CN112269022A - Competitive homogeneous phase chemiluminescence method porcine circovirus type 2 antibody detection kit, and preparation method and application thereof - Google Patents
Competitive homogeneous phase chemiluminescence method porcine circovirus type 2 antibody detection kit, 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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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses a competitive homogeneous chemiluminescence detection kit for porcine circovirus type 2 antibodies, which comprises a reagent R1, a reagent R2, a positive control and a negative control. The invention also discloses a preparation method of the kit. The donor microsphere can generate active oxygen in an excited state, and the acceptor microsphere can react with the active oxygen to generate a detectable chemiluminescent signal, so that the chemiluminescent signal can be directly detected without cleaning and separating during detection, and the detection of the porcine circovirus type 2 antibody 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 porcine circovirus type 2 antibody detection kit based on a competitive homogeneous chemiluminescence method, 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 antibodies 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 ELISA 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 an ELISA 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 time and labor waste and the like of the existing PCV2 antibody detection method, and provides a competitive homogeneous phase chemiluminescence method porcine circovirus type 2 antibody detection kit, and a preparation method and application thereof.
Therefore, in one aspect, the present invention provides a competitive homogeneous chemiluminescence method porcine circovirus type 2 antibody detection kit, which comprises a reagent R1 and a reagent R2, as well as a positive control and a negative control, wherein: the reagent R1 comprises donor microspheres and buffer solution of porcine circovirus type 2 Cap protein combined with the donor microspheres, wherein the donor microspheres can generate active oxygen in an excited state; the reagent R2 comprises a receptor microsphere and a buffer solution of an anti-porcine circovirus type 2 Cap protein monoclonal antibody combined with the receptor microsphere, wherein the receptor microsphere can react with active oxygen to generate a detectable chemiluminescent signal.
Preferably, the positive control is porcine serum with porcine circovirus type 2 IFA antibody titer of 1: 64; the negative control is porcine serum with porcine circovirus type 2 IFA antibody titer of 1: 4.
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 2mg of porcine circovirus type 2 Cap protein, 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 120mM Hepes buffer solution, 0.1% Proclin300, 5% NaCl, 0.1% Triton and the balance of purified water and pH 7.4 buffer solution to ensure that the final concentration is 5 mu g/mL, and performing ultrasonic dispersion and then storing at 4 ℃ for later use;
2) preparation of reagent R2:
a) preparing acceptor microspheres: weighing 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 the pH value 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 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 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 the pH value of 7.6 to ensure that the final concentration is 2 mu g/mL, and preserving at 4 ℃ for later use after ultrasonic dispersion.
In still another aspect, the present invention also provides a method for detecting porcine circovirus type 2 antibodies using the kit, the method comprising the steps of:
1) mixing the positive serum, the negative serum or the sample containing the porcine circovirus type 2 antibody with a reagent R1 and a reagent R2 in the kit according to the volume ratio of 10 muL to 50 muL, and incubating the mixed solution at 37 ℃ for 5 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 a blocking rate according to the chemiluminescence value, wherein the blocking rate is 1-sample chemiluminescence value/negative control chemiluminescence value; when the blocking rate is more than or equal to 35 percent, judging that the porcine circovirus type 2 antibody is positive; when the blocking rate is less than 35%, the porcine circovirus type 2 antibody is judged to be negative.
In another aspect, the invention also provides application of the kit in detecting porcine circovirus type 2 antibodies.
Has the advantages that: the donor microsphere can generate active oxygen in an excited state, and the acceptor microsphere can react with the active oxygen to generate a detectable chemiluminescent signal (the donor microsphere and the acceptor microsphere cannot emit the chemiluminescent signal in a dispersed state, and can only emit the detectable chemiluminescent signal when the donor microsphere and the acceptor microsphere are close to each other). Therefore, the chemiluminescent signal can be directly detected without cleaning and separation during detection, and the detection of the porcine circovirus type 2 antibody 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, in order to further improve the sensitivity of the kit, the streptavidin-biotin system is introduced on the basis of the prior invention, and the streptavidin-biotin system also belongs to the protection scope of the invention.
