combines, and forms antibody-antiantibody-fluorescent microsphere compound.Antibody in sample is by nitre
Canine coronavirus N expression albumen capture in acid cellulose film detection line, by fluorescent chromatographic analyzer quantitative detection sample
Canine coronavirus antibody titer.
Test strips of the invention have that high sensitivity, high specificity, at low cost, easy to operate, detection time is short, it is each to be suitble to
The advantages of kind unit uses, storage is simple, long shelf-life.Wherein, fluorescent microsphere label is effectively reduced on Streptavidin
Issuable steric hindrance, improves the joint efficiency of Ag-Ab when mark fluorescent microballoon, to improving detection sensitivity
There is larger help；And by the multi-level amplification of biotin-Streptavidin system, while improving sensitivity,
Do not increase nonspecific interference, can also reduce operating error, improves the accuracy of measurement.Test strips of the present invention can realize that dog is preced with
Shape antiviral antibody field quick detection, practical value and promotional value with higher.
Detailed description of the invention
Fig. 1 is test strips the schematic diagram of the section structure.
Fig. 2 is canine coronavirus antibody test canonical plotting.
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate this
Invention, and be not intended to limit the scope of the invention.In addition, the range that those skilled in the art limits in the appended claims
Interior to carry out various changes or modification to the present invention, these changes or modification should equally fall into protection scope of the present invention.
Embodiment 1: the composition (Fig. 1) of canine coronavirus antibody fluorescence test strip
The test strips are by bottom plate (8), sample absorption pad (1), conjugate release pad a (2), conjugate release pad b
(3), nitrocellulose filter (4), water absorption pad (5) composition；
The sample absorption pad (1), conjugate release pad a (2), conjugate release pad b (3), nitrocellulose filter (4),
Water absorption pad (5) is successively pasted onto order on PS bottom plate (8)；Conjugate release pad a (2) has 1/4 region by sample from starting point
Absorption pad (1) covering, conjugate release pad b (3) have 1/4 region to be combined object release pad a (2) covering, conjugate from starting point
The end of release pad b (3) is connect with the beginning of nitrocellulose filter (4), the end and water absorption pad (5) of nitrocellulose filter (4)
Beginning be connected, the beginning of sample absorption pad (1) is aligned with the beginning of PS bottom plate (8), the end of water absorption pad (5) and PS bottom plate
(8) end alignment；
There are detection line (6) and nature controlling line (7) on the nitrocellulose filter (4), detection line (T) and nature controlling line (C) are in
The perpendicular strip tape with the length of the test strips；Detection line (6) is located at the side close to the end of conjugate release pad b (3)；
Nature controlling line (7) is located remotely from the side of the end of conjugate release pad b (3).Test strips are cut into the small of 3.95mm wide with machine
Item is sealed in special plastics fabrication shell with aluminium foil bag, can be reserved for 12 months under the conditions of 2~30 DEG C.
Embodiment 2: the preparation of test strips described in embodiment 1
The preparation method of the test strips mainly comprises the steps that
1) Quality Control that preparation has the detection line for being coated with canine coronavirus N expression albumen and is coated with goat-anti chicken antiantibody
The nitrocellulose filter of line；
2) preparation is coated with the conjugate release pad a of the anti-dog antiantibody-biotin conjugates of chicken and to be coated with strepto- affine
Element-Fluorescent microsphere marker conjugate release pad b；
3) sample absorption pad, conjugate release pad a, conjugate release pad b, nitrocellulose filter, water absorption pad is sequentially solid
It is scheduled on bottom plate.
