CN106290868A - Fluorescent quantitation detection swine fever virus neutralizing antibody immune chromatography reagent kit - Google Patents
Fluorescent quantitation detection swine fever virus neutralizing antibody immune chromatography reagent kit Download PDFInfo
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- CN106290868A CN106290868A CN201610915797.7A CN201610915797A CN106290868A CN 106290868 A CN106290868 A CN 106290868A CN 201610915797 A CN201610915797 A CN 201610915797A CN 106290868 A CN106290868 A CN 106290868A
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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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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention discloses the immune chromatography reagent kit of a kind of fluorescent quantitation detection swine fever virus neutralizing antibody, including monolayer test strips and the composition that gets stuck, described get stuck by getting stuck upper cover and the lower cover that gets stuck is constituted, cover on getting stuck and offer well and display window, described display window is positioned at the surface of celluloid coated film, and the size of display window is equivalently-sized with celluloid coated film；Described test strips is sequentially overlapped to be pasted onto on vinyon base plate constituted by sample pad, labeling pad, celluloid coated film, absorbent paper, and is provided with detection band on celluloid coated film；The fluorescence immunoassay microsphere of coupling CSFV E 2 protein is contained in described labeling pad；It is coated with CSFV E 2 protein on described detection band.Test kit provided by the present invention has highly sensitive, high specificity, combining readout instrument can the advantage such as accurate quantitative analysis.
Description
Technical field
The invention belongs to animal medicine inspection field, relate to a kind of fluorescent quantitation detection swine fever virus neutralizing antibody immunity layer
Analysis test kit.
Background technology
Swine fever is a kind of Important Infectious Diseases of China's pig industry, belongs to a class animal epidemic, brings to the pig industry of China
Harm the most serious.Annual nearly 10,000,000,000 yuan of the economic loss caused to China, is to cause China's pig industry economic loss maximum
Disease.Due to its hazardness in the world and popularity, countries in the world scholar has carried out numerous studies, in the hope of setting up
A kind of quick, accurate, high sensitivity, easily operation and swine fever virus neutralizing antibody detection technique that can be quantitative.
Common correlation detection technology has: virus purification and qualification, newcastle disease virus strenuous test, the immunity of swine fever rabbit body
Mutual experiment, Immunofluorescent Antibody experiment, immune colloidal gold technique, indirect hemagglutination assay, neutralization test, enzyme linked immunological is inhaled
Adhesion test (ELISA), reverse transcription-polymerase chain reaction, fluorescent quantitative PCR technique, biochip technology.These detection skills existing
Art needs the laboratory operation personnel of specialty, is difficult to popularization and application, immune colloid gold skill in Chinese existing cultivation development scale layout
Although art simple and fast, but it cannot be quantitative, it is impossible to effectively instructs Swine Production.In order to break through the universal of existing detection technique
Bottleneck, match inspires confidence in biological invention and provides a kind of fluorescent quantitation detection hog cholera antibody immune chromatography reagent kit.
Summary of the invention
It is an object of the invention to break through the universal bottleneck of existing detection technique, it is provided that a kind of fluorescent quantitation detection swine fever virus
Neutralizing antibody immune chromatography reagent kit.The test kit detection high specificity of the present invention, highly sensitive, detection speed is fast, can realize
Detection by quantitative accurately.
For achieving the above object, present invention employs techniques below scheme:
The immune chromatography reagent kit of a kind of fluorescent quantitation detection swine fever virus neutralizing antibody, including monolayer test strips and getting stuck
Constitute, described get stuck by getting stuck upper cover and the lower cover that gets stuck fastens and constitutes, cover on getting stuck and offer well and display window,
Described well is positioned at the top of sample pad, and described display window is positioned at the surface of detecting pad, the size of display window and inspection
Survey the equivalently-sized of pad；Described test strips is sequentially overlapped by sample pad, labeling pad, detecting pad, absorbent paper and is pasted onto poly-second
Constitute on alkene plastics base plate, and on detecting pad, be provided with a detection band；Containing coupling hog cholera in described labeling pad
The fluorescence immunoassay microsphere of poison E2 albumen；It is coated with CSFV E 2 protein antigen on described detection band.
Further, the carboxyl modified that fluorescence immunoassay microsphere is 100～300nm of described coupling CSFV E 2 protein is micro-
Ball, the material of described sample pad be glass fibre, the base material of labeling pad be polyester fiber, the material of detecting pad is that nitric acid is fine
Dimension element coated film, the material of get stuck upper cover and the lower cover that gets stuck is plastics.
Further, excitation wavelength Ex of described fluorescent microsphere is 365nm, and launching wavelength Em is 620nm.
Further, described detection band be coated with concentration be 0.5mg/ml, consumption be the CSFV E 2 protein of 1 μ l/cm
Antigen.
