CN108845149A - The colloidal gold duplex test strips and preparation method thereof of porcine reproductive and respiratory syndrome virus and swine fever virus are detected simultaneously - Google Patents
The colloidal gold duplex test strips and preparation method thereof of porcine reproductive and respiratory syndrome virus and swine fever virus are detected simultaneously Download PDFInfo
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- CN108845149A CN108845149A CN201810732140.6A CN201810732140A CN108845149A CN 108845149 A CN108845149 A CN 108845149A CN 201810732140 A CN201810732140 A CN 201810732140A CN 108845149 A CN108845149 A CN 108845149A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/18—Togaviridae; Flaviviridae
- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
Abstract
The invention discloses a kind of colloidal gold duplex test strips and preparation method thereof for detecting porcine reproductive and respiratory syndrome virus and swine fever virus simultaneously.The test strips include support plate, nitrocellulose filter, sample pad, gold labeling antibody pad, blotting paper, detection line T2, detection line T1 and nature controlling line, gold labeling antibody pad mark the IgG mixture of anti-PRRSV and CSFV, and detection line T1 is coated with anti-PRRSV N protein monoclonal antibody, detection line T2 is coated with anti-CSFV E2 protein monoclonal antibody, and nature controlling line is coated with sheep anti-mouse igg.Provide while detecting the preparation method of the colloidal gold duplex test strips of porcine reproductive and respiratory syndrome virus and swine fever virus.The colloidal gold duplex test strips that porcine reproductive and respiratory syndrome virus and swine fever virus are detected while of the invention are quick, easy, specific good, technical support is provided for the monitoring of the combined immunization of porcine reproductive and respiratory syndrome and swine fever and epidemiological study, is had great significance to the prevention and control of swine disease.
Description
Technical field
The present invention relates to animal virus detection technique fields, and in particular to a kind of to detect porcine reproductive and respiratory syndrome simultaneously
The colloidal gold duplex test strips and preparation method thereof of virus and swine fever virus.
Background technique
Porcine reproductive and respiratory syndrome（Porcine Reproduetive and Respiratory Syndrome,
PRRS）It is that current world's pig breeding industry endangers viral disease the most serious.Infect porcine reproductive and respiratory syndrome virus
（PRRSV）Sow is mainly shown as premature labor, late abortion, stillborn foetus, the mummification of fetus, fever, anorexia characterized by dysgenesia afterwards
Deng the piglet of infection shows as respiratory symptom, also there is nervous symptoms.It will appear two ear skin cyanosis hair in pathogenic process
Purple, therefore also known as " blue otopathy ".There are two types of main serum types by PRRSV, are genotype Ⅰ respectively（Europe class）With II type（Beauty
Continent type）.I type is with Lelystad plants (LV plants) for representative, and II type is with VR2332 plants for representative.Infect pig and the sense of PRRSV
The pig of rehabilitation can toxin expelling after dye.It is infected by contact, the major transmission path that sperm is propagated, air borne is PRRSV, birds, mouse
Its medium for being all likely to become this disease of the haulagman such as class, the mankind.
Swine fever is by swine fever virus（CSFV）Caused one kind is acute, hot and high degree in contact viral infectious, should
Disease brings heavy loss to pig breeding industry characterized by popular wide, morbidity and mortality height.The disease is included in by international animal regulation
A class kind infectious disease is classified as a kind of infectious disease by China's animal health regulation.
Porcine reproductive and respiratory syndrome and swine fever are the main epidemic diseases for endangering China's pig breeding industry in recent years, wherein with blue otopathy
It endangers the most serious.In the U.S. and Europe, the flourishing country of some pig breeding industries has used " epidemic disease purification strategy ", that is, is directed to one
A little animal great epidemic diseases such as Schweineseuche, swine fever etc. stop using preventative vaccine, using stringent quarantine, monitoring and purification swinery
Equal measures give prevention and control.Currently, vaccine immunity and supervision of epidemic disease combination are still the major measure of China's swine disease prevention and control.
Enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) is that one kind is answered extensively
The immuno analytical method of albumen, antibody or hormone in measurement liquid sample, has sensitive, special, simple, quick, steady
The features such as determining and being easily operated automatically, suitable for being detected while a large amount of clinical samples.Oneself has been set up very both at home and abroad at present
ELISA method mostly about PRRSV and CSFV infection detection；But present inventor is the study found that these detection methods are big absolutely
Majority is all based on antibody test, and this method based on antibody test has certain limitation；For PRRSV, pig sense
Detectable antibody could be induced within earliest 7 days after dye PRRSV, individual pigs still can't detect anti-after even infecting PRRSV 3 weeks
Body, this situation that cannot detect PRRSV infection in time are caused to the subsequent large area outburst for taking steps to control PRRS
It is certain to hinder.
Therefore, currently needing to develop in infection early stage while can detect PRRSV and two boar of CSFV is total to the new of illness
Method, and then avoid virus infection early stage and the antibody blank phenomenon that occurs, it is more intuitive, fast to reach detection to virus infection
Speed, accurate detection purpose.
Summary of the invention
Porcine reproductive and respiratory syndrome virus and swine fever are detected simultaneously the technical problem to be solved in the present invention is to provide a kind of
The colloidal gold duplex test strips and preparation method thereof of virus, it is easy to operate quick to realize, it as a result clearly easily distinguishes, a step is examined more
Early detection.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
Design colloidal gold duplex test strips that are a kind of while detecting porcine reproductive and respiratory syndrome virus and swine fever virus, including branch
Fagging, nitrocellulose filter, sample pad, gold labeling antibody pad, blotting paper, detection line T2, detection line T1 and nature controlling line, the gold mark
Antibody pad is marked with the IgG mixture of anti-PRRSV and CFSV, and it is anti-that the detection line T1 is coated with anti-PRRSV N protein monoclonal
Body, the detection line T2 are coated with anti-CSFV E2 protein monoclonal antibody, and the nature controlling line is coated with sheep anti-mouse igg.
Preferably, the ELISA of the anti-PRRSV N protein monoclonal antibody or anti-CSFV E2 protein monoclonal antibody is imitated
Valence is not less than 105。
Preferably, the hypotype of the anti-PRRSV N protein monoclonal antibody is IgG1 type or IgG2a type.
Preferably, the hypotype of the anti-CSFV E2 protein monoclonal antibody is IgG1 type.
The preparation of colloidal gold duplex test strips that are above-mentioned while detecting porcine reproductive and respiratory syndrome virus and swine fever virus
Method includes the following steps：
（1）It is synthesized after carrying out codon optimization to PRRSV N protein and CSFV E2 protein gene respectively and is cloned into different expression
Carrier, prokaryotic expression N protein, baculovirus expression E2 albumen, albumen obtain external source N protein and E2 albumen after purification respectively；
（2）Prepare anti-PRRSV N protein monoclonal antibody and anti-CSFV E2 protein monoclonal antibody：
BALB/c mouse is immunized respectively using the N protein of purifying and E2 albumen as antigen, the hybridoma for the PRRSV for screening positive is thin
The hybridoma cell strain of born of the same parents strain and CSFV, the strong hybridoma cell strain of selective affinity prepares monoclonal antibody respectively；
（3）Prepare test strips：
I. colloidal gold solution is prepared using sodium citrate；
II. using the IgG mixture of the anti-PRRSV and CSFV as gold labeling antibody, colloidal gold and antibody are after stabilizer treatment
It is adsorbed on glass fibre membrane, is dried in vacuo, cuts to obtain label pad；
III. respectively by step（2）Resulting anti-PRRSV N protein monoclonal antibody and the spray of anti-CSFV E2 protein monoclonal antibody
Detection line T1 and T2 on to coated film, are sprayed onto nature controlling line for sheep anti-mouse igg, dry, envelope is spare；
IV. sequentially mutually overlap joint pasting sample pad, label pad, coated film and blotting paper obtains test paper plate on bottom liner, is cut into
Different in width obtains test strips.
