CN116559439A - Bovine coronavirus indirect ELISA antibody detection kit and application - Google Patents
Bovine coronavirus indirect ELISA antibody detection kit and application Download PDFInfo
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The invention discloses a bovine coronavirus indirect ELISA antibody detection kit and application thereof, belonging to the technical field of biological detection. The invention relates to a BCoV indirect ELISA antibody detection method which is established by utilizing a baculovirus expression system to express and prepare a BCoV recombinant S1 protein and taking the purified BCoV-S1 protein as an antigen to be detected. The BCoV-S1 protein prepared by the invention has higher purity and good antigenicity, and an indirect ELISA antibody detection method is established by using the BCoV-S1 for the first time, so that the method has the characteristics of sensitivity, specificity and high efficiency, and is suitable for clinical detection and diagnosis of the BCoV antibody.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a bovine coronavirus indirect ELISA antibody detection kit and application thereof.
Background
Bovine coronavirus (Bovine coronavirus, BCoV) belongs to the genus coronaviridae of the order monoviridae, is a enveloped single-stranded positive strand RNA virus, and can cause clinically hemorrhagic diarrhea, adult Niu Dong dysentery, and respiratory tract infection in calves. The virus is widely available worldwide and can infect wild ruminants in addition to cattle. In 1972, mebus et al in the United states detected BCoV pathogens for the first time in diarrhea lesions in calves and adult cattle. In 1985 Song Anlin, the existence of BCoV in China is reported for the first time, and then the BCoV is successfully detected for many times in each province in China, and the positive rate is up to 70%, which indicates that the infection rate of the BCoV is very high. In 1988, researchers isolated a strain closely related to the BCoV genome from a pediatric diarrhea sample in germany, suggesting that BCoV may be potentially spread across species. However, no specific therapeutic drug for bovine coronavirus exists at present, and the vaccine developed abroad has poor protective effect and poor broad spectrum.
Based on the above, rapid, specific, sensitive BCoV assays are important for disease monitoring and control. At present, the detection of BCoV in China is mainly performed by RT-PCR or fluorescence quantitative detection, the operation is complex, and the requirements on detection personnel are high; the established method for detecting the BCoV antibody is mainly an ELISA detection method established by prokaryotic expression of N protein, and the antigenicity of the method is greatly reduced. Therefore, the establishment of the virus antibody detection method with strong specificity, high sensitivity, simple operation and low cost has important significance.
Disclosure of Invention
The invention aims to provide a bovine coronavirus indirect ELISA antibody detection kit and application thereof, which are used for solving the problems in the prior art, and the virus antibody detection method which is strong in specificity, high in sensitivity, simple to operate and low in cost is established by taking the whole S1 length expressed by a baculovirus expression system as a coating antigen, so that the kit is suitable for on-site detection of bovine coronavirus antibodies.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a bovine coronavirus indirect ELISA antibody detection kit which is characterized by comprising a BCoV-S1 protein recombinant antigen coated on an ELISA plate.
Preferably, the amino acid sequence of the BCoV-S1 protein is shown as SEQ ID NO:1 is shown as follows: MKFLVNVALVFMVVYISYIYAFLILLISLPTAFAVIGDLKCTTVSINDVDTGVPSISTDTVDVTNGLGTYYVLDRVYLNTTLLLNGYYPTSGSTYRNMALKGTLLLSTLWFKPPFLSDFTNGIFAKVKNTKVIKDGVMYSEFPAITIGSTFVNTSYSVVVQPHTTNLDNKLQGFLEISVCQYTMCEYPNTICHPNLGNQRVELWHWDTGVVSCLYKRNFTYDVNADYLYFHFYQEGGTFYAYFTDTGVVTKFLFNVYLGTVLSHYYVMPLTCNSALTLEYWVTPLTSKQYLLAFNQDGVIFNAVDCKSDFMSEIKCKTLSIAPSTGVYELNGYTVQPIADVYRRIPNLPDCNIEAWLNDKSVPSPLNWERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSVSRFNPSTWNRRFGFTEQSVFKPQPAGVFTDHDVVYAQHCFKAPTNFCPCKLDGSLCVGSGSGIDAGYKHTGIGTCPAGTNYLTCHNAAQCDCLCTPDPITSKATGPYKCPQTKYLVGIGEHCSGLAIKSDHCGGNPCSCQPQAFLGWSVDSCLQGDRCNIFANFILHDVNSGTTCSTDLQKSNTDIILGVCVNYDLYGITGQGIFVEVNATYYNSWQNLLYDSNGNLYGFRDYLTNRTFMIRSCYSGRVSAAFHANSSEPALLFRNIKCNYVFNNTLLRQLQPINYFDSYLGCVVNADNSTSSVVQTCDLTVGSGYCVDYSTKRRSRRHHHHHHHH.