Invention also discloses the preparation of the immune chromatography reagent kit of a kind of fluorescent quantitation detection swine fever virus neutralizing antibody
Method, comprises the steps:
(1) microsphere activation
1.a. takes 100～200 μ L fluorescence immunoassay microspheres in 1.5mL centrifuge tube, by equal-volume pH=5, concentration is
The morpholino b acid solution centrifugal of 25mMol/l washs 1～2 time, removes supernatant；
1.b. deionized water prepares carbodiimide (EDC) solution and the 50mg/mL N-hydroxysuccinimidyl of 50mg/mL respectively
Acid imide (NHS) solution；
1.c. adds 10～50 μ L EDC solution and 10～50 μ L in the fluorescence immunoassay microsphere processed through step 1.a
NHS solution, is placed on blending instrument holding microsphere and suspends, room temperature activation 30～60min, and centrifuge washing removes supernatant, adds
100 μ L, pH=5, concentration are the MES solution of 25mMol/L, relaunder 1～2 time, add 100 μ L MES solution ultrasonic disperse
30-60s；
(2) microsphere and antigen coupling: CSFV E 2 protein is exempted from the fluorescence that step 1.c activates with the amount of 500 μ g/ml
Epidemic disease microsphere mixes, and on impeller, room temperature reaction 3-12h, addition concentration is 0.05Mol/L, containing 1% (w/v) Sanguis Bovis seu Bubali
The PBS solution 100 μ L of pure albumen (BSA), is placed on blending instrument holding microsphere and suspends, seal, react 30min；Microsphere is with dense
Degree is 0.05Mol/L, PBS solution centrifuge washing 3 containing 1% (w/v) BSA～5 times, removes supernatant；Adding concentration is
0.05Mol/L, containing 0.02% (w/v) Hydrazoic acid,sodium salt NaN3With the PBS solution 100 μ l of 1% (w/v) BSA as preserving liquid, 4
At DEG C preserve, standby；
(3) the coupling CSFV E 2 protein that step (2) is prepared immunofluorescence microsphere PH=9.0, containing 0.5%
The carbonate buffer solution of casein and 0.1% polysorbas20 dilutes 20-40 times, is sprayed onto mark with metal spraying machine with the discharge rate of 3～5 μ l/cm
In note thing pad (2), it is dried 6-12h, forms the labeling pad (2) of the immunofluorescence microsphere being marked with CSFV E 2 protein；
(4) CSFV E 2 protein MES is diluted to 0.5mg/ml, draws to detecting pad with the amount of 1 μ l/cm by Film-cutting machine
(3), on, detection band (4) is formed, will be with the detecting pad (3) of detection band (4) at 37 DEG C of dry 2-8h；
(5) by sample pad (1), the labeling pad (2) of the immunofluorescence microsphere being marked with CSFV E 2 protein, detecting pad
And absorbent paper (5) sequentially overlaps and is pasted onto on vinyon base plate (6) (3), after cutting out with strip cutting machine, with assembling of getting stuck, standby
With.
Further, in step 1.c, when in 1.a, the consumption of fluorescence immunoassay microsphere is 100 μ L, EDC solution and NHS solution
The optimal value of addition be 20 μ L.
Invention also discloses the detection of the immune chromatography reagent kit of a kind of fluorescent quantitation detection swine fever virus neutralizing antibody
Method, gathers porcine vein, and centrifugation serum 5min, rotating speed is 4000r/min；15 are diluted by the PBS solution of 0.05Mol/L
After Bei, take in the well of the immune chromatography reagent kit that 80 μ L add to fluorescent quantitation detection swine fever virus neutralizing antibody；Wait to chromatograph
After reaction 15min, by animal epidemic immunofluorescence instrument, fluorescence intensity, read testing result.
The Cleaning Principle of the present invention is: dual-antigen sandwich method, by fluorescence immunoassay microsphere and CSFV E 2 protein coupling, when
This immune microsphere coupling CSFV E 2 protein antigen forms complex with the swine fever virus neutralizing antibody in detection sample, and this is multiple
Compound moves the detection zone to coated film under chromatography effect, and the detection zone at coated film is coated with and swine fever virus neutralizing antibody
In conjunction with CSFV E 2 protein antigen, this antibodies in another epi-position of antigen, formed double antigens sandwich complex.
Utilize fluorescent quantitation spectrum monitoring system, detection zone on light source activation coated film the fluorescent microsphere built up, fluorescent glue suppurative mastitis
The fluorescence of injection is received by corresponding detecting system, by light-to-current inversion, optical signal changes into the signal of telecommunication, and by automatically controlling
Signal is exported by system, demonstrates final quantitative result.