Substep narration in detail below:
(1) preparation of each component
1. the expression and purifying of canine coronavirus N protein
(1) CCV N protein partial gene sequence selects
1. DNA sequence dna: ATGAATACTATACCTCTTTCATTCTTCAACCCCATTACCCTCCAACAAGGTTCAAA AT
TTTGGAACTTATGTCCGAGAGACTTTGTACCCAAAGGAATAGGTAACAAGGATCAACAGATTGGTTATTGGAATAGA
CAAAGCCGCTATCGCATGGTGAAGGGTCAGCGTAAAGAGCTTCCTGAAAGGTGGTTCTTCTACTACTTAGGTACTGG
TCCTCATGCTGATGCTAAATTTAAAGATAGAATAGATGGAGTTGTCTGGGTTGCCAAGGATGGTGCCATGAATAAAC
CAACTACACTTGGTAATCGTGGTGCTAATAATGAATCCAAAGCTTTGAAATTCGATGGTAAAGTACCAGGAGAATTT
CAACTTGAAGTGAACCAATCAAGGGACAATTCAAGATCACGCTCTCAATCTAGATCTCAGTCTAGAAATAGATCTCA
ATCTAGAGGAAGGCAACAATCCAATAACAAGAAGGATGACAGTGTAGAACAAGCTGTCCTTGCTGCACTCAAAAAGT
TAGGTGTTGACACAGAAAAACAACAACAACGCTCTCGTTCTAAATCTAAAGAACGTAGCAACTCTAAGACAAGAGAC
ACTACACCTAAGAATGAAAACAAACACACCTGGAAGAGAACTGCAGGTAAAGGTGATGTGACAAAATTTTATGGAGC
TAGAAGTAGTTCAGCCAATTTTGGTGACAGCGATCTCGTTGCCAATGGGAACGGTGCCAAGCATTACCCACAACTGG
CTGAATGTGTTCCATCTGTATCTAGCATTCTGTTTGGAAGCTATTGGACTGCAAAGGAAGATGGCGACCAGATTGAA
GTCACATTCACACATAAATACCACTTGCCAAAGGATGATCCTAAGACTGGACAATTCCTTCAGCAGATTAATGCCTA
TGCTCGTCCATCAGAGGTGGCAAAAGAACAGAGACAACGTAAAGCTCGTTCTAAATCTGCAGAAAGGGTAGAGCAAT
AA
2. protein sequence: MNTIPLSFFNPITLQQGSKFWNLCPRDFVPKGIGNKDQQIGYWNRQSRYRMVKGQR K
ELPERWFFYYLGTGPHADAKFKDRIDGVVWVAKDGAMNKPTTLGNRGANNESKALKFDGKVPGEFQLEVNQSRDNSR
SRSQSRSQSRNRSQSRGRQQSNNKKDDSVEQAVLAALKKLGVDTEKQQQRSRSKSKERSNSKTRDTTPKNENKHTWK
RTAGKGDVTKFYGARSSSANFGDSDLVANGNGAKHYPQLAECVPSVSSILFGSYWTAKEDGDQIEVTFTHKYHLPKD
DPKTGQFLQQINAYARPSEVAKEQRQRKARSKSAERVEQ
(2) expression and purification
1. direct gene synthesizes, connect on carrier to pET-28a, restriction enzyme site EcoR1, XhoI；
2. inductive condition: 37 DEG C are shaken OD600 or so, and the IPTG of 1mmol/L is added to induce, 22 DEG C, 160r/min induction 3h
Bacterium is received in left and right；
3. purifying protein condition: 100mL bacterium is resuspended with 200mL equilibrium liquid, ultrasonic (power 25%, ultrasonic 70min), centrifugation
(10000r/min, 20min) receives supernatant, crosses HIS column.
(3) process is purified
1. preparation of samples: sample suggests reducing impurity, raising protein purification efficiency by 0.45 μm of membrane filtration before loading
With prevent blocking pillar；
2. Sample Purification on Single: column being packed into chromatographic column, is balanced with the combination buffer of 5 times of column volumes；Sample is added
Into HIS nickel column balanced, repeatedly twice；It is cleaned with the miscellaneous buffer that washes of 5 times of column volumes；With 5~10 times of volumes
Elution buffer, collect eluent, i.e. destination protein component.
3. being concentrated by ultrafiltration: protein content is lower after purification, and with the ultrafiltration concentration pipe protein concentrate of 30kd and to reduce imidazoles dense
Degree.Former imidazoles is replaced with into PBS.
(4) SDS-PAGE is detected
Purification effect is detected using SDS-PAGE, discovery has apparent band together among 40~55kDa, with purpose egg
Bai great little is consistent, it was demonstrated that succeeded express express target protein.
2. the preparation of Streptavidin-Fluorescent microsphere marker
(1) activation of fluorescent microsphere
1% fluorescent microsphere of 4 DEG C of placements is taken, restores room temperature, takes 50 μ L to set in PE pipe with pipettor after shaking up, 450 μ are added
L ultrapure water carries out 10 times of dilutions (0.1%, volume fraction), this is working solution；Activator EDC, NHS are weighed, with selected ratio point
Oscillation Jia Ru not be protected from light in working solution, react 15min；15000r/min is centrifuged 10min；It discards supernatant, takes 500 μ L PBS multiple
It is molten, it shakes up.