Further, in the step（II）In：PH value control when the absorption is 7.8；The colloidal gold and described
The proportion of antibody is 25 μ g antibody/ml colloidal gold.
Further, in the step（III）In, the nature controlling line and two detection line interval 4mm.The anti-PRRSV
Concentration control when N protein monoclonal antibody or anti-CSFV E2 protein monoclonal antibody are coated with is 2mg/mL.
Compared with prior art, the beneficial technical effect of the present invention lies in：
1. test strips prepared by the present invention are the principles based on double crush syndrome antigen detection method, be for PRRSV and
The detection of CSFV antigen can be avoided the antibody blank phenomenon of virus infection early stage and appearance, thus the detection to virus infection
It is more intuitive, quick.
2. test strips prepared by the present invention can detect PRRSV simultaneously and two boar of CSFV is total to illness, quick, easy, special
Property is good, realizes accurately and rapidly testing goal.
3. the present invention realizes the detection method that a step is examined more, easy to operate quick, as a result clearly easily distinguish, suitable base pushes away
Extensively.
4. test strips of the invention are that the immunologic surveillance of porcine reproductive and respiratory syndrome and swine fever and epidemiological surveillance are ground
Study carefully and provide technical support, has great significance to the prevention and control of swine disease.
Detailed description of the invention
Fig. 1 is the SDS-PAGE electrophoresis and Western blot detection figure of N protein,
In figure, M Marker；1 is N protein；
Fig. 2 is the SDS-PAGE electrophoresis and Western blot detection figure of E2 albumen,
In figure, M Marker；1 is E2 albumen；
Fig. 3 is that the IPMA of hybridoma cell strain detects figure；
Fig. 4 is the structural schematic diagram of test strips,
In figure, 1 is support plate, and 2 be nitrocellulose filter, and 3 be sample pad, and 4 be gold labeling antibody pad, and 5 be blotting paper, and 6 be detection
Line T2,7 be detection line T1, and 8 be nature controlling line C；
Fig. 5 is test strips result judgement schematic diagram.
Specific embodiment
Illustrate a specific embodiment of the invention below with reference to embodiment, but following embodiment is used only to be described in detail
The present invention does not limit the scope of the invention in any way.
Related instrument and equipment is routine instrument device unless otherwise instructed in the examples below；It is related
Reagent is commercially available conventional reagent unless otherwise instructed；Related test method is unless otherwise instructed conventional method.
Embodiment one：Immunogene selection and preparation
1. the selection of immunogene
The long-term numerous studies discovery of inventor, in porcine reproductive and respiratory syndrome virus（PRRSV）The encoded albumen of genome
In, there is the nucleocapsid protein of ORF7 coding, i.e. N protein is highly conserved；Secondly, the immune response excited in body by N protein
Property is significantly larger than other albumen.
Based on the above two o'clock, the N protein is screened in research becomes the preferred target proteins of detection PRRSV.
2. the preparation of immunogene
A：The preparation of immunogene N protein
（1）Porcine reproductive andrespiratory syndrome virus N protein gene order is cloned, expression vector pET-28a is connected into, must recombinantly express
Carrier pET-28a-N；
（2）By recombinant expression carrier pET-28a-N CaCl2Method is transferred to competent cellE.coliIn BL21 (DE3), sun is obtained
Property recombination bacillus coli BL21 (DE3)；
（3）By positive restructuringE.coliBL21 (DE3) is inoculated in LB culture medium, under conditions of IPTG concentration is 0.5 mM,
30 DEG C after inducing expression 10 hours, are collected sample and carry out ultrasonication and Ni column purification；
（4）It albumen will be detected after purification through SDS-PAGE electrophoresis and Western blot；
As a result as shown in Figure 1.