Compared with prior art, the present invention has a following innovative point:
(1) swine fever virus neutralizing antibody fluorescence quantitative detection kit of the present invention and RT-PCR method, ELISA side
Method is compared, and has varying number pattern detection easy and simple to handle, applicable and detects the excellent of speed fast (about 15min can have result)
Point；Compared with colloidal gold immuno-chromatography test paper strip, the present invention has that sensitivity is higher, the range of linearity is wider, and can realize quantitatively
The advantages such as detection.
(2) swine fever virus neutralizing antibody fluorescence quantitative detection kit of the present invention, uses fluorescent microsphere to mark in advance
Remembering to labeling pad, along with sample-adding and sample jointly by the method for chromatography to district to be checked, the method makes reaction discharge with signal
More homogeneous, compared with other chromatography, the preci-sion and accuracy of its batch production reaches best effects.Coordinate the most glimmering
Light instrument, it is only necessary to swine fever virus neutralizing antibody can be made sensitive detection by quantitative result output by 15min.
Accompanying drawing illustrates:
Fig. 1 is the structural representation of test strips of the present invention；In figure, 1. sample pad；2. labeling pad；3. detection
Pad；4. detection band；5. adsorptive pads；6. vinyon base plate；
Fig. 2 is that the structure of fluorescent quantitation of the present invention detection swine fever virus neutralizing antibody chromatography test kit launches signal
Figure；In figure, 7. well；8. display window；9. test strips draw-in groove；10. get stuck upper cover；11. test strips；12. draw-in groove lower covers.
Detailed description of the invention
Embodiment 1 test kit
A kind of immune chromatography reagent kit of fluorescent quantitation detection swine fever virus neutralizing antibody, its structure such as Fig. 1 and Fig. 2 institute
Show, including monolayer test strips 11 and compositions that get stuck, described get stuck by getting stuck upper cover 10 and lower cover 12 fastening that gets stuck is constituted, at card
Offering well 7 and display window 8 on shell upper cover 10, described well 7 is positioned at the top of sample pad 1, described display window 8
Being positioned at the surface of detecting pad 3, the size of display window 8 is equivalently-sized with detecting pad 3；Described test strips 11 by sample pad 1,
Labeling pad 2, detecting pad 3, absorbent paper 5 sequentially overlap and are pasted onto on vinyon base plate 6 composition, and set on detecting pad 3
It is equipped with detection band 4；The fluorescence immunoassay microsphere of coupling CSFV E 2 protein is contained in described labeling pad 2；Described detection
It is coated with CSFV E 2 protein antigen on band (4).
Further, the carboxyl modified that fluorescence immunoassay microsphere is 100～300nm of described coupling CSFV E 2 protein is micro-
Ball, the material of described sample pad be glass fibre, the base material of labeling pad be polyester fiber, the material of detecting pad is that nitric acid is fine
Dimension element coated film, the material of get stuck upper cover and the lower cover that gets stuck is plastics.
Further, the excitation wavelength (Ex) of required fluorescent microsphere is 365nm, and launching wavelength (Em) is 620nm.
Further, being arranged on and being coated with concentration on the detection band (4) on detecting pad (3) is 0.5mg/ml, and consumption is 1 μ l/
The CSFV E 2 protein antigen of cm.
Embodiment 2: preparation method
The raw material sources that used of immune chromatography reagent kit preparing fluorescent quantitation detection swine fever virus neutralizing antibody are as follows:
It is limited that glass fibre, polyester fiber, nitrocellulose filter, cotton starch plate, PVC film are purchased from Shanghai gold mark biotechnology
Company；Get stuck purchased from Cangzhou Sheng Feng plasthetics company limited；Fluorescence immunoassay microsphere is purchased from the Nanjing limited public affairs of micrometering biotechnology
Department.
