CN110554199A - Hog cholera virus antibody detection kit - Google Patents
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- 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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Abstract
the invention discloses a hog cholera virus antibody detection kit, and belongs to the technical field of biological detection. The hog cholera virus antibody detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 is a buffer solution with the pH value of 6.0-8.0, and comprises a stabilizer, a high-molecular accelerator, a surfactant and a preservative; the reagent R2 is a classical swine fever virus antigen latex reagent, which comprises a stabilizer, classical swine fever virus E2 antigen latex particles, a preservative and a buffer solution. The kit has the advantages of high detection sensitivity and specificity, good stability, simple operation and short reaction time; the method can be applied to full-automatic biochemical analyzers, special protein analyzers and spectrophotometers, does not generate sediment after reaction, and is convenient for cleaning reaction cups.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a hog cholera virus antibody detection kit.
Background
Classical Swine Fever (CSF) is an acute, febrile, and contact infectious disease caused by Classical Swine Fever Virus (CSFV), which is classified as an animal epidemic disease by our country and as an a-class infectious disease by the world animal health organization. The disease has the advantages of high propagation speed, wide prevalence, high morbidity, high mortality and great harm to the pig industry in China. The clinical manifestations of swine fever are mostly sub-acute, chronic or atypical, and occasionally acute, which can occur all the year round, but frequently occur in spring, autumn and winter, and pigs of different ages, sexes and breeds are likely to occur, and only by mastering clinical diagnosis and scientific detection methods and accurately monitoring the immune antibody level and the antibody growth rule of a swinery, the epidemic of swine fever can be effectively restrained.
At present, methods for detecting the antibody of the hog cholera virus comprise HA, RT-PCR, ELISA, immunochromatography and the like. The HA method HAs low detection sensitivity, samples need to be inactivated, and the error of visual interpretation is large; RT-PCR, ELISA and other detection sensitivity and specificity are high, but expensive instruments and equipment are needed, the detection time is long, and professional technicians are needed for operation; the colloidal gold immunochromatography is simple to operate and short in detection time, but the accuracy is not high, the detection range is narrow, and accurate quantification cannot be achieved. With the development of monoclonal antibody technology and the popularization of full-automatic biochemical analyzers and protein analyzers, the immunoturbidimetric technology has developed various rapid immunoturbidimetric detection technologies and has been widely applied to the technical practices of clinical diagnosis, biological material analysis and the like.
The latex enhanced turbidimetry (PETIA) is a stable and accurate homogeneous phase immunoturbidimetry detection method. The PETIA process is broadly divided into two categories: one is a scattering turbidimetric assay and the other is a transmission turbidimetric assay. The basic principle of the two methods is very similar, and the monoclonal antibody is crosslinked on the surface of the polymer nano microsphere, and when the microsphere crosslinked with the antibody is combined with an antigen, the microsphere is rapidly aggregated together in a short time, so that the light-scattering property or the light-transmitting property of the reaction solution is changed. Moreover, the change of the light-scattering property or the light-transmitting property (i.e., the absorbance) of the reaction solution has strong correlation with the concentration of the antigen to be detected, and the concentration of the antigen to be detected can be reflected in a certain range. The PETIA detection method is to perform antigen and antibody reactions and result measurement in a homogeneous reaction system. After the reaction of antigen and antibody, the absorbance value of the reaction solution is directly measured, thus omitting the complicated operation steps of repeated incubation and plate washing of ELISA method, obtaining the result in a few minutes and saving time and labor. In addition, the simplification of PETIA operation steps also avoids the interference of a plurality of human operation factors and external factors such as reagents, environment and the like, has better stability and repeatability, and can reflect the existence condition of the tested substance more truly. The introduction of the nano-particles solves the problems of weak binding capacity to the monoclonal antibody, narrow linear range of clinical detection, more interference factors, poor experimental stability and the like, and greatly promotes the wide clinical application of the immunoturbidimetry.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a swine fever virus antibody detection kit, which has the advantages of high detection sensitivity and specificity, good stability, simple operation and short reaction time; the method can be applied to full-automatic biochemical analyzers, special protein analyzers and spectrophotometers, does not generate sediment after reaction, and is convenient for cleaning reaction cups.
In order to achieve the purpose, the invention adopts the technical scheme that:
a hog cholera virus antibody detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 is a buffer solution with the pH value of 6.0-8.0, and comprises a stabilizer, a high-molecular accelerator, a surfactant and a preservative; the reagent R2 is a classical swine fever virus antigen latex reagent, which comprises a stabilizer, classical swine fever virus E2 antigen latex particles, a preservative and a buffer solution.