In a homogeneous phase chemiluminescence detection reaction system of the porcine circovirus type 2 antibody based on a competition method, donor microspheres of porcine circovirus type 2 Cap protein are marked, and receptor microspheres of an anti-porcine circovirus type 2 Cap protein monoclonal antibody are marked. When no porcine circovirus type 2 antibody exists in the sample, a donor microsphere for marking porcine circovirus type 2 Cap protein and an acceptor microsphere for marking anti-porcine circovirus type 2 Cap protein monoclonal antibody in a sample pool to be detected are combined to form a complex of the donor microsphere for marking porcine circovirus type 2 Cap protein and the acceptor microsphere for marking anti-porcine circovirus type 2 Cap protein monoclonal antibody, under the irradiation of laser (680nm), the photosensitizer on the donor microsphere converts oxygen in the surrounding environment into more active monomer oxygen, the monomer oxygen diffuses to the acceptor microsphere, the chemiluminescent substrate reacts with a chemiluminescent agent on the receptor microsphere to further activate a luminescent group on the receptor microsphere to generate chemiluminescence, wherein the wavelength is 615nm, the luminescent value is the highest, the diffusible distance of 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 the specific reaction of the antigen and the antibody does not exist, namely if a donor microsphere for marking the porcine circovirus type 2 Cap protein-a receptor microsphere complex for marking the anti-porcine circovirus type 2 Cap protein monoclonal antibody is not formed, the monomer oxygen cannot diffuse to the receptor microsphere with a longer distance, and the chemiluminescence effect cannot be generated; if the sample contains the porcine circovirus type 2 antibody, the porcine circovirus type 2 antibody in the sample competes with the acceptor microsphere marked with the anti-porcine circovirus type 2 Cap protein monoclonal antibody for the binding site of the porcine circovirus type 2 Cap protein (donor microsphere), the porcine circovirus type 2 antibody existing in the sample is combined with the porcine circovirus type 2 Cap protein (donor microsphere), and the amount of the anti-porcine circovirus type 2 Cap protein monoclonal antibody (acceptor microsphere) combined with the porcine circovirus type 2 Cap protein (donor microsphere) is reduced, so that the number of the complex of the donor microsphere marked with the porcine circovirus type 2 Cap protein and the acceptor microsphere marked with the anti-porcine circovirus type 2 Cap protein monoclonal antibody formed in the solution is reduced, thereby reducing the chemiluminescence intensity and reducing the photon value read by the instrument. The principle of competitive homogeneous chemiluminescence detection of porcine circovirus type 2 antibodies is shown in figure 1.
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FIG. 1 is a schematic diagram of competitive homogeneous chemiluminescence detection of porcine circovirus type 2 antibodies.
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 described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention.
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. Monoclonal antibodies against the Cap protein of porcine circovirus type 2 were purchased from luoyang paleo biotechnology limited. Donor microspheres and acceptor microspheres are offered by Hangzhou Hongqiao Zhongke Gene technology, Inc.
Example 1: establishment of competitive homogeneous phase chemiluminescence method porcine circovirus type 2 antibody detection kit
1. Preparation of reagent R1:
a) preparation of donor microspheres: weighing 10mg of 150nm donor microspheres and 2mg of porcine circovirus type 2 Cap protein, 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 120mM Hepes buffer solution, 0.1% Proclin300, 5% NaCl, 0.1% Triton and the balance of purified water and pH 7.4 buffer solution to ensure that the final concentration is 5 mu g/mL, and performing ultrasonic dispersion and then storing at 4 ℃ for later use;
2. preparation of reagent R2:
a) preparing acceptor microspheres: weighing 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 the pH value 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 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 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 the pH value of 7.6 to ensure that the final concentration is 2 mu g/mL, and preserving at 4 ℃ for later use after ultrasonic dispersion.
3. Positive and negative control preparation
a) Preparing a positive control:
screening of piglets for immunization: and selecting the blood taken by the piglets with the age of 28 days for the following antibody screening, and selecting the piglets which are negative in antibody and meet the following standards as the healthy piglets for immunization. The standard is as follows: porcine epidemic diarrhea virus antibodies: detecting the negativity of the ELISA kit; porcine foot and mouth disease virus antibody (type O): detecting the negative by an ELISA detection kit; antibody against transmissible gastroenteritis virus of swine: detecting the negative by an ELISA detection kit; hog cholera virus antibodies: detecting the negative by an ELISA detection kit; porcine pseudorabies virus antibody: detecting the negative by an ELISA detection kit; porcine circovirus antibodies: detecting the negative by an ELISA detection kit; porcine reproductive and respiratory syndrome virus antibodies: and the ELISA detection kit detects negativity.
The immunization method comprises the following steps: 3 healthy piglets were injected with commercial porcine circovirus type 2 inactivated vaccine (purchased from Huanuowei biology, Shijiang poetry) at multiple points in neck muscles, and were boosted once in 14 days.