The preparation method of the immune chromatography reagent kit of fluorescent quantitation detection swine fever virus neutralizing antibody, comprises the steps:
(1) microsphere activation
1.a. takes 200 μ L, size in 100nm fluorescence immunoassay microsphere to 1.5mL centrifuge tube, by equal-volume pH=5, concentration
Morpholino b acid solution centrifugal for 25mMol/l washs 1 time, removes supernatant；
1.b. deionized water prepares the EDC solution of 50mg/mL and the NHS solution of 50mg/mL respectively；
1.c. adds 10 μ L EDC solution and 10 μ L NHS solution in the fluorescence immunoassay microsphere processed through step 1.a,
It is placed on blending instrument holding microsphere to suspend, room temperature activation 30min, centrifuge washing, removes supernatant, add 100 μ L, pH=5, dense
Degree is the MES solution of 25mMol/L, relaunders 2 times, adds 100 μ L MES solution ultrasonic disperse 30s；
(2) microsphere and antigen coupling: CSFV E 2 protein is exempted from the fluorescence that step 1.c activates with the amount of 500 μ g/ml
Epidemic disease microsphere mixes, and on impeller, room temperature reaction 3h, addition concentration is 0.05mol/l, PBS containing 1% (w/v) BSA
Solution 100 μ L, is placed on blending instrument holding microsphere and suspends, seal, react 30min；Microsphere concentration is 0.05mol/l, contains
The PBS solution centrifuge washing of 1% (w/v) BSA 3 times, removes supernatant；Addition concentration is 0.05mol/l, containing 0.02% (w/v)
Hydrazoic acid,sodium salt NaN3With the PBS solution 200 μ L of 1% (w/v) BSA as preserving liquid, preserve at 4 DEG C, standby；
(3) the coupling CSFV E 2 protein that step (2) is prepared immunofluorescence microsphere PH=9.0, containing 0.5%
The carbonate buffer solution of casein and 0.1% polysorbas20 dilutes 20 times, is sprayed onto labeling pad with metal spraying machine with the discharge rate of 5 μ l/cm
(2) on, it is dried 6h, forms the labeling pad (2) of the immunofluorescence microsphere being marked with CSFV E 2 protein；
(4) CSFV E 2 protein MES is diluted to 1mg/ml, draws to detecting pad with the amount of 1 μ l/cm by Film-cutting machine
(3) on, form detection band (4), 2h will be dried under 37 DEG C of temperature conditionss with the detecting pad (3) of detection band (4)；
(5) by sample pad (1), the labeling pad (2) of the immunofluorescence microsphere being marked with CSFV E 2 protein, detecting pad
And absorbent paper (5) sequentially overlaps and is pasted onto on vinyon base plate (6) (3), after cutting out with strip cutting machine, with assembling of getting stuck, standby
With.
Embodiment 3: preparation method
The raw material sources that used of immune chromatography reagent kit preparing fluorescent quantitation detection swine fever virus neutralizing antibody are as follows:
It is limited that glass fibre, polyester fiber, nitrocellulose filter, cotton starch plate, PVC film are purchased from Shanghai gold mark biotechnology
Company；Get stuck purchased from Cangzhou Sheng Feng plasthetics company limited；Fluorescence immunoassay microsphere is purchased from the Nanjing limited public affairs of micrometering biotechnology
Department.
The preparation method of the immune chromatography reagent kit of fluorescent quantitation detection swine fever virus neutralizing antibody, comprises the steps:
(1) microsphere activation
1.a. takes 100 μ L, size in 1.5mL centrifuge tube, uses equal-volume at the carboxyl modified fluorescence immunoassay microsphere of 200nm
PH=5, concentration are that the morpholino b acid solution centrifugal of 25mMol/l washs 2 times, remove supernatant；
1.b. deionized water prepares the EDC solution of 50mg/mL and the NHS solution of 50mg/mL respectively；
1.c. adds 20 μ L EDC solution and 20 μ L NHS solution in the fluorescence immunoassay microsphere processed through step 1.a,
It is placed on blending instrument holding microsphere to suspend, room temperature activation 60min, centrifuge washing, removes supernatant, add 100 μ L, pH=5, dense
Degree is the MES solution of 25mMol/L, relaunders 1 time, adds 100 μ L MES solution ultrasonic disperse 60s；
(2) microsphere and antigen coupling: CSFV E 2 protein is exempted from the fluorescence that step 1.c activates with the amount of 500 μ g/ml
Epidemic disease microsphere mixes, and on impeller, room temperature reaction 6h, addition concentration is 0.05mol/l, PBS containing 1% (w/v) BSA
Solution 100 μ L, is placed on blending instrument holding microsphere and suspends, seal, react 30min；Microsphere concentration is 0.05Mol/L, contains
The PBS solution centrifuge washing of 1% (w/v) BSA 4 times, removes supernatant；Addition concentration is 0.05mol/l, containing 0.02% (w/v)
Hydrazoic acid,sodium salt NaN3With the PBS solution 100 μ L of 1% (w/v) BSA as preserving liquid, preserve at 4 DEG C, standby；
(3) the coupling CSFV E 2 protein that step (2) is prepared immunofluorescence microsphere PH=9.0, containing 0.5%
The carbonate buffer solution of casein and 0.1% polysorbas20 dilutes 30 times, is sprayed onto labeling pad with metal spraying machine with the discharge rate of 3 μ l/cm
(2) on, it is dried 8h, forms the labeling pad (2) of the immunofluorescence microsphere being marked with CSFV E 2 protein；
(4) CSFV E 2 protein MES is diluted to 0.5mg/ml, draws to detecting pad with the amount of 1 μ l/cm by Film-cutting machine
(3) on, form detection band (4), 6h will be dried at 37 DEG C with the detecting pad (3) of detection band (4)；
(5) by sample pad (1), the labeling pad (2) of the immunofluorescence microsphere being marked with CSFV E 2 protein, detecting pad
And absorbent paper (5) sequentially overlaps and is pasted onto on vinyon base plate (6) (3), after cutting out with strip cutting machine, with assembling of getting stuck, standby
With.