Qualification result shows that the N protein purity of acquisition is higher；
B：The preparation of immunogene E2 albumen
（1）The building of shuttle vector：CSFV E 2 protein gene order is according to baculovirus expression system codon-bias
Optimize, its N-terminal introduced into signal peptide sequence and 6 × His label respectively, nucleotide sequence as shown in SEQ ID NO.1,
The synthesis of Hai Shenggong bioengineering Co., Ltd is served, to synthesize gene as template, is cloned into expression vector pFast-BacI
Carrier obtains recombinant expression carrier pFast-Bac-E2；By recombinant expression carrier pFast-Bac-E2 CaCl2Method is transferred to competence
Cell DH10BacE.coliIn, above pFast-Bac-E2 swivel base to Bacmid, picking hickie is screened through blue hickie, is obtained
Positive restructuring shuttle vector Bacmid-Bac-E2；
（2）Extract rod granule：Bacmid-Bac-E2 rod granule is extracted for insect cell transfection examination with a small amount of extraction agent boxes of Bacmid
It tests；
（3）The Primary Construction of recombinant baculovirus：The Bacmid-Bac-E2 DNA and liposome that purifying is obtained
Lipofectamine 2000（According to mass volume ratio（μg/μl）1：3）Sf9 cell is transfected, separates P1 for recombinant baculovirus
Ac-Bac-E2；P1It is inoculated with for recombinant baculovirus with the amount of MOI=0.1, connects and cultivate 72~96 hours for 27 DEG C after poison, collect cell
Supernatant is P2Generation virus, measures TCID50, cell conditioned medium is collected respectively and precipitating Preliminary detection E2 albumen is expressed in supernatant；
（4）Protein expression and purifying：Cell conditioned medium, Ni column purification E2 albumen, through SDS-PAGE are collected within 48 hours respectively after connecing poison
Electrophoresis and Western blot detection；
As a result as shown in Figure 2.
Qualification result shows that purity is up to 90% or more after E2 albumen is purified.
Embodiment two：Monoclonal antibody preparation and identification
1. animal immune
（1）Embodiment one is prepared into resulting immunogene N protein and E2 albumen is separately added into Freund's complete adjuvant（First immunisation makes
With）Or immunizing antigen is made in incomplete Freund's adjuvant, emulsification；
（2）By the method for dorsal sc multi-point injection, female BAl BIc/c mouse of 4~8 week old is immunized（4/group）, immunizing agent
Amount is respectively 20 μ g/, 40 μ g/, respectively exempts from 2；
（3）After first immunisation, every 3 weeks with after incomplete Freund's adjuvant and immunizing antigen emulsification in the same way and dosage
Booster immunization is carried out to BALB/c mouse；
（4）After four times immune, 3~4 days before cell fusion, by the method for tail vein injection, with the immunogene pair for being free of adjuvant
BALB/c mouse carry out booster immunization, immunizing dose be 50 μ g/only.
（5）Polyvalent antibody potency and sensitivity testing result：
Last time booster immunization latter week each mouse is carried out cutting tail blood sampling respectively, then by indirect elisa method and indirectly
Inhibition ELISA is respectively measured the potency of 3 mouse polyvalent antibodies and sensibility.The highest mouse of potency is selected to carry out
Cell fusion.
2. cell fusion and monoclonal antibody preparation
（1）Using the method for polyethylene glycol, the splenocyte of immune mouse and murine myeloma cell SP2/0 are pressed into cell quantity 8：
1 ratio carries out cell fusion, and fused cell is screened with HAT Selective agar medium；
（2）96 porocyte culture plates for being covered with feeder cells are divided in, 37 DEG C, 5% CO are set2Culture in incubator, after fusion
5th day every 50 μ L of hole supplemented medium, after 12 days, respectively using N protein or E2 albumen as envelope antigen, passes through indirect ELISA
Method carries out primary dcreening operation；And the synchronous corresponding PRRSV or CSFV standard strain of selection carries out immunopcroxidase monolayer assay
（IPMA）Hybridoma supernatant is screened, the positive hybridoma cell strain for selecting ELISA and IPMA while colour developing carries out
It is subcloned in next step；
（3）3~4 subclone screenings are carried out to positive hybridoma cell by limiting dilution assay, being finally obtained 6 plants can stablize
The hybridoma cell strain (1A3,5H5,2B10,2G3,1F4,3H1) of anti-N protein monoclonal antibody is secreted, 2 plants of energy stably excretings are anti-
The hybridoma cell strain (3B8,2G5) of CSFV monoclonal antibody.
