CN112255399A - Hog cholera virus detection kit based on double-antibody sandwich homogeneous phase chemiluminescence method, and preparation method and application thereof - Google Patents
Hog cholera virus detection kit based on double-antibody sandwich homogeneous phase chemiluminescence method, and preparation method and application thereof Download PDFInfo
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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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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- 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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- G01N2333/183—Flaviviridae, e.g. pestivirus, mucosal disease virus, bovine viral diarrhoea virus, classical swine fever virus (hog cholera virus) or border disease virus
Abstract
The invention discloses a hog cholera virus detection kit based on a double-antibody sandwich homogeneous chemiluminescence technology, which comprises a reagent R1, a reagent R2, a reagent R3, a positive control and a negative control, wherein donor microspheres in the reagent R1 can generate monomer oxygen under the excitation of 680nm light; the acceptor microspheres in the reagent R2 were able to react with monomeric oxygen to generate a detectable 615nm chemiluminescent signal. Therefore, the chemiluminescent signal can be directly detected without cleaning and separation during detection, and the detection of the classical swine fever virus is realized. In addition, the kit can complete antibody detection within 10min, and has good specificity, sensitivity and repeatability.
Description
Technical Field
The invention belongs to the technical field of biological diagnostic reagents, and particularly relates to a hog cholera virus detection kit based on a double-antibody sandwich chemiluminescence method and application thereof.
Background
Classical swine fever (CSF or Hog cholera, HC), also known as Classical swine fever or Classical swine fever, is a highly infectious disease of pigs. Swine fever can lead to fever, anorexia, diarrhea, death, etc. of affected pigs, and may be accompanied by neurological symptoms. Sows may miscarriage or lay dead piglets. Hog cholera is one of 16 legal infections listed in the world animal health organization under class A. Classical Swine Fever Virus (CSFV) is a genus of pestivirus of the family Flaviviridae. Viruses of the same genus are also Bovine Viral Diarrhea Virus (BVDV) and ovine Border Disease Virus (BDV) which infect ruminants. Pigs are the only host and source of transmission for classical swine fever viruses. Infected pigs are detoxified both before and during the onset of disease.
CSFV is enveloped virus, 40-60nm, single strand positive strand RNA. The CSFV genome is about 123kb in length and contains only one large Open Reading Frame (ORF) that is translated into a polyprotein of 3898 amino acid residues and a molecular weight of about 438kDa, and is further processed to the mature protein by viral and host cell proteases. All structural and non-structural proteins of CSFV are encoded by the ORF, which is flanked by a 5 '-untranslated region (5' -UTR) and a 3 '-untranslated region (3' -UTR), and has no cap structure at the 5 '-end and no poly (A) tail at the 3' -end. The large precursor protein is processed into structural protein and non-structural protein in a co-translated and post-translated form under the action of cellular protease and virus-specific protease, and the coding sequence of the structural protein and the non-structural protein on virus RNA is Npro, c, Erns (E0), E1, E2, P7, NS2-3, NS4A, NS4B, NS5A and NS 5B. NS2-3 can be processed into NS2, NS3(P80), except that c, E0, E1 and E2 are structural proteins.
E2 is the envelope glycoprotein of CSFV, is the main antigen protein of virus, and is the least conservative molecule among three virus glycoproteins. E2 is often present on the surface of virions and CSFV-infected cells as a homodimer of 100kDa and a heterodimer with E1 of 75 kDa. E2 induced the production of neutralizing antibodies against the virus in vitro and antibodies against the CSFV challenge in vivo. The protein backbone of E2 consisted of 370 amino acids (690-1060 amino acid residues encoded by the ORF.) and was anchored to the membrane with the 40 hydrophobic amino acids at its c-terminus. Due to the varying degree of glycosylation. The molecular weight of E2 may be 51-58 kDa. The 15 Cys residues in the E2 molecule are conserved within genus, wherein the 6 Cys residues at the N-terminus are involved in the formation of antigenic domains, and the 9 Cys residues at the c-terminus are involved in the formation of homo-and heterodimers. Therefore, the swine fever E2 protein can be used as a target protein for preventing and detecting swine fever.
Many methods for detecting classical swine fever viruses have been developed in many laboratories at home and abroad, such as enzyme-linked immunosorbent assay, colloidal gold immunochromatography, fluorescence immunochromatography, and the like. The fluorescence PCR detection method is time-consuming and labor-consuming; colloidal gold and fluorescence immunochromatography belong to qualitative and semi-quantitative detection, and result accuracy is not high. The need for accurate and rapid detection of products is urgent, and chemiluminescence is the best choice, but magnetic particle chemiluminescence costs more and has higher requirements for instrumentation, so homogeneous chemiluminescence is a suitable choice. Compared with a fluorescence PCR detection method, the technology has the advantages of simple operation, short reaction time, good sensitivity and accuracy and wide detection range.