Embodiment 4: preparation method
The raw material sources that used of immune chromatography reagent kit preparing fluorescent quantitation detection swine fever virus neutralizing antibody are as follows:
It is limited that glass fibre, polyester fiber, nitrocellulose filter, cotton starch plate, PVC film are purchased from Shanghai gold mark biotechnology
Company；Get stuck purchased from Cangzhou Sheng Feng plasthetics company limited；Fluorescence immunoassay microsphere is purchased from the Nanjing limited public affairs of micrometering biotechnology
Department.
The preparation method of the immune chromatography reagent kit of fluorescent quantitation detection swine fever virus neutralizing antibody, comprises the steps:
(1) microsphere activation
1.a. takes 100 μ L, size at the fluorescence immunoassay microsphere of 300nm in 1.5mL centrifuge tube, with equal-volume pH=5, dense
The morpholino b acid solution centrifugal that degree is 25mMol/l washs 2 times, removes supernatant；
1.b. deionized water prepares the EDC solution of 50mg/mL and the NHS solution of 50mg/mL respectively；
1.c. adds 50 μ L EDC solution and 50 μ L NHS solution in the fluorescence immunoassay microsphere processed through step 1.a,
It is placed on blending instrument holding microsphere to suspend, room temperature activation 45min, centrifuge washing, removes supernatant, add 100 μ L, pH=5, matter
Amount concentration is 25mMol/L MES solution, relaunders 2 times, adds 100 μ L MES solution ultrasonic disperse 45s；
(2) microsphere and antigen coupling: CSFV E 2 protein is exempted from the fluorescence that step 1.c activates with the amount of 500 μ g/ml
Epidemic disease microsphere mixes, and on impeller, room temperature reaction 12h, addition concentration is 0.05mol/l, containing 1% (w/v) BSA
PBS solution 100 μ L, is placed on blending instrument holding microsphere and suspends, seal, react 30min；Microsphere concentration is 0.05mol/L, contains
There is the PBS solution centrifuge washing 5 times of 1% (w/v) BSA, remove supernatant；Addition concentration is 0.05mol/L, containing 0.02% (w/
V) Hydrazoic acid,sodium salt NaN3With the PBS solution 100 μ L of 1% (w/v) BSA as preserving liquid, preserve at 4 DEG C, standby；
(3) the coupling CSFV E 2 protein that step (2) is prepared immunofluorescence microsphere PH=9.0, containing 0.5%
The carbonate buffer solution of casein and 0.1% polysorbas20 dilutes 40 times, is sprayed onto labeling pad with metal spraying machine with the discharge rate of 3 μ l/cm
(2) on, it is dried 12h, forms the labeling pad (2) of the immunofluorescence microsphere being marked with CSFV E 2 protein；
(4) CSFV E 2 protein MES is diluted to 0.5mg/ml, draws to detecting pad with the amount of 1 μ l/cm by Film-cutting machine
(3) on, form detection band (4), 8h will be dried at 37 DEG C with the detecting pad (3) of detection band (4)；
(5) by sample pad (1), the labeling pad (2) of the immunofluorescence microsphere being marked with CSFV E 2 protein, detecting pad
And absorbent paper (5) sequentially overlaps and is pasted onto on vinyon base plate (6) (3), after cutting out with strip cutting machine, with assembling of getting stuck, standby
With.
Embodiment 5: detection swine fever positive National reference
Use the immune chromatography reagent kit of fluorescent quantitation detection swine fever virus neutralizing antibody of embodiment 3 preparation as detection
Test kit.This test kit need to be with the animal epidemic immunofluorescence detection by quantitative of Zhengzhou Sai Fu biological engineering company limited independent development
Instrument (model: SF-100A type) supports the use；In fluorescent quantitative detector deposit have reagent calibration curve, this calibration curve with
Hyper-immune serum (enterprise's self-control) is standard substance, with standard substance prepare concentration be respectively 25SFU, 50SFU, 100SFU, 200SFU,
The standard pipe of 500SFU, 1000SFU is dense as vertical coordinate, with fluorescence calculating with the fluorescence intensity signals value that each standard pipe detects
The matched curve that semilog is abscissa of degree, this calibration curve can be with the fluorescent quantitation detection swine fever virus that the present invention relates to
The numerical value combination that neutralizing antibody immune chromatography reagent kit detects, quantitatively determines antibody concentration.(swine fever virus of the present invention
Neutralizing antibody immunochromatographytest test kit, in actual applications, can be available for luminoscope scanning recognition at the outer surface that gets stuck
Product information coding, write in ID chip after fluorescent quantitative detector scans.)