3. immunopcroxidase monolayer assay（IPMA）
IPMA operating process：
Marc145 cell or PK15 cell（PRRSV Marc145 cell, CSFV PK15 cell）96 porocyte plates are spread, to thin
Born of the same parents' convergence degree, which is grown to 70% or so, connects poison.Connect the DMEM culture medium that 1h after poison adds 2% fetal calf serum；Cell is fixed after 48h：It discards
Culture medium in 96 orifice plates, PBST are washed 2~3 times, and the hole 50uL/ adds the fixer of -20 DEG C of pre-coolings（Methanol）, it is stored at room temperature 10~
15min；Fixer is discarded, PBST is washed 3 times, and the hole 200uL/ adds defatted milk, and four spend night closing；Confining liquid is discarded, PBST washes 3
Secondary, 50 uL/hole adds the primary antibody of certain dilution, and 37 DEG C, 1h；Primary antibody is discarded, PBST is washed 5~6 times, patted dry, and 50 uL/hole adds
The diluted corresponding secondary antibody of certain multiple, 37 DEG C, 1h；Secondary antibody is discarded, PBST is washed 5~6 times, patted dry, 50 uL/hole, colour developing
10min.（AEC developing solution：Every 1ml ddw successively adds A liquid 1 to drip, and B liquid 2 drips, and C liquid 1 drips）.
As a result as shown in Figure 3：
It is screened through IPMA, only 1F4,5H5 can be in conjunction with PRRSV；3B8 and 2G5 can be in conjunction with CSFV.
4. inducing ascites method in body prepares monoclonal antibody
The female Balb/c mouse through producing is selected, 500 μ L of intraperitoneal injection sterilizing paraffin, after a week, intraperitoneal injection obtains again
The monoclonal hybridoma obtained, injection volume are 2 × 105A cell extracts abdomen after mouse web portion expands after after a week
Water takes supernatant after centrifugation, is purified with sad ammonium sulfate method to ascites.
5. the identification of odd contradictive hydroperitoneum
（1）Indirect ELISA measurement result shows that the potency of 2 plants of IPMA positive monoclonal antibodies reaches 106；
（2）Subtype identification：It is identified with hypotype of the subtype identification kit to monoclonal antibody, qualification result shows 5H5,1F4 Dan Ke
Grand antibody is belonging respectively to IgG1, IgG2a type, and 3B8,2G5 are IgG1 type.
6. repeated pruning
Involve the monoclonal hybridoma established in a criminal case continuous culture 3 months and liquid nitrogen cryopreservation is recovered repeatedly, to identify hybridization
The stability of oncocyte.
Embodiment three：The purifying of antibody
Caprylic acid-ammonium carries out antibody purification, and operating method is as follows：
（1）The odd contradictive hydroperitoneum or antiserum frozen is taken out, 6000 r/min are centrifuged 30min, obtain supernatant, use sodium acetate buffer
Liquid（0.06mol/L pH4.8）5 times of dilution.
（2）Adjusting pH with NaOH (5mol/L) is 4.5, and room temperature is slowly stirred 0.5 h, adds caprylic acid, ultimate density 25
μL/mL。
（3）6000 r/min are centrifuged 30min at 4 DEG C, retain supernatant.
（4）The supernatant being obtained by filtration with Medium speed filter paper, and 10 × PBS buffer solution (pH7.4) is added, volume 1/10 is mixed
It is 7.4 that pH is adjusted after closing uniformly.
（5）Solid ammonium sulfate is added into mixed liquor under the conditions of 4 DEG C, adding proportion is 0.2778 g/ mL, can be multiple
Addition, the interior addition of 30 min finish.