MES solution is prepared: it weighs 1.82g MES and is dissolved in 90mL ultrapure water, required pH is adjusted to 2mol/L NaOH,
It is settled to 100mL, packing freezes, and room temperature is returned back to before use.
Activator EDC is prepared: being weighed 10mg EDC, is drawn the MES solution 1mL dissolution of above-mentioned preparation, make final concentration of
10mg/mL is protected from light, ready-to-use.
Activator NHS is prepared: being weighed 10mg NHS, is drawn the MES solution 1mL dissolution of above-mentioned preparation, make final concentration of
10mg/mL is protected from light, ready-to-use.
(2) Streptavidin-Fluorescent microsphere marker preparation
Streptavidin is diluted to 1mg/mL with PBS, 20 μ L of addition are interior to the fluorescent microsphere (500 μ L) activated, mix
It is even, it is protected from light oscillation, marks 4h；15000r/min is centrifuged 10min, discards supernatant.
(3) Streptavidin-Fluorescent microsphere marker closing
It takes 500 μ L confining liquids to redissolve marked good fluorescent microsphere, is protected from light oscillation, close 1h；15000r/min centrifugation
10min is discarded supernatant.
Confining liquid: the 0.02mol/L phosphate buffer containing 5%BSA (mass fraction), pH 7.2.
(4) Streptavidin-Fluorescent microsphere marker redissolution
It takes 500 μ L to redissolve liquid to redissolve the Streptavidin closed-Fluorescent microsphere marker, mixes 4 DEG C of ice of postposition
Case is spare.
Redissolve liquid: the 0.02mol/L phosphate buffer containing 0.5%BSA (mass fraction), pH 7.2.
3. the preparation of conjugate release pad
Conjugate release pad is soaked in the 0.02mol/L phosphate-buffered containing 0.3%Cas (mass fraction), pH 7.4
In liquid, 2h is uniformly soaked, 37 DEG C are dried for standby.Film instrument is drawn by the anti-dog antiantibody-biotin conjugates of commercially available chicken with Bio dot
(conjugate release pad a), it is anti-that every 1cm conjugate release pad sprays the 0.7 anti-dog of μ L chicken to even application in conjugate release pad
Body-biotin conjugates is subsequently placed in 37 DEG C of environment (humidity < 20%) and takes out after 2h, is placed in dry environment (humidity <
20%) it is saved backup in.
Film instrument is drawn with Bio dot to release the Streptavidin prepared-Fluorescent microsphere marker even application in conjugate
Putting pad, above (conjugate release pad b), every 1cm conjugate release pad spray 0.5 μ L Streptavidin-Fluorescent microsphere marker, so
It is placed in 37 DEG C of environment (humidity < 20%) and is taken out after 2h, be placed in dry environment (humidity < 20%) and save backup.
4. the preparation of nitrocellulose filter
The canine coronavirus N protein of expression is coated on nitrocellulose filter and constitutes detection line, by goat-anti chicken antiantibody
It is coated on nitrocellulose filter and constitutes nature controlling line.
Coating process: being diluted to 5 μ g/mL for the canine coronavirus N protein of expression with phosphate buffer, is drawn with Bio dot
Film instrument is coated in the detection line (T) on nitrocellulose filter, and package amount is 1.0 μ L/cm；With 0.01mol/L, pH's 7.2
Goat-anti chicken antiantibody is diluted to 200 μ g/mL by phosphate buffer, is drawn film instrument with Bio dot and is coated in nitrocellulose
Nature controlling line (C) on film, package amount are 1.0 μ L/cm.Dry 16h under the conditions of the nitrocellulose filter being coated with is placed in 37 DEG C,
It is spare.
5. the preparation of sample absorption pad
0.02mol/L phosphate buffer of the sample absorption pad containing 0.5%BSA, pH 7.2 is impregnated into 2h, 37 DEG C of drying
2h is spare.