Disclosure of Invention
The invention aims to solve the problems of high cost, long time consumption, low precision and the like of the hog cholera virus diagnostic reagent in the current market, and provides a double-antibody sandwich homogeneous phase chemiluminescence method hog cholera virus detection kit.
Therefore, in one aspect, the present invention provides a double-antibody sandwich homogeneous phase chemiluminescence hog cholera virus detection kit, which comprises a reagent R1, a reagent R2, a reagent R3, a positive control and a negative control, wherein: the reagent R1 comprises a buffer solution of streptavidin-labeled donor microspheres capable of being excited to generate monomeric oxygen under 680nm light; the reagent R2 comprises a buffer solution of an acceptor microsphere for labeling the anti-classical swine fever virus monoclonal antibody, wherein the acceptor microsphere can react with monomer oxygen to generate a detectable 615nm chemiluminescent signal; the reagent R3 is a buffer solution of a biotinylated anti-classical swine fever virus monoclonal antibody.
Preferably, the receptor microsphere labeled with the anti-CSFV monoclonal antibody in the reagent R2 is the receptor microsphere labeled with the anti-CSFV E2 protein monoclonal antibody, the buffer solution is 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 5% sucrose, 0.1% Tween-20, the rest is purified water, and the pH is 7.2.
Preferably, the biotinylated anti-classical swine fever virus monoclonal antibody in the reagent R3 is a biotinylated anti-classical swine fever virus E2 protein monoclonal antibody, the concentration of the biotinylated anti-classical swine fever virus E2 protein monoclonal antibody is 0.5 μ g/mL, the buffer solution is 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 5% sucrose, 0.1% Tween-20, the balance is purified water, and the pH is 7.2.
Preferably, the positive control of the present invention is inactivated swine fever virus fluorescent PCR CT value of swine serum between 25 + -1; the negative control is inactivated hog serum without CT value by hog cholera virus fluorescence PCR.
Preferably, the donor microspheres and the acceptor microspheres of the present invention are both 150nm in diameter.
Preferably, the concentration of the donor microspheres in the reagent R1 is 5 mug/mL; the concentration of the acceptor microspheres in the reagent R2 is 2 mug/mL.
Preferably, the buffer component of the reagent R1 is 0.01M PBS buffer, 2% BSA, 0.1% Proclin300, 0.1% Tween-20, and the balance is purified water, and the pH value is 7.2.
In another aspect, the present invention also provides a method for preparing the kit, the method comprising the steps of:
1) preparation of reagent R1:
a) preparation of donor microspheres: weighing 10mg of 150nm donor microspheres and 1mg of streptavidin, quickly and uniformly mixing, and adding 0.05M MES buffer solution with the pH value of 6.0 to adjust the concentration of the donor microspheres to 10 mg/mL; b) reaction: adding 50mg/mL NaBH3CN solution prepared from 0.05M MES with the value of 100 mu LpH being 6.0, quickly mixing uniformly, and carrying out rotary reaction at room temperature for 12-16 h; c) and (3) sealing: adding 100mg/mL BSA solution prepared from 0.05M MES buffer solution with pH value of 6.0 and volume ratio of the BSA solution to the reaction solution of 1:4, rapidly mixing, and rotating at room temperature for reaction for 3 hours; d) cleaning: centrifuging the reacted solution at 4 ℃ and 12000rpm for 60min, discarding the supernatant, adding 2mL of 0.05M MES buffer solution with the pH value of 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the washed donor microspheres with 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 0.1% Tween-20 and the balance of purified water and a buffer solution with the pH value of 7.2 to ensure that the final concentration of the donor microspheres is 5 mug/mL, and preserving at 4 ℃ for later use after ultrasonic dispersion;
2) preparation of reagent R2:
a) preparing acceptor microspheres: weighing 10mg of 150nm receptor microsphere and 2mg of swine fever virus E2 protein resistant monoclonal antibody, quickly and uniformly mixing, and adding 0.05M MES buffer solution with the pH value of 6.0 to adjust the concentration of the receptor microsphere to 10 mg/mL; b) reaction: adding 100 mu L of NaBH3CN solution with the concentration of 50mg/mL prepared by 0.05M MES with the pH value of 6.0, uniformly mixing, and carrying out rotary reaction at room temperature for 12-16 h; c) and (3) sealing: adding 100mg/mL BSA solution prepared from 0.05M MES with pH value of 6.0, sealing, mixing with the reaction solution at a volume ratio of 1:4, rapidly mixing, and rotating at room temperature for 3 hr; d) cleaning: centrifuging the reacted solution at 4 ℃ and 12000rpm for 60min, discarding the supernatant, adding 2ml of 0.05M MES buffer solution with the pH value of 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the receptor microspheres prepared in the step by using 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 5% sucrose, 0.1% Tween-20 and the balance of purified water, using a buffer solution with the pH value of 7.2 to ensure that the final concentration of the receptor microspheres is 2 mu g/ml, and preserving at 4 ℃ for later use after ultrasonic dispersion;
3) preparation of reagent R3