And (3) measuring the serum titer: blood is collected every 7 days after secondary immunization, serum antibody detection is carried out by adopting an IFA method, blood is collected when the antibody is more than or equal to 1:128, serum is separated, and the blood is stored at 4 ℃ for detection.
Preparation of positive serum: carotid bleeding is carried out on experimental pigs meeting the conditions, blood of each pig is stored in a sterilized triangular flask, precipitated serum is transferred to a centrifugal flask, the centrifugal flask is centrifuged for 5 minutes at 3,000r/min, supernatant is taken, the supernatant is uniformly mixed, and then the mixture is filtered and sterilized by a 0.22 mu m filter membrane. Quantitatively subpackaging, 1 mL/bottle, marking as 'positive serum', noting harvest date and batch number, performing character inspection, sterile inspection, determining specificity and IFA titer, sealing after inspection, and storing at-20 deg.C.
Preparation of positive control: the positive serum qualified in the test was diluted to 4 dilutions of 1:50, 1:100, 1:200, and 1:400 with 2% BSA-containing PBST buffer (containing 0.1% Proclin300), each dilution was assayed with IFA, the highest dilution of IFA titer of 1:64 was selected, the positive serum was diluted with 2% BSA-containing PBST buffer (containing 0.1% Proclin300) by the dilution, mixed well, sterilized by filtration with a 0.22 μm filter, quantitatively dispensed (1 mL/tube, 3 mL/tube), and stored at-20 ℃ or lower.
b) Negative control preparation
Screening of negative pigs: and selecting the blood taken by the piglets with the age of 28 days for the following antibody screening, and selecting the piglets which are negative in antibody and meet the following standards as the healthy piglets for immunization. The standard is as follows: porcine epidemic diarrhea virus antibodies: detecting the negativity of the ELISA kit; porcine foot and mouth disease virus antibody (type O): detecting the negative by an ELISA detection kit; antibody against transmissible gastroenteritis virus of swine: detecting the negative by an ELISA detection kit; hog cholera virus antibodies: detecting the negative by an ELISA detection kit; porcine pseudorabies virus antibody: detecting the negative by an ELISA detection kit; porcine circovirus antibodies: detecting the negative by an ELISA detection kit; porcine reproductive and respiratory syndrome virus antibodies: and the ELISA detection kit detects negativity.
And (3) measuring the titer: and (3) collecting blood and separating serum of each experimental pig, and measuring the antibody titer by using IFA (intravenous injection) and using the pig for negative control preparation if the titer is less than or equal to 1: 4.
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 centrifuge bottle and centrifuged for 5 minutes at the speed of 3,000r/min, the supernatant is taken, the supernatant is mixed evenly and filtered through a 0.22 mu m filter membrane for sterilization, 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.
4. A method for detecting porcine circovirus type 2 antibodies using a kit prepared from 1-3, the method comprising the steps of:
1) mixing the positive serum, the negative serum or the sample with a reagent R1 and a reagent R2 in the kit according to the volume ratio of 10 muL to 50 muL, and incubating the mixture at 37 ℃ for 5-10 min; generally, the culture is incubated for 5 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 a blocking rate according to the chemiluminescence value, wherein the blocking rate is 1-sample chemiluminescence value/negative control chemiluminescence value; when the blocking rate is more than or equal to 35 percent, judging that the porcine circovirus type 2 antibody is positive; when the blocking rate is less than 35%, the porcine circovirus type 2 antibody is judged to be negative.
Example 2: performance verification of porcine circovirus type 2 antibody detection kit by competitive homogeneous chemiluminescence method
(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 antibody detection kit (purchased from Beijing Jinnuo organisms) 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, the blocking rate is less than 10% or less, no cross reaction occurs, and the kit specificity is good. The detection result is consistent with that of a commercial porcine circovirus type 2 antibody detection kit.
(2) Sensitivity test
The positive quality control sample is diluted by PBST according to the ratio of 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048 and 1:4096, and is respectively detected by the kit established in the embodiment 1 and the commercial porcine circovirus type 2 antibody detection kit, and is identified by a serum antibody IFA test to verify the sensitivity of the established method.
The established kit is consistent with the detection result of the IFA test, and the maximum dilution multiple of the kit can be detected to be 1:2048, while the commercial porcine circovirus type 2 antibody detection kit can only detect 1:1024 times diluted positive quality control sample.
(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 6-10% and 7-10%, respectively. The experimental result shows that the repeatability of the kit is good.