Preferably, the kit further comprises: blocking solution, washing solution, positive control, negative control, enzyme-labeled secondary antibody and chromogenic solution.
Preferably, the preparation method of the BCoV-S1 protein recombinant antigen comprises the following steps: after codon optimization of an S1 gene sequence of bovine coronavirus, adding a histidine tag at the 3' end, introducing BamHI and HindIII restriction enzyme cutting sites at the two ends of the S1 gene sequence, and transferring into a vector to construct a recombinant plasmid; and transfecting the recombinant plasmid into Sf9 insect cells, culturing to obtain virus supernatant, and separating and purifying to obtain the BCoV-S1 protein recombinant antigen.
Preferably, the optimal coating amount of the BCoV-S1 protein recombinant antigen is 0.125 mug/mL; the optimal sealing conditions are as follows: sealing for 3h at 37 ℃; the enzyme-labeled secondary antibody is a rabbit anti-bovine IgG-HRP antibody, the optimal dilution is 1:25000, and the incubation time is 45min at 37 ℃; the optimal color development condition is 13min at 37 ℃.
The invention also provides application of the detection kit in non-diagnosis purpose of identifying bovine coronavirus, and the detection kit is used for detecting a sample to be detected by adopting an indirect ELISA detection method so as to judge whether the sample to be detected contains bovine coronavirus.
Preferably, the detection of the sample to be detected by indirect ELISA specifically comprises the following steps:
(1) Incubation of serum to be detected: performing multiple ratio dilution and incubation on a sample to be tested; then washing;
(2) And (3) incubation of enzyme-labeled secondary antibodies: diluting rabbit anti-bovine IgG-HRP antibody, and incubating; then washing;
(3) Developing a substrate: adding a color development liquid in a dark place, adding a stop solution to stop the reaction after the incubation is completed, and measuring the OD of the sample to be measured 450nm Values and P/N values to determine whether bovine coronavirus is present.
Preferably, in steps (1) - (2), the wash solution is a PBST solution of 0.05% tween 20.
Preferably, the criterion for judging whether the bovine coronavirus is contained is as follows: critical value OD of negative and positive 450nm More than or equal to 0.297 positive, i.e. negative, OD 450nm And < 0.297 is judged negative.
The invention discloses the following technical effects:
the key point of serological diagnosis is necessary conformational folding and modification of target antigen, the S1 subunit in bovine coronavirus (BCoV) S protein has main neutralizing epitope, which can induce the generation of neutralizing antibody, and provides basis for bovine coronavirus detection and diagnosis, in particular, the BCoV-S1 is obtained by expression of baculovirus expression system, and compared with the prokaryotic expression of BCoV-N protein in the existing detection method, the antigenicity of the BCoV-S1 is greatly improved.
The sensitivity of the indirect ELISA antibody detection method established by the invention is as high as 1:3200; has no cross reaction with bovine viral diarrhea, bovine rotavirus, bovine astrovirus, bovine infectious rhinotracheitis virus and bovine adenovirus positive serum; repeatability experiments show that the variation coefficient of the detected samples in different batches is between 1.20 and 7.12 percent and is smaller than 10 percent, and the variation coefficient of the detected samples in the same batch in different time periods is between 0.05 and 6.67 percent and is smaller than 10 percent, which indicates that the method has good batch and batch-to-batch repeatability.