a) Biotin activation: weighing 1mg of biotin, adding the biotin into 100 mu L of DMF solution, fully and uniformly mixing, then diluting to 0.5mL by using PBS solution, accurately weighing 2mg of EDC, adding the EDC into the solution, and activating for 1h at room temperature; b) Reaction: measuring a certain amount of swine fever virus E2 protein-resistant monoclonal antibody, dialyzing overnight at 4 ℃ in a PBS solution, measuring the concentration of the antibody and adjusting to 2mg/mL, adding the activated biotin solution into the swine fever virus E2 protein-resistant monoclonal antibody solution, fully and uniformly mixing, and reacting at room temperature for 6 hours; c) and (3) purification: adding a biotinylated anti-CSFV E2 protein monoclonal antibody solution into a dialysis bag with a retention molecular weight of 10KD, dialyzing for 24 hours at 4 ℃ by using a PBS solution as a buffer solution, sampling and determining the antibody concentration, adjusting the concentration of the biotinylated anti-CSFV E2 protein monoclonal antibody qualified by quality inspection to 0.5 mu g/mL by using 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 5% sucrose, 0.1% Tween-20 and the balance of purified water and a buffer solution with the pH of 7.2, and storing for later use at 4 ℃.
In still another aspect, the present invention provides a detection method using the detection kit for swine fever virus, wherein the method comprises the following steps:
1) mixing the detection sample or the positive control or the negative control with the reagent R1, the reagent R2 and the reagent R3 according to the volume ratio of 10 muL to 30 muL, and placing the mixed solution at 37 ℃ and keeping out of the sun for incubation for 10 min;
2) immediately after incubation, the chemiluminescence signal of the solution was detected by a photomultiplier tube for 3s, and the chemiluminescence values of the positive control, the negative control and the sample were recorded;
3) calculating an S/P value from the chemiluminescence value, wherein S/P value ═ (sample chemiluminescence value-negative control chemiluminescence value)/(positive control chemiluminescence value-negative control chemiluminescence value); when the S/P value is more than or equal to 0.4, the swine fever virus is judged to be positive; when the S/P value is less than 0.4, the swine fever virus is judged to be negative.
In still another aspect, the invention further provides an application of the kit in detecting classical swine fever viruses.
The donor microsphere can generate active oxygen in an excited state; the acceptor microspheres are capable of reacting with reactive oxygen species to generate a detectable chemiluminescent signal (the donor and acceptor microspheres are incapable of emitting a chemiluminescent signal when in a dispersed state and capable of emitting a detectable chemiluminescent signal only when in close proximity). Therefore, the chemiluminescent signal can be directly detected without cleaning and separation during detection, and the detection of the classical swine fever virus is realized. In addition, the kit can complete antibody detection within 10min, and has good specificity, sensitivity and repeatability. In addition, the kit provided by the invention can be applied to tubular chemiluminescence and plate chemiluminescence. Of course, the deletion of the streptavidin-biotin system based on the present invention is also within the scope of the present invention.
A homogeneous chemiluminescent detection reaction system for classical swine fever viruses based on a competition method comprises donor microspheres for marking streptavidin, acceptor microspheres for marking monoclonal antibodies against classical swine fever viruses E2 protein, and biotinylated monoclonal antibodies against classical swine fever viruses E2 protein. When a double-antibody sandwich method (two antibodies recognize different antigenic sites) is adopted to detect the CSFV in a sample, when the CSFV exists in the sample, the biotinylated anti-CSFV E2 protein monoclonal antibody in a sample pool to be detected can be combined with the CSFV, and then is combined with an acceptor microsphere of the monoclonal antibody which is marked with the anti-CSFV E2 protein monoclonal antibody, then biotin can be combined with a streptavidin donor microsphere, finally a streptavidin-marked acceptor microsphere-biotinylated anti-CSFV E2 protein-CSFV-marked anti-CSFV E2 protein donor microsphere is formed, under the irradiation of laser (680nm), the photosensitizer on the donor microsphere converts the oxygen in the surrounding environment into more active monomer oxygen, and the monomer oxygen is diffused to the acceptor microsphere to react with a chemiluminescent agent on the acceptor microsphere, further activating the luminescent groups on the receptor microsphere to generate chemiluminescence, wherein the wavelength is 615nm, the luminescent value is the highest, the diffusible distance of monomer oxygen in the half-life period of 4 mu s is about 200nm, and the optical signal can be detected by a high-throughput chemiluminescence detector; if there is no specific reaction of antigen-antibody, i.e. if a complex of streptavidin-labeled acceptor microspheres-biotinylated anti-classical swine fever virus E2 protein monoclonal antibody-classical swine fever virus-labeled anti-classical swine fever virus E2 protein monoclonal antibody donor microspheres is not formed, the monomeric oxygen will not diffuse to the distant acceptor microspheres, and no chemiluminescence will occur. The principle of double-antibody sandwich homogeneous chemiluminescence detection of CSFV is shown in figure 1.