(2) assembling of each component
Sample absorption pad, conjugate release pad a, conjugate release pad b, nitrocellulose filter, water absorption pad are successively pressed suitable
Sequence is pasted on PS bottom plate；Conjugate release pad a has 1/4 region to be absorbed by the sample pad covering, conjugate release pad b from starting point
There is 1/4 region to be combined object release pad a covering, the end of conjugate release pad b and the beginning of nitrocellulose filter from starting point
Connection, the end of nitrocellulose filter are connected with the beginning of water absorption pad, and the beginning of sample absorption pad is aligned with the beginning of PS bottom plate,
The end of water absorption pad is aligned with the end of PS bottom plate；Have detection line (T) and nature controlling line (C) on nitrocellulose filter, detection line and
Nature controlling line is in the strip tape perpendicular with the length of the test strips；Detection line is located at close to the end of conjugate release pad b
Side；Nature controlling line is located remotely from the side of the end of conjugate release pad b.Test strips are cut into the small of 3.95mm wide with machine
Item is sealed in special plastics fabrication shell with aluminium foil bag, can be reserved for 12 months under the conditions of 2~30 DEG C.
Embodiment 3: the use of test strips described in embodiment 1
1. the preparation of sample
(1) plasma sample: with anticoagulant blood sampling tube, or being first added EDTA anti-coagulants in heparin tube, and acquisition blood is added
And slowly shake up, 3000~5000r/min room temperature is centrifuged 10~30min (adjusting in right amount according to blood sampling volume).It is clear to draw upper layer yellow
Bright liquid is plasma sample.
(2) serum sample: acquisition blood is added in heparin tube and (is free of anti-coagulants), room temperature slant setting has to upper layer
3000~5000r/min room temperature centrifugation 10~30min (being adjusted in right amount according to blood sampling volume) when clear yellow liquid is precipitated.Draw upper layer
Clear yellow liquid is serum sample.
(3) Sample Dilution: taking the above-mentioned sample restored to room temperature, 5 μ L is accurately drawn with pipettor, Sample Dilution is added
It manages in (the 0.02mol/L phosphate buffer containing 1000 μ L pH 7.2), mixes well, this is prepare liquid, it is disposed vertically, to
It surveys.
2. test strips detect
It draws 70 μ L of prepare liquid with pipettor vertically to drip in well, liquid flowing starts timing, reacts 10min, adds
Outside, by the detection hole of test strips insertion fluorescent chromatographic analyzer, Pen-down detection key will automatically carry out test strips sample nose end
Interpretation.If not occurring C line, shows that incorrect operating process or test strips have been failed, retested using new test strips.
3. testing result interpretation
Canine coronavirus antibody standard substance is configured to series of concentrations with Sample dilution: 0,1/1024,1/256,1/64,
1/16,1/4, it takes 70 μ L to be added drop-wise in the well of test strips, detection line and matter is read by fluorescent chromatographic analyzer after 10min
The ratio of line signal strength, i.e. T/C value (each concentration point measures 4 times respectively, is averaged) are controlled, corresponding standard curve is drawn
(Fig. 2).
Using above-mentioned standard curve detection clinical sample, by the standard curve Y=(a-d) of drafting/[1+ (x/c)b] in+d
A, b, c, d value input fluorescent chromatographic analyzer can immediately arrive at canine coronavirus in actual sample when measuring actual sample
The titre of antibody.
As a result it explains:
| Testing result | Clinical application reference |
| <1 | Antibody level is extremely low, such as animal health, it is proposed that vaccine inoculation immediately |
| 1~3 | Antibody level is lower, such as animal health, it is proposed that in due course vaccine inoculation |
| 3~4 | Antibody level is moderate, and animal is centainly protected, it is proposed that periodic monitoring antibody level |
| 4~5 | Antibody level is higher, and animal is centainly protected, and the wild poison of contact should be avoided |
| 5~6 | Antibody level is high, and animal is protected, and the wild poison of contact should be avoided |
| >6 | Antibody level is very high, and animal has the wild malicious risk of contact in the recent period, needs close observation |
Embodiment 4: the evaluation of test strips described in embodiment 1
1. precision measures
Canine coronavirus antibody standard substance is configured to series of concentrations with Sample dilution: 1/1024,1/64,1/4, respectively
(each concentration every batch of detects 24 parallel, detections three batches altogether respectively) is detected with test strips.
Crowd interior maximum CVs of each concentration is 7.8%, and average CVs is 6.5%；Each concentration batch between maximum CVs be 8.6%,
Average CVs is 7.6%, shows that test strips provided by the present invention have good repeatability.
2. Stability Determination
By the test strips prepared be placed on 4 DEG C, 25 DEG C, 37 DEG C, carry out accelerated test in 45 DEG C of environment.Respectively 7d,
14d, 21d and 28d take out, and the titre of canine coronavirus antibody in actual sample is detected with it.To measured concentration and practical sample
Product concentration values carry out error analysis, obtain CV value within 10% as a result, showing that test strips provided by the present invention have
Good stability.
