CN112326635A - Double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit, and preparation method and application thereof - Google Patents
Double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit, and a preparation method and application thereof, wherein the kit comprises a reagent R1, a reagent R2, a reagent R3, a positive control and a negative control, and donor microspheres in the reagent R1 can generate monomer oxygen under the excitation of 680nm light; the receptor microsphere in the reagent R2 can react with the monomer oxygen to generate a detectable 615nm chemiluminescent signal, so that the chemiluminescent signal can be directly detected without being cleaned and separated during detection, the detection of the porcine circovirus type 2 is realized, in addition, the detection can be completed within 10min, and the specificity, the sensitivity and the repeatability of the kit are good.
Description
Technical Field
The invention belongs to the technical field of biological diagnostic reagents, and particularly relates to a double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit, and a preparation method and application thereof.
Background
Porcine circovirus disease is a disease which has attracted much attention in recent years and is widely spread around the world, and is a general term for a series of diseases caused by porcine circovirus type. Porcine Circovirus (PCV) was first discovered in 1974 et al. The genus circovirus, is one of the smallest animal viruses known. The porcine circovirus type 2 (PCV2) has extremely high pathogenic virus, mainly infects pigs with the porcine circovirus type II, is widely popularized in the swinery of China at present, causes huge economic loss to the pig raising industry, seriously jeopardizes the development of the pig raising industry, and needs to draw high attention.
The genome of PCV2 is very small, the whole length of the genome is PCV2 of 1767-1768bp, PCV2 contains 11 ORFs which respectively code proteins of 1.8-35.8kD, ORF1 and ORF2 are two main open reading frames of PCV2, ORF1 which reversely codes two proteins codes virus replication-related proteins (Rep), and ORF2 codes virus capsid proteins (Cap), wherein the latter contains an antigen table with immunogenicity, so that the PCV2 virus detection kit can be used for detecting PCV2 virus. ORF2 is the second largest reading frame in the PCV genome, encodes Cap protein (also called nucleocapsid protein), is the main structural protein of virus, contains the main antigen epitope of virus, consists of 233 amino acids and has the molecular weight of about 30 KD. Using monoclonal antibodies and pig positive serum detection assays, ORF2 gene contains multiple antigen binding sites: 69-83aa, 117-131aa, 132-146aa, 156-162aa, 169-183aa, 175-192aa, 195-202aa and 230-233 aa.
Many methods for detecting porcine circovirus type 2 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 a PCV2 diagnostic reagent in the current market, and provides a double-antibody sandwich homogeneous phase chemiluminescence method porcine circovirus type 2 detection kit and a preparation method thereof.
Therefore, in one aspect, the invention provides a double-antibody sandwich homogeneous chemiluminescence porcine circovirus type 2 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 a receptor microsphere for labeling the porcine circular ring type 2 Cap protein monoclonal antibody, and the receptor microsphere can react with the monomer oxygen to generate a detectable 615nm chemiluminescent signal; the reagent R3 is a buffer solution of the porcine circovirus type 2 Cap protein monoclonal antibody.
Preferably, the concentration of the biotinylated anti-porcine circovirus type 2 Cap protein monoclonal antibody in the reagent R3 is 0.5 mu g/mL, the buffer solution is 100mM Hepes buffer solution, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% triton, the balance is purified water, and the pH value is 7.6.
Preferably, the positive control is inactivated porcine serum with porcine circovirus type 2 fluorescent PCR CT value of 25 +/-1; the negative control is inactivated porcine serum without CT value of porcine circovirus type 2 fluorescent PCR.
Preferably, the concentration of the biotinylated anti-porcine circovirus type 2 Cap protein monoclonal antibody is 0.5 mug/mL.
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.
Preferably, the concentration of the acceptor microsphere in the reagent R2 is 2 mug/mL.
Preferably, the buffer component of the reagent R1 is 120mM Hepes buffer, 0.1% Proclin300, 5% NaCl, 0.1% triton, and the balance is purified water, and the pH value is 7.4.
Preferably, the buffer component of the reagent R2 is 100mM Hepes buffer, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% triton, and the balance of purified water, and the pH is 7.6.