In the present embodiment, by swine fever positive serum National reference (rabbit body neutralization test；Production code member: S07500-3；Rule
Lattice: 1ml；Content: neutralization titer is 1:847 ± 1) (purchased from China Veterinery Drug Inspection Office).This reference material is examined through hog cholera antibody
Test agent box (euzymelinked immunosorbent assay (ELISA)) (deriving from Zhengzhou Sai Fu biological engineering company limited) detects, and OD value is 0.68.By this reference
Product make 1:15,1:30,1:60 dilution respectively, and the animal epidemic immunity with Zhengzhou Sai Fu biological engineering company limited independent development is glimmering
Light instrument fluorescence intensity, reads testing result as shown in table 1.
Table 1 swine fever positive serum National reference different proportion antibody concentration testing result
| Dilution ratio | 1:15 | 1:30 | 1:60 |
| Antibody concentration (SFU) | 218 | 116 | 63 |
The value for antibody of the swine fever positive serum of comparison and detection difference dilution ratio understands, and the calibration that the present embodiment is set up is bent
Line is capable of detecting when fixed swine fever positive serum, and along with the increase of dilution ratio, its detection antibody concentration is gradually lowered,
Illustrating that the antibody that this test kit detects is swine fever virus neutralizing antibody, testing result is consistent with actual, has actually used meaning.
Embodiment 6: swine fever virus neutralizing antibody in detection different phase pig body
Use the immune chromatography reagent kit of fluorescent quantitation detection swine fever virus neutralizing antibody of embodiment 3 preparation as detection
Test kit.
Choose 20 ages in days, 40 ages in days, 65 ages in days, 90 ages in days, 105 ages in days, 120 ages in days, replacement gilt and multiparity respectively female
Eight stages of pig respectively take 10 and gather venous blood, and centrifugation serum 5min, 4000r/min are dilute with 0.05Mol/LPBS solution
After releasing 15 times, take 80 μ L and add in well；After chromatography reaction carries out 15min, with Zhengzhou Sai Fu biological engineering company limited
The animal epidemic immunofluorescence instrument of independent development, fluorescence intensity, reads testing result.
The present invention is by detecting 80 example samples in embodiment, simultaneously with Enzyme-linked Immunosorbent Assay (ELISA) reagent
Box carries out detection and compares.Detecting the sample of 80 parts of pig different phases altogether, wherein 8 parts are detected suspicious for Elisa, remove suspicious sample
This, two kinds of method testing results of 4 parts of samples are inconsistent, and concrete outcome is as shown in table 2:
Table 2 the method for the invention compares with ELISA testing result
Table 3 the method for the invention compares detailed results with ELISA testing result
From table 2 " the method for the invention compares with ELISA testing result ", from the present embodiment result and ELISA side
Finding in method contrast, total coincidence rate between the two is up to 94.4%, it was demonstrated that detection method provided by the present invention and ELISA side
Method testing result coincidence rate is higher, illustrates that the testing result measured by detection method provided by the present invention is true and reliable.Due to
The enzyme labelled antibody of indirect elisa method is the antibody of anti-pig IgG, is the two anti-combinations with antibody, has certain limitation, exist
A certain proportion of false positive, and the fluorescent microsphere quantitative detecting method that this experiment is set up, based on dual-antigen sandwich method, by two
Combining with the hog cholera antibody in sample to be tested of individual antigenic specificity so that reagent is more directly more special to the combination of pattern detection
One, thus ensure that the specificity of product.It addition, fluorescent microsphere itself has highly sensitive feature, can fundamentally carry again
Rise the sensitivity of this Product checking.
Known in those skilled in the art, piglet immunological is successfully it is crucial that get rid of maternal antibody interference, and it is suitable to determine
First immune day-age.The existence of maternal antibody, is protected in can making piglet certain time passively, meanwhile, when in piglet body
When there is a certain amount of maternal antibody, after vaccination, a part of vaccine virus is neutralized by maternal antibody, makes to note by former dosage
The vaccine virus penetrated does not reaches antigen threshold value and causes immuning failure.Fan Xuezheng etc. measure 10 nest piglets by Elisa method, and (every nest selects
5) the maternal antibody level of pig different days carry out counteracting toxic substances Protection research in 81D and 35D, result shows, 81
Age, piglet can not resist strong virus attack, and 35 age in days piglet major parts can resist strong virus attack, but maternal antibody can not hinder completely
Disconnected swine fever strong virus attack, during 35 age in days, maternal antibody is in critical level.During the immunity safety of whole colony, head exempts from day
Should determine that to be 25～35 ages in days age.Further by table 3 " the method for the invention compares detailed results with ELISA testing result "
Demonstrating the research of model political affairs et al., from 20 ages in days to 40 ages in days, the level of hog cholera antibody is decreased obviously.After head exempts from, by 65 days
During age, antibody horizontal is significantly improved, and further with the passage of time, hog cholera antibody presents again significantly to decline and
Gesture.