（6）6000 r/min are centrifuged 20min, throw away supernatant.The PBS (pH7.2) of addition starting ascites volume 1/3 is carried out
It is resuspended, with PBS buffer solution 24 h of dialysis at 4 DEG C, during which changes liquid 3~4 times, collect, packing, -20 DEG C of preservations.
Example IV：Preparation while the colloidal gold duplex test paper for detecting porcine reproductive and respiratory syndrome virus and swine fever virus
Item
1. the preparation of colloidal gold solution
Colloidal gold is prepared using sodium citrate, method is as follows：
It takes 99mL ddw to be put into 200mL triangular flask with a scale, triangular flask is placed on heating magnetic stirring apparatus and heats and stirs
It mixes, the gold chloride of 1m L 1% is then added, heating stirring is then quickly added into the trisodium citrate of 1.6m L 1% to boiling,
Continuous heating stirring, observation color are gradually become peony and are stirred under heating 5min after no longer changing to color by light yellow,
Then it is cooling to remove triangular flask, is settled to 100mL with sterile ddw after room temperature to be restored, determines absorption maximum using UV scanning
4 DEG C of preservations after a length of 530nm of spike.
2. the preparation of colloidal gold labeled monoclonal antibody
Using the IgG mixture of anti-PRRSV and CSFV as gold labeling antibody, best combination pH value is 7.8, colloidal gold and antibody
Proportion is 25 μ g antibody/ml colloidal gold.Colloidal gold is marked to press the measurement of 60 μ l every square centimeter after 1%BSA stabilizer treatment
Marker solution uniform adsorption is on glass fibre membrane, and 37 DEG C are dried in vacuo 1 hour, and cutting width is 7mm, obtains label pad.
3. the preparation of coated film
Anti- N protein monoclonal antibody 5H5 that embodiment three purifies and anti-CSFV antibody 3B8 are adjusted to coating buffer respectively
Concentration is 2mg/mL, and it is 1.8mg/mL that sheep anti-mouse igg, which is adjusted to concentration with coating buffer, and monoclonal antibody 5H5 and 3B8 are sprayed respectively
Sheep anti-mouse igg is sprayed onto the quality control region C line on coated film, the monoclonal antibody by detection line T1 and T2 on to coated film
The dosage of 5H5, monoclonal antibody 3B8 and sheep anti-mouse antibody are 1 μ l/cm by coating coating liquid measure.Nature controlling line and two detections
Line interval 4mm, in humidity<30%, it dries 10 hours in 37 DEG C of baking ovens of temperature, envelope is spare.
4. sequentially mutually sample pad is pasted (having a size of 18*300mm, glass in overlap joint ground on bottom liner (having a size of 60*300mm)
Glass cellucotton material, first the Tris-HCl buffer impregnation 2h through 50mM, pH8.0), label pad (having a size of 7*300mm,
Glass fibre cotton material), coated film (having a size of 25*300mm, nitrocellulose material) and blotting paper (having a size of 16*300mm)
Test paper plate is obtained, is cut into the test strips of different in width as requested.
The structure of test strips is as shown in Figure 4：
1 is support plate, and 2 be nitrocellulose filter, and 3 be sample pad, and 4 be gold labeling antibody pad, and 5 be blotting paper, and 6 be detection line T2,7
It is nature controlling line C for detection line T1,8.
4. application method
The colloidal gold duplex test strips that porcine reproductive and respiratory syndrome virus and swine fever virus are detected while of the invention, are using
When, it is assembled in plastic shell made of being fastened as plastics upper casing and plastics lower casing, plastics upper casing is set there are two aperture, is loaded window
And show hole, sample-adding window correspond to detection PRRSV and CSFV the colloidal gold dual card test strips sample pad.