The two monoclonal antibodies used in the present invention are monoclonal antibodies against classical swine fever virus E2 protein, and as a person skilled in the art, similar schemes for substituting other monoclonal antibodies against classical swine fever virus are also within the scope of the present invention.
Drawings
FIG. 1: schematic diagram of double-antibody sandwich homogeneous-phase chemiluminescent hog cholera virus detection kit.
FIG. 2: classical swine fever virus E2 protein standard curve diagram.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It is to be understood that the embodiments described are only some of the embodiments of the invention, and not all of them.
The chemical reagents related to the invention are all made in China. Classical swine fever virus E2 protein was offered by Zhejiang Hirong Biotech, Inc. Both swine fever virus-resistant monoclonal antibodies are produced by Hangzhou Hongshen sanden. Donor microspheres and acceptor microspheres are offered by Hangzhou Hongqiao Zhongke Gene technology, Inc. Streptavidin and biotin were purchased from Beijing Soilebao.
Example 1 establishment of double-antibody sandwich homogeneous-phase chemiluminescence hog cholera virus detection kit
1 reagent R1 preparation:
a) preparation of donor microspheres: weighing 10mg of 150nm donor microspheres and 1mg of streptavidin, quickly and uniformly mixing, and adding 0.05M MES buffer solution with the pH value of 6.0 to adjust the concentration of the donor microspheres to 10 mg/mL; b) reaction: adding 50mg/mL NaBH3CN solution prepared from 0.05M MES with the value of 100 mu LpH being 6.0, quickly mixing uniformly, and carrying out rotary reaction at room temperature for 12-16 h; c) and (3) sealing: adding 100mg/mL BSA solution prepared from 0.05M MES buffer solution with pH value of 6.0 and volume ratio of the BSA solution to the reaction solution of 1:4, rapidly mixing, and rotating at room temperature for reaction for 3 hours; d) cleaning: centrifuging the reacted solution at 4 ℃ and 12000rpm for 60min, discarding the supernatant, adding 2mL of 0.05M MES buffer solution with the pH value of 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the washed donor microspheres with 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 0.1% Tween-20 and the balance of purified water and a buffer solution with the pH value of 7.2 to ensure that the final concentration of the donor microspheres is 5 mug/mL, and preserving at 4 ℃ for later use after ultrasonic dispersion;
2 reagent R2 preparation:
a) preparing acceptor microspheres: weighing 10mg of 150nm receptor microsphere and 2mg of swine fever virus E2 protein resistant monoclonal antibody, quickly and uniformly mixing, and adding 0.05M MES buffer solution with the pH value of 6.0 to adjust the concentration of the receptor microsphere to 10 mg/mL; b) reaction: adding 100 mu L of NaBH3CN solution with the concentration of 50mg/mL prepared by 0.05M MES with the pH value of 6.0, uniformly mixing, and carrying out rotary reaction at room temperature for 12-16 h; c) and (3) sealing: adding 100mg/mL BSA solution prepared from 0.05M MES with pH value of 6.0, sealing, mixing with the reaction solution at a volume ratio of 1:4, rapidly mixing, and rotating at room temperature for 3 hr; d) cleaning: centrifuging the reacted solution at 4 ℃ and 12000rpm for 60min, discarding the supernatant, adding 2ml of 0.05M MES buffer solution with the pH value of 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the receptor microspheres prepared in the step by using 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 5% sucrose, 0.1% Tween-20 and the balance of purified water, using a buffer solution with the pH value of 7.2 to ensure that the final concentration of the receptor microspheres is 2 mu g/ml, and preserving at 4 ℃ for later use after ultrasonic dispersion;
3 preparation of reagent R3
a) Biotin activation: weighing 1mg of biotin, adding the biotin into 100 mu L of DMF solution, fully and uniformly mixing, then diluting to 0.5mL by using PBS solution, accurately weighing 2mg of EDC, adding the EDC into the solution, and activating for 1h at room temperature; b) Reaction: measuring a certain amount of swine fever virus E2 protein-resistant monoclonal antibody, dialyzing overnight at 4 ℃ in a PBS solution, measuring the concentration of the antibody and adjusting to 2mg/mL, adding the activated biotin solution into the swine fever virus E2 protein-resistant monoclonal antibody solution, fully and uniformly mixing, and reacting at room temperature for 6 hours; c) and (3) purification: adding a biotinylated anti-CSFV E2 protein monoclonal antibody solution into a dialysis bag with a retention molecular weight of 10KD, dialyzing for 24 hours at 4 ℃ by using a PBS solution as a buffer solution, sampling and determining the antibody concentration, adjusting the concentration of the biotinylated anti-CSFV E2 protein monoclonal antibody qualified by quality inspection to 0.5 mu g/mL by using 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 5% sucrose, 0.1% Tween-20 and the balance of purified water and a buffer solution with the pH of 7.2, and storing for later use at 4 ℃.
