CN109856407A - A kind of canine distemper virus antibody fluorescence test strip and its preparation method and application - Google Patents
A kind of canine distemper virus antibody fluorescence test strip and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of canine distemper virus 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 distemper virus 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 distemper virus antibody scene rapid quantitative detection.
Description
Technical field
The present invention relates to the detections of canine distemper virus antibody, and in particular to a kind of canine distemper virus antibody fluorescence Test paper
Item and its preparation method and application.
Technical background
Canine distemper (Canine Distemper, CD) is by canine distemper virus (Canine Distemper Virus, CDV)
A kind of acute, highly contagious disease caused by infection, is endangered to canine farming, fur animal farming and the conservation of wildlife
One of maximum epidemic disease of evil.CDV belongs to Paramyxoviridae Morbillivirus, is the ameristic RNA virus of minus strand sub-thread.CDV master
It will be by 6 hatching eggs such as nucleocapsid protein N, phosphoprotein P, matrix membrane protein M, fusion protein F, attachment or hemagluttinin proteins H, large protein L
White composition, wherein nucleocapsid protein N, fusion protein F and hemagluttinin proteins H are the main purpose antigen of dog immune system, are to produce
The important antigen of raw neutralizing antibody.Nucleocapsid protein N is the stronger immunogenic protein of conservative, removes epitope containing B cell, also contains
There is t cell epitope, plays a major role in the early immune response of canine distemper infection, strong antibody response can be caused.
Canine distemper virus 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 CDV 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 distemper virus antibody clinical, therefore, the invention patent
Purpose is exactly that it is anti-to establish a kind of canine distemper virus easy to operate, sensitive, special, quick the shortcomings that overcoming the above several method
Body quantitative detecting method, applied to the monitoring of canine distemper virus immune effect, to limit the prevalence of canine distemper virus.
Summary of the invention
It is an object of the present invention to provide a kind of canine distemper virus antibody fluorescence test strips.
Canine distemper virus 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 distemper virus 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 distemper virus antibody fluorescence test strip
Method comprising step:
1) Quality Control that preparation has the detection line for being coated with canine distemper virus 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 distemper virus N protein；
2) 1) the canine distemper virus 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 distemper virus antibody fluorescence test strip in detection canine distemper virus antibody is also this hair
The range of bright protection.
Canine distemper virus 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 distemper virus 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 distemper virus N expression albumen capture in acid cellulose film detection line, by fluorescent chromatographic analyzer quantitative detection sample
Canine distemper virus 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 hundstaupe
Fever virus 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 distemper virus 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 distemper virus 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 distemper virus 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 distemper virus N protein
(1) sequence information
1. CDV-N 275-523 gene order: tttggcattgaaactatgtatccggctcttgggttgcatgagtt
ttccggagagttaacaactattgaatcccttatgatgctatatcaacagatgggtgaaacagcaccgtacatggtt
attctggaaaattctgttcagaacaaatttagtgcaggatcctacccactgctctggagttatgctatgggagttg
gtgttgaacttgaaaactccatgggagggttaaatttcggtagatcctactttgatccggcctatttcaggctcgg
gcaagaaatggtgagaagatctgccggcaaagtaagctctgcacttgccgccgagcttggcatcaccaaggaagag
gctcagctagtgtcagaaatagcatccaagacaacggaggaccggacgattcgcactgctggtcccaagcaatctc
aaatcacttttctgcactcagaaagatccgaagtcactaatcaacaacccccaaccatcaacaagaggtccgaaaa
ccaaggaggagacaaataccccatccacttcaatgatgaacggtttccagggtacacccctgatgtcaacagctcc
gaatggagtgaatcacgctatgatacccagactattcaagatgatggaaacgacgatgaccggaaatcgatggaag
caatcgccaagatgagaatgcttactaagatgctcagtcaacctgggaccagtgaagagagttctcctgtctataa
tgatagagagctactcaat
2. amino acid sequence: PLGSPEFFGIETMYPALGLHEFSGELTTIESLMMLYQQMGETAPYMVILENSVQN
KFSAGSYPLLWSYAMGVGVELENSMGGLNFGRSYFDPAYFRLGQEMVRRSAGKVSSALAAELGITKEEAQLVSEIAS
KTTEDRTIRTAGPKQSQITFLHSERSEVTNQQPPTINKRSENQGGDKYPIHFNDERFPGYTPDVNSSEWSESRYDTQ
TIQDDGNDDDRKSMEAIAKMRMLTKMLSQPGTSEESSPVYNDRELLN
(2) gene chemical synthesis and vector construction
According to Gene Bank (Sequence ID:JN896331) sequence, the 275-523 amino acids sequence of N protein is synthesized
Column, and construct by restriction enzyme site of Nde1 and Xho1 into PET-28A plasmid.
(3) plasmid is converted into BL21 (DE3) competent cell
1. 2 μ L of plasmid is added in 100 μ L competent bacterias, 30min on ice is set.