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 pH of 6.0 to adjust the concentration of the donor microspheres to 10 mg/mL; b) reaction: adding 50mg/mL NaBH3CN solution prepared from 100 mu LpH 6.0.0 of 0.05M MES, 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 of 6.0 and the volume ratio of the BSA solution to the reaction solution being 1:4, rapidly mixing the solution uniformly, and carrying out rotary reaction for 3 hours at room temperature; d) cleaning: centrifuging the reacted solution at 4 ℃ and 12000rpm for 60min, discarding the supernatant, adding 2mL of 0.05M MES buffer solution with pH 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the cleaned donor microspheres by using 120mM Hepes buffer solution, 0.1% Proclin300, 5% NaCl, 0.1% Triton and the balance of purified water, using the buffer solution with pH7.4 to ensure that the final concentration is 5 mu g/mL, ultrasonically dispersing, and storing at 4 ℃ for later use;
2) preparation of reagent R2:
a) preparing acceptor microspheres: measuring 10mg of 150nm receptor microspheres and 2mg of anti-porcine circovirus type 2 Cap protein monoclonal antibody, quickly and uniformly mixing, and adding 0.05M MES buffer solution with pH of 6.0 to adjust the concentration of the receptor microspheres to 10 mg/mL; b) reaction: adding 100 mu L of NaBH3CN solution with the concentration of 50mg/mL and 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 a BSA solution with the concentration of 100mg/mL prepared from 0.05M MES with the pH value of 6.0 for blocking, wherein the volume ratio of the BSA solution to the reaction solution is 1:4, quickly and uniformly mixing, and carrying out rotary reaction at room temperature 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 pH 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the receptor microspheres prepared in the step by using 100mM Hepes buffer solution, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% Triton and the balance of purified water and the buffer solution with pH 7.6 to ensure that the final concentration 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: weighing a certain amount of anti-porcine circovirus type 2 Cap protein monoclonal antibody, dialyzing the monoclonal antibody in a PBS solution, measuring the antibody concentration, adjusting the antibody concentration to 2mg/mL, adding the activated biotin solution into the dialyzed porcine circovirus type 2 Cap protein solution, fully and uniformly mixing, and reacting at room temperature for 6 hours; c) and (3) purification: adding a biotinylated porcine circovirus type 2 Cap protein clone 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 porcine circovirus type 2 Cap protein clone antibody qualified by quality inspection to 0.5 mu g/mL by using 100mM Hepes buffer solution, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% Triton and the balance of purified water, adjusting the pH value of the buffer solution to 7.6, and storing at 4 ℃ for later use.
In still another aspect, the present invention also provides a detection method for detecting porcine circovirus type 2 using the kit, the 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 0.3, judging the porcine circovirus type 2 is positive; when the S/P value is less than 0.3, the porcine circovirus type 2 is judged to be negative.
In another aspect, the invention also provides an application of the kit in detecting porcine circovirus type 2.
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 porcine circovirus type 2 is realized. In addition, the kit can complete 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.