4. Positive and negative control preparation
(1) Positive control
Screening of piglets for immunization: the 28-day-old piglets are selected to take blood for the following antigen screening, and the negative antigen piglets meeting the following standards are selected as healthy piglets. The standard is as follows: porcine epidemic diarrhea virus: detecting the negativity by using a fluorescent PCR kit; porcine foot and mouth disease virus (type O): detecting the negative by a fluorescent PCR detection kit; porcine transmissible gastroenteritis virus: detecting the negative by a fluorescent PCR detection kit; hog cholera virus: detecting the negative by a fluorescent PCR detection kit; porcine pseudorabies virus: detecting the negative by a fluorescent PCR detection kit; porcine circovirus: detecting the negative by a fluorescent PCR detection kit; porcine reproductive and respiratory syndrome virus: the fluorescence PCR detection kit detects the negativity. The kits were purchased from guangzhou daan gene.
The toxin counteracting method comprises the following steps: taking 3 healthy piglets, injecting hog cholera virus (Zhejiang poetry Huanuobei biological Huifeng) at multiple points in neck muscle.
Serum determination: collecting blood every 7 days after counteracting toxic substance, detecting by fluorescence PCR, collecting blood when CT value is greater than 20, separating serum, and storing at 4 deg.C.
Preparation of positive serum: performing carotid artery exsanguination on experimental pigs meeting the conditions, storing blood of each pig in a sterilized triangular flask, transferring separated serum into a centrifugal flask, centrifuging for 5 minutes at 3,000r/min, taking supernatant, inactivating the supernatant at 56 ℃ for 30 minutes, uniformly mixing the supernatant, and filtering and sterilizing by using a 0.22-micrometer filter membrane. Quantitatively subpackaging, 1 mL/bottle, marking as 'positive serum', noting harvest date and batch number, performing character inspection, sterile inspection, specificity and fluorescence PCR determination, sealing after inspection, and storing at-20 deg.C.
Preparation of positive control: diluting qualified positive serum with PBST buffer solution containing 2% BSA (containing 0.1% Proclin300) to 4 dilutions of 1:50, 1:100, 1:200, and 1:400, detecting each dilution by PCR, selecting the highest dilution with CT value of 25 + -1, diluting the positive serum with PBST buffer solution containing 2% BSA (containing 0.1% Proclin300) according to the dilution, mixing well, filtering with 0.22 μm filter membrane for sterilization, quantitatively packaging (1 mL/tube, 3 mL/tube), and storing at below-20 deg.C.
(2) Negative control
Screening of negative pigs: the 28-day-old piglets are selected to take blood for the following antigen screening, and the negative piglets meeting the following standards are selected as healthy piglets. The standard is as follows: porcine epidemic diarrhea virus: detecting the negativity by using a fluorescent PCR kit; porcine foot and mouth disease virus (type O): detecting the negative by a fluorescent PCR detection kit; porcine transmissible gastroenteritis virus: detecting the negative by a fluorescent PCR detection kit; hog cholera virus: detecting the negative by a fluorescent PCR detection kit; porcine pseudorabies virus: detecting the negative by a fluorescent PCR detection kit; porcine circovirus: detecting the negative by a fluorescent PCR detection kit; porcine reproductive and respiratory syndrome virus: the fluorescence PCR detection kit detects the negativity.
And (3) determination: serum is separated from each experimental pig blood and detected by fluorescence PCR, and if no CT value exists, the pig is used for preparing negative control.
Negative control preparation: the pig is killed by carotid artery exsanguination, the blood is placed in a sterilized and clean triangular flask, the separated serum is transferred to a centrifugal flask, the centrifugal flask is centrifuged for 5 minutes at 3,000r/min, the supernatant is taken, the supernatant is uniformly mixed and then inactivated for 30 minutes at 56 ℃, then the mixture is filtered and sterilized by a 0.22 mu m filter membrane, and 0.1 percent of Proclin300 is added. Quantitatively subpackaging (1 mL/tube, 3 mL/tube), noting lot number, harvesting date, etc., and storing below-20 deg.C for use.