Its testing result determination method：
After being filtered on sample load sample pad to be detected, if containing PRRSV antigen in determinand, with colloid in test strips
The anti-PRRSV monoclonal antibody of gold label forms corresponding complexes, and uplink is coated on the anti-PRRSV N egg on nitrocellulose membrane
White monoclonal antibody 5H5 intercepts to form compound, i.e., red stripes are formed at T1；If containing CSFV antigenic substance in sample,
The compound that the anti-CSFV monoclonal antibody of itself and colloid gold label in test strips is formed continues uplink, is coated on cellulose nitrate
Anti- CSFV E2 protein monoclonal antibody 3B8 on film intercepts to form compound, i.e., red stripes are formed at T2.Regardless of whether
Containing PRRSV or CSFV antigen, the antibody of colloid gold label will all chromatograph the sheep anti-mouse igg being coated on NC film upwards and block
It cuts, forms red stripes on nature controlling line at the C.This band is nature controlling line, if this line does not occur, illustrates that colloidal gold strip loses
Effect.
As shown in figure 5, two bonding wires then occurs in the observation result positive, T1 colour developing illustrates there is PRRSV in sample；T2 colour developing
Illustrate there is CSFV in sample, T1, T2, which are shown, illustrates to have in sample PRRSV and CSFV, determines that a matter only occurs in result feminine gender
Line C is controlled, reagent strip is invalid if C band does not develop the color.
The present invention is described in detail above in conjunction with embodiment, still, person of ordinary skill in the field can
Understand, without departing from the purpose of the present invention, each design parameter in above-described embodiment can also be changed, shape
At multiple specific embodiments, it is common variation range of the invention, is no longer described in detail one by one herein.
SEQUENCE LISTING
<110>Zhengzhou University, Henan Zhong Ze bioengineering Co., Ltd
<120>
The colloidal gold duplex test strips and its preparation of porcine reproductive and respiratory syndrome virus and swine fever virus are detected simultaneously
Method
<130> 2018
<160> 1
<170> PatentIn version 3.2
<210> 1
<211> 1158
<212> DNA
<213> Sus scrofa
<400> 1
atgctactag taaatcagtc acaccaaggc ttcaataagg aacacacaag caagatggta 60
agcgctattg ttttatatgt gcttttggcg gcggcggcgc attctgcctt tgcgcatcac 120
catcaccatc accgcctggc ctgcaaggag gactaccgct acgccatcag cagcaccaac 180
gagatcggcc tgctgggcgc cggcggcctg accaccacct ggaaggagta cagccacgac 240
ctgcagctga acgacggcac cgtgaaggcc atctgcgtgg ccggcagctt caagatcacc 300
gccctgaacg tggtgagccg ccgctacctg gccagcctgc acaagggcgc cctgctgacc 360
agcgtgacct tcgagctgct gttcgacggc accaacccca gcaccgagga gatgggcgac 420
gacttcggct tcggcctgtg ccccttcgac accagccccg tggtgaaggg caagtacaac 480
accaccctgc tgaacggcag cgccttctac ctggtgtgcc ccatcggctg gaccggcgtg 540
atcgagtgca ccgccgtgag ccccaccacc ctgcgcaccg aggtggtgaa gaccttccgc 600
cgcgagaagc ccttccccca ccgcatggac tgcgtgacca ccaccgtgga gaacgaggac 660
ctgttctact gcaagctggg cggcaactgg acctgcgtga agggcgagcc cgtggtgtac 720
accggcggcc aggtgaagca gtgcaagtgg tgcggcttcg acttcaacga gcccgacggc 780
ctgccccact accccatcgg caagtgcatc ctggccaacg agaccggcta ccgcatcgtg 840
gacagcaccg actgcaaccg cgacggcgtg gtgatcagcg ccaagggcag ccacgagtgc 900
ctgatcggca acaccaccgt gaaggtgcac gccagcgacg agcgcctggg ccccatgccc 960
tgccgcccca aggagatcgt gagcagcgcc ggccccgtgc gcaagaccag ctgcaccttc 1020
aactacgcca agaccctgaa gaacaagtac tacgagcccc gcgacagcta cttccagcag 1080
tacatgctga agggcgagta ccagtactgg ttcgacctgg acgtgaccga ccgccacagc 1140
ggctacttcg ccgagttc 1158
Claims (8)
1. a kind of colloidal gold duplex test strips for detecting porcine reproductive and respiratory syndrome virus and swine fever virus simultaneously, including branch
Fagging, nitrocellulose filter, sample pad, gold labeling antibody pad, blotting paper, detection line T2, detection line T1 and nature controlling line, feature exist
In the gold labeling antibody pad is marked with the mixture of the IgG of the IgG and anti-CSFV of anti-PRRSV, and the detection line T1 is coated with anti-
PRRSV N protein monoclonal antibody, the detection line T2 are coated with anti-CSFV E2 protein monoclonal antibody, the nature controlling line packet
There is sheep anti-mouse igg.