On the basis of the scheme, the swine fever virus E2 antigen latex particles of the reagent R2 are gold nanoparticles with the particle size of 60 +/-1.5 nm and marked with the swine fever virus E2 recombinant antigen.
On the basis of the scheme, the reagent R2 is prepared by the following method:
step 1), amplifying a gene sequence of a classical swine fever virus E2 gene, and connecting the gene sequence to a eukaryotic expression vector pEGFP-C1 vector by a clonex technology; transfecting the recombinant plasmid into a porcine kidney epithelial cell PK-15 by a DEAE-dextran method, crushing the cell under high pressure after culturing, and purifying and identifying cell supernatant by an affinity chromatography method to obtain classical swine fever virus E2 protein;
step 2), taking 100mL of nano gold solution, adjusting the pH value to 7.5-10.0 by using 1% K 2 CO 3, slowly adding the CSFV E2 recombinant protein according to a certain proportion, reacting for 10min under a magnetic stirrer, adding the BSA solution until the final concentration is 1%, continuously stirring for 5min to obtain CSFV E2 antigen latex particles, and standing overnight at 4 ℃;
and 3) centrifuging the CSFV E2 antigen latex particles at 3000rpm for 30min, removing polymeric agglutination impurities, centrifuging at 12000rpm and 4 ℃ for 30min, discarding the supernatant, redissolving with a heavy suspension (0.01M PBS solution containing 1% BSA), repeatedly washing for 3 times, collecting the precipitate, resuspending with a buffer solution containing a stabilizer and a preservative, precipitating, adjusting the concentration to OD 540nm to 0.2, and subpackaging.
On the basis of the scheme, in the step 2), the mass ratio of the nanogold to the classical swine fever virus E2 recombinant protein is 1:0.8-1: 1.6.
on the basis of the scheme, the nano gold solution in the step 2) is prepared by the following method: adding 100mL of ultrapure water into a clean round-bottom flask, adding 1mL of 1% chloroauric acid solution, stirring at a high speed of 600rpm, heating until the solution is boiled, quickly adding 1.5mL of 1% trisodium citrate solution, continuing to heat and stir for 10min, turning the solution color black and then red, stopping heating, continuing to stir for 10min, stopping stirring, returning to room temperature, and returning to 100mL with the ultrapure water; preparing nano gold solution, and ultrafiltering to remove small molecules for later use.
On the basis of the above-mentioned scheme,
the buffer solution of the reagent R2 comprises one or more of phosphate buffer solution, boric acid buffer solution, glycine buffer solution, Tris-Cl buffer solution and HEPS buffer solution;
the stabilizer of the reagent R2 comprises one or more of calf serum, BSA, skim milk, gelatin, sucrose and glycerol;
The preservative of the reagent R2 comprises one or more of sodium azide, phenol, Proclin and p-hydroxybenzoic acid.
on the basis of the scheme, the stabilizing agent of the reagent R2 is sucrose with the mass-volume ratio of 10-20%, BSA with the mass-volume ratio of 0.5-2% and glycerol with the mass-volume ratio of 3-10%;
the preservative of the reagent R2 is Proclin300 with the mass volume ratio of 0.1-0.5%;
The buffer solution of the reagent R2 is phosphate buffer solution with pH value of 6.0-8.0.
on the basis of the above-mentioned scheme,
the buffer solution of the reagent R1 comprises one or more of phosphate buffer solution, TRIS buffer solution, borax-sodium hydroxide buffer solution and glycine buffer solution;
The stabilizer of the reagent R1 comprises one or more of disodium ethylene diamine tetraacetate, sodium diethylenetriamine pentaacetate, mannose, glucose, chitosan, sorbitol and bovine serum albumin;
The preservative of the reagent R1 comprises one or more of sodium azide, phenol, Proclin and p-hydroxybenzoic acid;
the high molecular accelerator of the reagent R1 comprises one or more of polyvinylpyrrolidone, sodium polyacrylate and polyethylene glycol;
The surfactant of the reagent R1 comprises one or more of Triton series, Tween series, lauryl ether and polyoxyethylene alkyl phenyl ether.