A method for detecting classical swine fever virus using a kit prepared according to 1-4, said method comprising the steps of:
1) mixing the detection sample or the positive control or the negative control with the reagent R1, the reagent R2 and the reagent R3 according to the volume ratio of 10 muL to 30 muL, and placing the mixed solution at 37 ℃ and keeping out of the sun for incubation for 10 min;
2) immediately after incubation, the chemiluminescence signal of the solution was detected by a photomultiplier tube for 3s, and the chemiluminescence values of the positive control, the negative control and the sample were recorded;
3) calculating an S/P value from the chemiluminescence value, wherein S/P value ═ (sample chemiluminescence value-negative control chemiluminescence value)/(positive control chemiluminescence value-negative control chemiluminescence value); when the S/P value is more than or equal to 0.4, the swine fever virus is judged to be positive; when the S/P value is less than 0.4, the swine fever virus is judged to be negative.
Example 2 preparation and detection of double antibody Sandwich homogeneous phase chemiluminescence method CSFV detection kit calibrator
(1) The CSFV E2 protein calibrator was diluted in duplicate with PBS (0.01mol/L pH7.4) buffer containing 2% BSA and 0.1Proclin, and the stock CSFV E2 protein calibrator was diluted to the concentrations of each gradient (0ng/mL, 2ng/mL, 5ng/mL, 25ng/mL, 100ng/mL, 500ng/mL, 3000ng/mL) required for the assay.
(2) The calibrator, the reagent R1, the reagent R2 and the reagent R3 were added to the reaction tube, and the reaction tube was placed in a constant temperature oscillator and incubated at 37 ℃ for 10min under dark conditions.
(3) Immediately after incubation, the chemiluminescent signal of the solution was detected by a photomultiplier for 3 seconds, and the chemiluminescence value of each calibrator was recorded and plotted on the abscissa and the ordinate as the concentration of calibrator, the standard curve of which is shown in FIG. 2. From the results, the standard curve is good, the lowest detection limit can reach 2ng/mL, and the linear range of the kit is good.
Example 3 Performance verification of double-antibody sandwich homogeneous-phase chemiluminescence hog cholera virus detection kit
(1) Experiment of specificity
The kit established in example 1 is used for detecting 1 part of each of PPV, PRV, CSFV and PRRSV positive serum and 2 parts of escherichia coli positive serum, and a commercial classical swine fever virus fluorescent PCR detection kit (purchased from Guangzhou daan gene) is used for parallel detection to verify the specificity of the method.
The established kit detects that PPV, PRV, CSFV, PRRSV and Escherichia coli positive serum are all negative, S/P values are all less than 0.2, no cross reaction occurs, and the kit specificity is good. The detection result is consistent with that of a commercial hog cholera virus detection kit.
(2) Sensitivity test
The positive quality control sample is diluted by PBST according to the ratio of 1:10, 1:100, 1:1000, 1:10000, 1:100000 and 1:1000000, and is respectively detected by the kit established in the example 1 and the commercial swine fever virus fluorescent PCR detection kit, and the sensitivity of the established method is verified.
The established kit can detect the positive quality control sample with the maximum dilution multiple of 1:100000, and the commercial hog cholera virus detection kit can also detect the positive quality control sample with the dilution of 1: 100000. This indicates that the sensitivity of the kit is substantially identical to that of the commercial kit.
(3) Repeatability test
Performing repeated tests on known strong positive serum, weak positive serum and negative serum (5 parts of each) according to the 3 batches of kit prepared in the example 1 and the detection method thereof, and calculating the intra-batch variation coefficient; and randomly selecting 2 kits from the 3 kits according to batches, and performing repeated test on known strong positive serum, weak positive serum and negative serum by each kit to calculate the inter-batch variation coefficient. The result shows that the 3 batches of the kit have the intra-batch variation coefficient of 3-7 percent and the inter-batch variation coefficient of 5-9 percent on the detection result of the positive serum; the intra-batch variation coefficient and the inter-batch variation coefficient of the negative serum detection result are 6-11% and 7-12%, respectively. The experimental result shows that the repeatability of the kit is good.
Example 3 comparison test of double-antibody Sandwich homogeneous-phase chemiluminescence hog cholera virus detection kit
(1) 1220 samples to be detected, positive control and negative control are respectively mixed with a reagent R1, a reagent R2 and a kit R3 in a reaction tube according to the volume ratio of 10 muL to 30 muL, the mixture is placed in a constant temperature oscillator, and incubation is carried out for 10min in a dark place at the temperature of 37 ℃.