3. relativity determination
Using the kit of test strips of the invention and control company A, 56 parts of dog serum samples are measured simultaneously, it is right
Measured value carries out correlation analysis, the results show that the related coefficient of two methods is R2=0.924, show test paper of the invention
Item and the correlation of import reagent measurement result are good, have specificity and accuracy well.
Sequence table
<110>Beijing Kwinbon Biotechnology Co., Ltd.
<120>a kind of canine coronavirus antibody fluorescence test strip and its preparation method and application
<140> 201910083502.8
<141> 2019-01-29
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 984
<212> DNA
<213> Canine coronavirus
<400> 1
atgaatacta tacctctttc attcttcaac cccattaccc tccaacaagg ttcaaaattt 60
tggaacttat gtccgagaga ctttgtaccc aaaggaatag gtaacaagga tcaacagatt 120
ggttattgga atagacaaag ccgctatcgc atggtgaagg gtcagcgtaa agagcttcct 180
gaaaggtggt tcttctacta cttaggtact ggtcctcatg ctgatgctaa atttaaagat 240
agaatagatg gagttgtctg ggttgccaag gatggtgcca tgaataaacc aactacactt 300
ggtaatcgtg gtgctaataa tgaatccaaa gctttgaaat tcgatggtaa agtaccagga 360
gaatttcaac ttgaagtgaa ccaatcaagg gacaattcaa gatcacgctc tcaatctaga 420
tctcagtcta gaaatagatc tcaatctaga ggaaggcaac aatccaataa caagaaggat 480
gacagtgtag aacaagctgt ccttgctgca ctcaaaaagt taggtgttga cacagaaaaa 540
caacaacaac gctctcgttc taaatctaaa gaacgtagca actctaagac aagagacact 600
acacctaaga atgaaaacaa acacacctgg aagagaactg caggtaaagg tgatgtgaca 660
aaattttatg gagctagaag tagttcagcc aattttggtg acagcgatct cgttgccaat 720
gggaacggtg ccaagcatta cccacaactg gctgaatgtg ttccatctgt atctagcatt 780
ctgtttggaa gctattggac tgcaaaggaa gatggcgacc agattgaagt cacattcaca 840
cataaatacc acttgccaaa ggatgatcct aagactggac aattccttca gcagattaat 900
gcctatgctc gtccatcaga ggtggcaaaa gaacagagac aacgtaaagc tcgttctaaa 960
tctgcagaaa gggtagagca ataa 984
Claims (5)
1. a kind of canine coronavirus antibody fluorescence test strip, including sample absorption pad, conjugate release pad a, conjugate are released
Put pad b, nitrocellulose filter, water absorption pad and bottom plate, it is characterised in that: it is anti-that the anti-dog of chicken is coated on the conjugate release pad a
Antibody-biotin conjugate；Streptavidin-Fluorescent microsphere marker is coated on the conjugate release pad b；The nitric acid
There are detection line and nature controlling line on cellulose membrane, canine coronavirus N expression albumen is coated in the detection line, on the nature controlling line
It is coated with goat-anti chicken antiantibody.
2. canine coronavirus antibody fluorescence test strip according to claim 1, it is characterised in that: the conjugate is released
The anti-dog antiantibody of chicken used on pad a is put as the IgY of the anti-dog IgG of chicken, the goat-anti that nature controlling line uses on the nitrocellulose filter
Chicken antiantibody is the IgG of goat-anti chicken IgY.
3. canine coronavirus antibody fluorescence test strip according to claim 1 to 2, it is characterised in that: the sample
Product absorption pad, conjugate release pad a, conjugate release pad b, nitrocellulose filter, water absorption pad are sequentially fixed on the bottom plate.
4. a kind of preparation method of any canine coronavirus antibody fluorescence test strip of claim 1-3, feature
It is: includes the following steps:
1) preparation is with the detection line and the nature controlling line for being coated with goat-anti chicken antiantibody for being coated with canine coronavirus N expression albumen
Nitrocellulose filter；
2) preparation is coated with the conjugate release pad a of the anti-dog antiantibody-biotin conjugates of chicken and to be coated with Streptavidin-glimmering
The conjugate release pad b of light microballoon marker；
3) sample absorption pad, conjugate release pad a, conjugate release pad b, nitrocellulose filter, water absorption pad are sequentially fixed on
On bottom plate.