In summary, the invention uses baculovirus expression system to express S1 full length to prepare BCoV recombinant S1 protein (BCoV-S1), and the purified BCoV-S1 protein is used as a coating antigen, and experiments show that the prepared recombinant BCoV-S1 protein has good antigenicity, and the indirect ELISA antibody detection method established based on the recombinant BCoV-S1 protein has the characteristics of sensitivity, specificity and high efficiency, and is suitable for on-site detection of BCoV antibodies.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a pFastBac TM 1-S1 double enzyme digestion identification map; 1 double digested plasmid pFastBac TM 1-S1；M：1kb DNA Ladder；
FIG. 2 shows PCR identification results of M13 bacterial liquid of recombinant Bacmid, wherein 1-8: recombinant bacteria of BCoV-S1; m: DL5000DNA Marker;
FIG. 3 is an identification of protein expression of recombinant protein S1 in SF9 insect cells, wherein a: IFA identified recombinant protein S1 expressed in SF9 insect cells; b: protein immunoblotting identifies S1 protein expression, 1: SF9 cells after virus inoculation; 2: non-toxic SF9 cells; 3: SF9 supernatant after virus inoculation; 4: non-toxic SF9 supernatant; m: pre-dyeing a protein Marker;
FIG. 4 shows SDS-PAGE analysis after protein purification of recombinant protein S1 expressed in High5 cells (a) and a protein immunoblot analysis (b); 1: a stock solution; 2: flowing through liquid; 3-6: washing impurities with 5mM imidazole buffer; 7-11: eluting with 500mM imidazole buffer;
FIG. 5 is a diagram of ultrafiltration tube concentration analysis after purification of recombinant protein S1; 1: purified protein; 2: purifying the concentrated protein; m: a Marker;
fig. 6 is a determination of the threshold value.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
Example 1 preparation method of eukaryotic BCoV-S1 protein recombinant antigen
1. Codon optimization and Gene Synthesis of the major Domain of BCoV-S1
Codon optimization was performed based on the S1 Gene of the BCoV-China/SWUN/A1/2018 strain ((Gene bank: QOV 05164.1), optimized synthesis was performed by Nanjing Jinsi Biotechnology Co., ltd.; the C-terminal of the optimized Gene sequence had 6 histidine (His) tags, the N-terminal had a propolis secretion signal peptide (Honeybee melittin signal peptide, mels), and the optimized protein sequence was shown as SEQ ID No. 2. Gene cloning to pFastBac using two cleavage sites of BamHI and HindIII TM 1 construction of pFastBac containing optimized Gene sequences on vector TM 1-S1 plasmid. Plasmid pFastBac TM 1-S1 double enzyme cleavage was verified, FIG. 1 is pFastBac TM 1-S1 double enzyme digestion result graph.
SEQ ID No:2 is as follows:
MKFLVNVALVFMVVYISYIYAFLILLISLPTAFAVIGDLKCTTVSINDVDTGVPSISTDTVDVT
NGLGTYYVLDRVYLNTTLLLNGYYPTSGSTYRNMALKGTLLLSTLWFKPPFLSDFTNGIFA
KVKNTKVIKDGVMYSEFPAITIGSTFVNTSYSVVVQPHTTNLDNKLQGFLEISVCQYTMCE
YPNTICHPNLGNQRVELWHWDTGVVSCLYKRNFTYDVNADYLYFHFYQEGGTFYAYFTDT
GVVTKFLFNVYLGTVLSHYYVMPLTCNSALTLEYWVTPLTSKQYLLAFNQDGVIFNAVDC
KSDFMSEIKCKTLSIAPSTGVYELNGYTVQPIADVYRRIPNLPDCNIEAWLNDKSVPSPLNW
ERKTFSNCNFNMSSLMSFIQADSFTCNNIDAAKIYGMCFSSITIDKFAIPNGRKVDLQLGNLGYLQSFNYRIDTTATSCQLYYNLPAANVSVSRFNPSTWNRRFGFTEQSVFKPQPAGVFTDHDVVYAQHCFKAPTNFCPCKLDGSLCVGSGSGIDAGYKHTGIGTCPAGTNYLTCHNAAQCDCLCTPDPITSKATGPYKCPQTKYLVGIGEHCSGLAIKSDHCGGNPCSCQPQAFLGWSVDSCLQGDRCNIFANFILHDVNSGTTCSTDLQKSNTDIILGVCVNYDLYGITGQGIFVEVNATYYNSWQNLLYDSNGNLYGFRDYLTNRTFMIRSCYSGRVSAAFHANSSEPALLFRNIKCNYVFNNTLLRQLQPINYFDSYLGCVVNADNSTSSVVQTCDLTVGSGYCVDYSTKRRSRRHHHHHHHH*.
2. construction of recombinant Bacmid
pFast Bac1-S1 plasmid was transformed into E.coli DH10Bac E.coli competent cells (purchased from Shanghai Weidi Biotechnology Co., ltd.) respectively, and the competent cells were plated on blue-white plaque plates (X-Gal, gentamicin, kanamycin, tetracycline, IPTG) containing 3 antibiotics, and after 48 hours, white monoclonal colonies were streaked on Lan Baiban; after 24h blue-white spot purification, single bacteria are selected and shaken in a liquid LB containing gentamicin, kanamycin and tetracycline for 1h, and then M13 primers are used for bacterial liquid identification of recombinant S1-Bacmid, as shown in a graph of the PCR identification result of the M13 bacterial liquid of recombinant S1-Bacmid. The correct nucleic acid gel for the cut strip was sent to Shanghai Bioengineering Co.Ltd for sequencing. And (3) after sequencing is successful, shaking the small plasmid for 16 hours to obtain the S1-Bacmid recombinant plasmid.