(2) Immediately after the incubation was complete, the chemiluminescent signal of the solution was detected by a photomultiplier tube for a period of 3 seconds and the luminescence value recorded for each sample.
(3) Calculating an S/P value from the chemiluminescence value, wherein S/P value ═ (sample chemiluminescence value-negative control chemiluminescence value)/(positive control chemiluminescence value-negative control chemiluminescence value); when the S/P value is more than or equal to 0.4, the swine fever virus is judged to be positive; when the S/P value is less than 0.4, the swine fever virus is judged to be negative.
(4) 1220 samples to be detected are detected by a commercial fluorescent PCR kit (purchased from the Guangzhou Daan gene), and the detection method and the detection standard are strictly operated according to the kit instructions. Detecting the result and recording the result in detail.
(5) The detection result of the double-antibody sandwich homogeneous phase chemical light-emitting classical swine fever virus kit is compared with the detection result of a commercially available classical swine fever virus fluorescent PCR kit for analysis.
The double-antibody sandwich homogeneous-phase chemiluminescent hog cholera virus detection kit provided by the invention is used for detecting a sample, wherein the positive coincidence rate of the detection result and the PCR detection result is 151/160-94.4%, the negative coincidence rate is 1060/1060-100%, and the total coincidence rate is 99.3%. The double-antibody sandwich homogeneous phase chemiluminescent classical swine fever virus detection kit has high coincidence rate with fluorescent PCR.
In conclusion, the kit disclosed by the invention is simple to operate, can be used for outputting a detection report within about 10 minutes, greatly shortens the turnover time of clinical examination specimens, and is suitable for the emergency detection requirements of veterinarians. The kit provided by the invention is used for detecting the classical swine fever virus on the basis of a double-antibody sandwich homogeneous phase chemiluminescence immune competition method, and has the advantages of accurate result, high precision and short time consumption. When the sample is detected, the sample does not need to be diluted, and the HOOK effect does not exist.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A hog cholera virus detection kit based on a double-antibody sandwich homogeneous chemiluminescence method is characterized by comprising a reagent R1, a reagent R2, a reagent R3, a positive control and a negative control, wherein:
the reagent R1 comprises a buffer solution of streptavidin-labeled donor microspheres capable of being excited to generate monomeric oxygen under 680nm light;
the reagent R2 comprises a buffer solution of an acceptor microsphere for labeling the anti-classical swine fever virus monoclonal antibody, wherein the acceptor microsphere can react with monomer oxygen to generate a detectable 615nm chemiluminescent signal;
the reagent R3 is a buffer solution of a biotinylated anti-classical swine fever virus monoclonal antibody.
2. The kit according to claim 1, wherein the receptor microsphere labeled with the anti-CSFV monoclonal antibody in the reagent R2 is the receptor microsphere labeled with the anti-CSFV E2 protein monoclonal antibody, and the buffer components are 0.01M PBS buffer, 2% BSA, 0.1% Proclin300, 5% sucrose, 0.1% Tween-20, and the balance being purified water, and the pH value is 7.2.
3. The kit according to claim 1, wherein the biotinylated anti-classical swine fever virus monoclonal antibody in the reagent R3 is a biotinylated anti-classical swine fever virus E2 protein monoclonal antibody, the biotinylated anti-classical swine fever virus E2 protein monoclonal antibody is at a concentration of 0.5 μ g/mL, the buffer components are 0.01M PBS buffer, 2% BSA, 0.1% Proclin300, 5% sucrose, 0.1% Tween-20, and the balance is purified water, and the pH is 7.2.
4. The kit according to claim 1, wherein the positive control is inactivated hog cholera virus fluorescent PCR CT value of 25 ± 1 in swine serum; the negative control is inactivated hog serum without CT value by hog cholera virus fluorescence PCR.
5. The method of claim 1, wherein the donor and acceptor microspheres are each 150nm in diameter.
6. The detection method according to claim 1, wherein the concentration of the donor microspheres in the reagent R1 is 5 μ g/mL; the concentration of the acceptor microspheres in the reagent R2 is 2 mug/mL.
7. The detection method as claimed in claim 1, wherein the buffer component of the reagent R1 is 0.01M PBS buffer, 2% BSA, 0.1% Proclin300, 0.1% Tween-20, and the rest is purified water, pH 7.2.