In a PCV2 homogeneous phase chemiluminescence detection reaction system based on a competition method, donor microspheres for marking streptavidin, receptor microspheres for marking anti-porcine circovirus type 2 Cap protein monoclonal antibodies, and biotinylated anti-porcine circovirus type 2 Cap protein monoclonal antibodies are included. When a double-antibody sandwich method (two monoclonal antibodies recognize different antigen sites) is adopted to detect the porcine circovirus type 2 in a sample, when the porcine circovirus type 2 exists in the sample, the biotinylated anti-porcine circovirus type 2 Cap protein monoclonal antibody in a sample pool to be detected can be combined with the porcine circovirus type 2, then combined with an acceptor microsphere of the monoclonal antibody marked with the anti-porcine circovirus type 2 Cap protein, then biotin can be combined with a streptavidin donor microsphere, finally a streptavidin-marked acceptor microsphere-biotinylated anti-porcine circovirus type 2 Cap protein monoclonal antibody-porcine circovirus type 2-donor microsphere of the monoclonal antibody marked with the porcine circovirus type 2 is formed, under the irradiation of laser (680nm), a photosensitizer on the donor microsphere converts oxygen in the surrounding environment into more active monomer oxygen, the monomer oxygen diffuses to the receptor microsphere to react with a chemiluminescence agent on the receptor microsphere, so that a luminescent group on the receptor microsphere is further activated to generate chemiluminescence, the wavelength is 615nm, the luminescent value is the highest, the diffusible distance of the monomer oxygen within the half-life period of 4 mu s of the monomer oxygen is about 200nm, and an optical signal can be detected by a high-throughput chemiluminescence detector; if there is no specific reaction of antigen antibody, that is, if a complex of streptavidin-labeled acceptor microsphere-biotinylated anti-porcine circovirus type 2 Cap protein monoclonal antibody-porcine circovirus type 2-labeled donor microsphere of anti-porcine circovirus type 2 Cap protein monoclonal antibody is not formed, the monomer oxygen will not diffuse to the acceptor microsphere with a longer distance, and no chemiluminescence effect will be generated. The principle of double-antibody sandwich homogeneous chemiluminescence detection of porcine circovirus type 2 Cap protein is shown in FIG. 1.
Drawings
FIG. 1: schematic diagram of a double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit.
FIG. 2: calibration standard curve chart.
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. Porcine circovirus type 2 Cap protein was offered as a gift by Zhejiang Synong Biotechnology Ltd. Both anti-porcine circovirus type 2 Cap protein monoclonal antibodies are offered by Hangzhou Hongshan bioscience and technology Limited. 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 porcine circovirus type 2 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 pH of 6.0 to adjust the concentration of the donor microspheres to 10 mg/mL; b) reaction: adding 50mg/mL NaBH3CN solution prepared from 100 mu LpH 6.0.0 of 0.05M MES, 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 of 6.0 and the volume ratio of the BSA solution to the reaction solution being 1:4, rapidly mixing the solution uniformly, and carrying out rotary reaction for 3 hours at room temperature; d) cleaning: centrifuging the reacted solution at 4 ℃ and 12000rpm for 60min, discarding the supernatant, adding 2mL of 0.05M MES buffer solution with pH 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the cleaned donor microspheres by using 120mM Hepes buffer solution, 0.1% Proclin300, 5% NaCl, 0.1% Triton and the balance of purified water, using the buffer solution with pH7.4 to ensure that the final concentration is 5 mu g/mL, ultrasonically dispersing, and storing at 4 ℃ for later use;
2 reagent R2 preparation:
a) preparing acceptor microspheres: measuring 10mg of 150nm receptor microspheres and 2mg of anti-porcine circovirus type 2 Cap protein monoclonal antibody, quickly and uniformly mixing, and adding 0.05M MES buffer solution with pH of 6.0 to adjust the concentration of the receptor microspheres to 10 mg/mL; b) reaction: adding 100 mu L of NaBH3CN solution with the concentration of 50mg/mL and 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 a BSA solution with the concentration of 100mg/mL prepared from 0.05M MES with the pH value of 6.0 for blocking, wherein the volume ratio of the BSA solution to the reaction solution is 1:4, quickly and uniformly mixing, and carrying out rotary reaction at room temperature 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 pH 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the receptor microspheres prepared in the step by using 100mM Hepes buffer solution, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% Triton and the balance of purified water and the buffer solution with pH 7.6 to ensure that the final concentration 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: weighing a certain amount of anti-porcine circovirus type 2 Cap protein monoclonal antibody, dialyzing the monoclonal antibody in a PBS solution, measuring the antibody concentration, adjusting the antibody concentration to 2mg/mL, adding the activated biotin solution into the dialyzed porcine circovirus type 2 Cap protein solution, fully and uniformly mixing, and reacting at room temperature for 6 hours; c) and (3) purification: adding a biotinylated porcine circovirus type 2 Cap protein clone 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 porcine circovirus type 2 Cap protein clone antibody qualified by quality inspection to 0.5 mu g/mL by using 100mM Hepes buffer solution, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% Triton and the balance of purified water, adjusting the pH value of the buffer solution to 7.6, and storing at 4 ℃ for later use.