2. 42 DEG C of heat shock 90s, set rapidly 5min in ice；500 μ L LB culture solutions are added.
3. 37 DEG C, 220r/min shaking 1h, are all coated on the LB plate of that resistance containing card after centrifugation, 37 DEG C of inversions trainings
It supports overnight.
(4) expression identification
1. monoclonal is inoculated in the test tube of 4mL LB culture solution of that resistance containing card on 11 plates of picking.
2. 37 DEG C, 220r/min shakes to thallus OD600It is 0.6~0.8.
3. taking out 1mL culture, 12000g room temperature is centrifuged 5min, abandons supernatant, and thallus is resuspended with 80 μ 1 × PBS buffer solution of L
Precipitate and be added 5 × Loading Buffer of 20 μ L.
4. IPTG to final concentration of 0.5mmol/L is added into remaining culture, 4h is shaken in 37 DEG C, 220r/min,
Induced fusion protein expression.
5. taking out 0.5mL culture, 12000g room temperature is centrifuged 5min, abandons supernatant, and bacterium is resuspended with 80 μ 1 × PBS buffer solution of L
Body precipitates and 5 × Loading Buffer of 20 μ L is added.
6. carrying out 12%SDS-PAGE analysis, expressing fusion protein is obvious.
(5) amplification expression and affinity purification
1. take optimal clone conservation bacterium to be seeded in the LB culture medium of 1L that resistance containing card, 37 DEG C, 220r/min shake to
OD600=0.6~0.8, after final concentration of 0.1mmol/L IPTG is added, 37 DEG C, 220r/min shaking 4h.
2. 8000r/min is centrifuged 15min, discards supernatant, collect all thallus.
3. using Buffer A (20mmol/L Imidazole, 20mmol/L Tris, 500mmol/L NaCl, pH8.0)
Thallus, ultrasonication (Φ 10,20%, 3s/4s, 30min) is resuspended, 8000r/min is centrifuged 15min, collects supernatant.
4. remove supernatant, inclusion body protein with Buffer B (20mmol/L Imidazole, 20mmol/L Tris,
500mmol/L NaCl, 8mol/L Urea, pH 8.0) soluble protein.
5. crossing column using the Buffer A of 40mL or so after activating nickel column.
6. inclusion body protein crosses 3 nickel columns repeatedly.
7. crossing column with the Buffer B of 20mL or so, foreign protein is washed away.
8. utilizing Buffer C (250mmol/L Imidazole, 20mmol/L Tris, 500mmol/L NaCl, 8mol/
L Urea, pH 8.0) elution, collect eluent.
9. SDS-PAGE electrophoresis detection.
(6) protein renaturation dialysis after purification
1. the sample cell after purification containing albumen is merged into two dialysis bands.
2. gradient dialysis renaturation, 8mol/L Urea-4mol/L Urea-2mol/L Urea-0.2mol/L PBS, every rank
Section dialysis 3h.
3. 5% or so glycerol are added, -20 DEG C of preservations.
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 distemper virus 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: the canine distemper virus N protein of expression is diluted to 500 μ g/mL with phosphate buffer, with Bio dot
It draws film instrument and is coated in the detection line (T) on nitrocellulose filter, package amount is 1.0 μ L/cm；With 0.01mol/L, pH 7.2
Phosphate buffer goat-anti chicken antiantibody is diluted to 300 μ g/mL, with Bio dot draw film instrument be coated in cellulose nitrate
Nature controlling line (C) on plain film, package amount are 1.0 μ L/cm.It is dry under the conditions of the nitrocellulose filter being coated with is placed in 37 DEG C
16h, 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 distemper virus 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 distemper virus 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 distemper virus 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 9.3%, and average CVs is 7.2%；Each concentration batch between maximum CVs be
10.3%, average CVs is 8.5%, 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 distemper virus 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
Comb enzyme using test strips of the invention and control ICL International Computer Limited exempts from kit, simultaneously to 64 parts of dog serum samples
It is measured, correlation analysis is carried out to measured value, the results show that the related coefficient of two methods is R2=0.932, show this
The test strips of invention and the correlation of import reagent measurement result are good, have specificity and accuracy well.
Claims (5)
1. a kind of canine distemper virus 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 distemper virus N expression albumen is coated in the detection line, on the nature controlling line
It is coated with goat-anti chicken antiantibody.
2. canine distemper virus 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 distemper virus 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 distemper virus 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 distemper virus 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 distemper virus antibody fluorescence test strip of claim 1-3 is in detection canine distemper virus antibody
Application.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN110699493A (en) * | 2019-12-04 | 2020-01-17 | 军事科学院军事医学研究院军事兽医研究所 | Primer, probe and kit for detecting panda canine distemper virus |
| CN112946252A (en) * | 2021-02-01 | 2021-06-11 | 福州海关技术中心 | Fluorescent test strip for identifying duck meat doped in beef and mutton and preparation method and application thereof |
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