Another notable rule of hog cholera antibody level is exactly to improve along with the increase of vaccine immunity number of times.Standby is female
The immune time of pig and Suprapubic arch sling is significantly more than piglet and growing and fattening pigs.So, the result embodied as table 3, replacement gilt
With the hog cholera antibody level in Suprapubic arch sling the two stage apparently higher than the integral level in other stage.
From table 3 " the method for the invention compares detailed results with ELISA testing result ", inspection provided by the present invention
Survey method, the result detected, both met maternal antibody Fluctuation and also complied with immunologic rule simultaneously, had stronger
Actual application value.
At present, the operation of the test kit of the application ELISA method detection food swine fever virus neutralizing antibody of commercial type needs
Wanting the substantial amounts of time, by contrast, present invention incorporates immune microsphere beneficiation technologies and fluorescent quantitation detection technique, 15min is i.e.
Can obtain and detect data accurately and reliably, and easy and simple to handle.
It is above illustrating of the feasible embodiment for the present invention, when this embodiment and be not used to limit this
Bright the scope of the claims, all equivalences done without departing from skill spirit of the present invention are implemented or change, are intended to be limited solely by the special of the present invention
In profit scope.
Claims (7)
1. the immune chromatography reagent kit of a fluorescent quantitation detection swine fever virus neutralizing antibody, it is characterised in that include that monolayer tries
Paper slip (11) and the composition that gets stuck, described getting stuck is made up of, at the upper cover that gets stuck the upper cover that gets stuck (10) and the lower cover that gets stuck (12) fastening
(10) offering well (7) and display window (8) on, described well (7) is positioned at the top of sample pad (1), and described is aobvious
Showing that window (8) is positioned at the surface of detecting pad (3), the size of display window (8) is equivalently-sized with detecting pad (3)；Described reagent paper
Bar (11) is sequentially overlapped by sample pad (1), labeling pad (2), detecting pad (3), absorbent paper (5) and is pasted onto vinyon base plate
(6) upper composition, and on detecting pad (3), it is provided with detection band (4)；Described labeling pad (2) is upper containing coupling swine fever
The fluorescence immunoassay microsphere of virus E2 albumen；It is coated with CSFV E 2 protein antigen on described detection band (4).
The immune chromatography reagent kit of fluorescent quantitation the most according to claim 1 detection swine fever virus neutralizing antibody, its feature
It is,
The carboxyl modified microsphere that fluorescence immunoassay microsphere is 100 ~ 300 nm of described coupling CSFV E 2 protein, described sample
The material of pad (1) be glass fibre, the base material of labeling pad (2) be polyester fiber, the material of detecting pad (3) is celluloid
Coated film, the material of the upper cover that gets stuck (10) and the lower cover that gets stuck (12) is plastics.
The immune chromatography reagent kit of fluorescent quantitation the most according to claim 1 detection swine fever virus neutralizing antibody, its feature
It is,
Excitation wavelength Ex of described fluorescent microsphere is 365 nm, and launching wavelength Em is 620 nm.
The immune chromatography reagent kit of fluorescent quantitation the most according to claim 1 detection swine fever virus neutralizing antibody, its feature
It is,
It is 0.5mg/ml that described detection band (4) is coated with concentration, and consumption is the CSFV E 2 protein antigen of 1 μ l/cm.
5. the immune chromatography reagent kit of the fluorescent quantitation detection swine fever virus neutralizing antibody as described in claim 1-4 is arbitrary
Preparation method, it is characterised in that comprise the steps:
(1) microsphere activation
1.a. takes in 100 ~ 200 μ L fluorescence immunoassay microspheres to 1.5 ml centrifuge tubes, is 25 by equal-volume pH=5, concentration
Morpholino b acid solution (MES, the lower same) centrifuge washing of mMol/L 1 ~ 2 time, removes supernatant；
1.b. deionized water prepares carbodiimide (EDC) solution and the 50 mg/ ml N-hydroxyl ambers of 50 mg/ ml respectively
Amber acid imide (NHS) solution；
1.c. adds 10 ~ 50 μ L EDC solution in the fluorescence immunoassay microsphere processed through step 1.a and 10 ~ 50 μ L NHS are molten
Liquid, is placed on blending instrument holding microsphere and suspends, room temperature activation 30 ~ 60 min, and centrifuge washing removes supernatant, adds 100 μ L
, pH=5, concentration be the MES solution of 25 mMol/L, relaunder 1 ~ 2 time, add 100 μ L MES solution ultrasonic disperse 30-