4. Positive and negative control preparation
(1) Positive control
Screening of piglets for immunization: the method comprises the steps of selecting a 28-day-old piglet for blood sampling, carrying out the following antigen screening, and selecting an antigen-negative piglet meeting the following standard as a healthy piglet for immunization. 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: 3 healthy piglets were taken, and porcine circovirus type 2 (a biological gift from Huanuobewei Shijiang poetry) was injected intramuscularly in the neck.
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.
5 a method for detecting porcine circovirus type 2 using a kit prepared from 1-4, the 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 0.3, judging the porcine circovirus type 2 is positive; when the S/P value is less than 0.3, the porcine circovirus type 2 is judged to be negative.
Example 2 preparation and detection of double antibody Sandwich homogeneous chemiluminescence porcine circovirus type 2 detection kit calibrator
(1) The porcine circovirus type 2 Cap protein calibrator was diluted in duplicate with PBS (0.01mol/L pH7.4) buffer containing 2% BSA and 0.1Proclin, and the stock solution of the porcine circovirus type 2 Cap protein calibrator was diluted to the concentrations of each gradient (0ng/mL, 1ng/mL, 5ng/mL, 20ng/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 1ng/mL, and the linear range of the kit is good.
Example 3 Performance verification of double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit
(1) Experiment of specificity
The kit established in the embodiment 1 is used for respectively detecting 1 part of PPV, PRV, CSFV and PRRSV positive serum and 2 parts of escherichia coli positive serum, and a commercial porcine circovirus type 2 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 porcine circovirus type 2 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 porcine circovirus type 2 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 porcine circovirus type 2 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 2 to 6 percent and the inter-batch variation coefficient of 3 to 9 percent for 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 respectively 5 to 10 percent and 7 to 11 percent. The experimental result shows that the repeatability of the kit is good.
Example 4 comparison test of double-antibody Sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit
(1) 1236 samples to be detected, positive control and negative control are respectively mixed with the reagent R1, the reagent R2 and the kit R3 according to the volume ratio of 10 muL to 30 muL in a reaction tube, and the mixture is placed in a constant temperature oscillator and incubated 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 0.3, judging the porcine circovirus type 2 is positive; when the S/P value is less than 0.3, the porcine circovirus type 2 is judged to be negative.
(4) 1236 samples to be detected are detected by a commercial fluorescent PCR kit (purchased from the gene Daan, Guangzhou), 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 chemiluminescence porcine circovirus type 2 kit is compared with the detection result of a commercially available porcine circovirus type 2 fluorescent PCR kit for analysis.
The double-antibody sandwich homogeneous-phase chemiluminescent porcine circovirus type 2 detection kit provided by the invention detects a sample, wherein the positive coincidence rate of the detection result and the PCR detection result is 361/380-95%, the negative coincidence rate is 856/856-100%, and the total coincidence rate is 98.5%. The double-antibody sandwich homogeneous phase chemiluminescence porcine circovirus type 2 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 porcine circovirus type 2 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 double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit 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 a receptor microsphere for labeling the porcine circovirus type 2 monoclonal antibody, and the receptor microsphere can react with monomer oxygen to generate a detectable 615nm chemiluminescent signal;
the reagent R3 is a buffer solution of a biotinylated anti-porcine circovirus type 2 Cap protein monoclonal antibody.
2. The double-antibody sandwich homogeneous-phase chemiluminescence method porcine circovirus type 2 detection kit according to claim 1, wherein the concentration of the biotinylated anti-porcine circovirus type 2 Cap protein monoclonal antibody in the reagent R3 is 0.5 μ g/mL, the buffer solution is 100mM Hepes buffer solution, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% triton, the balance is purified water, and the pH is 7.6.
3. The double-antibody sandwich homogeneous-phase chemiluminescence detection kit for porcine circovirus type 2 according to claim 1, wherein the positive control is inactivated porcine serum with a fluorescent PCR CT value of porcine circovirus type 2 within 25 ± 1; the negative control is inactivated porcine serum without CT value of porcine circovirus type 2 fluorescent PCR.