5. any canine coronavirus antibody fluorescence test strip of claim 1-3 is in detection canine coronavirus antibody
Application.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111233985A (en) * | 2020-02-09 | 2020-06-05 | 北京丹大生物技术有限公司 | Preparation method of novel coronavirus IgA antibody rapid detection test strip |
| CN111273017A (en) * | 2020-03-02 | 2020-06-12 | 江苏扬新生物医药有限公司 | Fluorescence immunochromatography kit for rapidly detecting novel coronavirus |
| CN111303297A (en) * | 2020-02-14 | 2020-06-19 | 中国人民解放军总医院 | Recombinant protein for detecting 2019 novel coronavirus antibody by double-antigen sandwich method, test strip, preparation method and application |
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| CN111665365A (en) * | 2020-05-22 | 2020-09-15 | 无锡市孚维尔生物医疗科技有限公司 | Novel coronavirus 2019-nCoV antibody spectrum detection kit |
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| CN111948381A (en) * | 2020-08-21 | 2020-11-17 | 苏州阿科索生物科技有限公司 | Canine coronavirus fluorescence aptamer test strip |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4567042A (en) * | 1983-06-15 | 1986-01-28 | American Home Products Corporation | Inactivated canine coronavirus vaccine |
| CN103687615A (en) * | 2011-06-28 | 2014-03-26 | 尼古拉·德卡罗 | Canine coronavirus vaccine |
| CN103675261A (en) * | 2013-08-12 | 2014-03-26 | 南昌大学 | Method for marking clenbuterol monoclonal antibody by fluorescent microsphere |
| CN107831317A (en) * | 2017-11-01 | 2018-03-23 | 杭州微瑞科技有限公司 | Canine coronavirus antibody Quantitative detection card and application method |
| CN108414748A (en) * | 2018-01-30 | 2018-08-17 | 深圳市伯劳特生物制品有限公司 | A kind of test strip and detection method of THSD7A antibody |
-
2019
- 2019-01-29 CN CN201910083502.8A patent/CN109765384B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4567042A (en) * | 1983-06-15 | 1986-01-28 | American Home Products Corporation | Inactivated canine coronavirus vaccine |
| CN103687615A (en) * | 2011-06-28 | 2014-03-26 | 尼古拉·德卡罗 | Canine coronavirus vaccine |
| CN103675261A (en) * | 2013-08-12 | 2014-03-26 | 南昌大学 | Method for marking clenbuterol monoclonal antibody by fluorescent microsphere |
| CN107831317A (en) * | 2017-11-01 | 2018-03-23 | 杭州微瑞科技有限公司 | Canine coronavirus antibody Quantitative detection card and application method |
| CN108414748A (en) * | 2018-01-30 | 2018-08-17 | 深圳市伯劳特生物制品有限公司 | A kind of test strip and detection method of THSD7A antibody |
Non-Patent Citations (1)
| Title |
|---|
| 卓国荣 等: "快速胶体金免疫层析试纸条检测犬血清弓形虫IgG抗体的研究", 《黑龙江畜牧兽医》 * |
Cited By (13)
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|---|---|---|---|---|
| CN111856019A (en) * | 2020-02-04 | 2020-10-30 | 潍坊市康华生物技术有限公司 | Test strip, kit and detection method for detecting novel coronavirus (2019-nCoV) antigen |
| CN111233985A (en) * | 2020-02-09 | 2020-06-05 | 北京丹大生物技术有限公司 | Preparation method of novel coronavirus IgA antibody rapid detection test strip |
| CN111303297B (en) * | 2020-02-14 | 2021-08-31 | 中国人民解放军总医院 | Recombinant protein for detecting 2019 novel coronavirus antibody by double-antigen sandwich method, test strip, preparation method and application |
| CN111303297A (en) * | 2020-02-14 | 2020-06-19 | 中国人民解放军总医院 | Recombinant protein for detecting 2019 novel coronavirus antibody by double-antigen sandwich method, test strip, preparation method and application |
| CN111273017A (en) * | 2020-03-02 | 2020-06-12 | 江苏扬新生物医药有限公司 | Fluorescence immunochromatography kit for rapidly detecting novel coronavirus |
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| CN111948381A (en) * | 2020-08-21 | 2020-11-17 | 苏州阿科索生物科技有限公司 | Canine coronavirus fluorescence aptamer test strip |
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