2. colloidal gold that is according to claim 1 while detecting porcine reproductive and respiratory syndrome virus and swine fever virus is double
Joint-trial paper slip, which is characterized in that the anti-PRRSV N protein monoclonal antibody or anti-CSFV E2 protein monoclonal antibody
ELISA potency is not less than 105。
3. colloidal gold that is according to claim 1 while detecting porcine reproductive and respiratory syndrome virus and swine fever virus is double
Joint-trial paper slip, which is characterized in that the hypotype of the anti-PRRSV N protein monoclonal antibody is IgG1 type or IgG2a type.
4. colloidal gold that is according to claim 1 while detecting porcine reproductive and respiratory syndrome virus and swine fever virus is double
Joint-trial paper slip, which is characterized in that the hypotype of the anti-CSFV E2 protein monoclonal antibody is IgG1 type.
5. the colloidal gold duplex examination of porcine reproductive and respiratory syndrome virus and swine fever virus is detected described in a kind of claim 1 simultaneously
The preparation method of paper slip, which is characterized in that include the following steps：
（1）Codon optimization rear clone carried out to PRRSV N protein and CSFV E2 protein gene respectively, prokaryotic expression N protein,
Baculovirus expression E2 albumen, albumen obtain external source N protein and E2 albumen after purification respectively；
（2）Prepare anti-PRRSV N protein monoclonal antibody and anti-CSFV E2 protein monoclonal antibody：
BALB/c mouse is immunized respectively using the N protein of purifying and E2 albumen as antigen, screening obtains the hybridoma of PRRSV
Strain, the hybridoma cell strain of CSFV compare the strong hybridoma cell strain of selective affinity respectively and prepare monoclonal antibody；
（3）Prepare test strips：
I. colloidal gold solution is prepared using sodium citrate；
II. using the IgG mixture of the anti-PRRSV and anti-CSFV as gold labeling antibody, colloidal gold and antibody are through stabilizer treatment
After be adsorbed on glass fibre membrane, be dried in vacuo, cut to obtain label pad；
III. respectively by step（2）Resulting anti-PRRSV N protein monoclonal antibody and the spray of anti-CSFV E2 protein monoclonal antibody
Detection line T1 and T2 on to coated film, sheep anti-mouse igg is sprayed/nature controlling line is printed to, it dries, envelope is spare；
IV. sequentially mutually overlap joint pasting sample pad, label pad, coated film and blotting paper obtains test paper plate on bottom liner, is cut into
Different in width.
6. preparation method according to claim 5, which is characterized in that in the step（II）In：The pH when absorption
Value control is 7.8；The proportion of the colloidal gold and the antibody is 25 μ g antibody/ml colloidal gold.
7. preparation method according to claim 5, which is characterized in that in the step（III）In, the nature controlling line and two
Detection line interval 4mm.
8. preparation method according to claim 5, which is characterized in that in the step（III）In, the anti-PRRSV N
Concentration control when protein monoclonal antibody or anti-CSFV E2 protein monoclonal antibody are coated with is 2mg/mL.
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| CN111707821A (en) * | 2020-07-28 | 2020-09-25 | 郑州大学 | Colloidal gold test paper for detecting IBV (infectious bronchitis Virus) and preparation method thereof |
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