8. A method of making the kit of claim 1, comprising the steps of:
8-1) preparation of reagent R1:
a) preparation of donor microspheres: weighing 10mg of 150nm donor microspheres and 1mg of streptavidin, quickly and uniformly mixing, and adding 0.05M MES buffer solution with the pH value of 6.0 to adjust the concentration of the donor microspheres to 10 mg/mL;
b) reaction: adding 100 mu L of 50mg/mL NaBH3CN solution prepared from 0.05M MES with the pH value of 6.0, quickly mixing uniformly, and carrying out rotary reaction at room temperature for 12-16 h;
c) and (3) sealing: adding 100mg/mL BSA solution prepared from 0.05M MES buffer solution with pH value of 6.0 and volume ratio of the BSA solution to the reaction solution of 1:4, rapidly mixing, and rotating at room temperature for reaction for 3 hours;
d) cleaning: centrifuging the reacted solution at 4 ℃ and 12000rpm for 60min, discarding the supernatant, adding 2mL of 0.05M MES buffer solution with the pH value of 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the washed donor microspheres with 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 0.1% Tween-20 and the balance of purified water and a buffer solution with the pH value of 7.2 to ensure that the final concentration of the donor microspheres is 5 mug/mL, and preserving at 4 ℃ for later use after ultrasonic dispersion;
8-2) preparation of reagent R2:
a) preparing acceptor microspheres: weighing 10mg of 150nm receptor microsphere and 2mg of swine fever virus E2 protein resistant monoclonal antibody, quickly and uniformly mixing, and adding 0.05M MES buffer solution with the pH value of 6.0 to adjust the concentration of the receptor microsphere to 10 mg/mL;
b) reaction: adding 100 mu L of NaBH3CN solution with the concentration of 50mg/mL prepared by 0.05M MES with the pH value of 6.0, uniformly mixing, and carrying out rotary reaction at room temperature for 12-16 h;
c) and (3) sealing: adding 100mg/mL BSA solution prepared from 0.05M MES with pH value of 6.0, sealing, mixing with the reaction solution at a volume ratio of 1:4, rapidly mixing, and rotating at room temperature for 3 hr;
d) cleaning: centrifuging the reacted solution at 4 ℃ and 12000rpm for 60min, discarding the supernatant, adding 2ml of 0.05M MES buffer solution with the pH value of 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the receptor microspheres prepared in the step by using 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 5% sucrose, 0.1% Tween-20 and the balance of purified water, dissolving the receptor microspheres with the buffer solution with the pH value of 7.2 to ensure that the final concentration of the receptor microspheres is 2 mu g/ml, and preserving at 4 ℃ for later use after ultrasonic dispersion;
8-3) preparation of reagent R3
a) Biotin activation: weighing 1mg of biotin, adding the biotin into 100 mu L of DMF solution, fully and uniformly mixing, then diluting to 0.5mL by using PBS solution, accurately weighing 2mg of EDC, adding the EDC into the solution, and activating for 1h at room temperature;
b) reaction: measuring a certain amount of swine fever virus E2 protein-resistant monoclonal antibody, dialyzing overnight at 4 ℃ in a PBS solution, measuring the concentration of the antibody and adjusting to 2mg/mL, adding the activated biotin solution into the swine fever virus E2 protein-resistant monoclonal antibody solution, fully and uniformly mixing, and reacting at room temperature for 6 hours;
c) and (3) purification: adding a biotinylated anti-CSFV E2 protein monoclonal antibody solution into a dialysis bag with a retention molecular weight of 10KD, dialyzing for 24 hours at 4 ℃ by using a PBS solution as a buffer solution, sampling and determining the antibody concentration, adjusting the concentration of the biotinylated anti-CSFV E2 protein monoclonal antibody qualified by quality inspection to 0.5 mu g/mL by using 0.01M PBS buffer solution, 2% BSA, 0.1% Proclin300, 5% sucrose, 0.1% Tween-20 and the balance of purified water, adjusting the pH value of the buffer solution to 7.2, and storing at 4 ℃ for later use.
9. A method of detecting a hog cholera virus with the use of the test kit according to any of claims 1-8, comprising the steps of:
9-1) mixing the detection sample or the positive control or the negative control with the reagent R1, the reagent R2 and the reagent R3 according to the volume ratio of 10 muL to 30 muL, and placing the mixed solution at 37 ℃ and keeping away from light for incubation for 10 min;
9-2) immediately after incubation, detecting the chemiluminescence signal of the solution by a photomultiplier for 3s, and recording the chemiluminescence values of a positive control, a negative control and a sample;
9-3) calculating S/P values from the chemiluminescence values, wherein S/P value ═ (sample chemiluminescence value-negative control chemiluminescence value)/(positive control chemiluminescence value-negative control chemiluminescence value); when the S/P value is more than or equal to 0.4, the swine fever virus is judged to be positive; when the S/P value is less than 0.4, the swine fever virus is judged to be negative.
10. Use of a kit according to any one of claims 1 to 8 for the detection of classical swine fever virus.
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