3. Expression and identification of BCoV-S1 protein
The recombinant plasmid S1-Bacmid is passed through transfection reagent X-tremeGENE TM The HP DNA is transfected into Sf9 insect cells, after the cells are cultured for 72 hours, virus supernatant is harvested and stored at 4 ℃ in a dark place; sf9 cells with good growth status were inoculated into 24-well plates one day in advance, with a cell density of about 1.5-2.5X10 6 Performing stationary culture on cells/mL in a constant temperature incubator at 27 ℃ for 24 hours to adhere cells; cells with confluence of about 70% were washed 2 times with PBS and fresh medium was changed. Inoculating recombinant baculovirus into SF9 cells according to the inoculation amount of 5%, and placing a cell plate in a constant temperature incubator at 27 ℃ for culturing; and after the virus inoculation is carried out for 72 hours, whether obvious lesions appear on cells or not is observed, and the supernatant is collected and stored at the temperature of 4 ℃ until the obvious lesions appear.
After obvious lesions appear, 6 holes are paved on a 24-hole plate, 4 holes are used for receiving virus supernatant, 2 holes are not used for receiving virus as a control, the supernatant is collected for 4 ℃ for preservation after 72 hours of lesions appear, a small amount of PBS is added to cover the cell surface for rinsing, and the PBS is discarded. Adding 80 mu L of RIPA protein lysate containing PMSF into 2 toxin receiving holes and one control hole of a 24-hole cell plate, blowing the two holes by a pipetting gun to make the lysate fully contact with cells, putting the cell plate on ice for cracking for 30min, transferring the cell lysate into a 1.5mL centrifuge tube after fully cracking, separating 14000g from 5min, taking 80 mu L of the supernatant after cracking, adding 20 mu L of 5 xSDS-PAGE Loading Buffer, uniformly mixing, and carrying out water bath at 100 ℃ for 10min to denature proteins for subsequent Western blot. And (3) after the remaining 3 holes are inoculated with the toxin for 72 hours, collecting the supernatant and preserving at 4 ℃. Cells were washed three times with PBST, and absolute ethanol was added in an amount of 500. Mu.L/well and fixed at 4℃for 45min. After the fixation, the cells were washed three times with PBST, and 500. Mu.L of mouse His tag monoclonal antibody was added thereto, followed by incubation in a incubator at 37℃for 1 hour. After the incubation, the cells were washed three times with PBST, 500. Mu.L of FITC-labeled goat anti-mouse monoclonal antibody was added, incubated in a 37℃incubator for 50min, washed three times with PBST, and fluorescence was observed using an inverted fluorescence microscope.
As shown in fig. 3, which is an identification chart of protein expression of recombinant protein S1 in SF9 insect cells, western blot results indicate that: a specific band appears at the upper part of 100kDa, and the control cells have no band, thus proving that the recombinant protein is successfully expressed in Sf9 insect cells; s1 is expressed in both supernatant and cells, and the secretion of foreign proteins in supernatant is small. The indirect immunofluorescence results indicate that: obvious green fluorescence appears in the Sf9 insect cells after the inoculation, and the Sf9 insect cells without inoculation have no green fluorescence, thus proving that the recombinant protein is successfully expressed in the Sf9 insect cells.
4. Large scale expression and purification of BCoV-S1 proteins
2 bottles of 1000mL high pressure Erlenmeyer flasks were prepared, each flask containing 300mL of a density of 2X 10 6 The cells/mL of High5 cells were inoculated with a 2% virus loading dose of baculovirus, mixed well and incubated in a constant temperature shaker at 27℃and 110 rpm. After 96 hours of culture, the cell viability was about 70%, the cell suspension was placed in a 50mL centrifuge tube, centrifuged at 2500rpm for 10 minutes, and the supernatant medium was collected for protein purification.
Preparation of the solution before purification: all the purified solutions were passed through a 0.22 μm filter and the culture supernatant solution to be purified was passed through a 0.45 μm filter.
Next, 5mL Cytiva His Trap is employed TM The protein obtained above was further purified by the HP nickel column, and the specific procedure was as follows:
(1) Respectively using ddH of 5 times column volume 2 O and 10 column volumes of 5mM imidazole buffer (50 mM NaH 2 PO 4 300mM NaCl, 5mM imidazole) equilibriumA nickel column;
(2) Placing the protein solution on ice, and passing through a nickel column by an NGC system through an A pump;
(3) 5mM imidazole buffer (50 mM NaH) was used at 5 column volumes 2 PO 4 Rinsing with 300mM NaCl and 5mM imidazole by passing through a nickel column through a B pump to wash out the impurity protein with weak binding;
(4) 10 column volumes of 500mM imidazole buffer (50 mM NaH were used 2 PO 4 300mM NaCl, 500mM imidazole) to elute the protein of interest on the column at a flow rate of 1mL/min, the collected eluate was stored in 1.5mL EP tubes, and 1mL each tube was collected separately.