4. The double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit according to claim 1, wherein the donor microspheres and the acceptor microspheres are both 150nm in diameter.
5. The double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit according to claim 1, wherein the concentration of 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.
6. The double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit according to claim 1, wherein the buffer components of the reagent R1 are 120mM Hepes buffer, 0.1% Proclin300, 5% NaCl, 0.1% triton, and the balance purified water, pH 7.4.
7. The double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit according to claim 1, wherein the buffer solution component of the reagent R2 is 100mM Hepes buffer solution, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% triton, and the balance purified water, and the pH is 7.6.
8. A preparation method for preparing the double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit according to claim 1, wherein the preparation method comprises the following steps:
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 pH of 6.0 to adjust the concentration of the donor microspheres to 10 mg/mL;
b) reaction: adding 50mg/mL NaBH3CN solution prepared from 100 mu LpH 6.0.0 of 0.05M MES, 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 of 6.0 and the volume ratio of the BSA solution to the reaction solution being 1:4, rapidly mixing the solution uniformly, and carrying out rotary reaction for 3 hours at room temperature;
d) cleaning: centrifuging the reacted solution at 4 ℃ and 12000rpm for 60min, discarding the supernatant, adding 2mL of 0.05M MES buffer solution with pH 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the cleaned donor microspheres by using 120mM Hepes buffer solution, 0.1% Proclin300, 5% NaCl, 0.1% Triton and the balance of purified water, using the buffer solution with pH7.4 to ensure that the final concentration is 5 mu g/mL, ultrasonically dispersing, and storing at 4 ℃ for later use;
8-2) preparation of reagent R2:
a) preparing acceptor microspheres: measuring 10mg of 150nm receptor microspheres and 2mg of anti-porcine circovirus type 2 Cap protein monoclonal antibody, quickly and uniformly mixing, and adding 0.05M MES buffer solution with pH of 6.0 to adjust the concentration of the receptor microspheres to 10 mg/mL;
b) reaction: adding 100 mu L of NaBH3CN solution with the concentration of 50mg/mL and 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 a BSA solution with the concentration of 100mg/mL prepared from 0.05M MES with the pH value of 6.0 for blocking, wherein the volume ratio of the BSA solution to the reaction solution is 1:4, quickly and uniformly mixing, and carrying out rotary reaction at room temperature 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 pH 6.0 for ultrasonic suspension, centrifuging again, discarding the supernatant, finally dissolving the receptor microspheres prepared in the step by using 100mM Hepes buffer solution, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% Triton and the balance of purified water and the buffer solution with pH 7.6 to ensure that the final concentration 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: weighing a certain amount of anti-porcine circovirus type 2 Cap protein monoclonal antibody, dialyzing the monoclonal antibody in a PBS solution, measuring the antibody concentration, adjusting the antibody concentration to 2mg/mL, adding the activated biotin solution into the dialyzed porcine circovirus type 2 Cap protein solution, fully and uniformly mixing, and reacting at room temperature for 6 hours;
c) and (3) purification: adding a biotinylated porcine circovirus type 2 Cap protein clone 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 porcine circovirus type 2 Cap protein clone antibody qualified by quality inspection to 0.5 mu g/mL by using 100mM Hepes buffer solution, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% Triton and the balance of purified water, adjusting the pH value of the buffer solution to 7.6, and storing at 4 ℃ for later use.
9. A method for detecting porcine circovirus type 2 using the double-antibody sandwich homogeneous chemiluminescent porcine circovirus type 2 detection kit of any one of claims 1 to 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 0.3, judging the porcine circovirus type 2 is positive; when the S/P value is less than 0.3, the porcine circovirus type 2 is judged to be negative.