On the basis of the above-mentioned scheme,
the buffer solution of the reagent R1 is phosphate buffer solution with pH value of 6.0-8.0;
The stabilizer of the reagent R1 is bovine serum albumin with the mass volume ratio of 0.2-2%;
the preservative of the reagent R1 is Proclin300 with the mass volume ratio of 0.1-0.5%;
The macromolecular accelerator of the reagent R1 is polyethylene glycol PEG8000 with the mass volume ratio of 0.2-0.5%;
The surfactant of the reagent R1 is Tween20 with the mass-volume ratio of 0.05-0.1%.
on the basis of the scheme, the preparation method of the reagent R1 is as follows:
0.5g BSA and 0.5g PEG8000 were added to 90mL phosphate buffer (pH7.2) under magnetic stirring, after complete dissolution, 100uL Proclin300 and 0.08% Tween20 were added, and finally 100mL was made up with phosphate buffer (pH7.2), and the volume was filled up to 100mL and stored.
the invention has the following beneficial effects:
1) the kit has the advantages of high detection sensitivity and specificity, good stability, simple operation and short reaction time which only needs 10 min;
2) The kit can be suitable for full-automatic biochemical analyzers, special protein analyzers and spectrophotometers, does not generate precipitates after reaction, and is convenient for cleaning reaction cups;
3) The kit provided by the invention has no significant difference in statistical analysis of CSFV antibody detection data in a sample with methods such as RT-PCR, ELISA, immunochromatography and the like, and has reliable detection results and larger market competitiveness;
4) And judging whether the antibody of the classical swine fever virus exists or not through the turbidity change of the solution before and after the reaction of the classical swine fever virus antigen marked by the latex microspheres by using the nanogold and the sample to be detected. The method does not need to pretreat the sample, has simple and convenient operation, accuracy and reliability and good repeatability, and can be used on a full-automatic biochemical analyzer, a special protein analyzer and a spectrophotometer.
Detailed Description
Terms used in the present invention have generally meanings as commonly understood by one of ordinary skill in the art, unless otherwise specified.
the present invention will be described in further detail with reference to the following data in conjunction with specific examples. The following examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.
Example 1
A hog cholera virus antibody detection kit comprises a reagent R1 and a reagent R2, wherein the reagent R1 is a buffer solution with the pH value of 6.0-8.0, and comprises a stabilizer, a high-molecular accelerator, a surfactant and a preservative; the reagent R2 is a classical swine fever virus antigen latex reagent, which comprises a stabilizer, classical swine fever virus E2 antigen latex particles, a preservative and a buffer solution.
1. Preparation of latex microspheres
The preparation method of the latex microsphere with the nano gold comprises the following steps: adding 100mL of ultrapure water into a clean 1000mL round-bottom flask, adding 1 piece of magnetic stirrer, adding 1mL of 1% chloroauric acid solution, stirring at a high speed of 600rpm, heating until the solution is boiled, rapidly adding 1.5mL of 1% trisodium citrate solution, continuing to heat and stir for 10min, enabling the solution to turn black and red, stopping heating, continuing to stir for 10min, stopping stirring, returning to room temperature, and returning to 100mL with the ultrapure water. The particle size of the prepared colloidal gold is about 40 +/-1.2 nm by electron microscope observation, and micromolecules are removed by ultrafiltration for later use.
2. Preparation of reagent R2
1) the suspected swine fever dead pigs are diagnosed as the swine fever after clinical symptom observation, pathological anatomy change, gold-labeled card detection, rabbit body interaction test and RT-PCR experimental diagnosis. Then, a pair of specific primers is designed according to the gene sequences of classical swine fever virus shimen (GenBank accession number is AF092449) and E2 gene of C strain (GenBank accession number is Z46258):
P1:5-TGGCTAGTTAATGCAGACA-3
P2:5-AAATTACCTTAGTCCAACT-3；
collecting blood, tonsil, lymph node, spleen, kidney and other tissues of dead pig, extracting total RNA, RT-PCR amplifying, electrophoresis detecting and sequencing to obtain expected target DNA band, purifying, connecting with pMD18-T vector, transforming into TOP10 sensing cell, adding IPTG and X-gal into Amp-containing agar culture medium, inducing culture, selecting white colony culture to extract plasmid, digestion identifying and sequencing with BamHI and HindIII, analyzing the sequencing result with DNASAR and DNAMN software to obtain DNA with nucleotide homology of 98.9% and 94.9% and amino acid homology of 99.7% and 98.1%. Connecting the amplified classical swine fever virus E2 gene into a eukaryotic expression vector pEGFP-C1 vector through a clonExpress technology, transforming DH5 alpha competent cells, selecting a monoclonal extraction plasmid on an LB plate, and performing sequencing identification; transfecting the recombinant plasmid pEGFP-E2 to porcine kidney epithelial cells PK-15 by a DEAE-dextran method, crushing the cells under high pressure after culturing, and purifying and identifying cell supernatant by an affinity chromatography method to obtain the classical swine fever virus E2 protein.