Example 3: comparison test of hog cholera virus antibody detection kit based on homogeneous chemiluminescence method
1) Mixing 1027 parts of sample to be detected, positive control and negative control with a reagent R1 and a reagent R2 in the kit according to the volume ratio of 10 muL to 50 muL, and incubating the mixed solution at 37 ℃ for 5-10 min; generally, the culture is incubated for 5 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 a blocking rate according to the chemiluminescence value, wherein the blocking rate is 1-sample chemiluminescence value/negative control chemiluminescence value; when the blocking rate is more than or equal to 35 percent, judging that the porcine circovirus type 2 antibody is positive; when the blocking rate is less than 35%, the porcine circovirus type 2 antibody is judged to be negative. .
4) 1116 samples to be detected were detected by a commercially available kit (purchased from kino, beijing), and the detection method and detection standard thereof were strictly performed according to the kit instructions. Detecting the result and recording the result in detail.
5) The detection result of the competitive homogeneous phase chemiluminescence porcine circovirus type 2 antibody detection kit is compared with the detection result of a commercially available kit for analysis.
The competitive homogeneous phase chemiluminescence porcine circovirus type 2 antibody detection kit provided by the invention detects samples, the positive coincidence rate of the detection result and the detection result of a commercial kit is 820/820-100%, the negative coincidence rate is 200/207-96.2%, and the total coincidence rate is 99.3%. The coincidence rate of the kit disclosed by the invention and a commercialized kit is very high. For 7 samples out of compliance, the inventors believe that the sensitivity of the kit of the invention is higher, the 7 samples tested are weakly positive, the test results may not be very accurate using commercial kits, but there is currently no more sensitive method to verify.
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 antibody on the basis of a competitive homogeneous 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 (9)
1. A competitive homogeneous chemiluminescence method porcine circovirus type 2 antibody detection kit is characterized by comprising a reagent R1, a reagent R2, a positive control and a negative control, wherein:
the reagent R1 comprises donor microspheres and buffer solution of porcine circovirus type 2 Cap protein combined with the donor microspheres, wherein the donor microspheres can generate active oxygen in an excited state;
the reagent R2 comprises a receptor microsphere and a buffer solution of an anti-porcine circovirus type 2 Cap protein monoclonal antibody combined with the receptor microsphere, wherein the receptor microsphere can react with active oxygen to generate a detectable chemiluminescent signal.
2. The kit of claim 1, wherein the positive control is porcine serum having an IFA type 2 porcine circovirus antibody titer of 1: 64; the negative control is porcine serum with porcine circovirus type 2 IFA antibody titer of 1: 4.
3. The kit of claim 1, wherein the donor and acceptor microspheres are each 150nm in diameter.
4. The kit 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.
5. The kit according to claim 1 or 4, characterized in that the buffer components of the reagent R1 are 120mM Hepes buffer, 0.1% Proclin300, 5% NaCl, 0.1% Triton, the rest being purified water, pH 7.4.
6. The kit according to claim 1 or 4, wherein the buffer component of the reagent R2 is 100mM Hepes buffer, 0.1% Proclin300, 5% sucrose, 5% NaCl, 0.1% Triton, and the balance purified water, and the pH is 7.6.
7. A method of making the kit of claim 1, comprising the steps of:
7-1) preparation of reagent R1:
a) preparation of donor microspheres: measuring 10mg of 150nm donor microspheres and 2mg of porcine circovirus type 2 Cap protein, 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 100 mu L of NaBH3CN solution of 50mg/mL prepared by 0.05M MES with pH of 6.0, rapidly mixing uniformly, and carrying out a 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 pH 7.4 to ensure that the final concentration is 5 mu g/mL, ultrasonically dispersing, and storing at 4 ℃ for later use;
7-2) 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, removing the supernatant, adding 2mL of 0.05M MES buffer solution with pH 6.0 for ultrasonic suspension, centrifuging again, removing 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 a 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. A method for detecting antibodies of porcine circovirus type 2 using a kit according to any one of claims 1 to 7, comprising the steps of:
8-1) mixing the positive serum, the negative serum or the sample with a reagent R1 and a reagent R2 in the kit according to the volume ratio of 10 muL to 50 muL, and incubating the mixed solution at 37 ℃ for 5 min;
8-2) 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;
8-3) calculating a blocking rate according to the chemiluminescence value, wherein the blocking rate is 1-sample chemiluminescence value/negative control chemiluminescence value; when the blocking rate is more than or equal to 35 percent, judging that the porcine circovirus type 2 antibody is positive; when the blocking rate is less than 35%, the porcine circovirus type 2 antibody is judged to be negative.
9. Use of a kit according to any one of claims 1 to 7 for the detection of antibodies to porcine circovirus type 2.
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