60s；
(2) microsphere and antigen coupling: the fluorescence immunoassay that CSFV E 2 protein is activated with step 1.c with the amount of 500 μ g/ml
Microsphere mixes, on impeller, and room temperature reaction 3-12h, addition concentration is 0.05Mol/L, containing 1%(w/v) Ox blood serum
The PBS solution 100 μ L of albumin (BSA), is placed on blending instrument holding microsphere and suspends, seal, react 30 min；Microsphere is used
Concentration is 0.05Mol/L, containing 1%(w/v) the PBS solution centrifugal of BSA washs 3 ~ 5 times, removes supernatant；Adding concentration is
0.05Mol/L, containing 0.02%(w/v) Hydrazoic acid,sodium salt NaN3And 1%(w/v) BSA PBS solution 100 μ l as preserve liquid,
At 4 DEG C preserve, standby；
(3) the coupling CSFV E 2 protein that step (2) is prepared immunofluorescence microsphere PH=9.0, containing 0.5% casein
Dilute 20-40 times with the carbonate buffer solution of 0.1% polysorbas20, be sprayed onto labeling pad (2) with metal spraying machine with the discharge rate of 3 ~ 5 μ l/cm
On, it is dried 6-12 h, forms the labeling pad (2) of the immunofluorescence microsphere being marked with CSFV E 2 protein；
(4) CSFV E 2 protein MES is diluted to 0.5 mg/ml, draws to detecting pad (3) with the amount of 1 μ l/cm by Film-cutting machine
On, form detection band (4), 2-8h will be dried at 37 DEG C with the detecting pad (3) of detection band (4)；
(5) by sample pad (1), the labeling pad (2) of the immunofluorescence microsphere being marked with CSFV E 2 protein, detecting pad (3)
And absorbent paper (5) sequentially overlaps and is pasted onto on vinyon base plate (6), after cutting out with strip cutting machine, with assembling of getting stuck, standby.
The preparation side of the immune chromatography reagent kit of fluorescent quantitation the most according to claim 5 detection swine fever virus neutralizing antibody
Method, it is characterised in that in step 1.c, when in 1.a, the consumption of fluorescence immunoassay microsphere is 100 μ L, EDC solution and NHS solution
Addition be 20 μ L.
7. the immune chromatography reagent kit of the fluorescent quantitation detection swine fever virus neutralizing antibody as described in claim 1-4 is arbitrary
Detection method, it is characterised in that gather porcine vein, centrifugation serum 5 min, rotating speed is 4000r/min；With 0.05
After the PBS solution of Mol/L dilutes 15 times, take 80 μ L and add to the immunity-chromatography test of fluorescent quantitation detection swine fever virus neutralizing antibody
In the well (7) of agent box；After chromatography reaction 15 min, inspire confidence in biological animal epidemic immunofluorescence instrument, inspection with match
Surveying fluorescence intensity, the calibration curve of lot number relevant to instrument internal carries out internal calculation and obtains detectable concentration.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106896224A (en) * | 2017-03-15 | 2017-06-27 | 广州万联生物科技有限公司 | A kind of swine fever novel antibodies ELISA detection kit |
| CN107589253A (en) * | 2017-09-06 | 2018-01-16 | 詹爱军 | A kind of H9 subtype avian influenza virus fluorescent chromatographic test strips and its application |
| CN108490196A (en) * | 2018-05-16 | 2018-09-04 | 江苏维尔生物科技有限公司 | Test-strips of filaria volvulus metabolin NATOG and preparation method thereof, test card in a kind of detection urine |
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| CN105548535A (en) * | 2016-01-21 | 2016-05-04 | 成都微瑞生物科技有限公司 | Classical swine fever antibody detection card and preparation method thereof |
| CN106018800A (en) * | 2016-05-25 | 2016-10-12 | 南方医科大学 | Detecting device for Brucella infection |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105548535A (en) * | 2016-01-21 | 2016-05-04 | 成都微瑞生物科技有限公司 | Classical swine fever antibody detection card and preparation method thereof |
| CN106018800A (en) * | 2016-05-25 | 2016-10-12 | 南方医科大学 | Detecting device for Brucella infection |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106896224A (en) * | 2017-03-15 | 2017-06-27 | 广州万联生物科技有限公司 | A kind of swine fever novel antibodies ELISA detection kit |
| CN107589253A (en) * | 2017-09-06 | 2018-01-16 | 詹爱军 | A kind of H9 subtype avian influenza virus fluorescent chromatographic test strips and its application |
| CN108490196A (en) * | 2018-05-16 | 2018-09-04 | 江苏维尔生物科技有限公司 | Test-strips of filaria volvulus metabolin NATOG and preparation method thereof, test card in a kind of detection urine |
| CN109777890A (en) * | 2019-01-23 | 2019-05-21 | 华南农业大学 | A kind of PCV2, CSFV IgG antibody double fluorescent microballoon immunological detection method |
| CN113391064A (en) * | 2020-03-13 | 2021-09-14 | 科美诊断技术股份有限公司 | Receptor reagent for detecting novel coronavirus neutralizing antibody and application thereof |
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