2) adjusting the pH value of 100mL of colloidal gold solution to 9.6 by using 1% K 2 CO 3, slowly adding 10mg of CSFV E2 recombinant antigen, reacting for 10min under a magnetic stirrer, adding BSA solution until the final concentration is 1%, continuously stirring for 5min to obtain CSFV E2 antigen latex particles, standing overnight at 4 ℃, centrifuging for 30min at 3000rpm the next day to remove aggregated impurities, centrifuging for 30min at 12000rpm4 ℃, discarding supernatant, redissolving by using a heavy suspension (1% BSA +0.01M PBS), repeatedly washing for 3 times, collecting precipitates, resuspending the precipitates by using 20% sucrose by mass/volume, 2% BSA by mass/volume, 10% glycerol by mass/volume and 0.5% Proclin300 by mass/volume as a stabilizer in 0.01M PBS buffer (pH7.2), adjusting the concentration to 0.2, and subpackaging 540nm.
3. Preparation of reagent R1
Adding 0.5g BSA and 0.5g PEG8000 into 90mL phosphate buffer (pH7.2) under magnetic stirring, completely dissolving, adding 100uL Proclin300, diluting to 100mL with phosphate buffer (pH7.2), and packaging.
4. the determination method comprises the following steps: end point method, main wavelength 340nm, sub-wavelength 700 nm. And (3) sucking 300uL of reagent R1 and 10uL of sample to be detected into a reaction cup, mixing uniformly, reading a blank after reacting for 180s, adding 100uL of reagent R2, mixing uniformly, and reading reaction absorbance after reacting for 300 s. The antibody levels in the samples were calculated by analysis with statistical software SPSS 11.5.
5. Measurement of Performance
5.1 reliability analysis 30 clinical serum samples were tested in parallel with the IDEXX kit, with 100% consistency. The results are as follows:
TABLE 1 comparison of the test results of the kit of the invention and IDEXX kit
5.2 assay sensitivity assay the 3# positive specimen in the 5.1 test was assayed at 1:2, 1:4, 1:8, 1:16. gradient dilution, the assay results of the kit of the invention were essentially identical to the IDEXX kit, and the assay sensitivity of the kit of the invention to the sample was higher than the control kit.
TABLE 2 assay of the detection sensitivity of the kit of the invention
5.3 precision analysis 3 portions of immunoglobulin with concentrations of 3.5mg/L, 47mg/L, 123mg/L respectively as experimental samples, each repeat 20 times to calculate the average value, standard deviation and coefficient of variation to obtain the data of the intra-batch precision related system, then measure the 3 samples 2 times per day (each time in the morning and afternoon), each time measure the double-point repeat, measure 20d, calculate the average value, standard deviation and coefficient of variation to obtain the data of the inter-batch precision related system, the results are as follows:
TABLE 3 results of the precision analysis of the kit of the present invention
the foregoing is directed to preferred embodiments of the present invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope of the technical solution of the present invention.
Claims (10)
1. A hog cholera virus antibody detection kit comprises a reagent R1 and a reagent R2, and is characterized in that: the reagent R1 is a buffer solution with pH of 6.0-8.0, and comprises a stabilizer, a high molecular accelerator, a surfactant and a preservative; the reagent R2 is a classical swine fever virus antigen latex reagent, which comprises a stabilizer, classical swine fever virus E2 antigen latex particles, a preservative and a buffer solution.
2. the hog cholera virus antibody detection kit of claim 1, wherein:
The swine fever virus E2 antigen latex particles of the reagent R2 are gold nanoparticles marked with swine fever virus E2 recombinant antigen and having the particle size of 60 +/-1.5 nm.
3. the swine fever virus antibody detection kit according to claim 2, wherein: the reagent R2 is prepared by the following method:
step 1), amplifying a gene sequence of a classical swine fever virus E2 gene, and connecting the gene sequence to a eukaryotic expression vector pEGFP-C1 vector by a clonex technology; transfecting the recombinant plasmid into a porcine kidney epithelial cell PK-15 by a DEAE-dextran method, crushing the cell under high pressure after culturing, and purifying and identifying cell supernatant by an affinity chromatography method to obtain classical swine fever virus E2 protein;
step 2), taking 100mL of nano gold solution, adjusting the pH value to 7.5-10.0 by using 1% K 2 CO 3, slowly adding the CSFV E2 recombinant protein according to a certain proportion, reacting for 10min under a magnetic stirrer, adding the BSA solution until the final concentration is 1%, continuously stirring for 5min to obtain CSFV E2 antigen latex particles, and standing overnight at 4 ℃;
and 3) centrifuging the CSFV E2 antigen latex particles at 3000rpm for 30min, removing polymeric agglutination impurities, centrifuging at 12000rpm and 4 ℃ for 30min, discarding the supernatant, redissolving with a heavy suspension (0.01M PBS solution containing 1% BSA), repeatedly washing for 3 times, collecting the precipitate, resuspending with a buffer solution containing a stabilizer and a preservative, precipitating, adjusting the concentration to OD 540nm to 0.2, and subpackaging.