10. The application of the double-antibody sandwich homogeneous-phase chemiluminescence porcine circovirus type 2 detection kit disclosed in any one of claims 1-8 in detection of porcine circovirus type 2.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103499689A (en) * | 2013-10-11 | 2014-01-08 | 重庆出入境检验检疫局检验检疫技术中心 | Competitive Alpha LISA (linked immuno sorbent assay) detection kit for porcine circovirus (PCV) 2 antibody and detection method thereof |
| CN106353507A (en) * | 2016-08-24 | 2017-01-25 | 武汉生之源生物科技股份有限公司 | Kit for detecting serum amyloid protein and application thereof |
| CN108627652A (en) * | 2018-05-31 | 2018-10-09 | 宁波海壹生物科技有限公司 | It is a kind of to detect based on simple grain diameter and simultaneously the kit of RBP ELISA in serum and urine specimen |
| CN109444414A (en) * | 2018-09-10 | 2019-03-08 | 珠海国际旅行卫生保健中心 | Using the method and corresponding composition, kit of AlphaLISA detection dengue virus |
| CN109799351A (en) * | 2018-09-19 | 2019-05-24 | 天津瑞普生物技术股份有限公司 | Porcine circovirus 2 type double-antibody sandwich elisa kit and its application |
| WO2019223692A1 (en) * | 2018-05-21 | 2019-11-28 | 博阳生物科技（上海）有限公司 | Chemical luminescence analysis and measurement method, system using same, and kit |
| CN110736734A (en) * | 2018-07-18 | 2020-01-31 | 博阳生物科技（上海）有限公司 | cTnI homogeneous phase chemiluminescence detection kit, detection method and device |
| WO2020034940A1 (en) * | 2018-08-13 | 2020-02-20 | 博阳生物科技（上海）有限公司 | Homogeneous chemiluminescence detection kit and application thereof |
| CN111381025A (en) * | 2018-12-29 | 2020-07-07 | 深圳市帝迈生物技术有限公司 | Immunoassay kit for multiplex detection, application and multiplex detection method |
-
2020
- 2020-10-20 CN CN202011123219.2A patent/CN112326635A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103499689A (en) * | 2013-10-11 | 2014-01-08 | 重庆出入境检验检疫局检验检疫技术中心 | Competitive Alpha LISA (linked immuno sorbent assay) detection kit for porcine circovirus (PCV) 2 antibody and detection method thereof |
| CN106353507A (en) * | 2016-08-24 | 2017-01-25 | 武汉生之源生物科技股份有限公司 | Kit for detecting serum amyloid protein and application thereof |
| WO2019223692A1 (en) * | 2018-05-21 | 2019-11-28 | 博阳生物科技（上海）有限公司 | Chemical luminescence analysis and measurement method, system using same, and kit |
| CN108627652A (en) * | 2018-05-31 | 2018-10-09 | 宁波海壹生物科技有限公司 | It is a kind of to detect based on simple grain diameter and simultaneously the kit of RBP ELISA in serum and urine specimen |
| CN110736734A (en) * | 2018-07-18 | 2020-01-31 | 博阳生物科技（上海）有限公司 | cTnI homogeneous phase chemiluminescence detection kit, detection method and device |
| WO2020034940A1 (en) * | 2018-08-13 | 2020-02-20 | 博阳生物科技（上海）有限公司 | Homogeneous chemiluminescence detection kit and application thereof |
| CN109444414A (en) * | 2018-09-10 | 2019-03-08 | 珠海国际旅行卫生保健中心 | Using the method and corresponding composition, kit of AlphaLISA detection dengue virus |
| CN109799351A (en) * | 2018-09-19 | 2019-05-24 | 天津瑞普生物技术股份有限公司 | Porcine circovirus 2 type double-antibody sandwich elisa kit and its application |
| CN111381025A (en) * | 2018-12-29 | 2020-07-07 | 深圳市帝迈生物技术有限公司 | Immunoassay kit for multiplex detection, application and multiplex detection method |
Non-Patent Citations (2)
| Title |
|---|
| 原霖;蔡林;倪建强;顾小雪;张硕;张淼洁;王传彬;遇秀玲;翟新验;田克恭;: "猪圆环病毒2型IgM抗体间接ELISA的建立", 中国畜牧兽医, no. 07 * |
| 赵爽;周宇捷;相平;李会强;孙辉;: "血清糖链抗原-125均相化学发光免疫检测方法的初建", 免疫学杂志, no. 05 * |
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