4. The hog cholera virus antibody detection kit of claim 3, wherein: in the step 2), the mass ratio of the nanogold to the classical swine fever virus E2 recombinant protein is 1:0.8-1: 1.6.
5. The hog cholera virus antibody detection kit of claim 3, wherein: the nano gold solution in the step 2) is prepared by the following method: adding 100mL of ultrapure water into a clean round-bottom flask, adding 1mL of 1% chloroauric acid solution, stirring at a high speed of 600rpm, heating until the solution is boiled, quickly adding 1.5mL of 1% trisodium citrate solution, continuing to heat and stir for 10min, turning the solution color black and then red, stopping heating, continuing to stir for 10min, stopping stirring, returning to room temperature, and returning to 100mL with the ultrapure water; preparing nano gold solution, and ultrafiltering to remove small molecules for later use.
6. the hog cholera virus antibody detection kit according to any one of claims 1-5, wherein:
The buffer solution of the reagent R2 comprises one or more of phosphate buffer solution, boric acid buffer solution, glycine buffer solution, Tris-Cl buffer solution and HEPS buffer solution;
The stabilizer of the reagent R2 comprises one or more of calf serum, BSA, skim milk, gelatin, sucrose and glycerol;
the preservative of the reagent R2 comprises one or more of sodium azide, phenol, Proclin and p-hydroxybenzoic acid.
7. the hog cholera virus antibody detection kit of claim 6, wherein:
The stabilizer of the reagent R2 is sucrose with the mass-volume ratio of 10-20%, BSA with the mass-volume ratio of 0.5-2% and glycerol with the mass-volume ratio of 3-10%;
The preservative of the reagent R2 is Proclin300 with the mass volume ratio of 0.1-0.5%;
The buffer solution of the reagent R2 is phosphate buffer solution with pH value of 6.0-8.0.
8. the hog cholera virus antibody detection kit according to claim 1, wherein:
the buffer solution of the reagent R1 comprises one or more of phosphate buffer solution, TRIS buffer solution, borax-sodium hydroxide buffer solution and glycine buffer solution;
the stabilizer of the reagent R1 comprises one or more of disodium ethylene diamine tetraacetate, sodium diethylenetriamine pentaacetate, mannose, glucose, chitosan, sorbitol and bovine serum albumin;
The preservative of the reagent R1 comprises one or more of sodium azide, phenol, Proclin and p-hydroxybenzoic acid;
The high molecular accelerator of the reagent R1 comprises one or more of polyvinylpyrrolidone, sodium polyacrylate and polyethylene glycol;
The surfactant of the reagent R1 comprises one or more of Triton series, Tween series, lauryl ether and polyoxyethylene alkyl phenyl ether.
9. the hog cholera virus antibody detection kit of claim 8, wherein:
The buffer solution of the reagent R1 is phosphate buffer solution with pH value of 6.0-8.0;
the stabilizer of the reagent R1 is bovine serum albumin with the mass volume ratio of 0.2-2%;
The preservative of the reagent R1 is Proclin300 with the mass volume ratio of 0.1-0.5%;
The macromolecular accelerator of the reagent R1 is polyethylene glycol PEG8000 with the mass volume ratio of 0.2-0.5%;
the surfactant of the reagent R1 is Tween20 with the mass-volume ratio of 0.05-0.1%.
10. The hog cholera virus antibody detection kit of claim 8 or 9, wherein: the preparation method of the reagent R1 is as follows:
0.5g BSA and 0.5g PEG8000 were added to 90mL phosphate buffer (pH7.2) under magnetic stirring, after complete dissolution, 100uL Proclin300 and 0.08% Tween20 were added, and finally 100mL was made up with phosphate buffer (pH7.2), and the volume was filled up to 100mL and stored.
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| CN108152501A (en) * | 2018-01-08 | 2018-06-12 | 山东畜牧兽医职业学院 | A groups of bovine rota detection kits and the antiviral antibody colloid gold reagent preparation method |
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| CN111665355A (en) * | 2020-05-06 | 2020-09-15 | 量准（上海）医疗器械有限公司 | Kit based on nano plasma resonance molecules and testing method |
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