(5) Concentrating and changing the protein by using an ultrafiltration tube. 5mL of 50KD ultrafiltration tube was added with no more than 5mL of ddH 2 O, balancing 3000rpm, centrifuging for 10min to wet the filter membrane, and discarding ddH in the centrifuge tube 2 O, adding 3-tube solution with target protein bands in SDS-PAGE (shown in figure 3 a), centrifuging at 3000rpm for 10min, adding 5% glycerol in PBS solution to the original position, centrifuging, continuing adding, repeating liquid exchange for multiple times to reduce the salt ion content in the protein solution, and finally prolonging the centrifuging time to ensure that the solution only remains 500 mu L, and separating out the protein after liquid exchange and concentration by using a pipette.
(6) After the concentration of the purified S1 protein was measured by BCA method, the purified S1 protein was dispensed at-80 ℃.
As shown in FIG. 4, the purification results showed that most of the hetero proteins could be eluted with low concentration imidazole (5 mmol/L), and the target proteins could be eluted with high concentration imidazole (500 mmol/L), and the protein purity after concentration was improved.
As shown in FIG. 5, which is an ultrafiltration concentration analysis chart of recombinant protein S1 protein after concentration and liquid exchange by using an ultrafiltration tube, the concentration of the protein after concentration is higher.
Example 2 establishment of an Indirect ELISA antibody detection method based on eukaryotic expression of BCoV-S1 protein
1. Determination of optimal antigen coating concentration and serum dilution
The optimal coating concentration and serum dilution of the BCoV-S1 protein antigen were determined using a matrix method, and the BCoV-S1 protein was subjected to double dilution using sodium bicarbonate coating buffer, and then subjected to double dilutionCoating concentration was diluted to 2, 1, 0.5, 0.25, 0.125, 0.0625. Mu.g/mL, 100. Mu.L per well (sequential transverse coating), coating overnight at 4 ℃; discarding the liquid in the holes, washing with 200 mu L of PBST in each hole for 3 times every 5min, and finally beating the liquid on the water-absorbent paper; preparing 5% skim milk sealing buffer solution, adding 200 μl sealing buffer solution into each hole, and sealing at 37deg.C for 2 hr; discarding, washing the plate 3 times by using PBST for 5min each time, and finally drying the liquid on the water absorbing paper; bovine positive and negative serum (negative control) were diluted in a ratio of 1:50, 1:100, 1:200, 1:400,4 dilutions, 100 μl per well (added in vertical rows in order), and incubated for 1h at 37 ℃; discarding, washing the plate 3 times by using PBST for 5min each time, and finally drying the liquid on the water absorbing paper; the rabbit anti-bovine IgG-HRP antibody was diluted 1:20000 with PBST and incubated at 37℃for 45min at 100. Mu.L per well; discarding, washing the plate 3 times by using PBST for 5min each time, and finally drying the liquid on the water absorbing paper; adding 100 mu L of TMB color developing solution into each hole in dark place, and incubating for 15min at 37 ℃; 100 mu L of 1M sulfuric acid stop solution is added into each hole; OD of negative and positive serum of BCoV was compared using absorbance at 450mm with an ELISA reader 450nm Values and P/N values to determine the optimal antigen coating concentration and serum dilution.
2. Determination of optimal closure conditions
And (3) optimizing the closed condition by using the experimental condition optimized in the previous step. ELISA plates were coated with wells and blocked with 5% skim milk, 2.5% skim milk, 1% gelatin, 1% BSA, 5% BSA at 37℃for 30min, 60min, 90min, 120min, 180min, respectively. Comparison of the groups of BCoV negative and positive serum OD 450nm Values and P/N values to determine the optimal closed condition.
3. Determination of optimal conditions for serum
And (3) optimizing the serum optimal action condition by using the experimental condition optimized in the previous step. ELISA plates are coated and grouped, and after serum is diluted according to proportion, the serum is respectively incubated for 30min, 45min, 60min, 75min and 90min at 37 ℃. Comparison of the groups of BCoV negative and positive serum OD 450nm Values and P/N values to determine the optimal conditions for serum.
4. Determination of optimal dilution of second enzyme-labeled antibody and incubation conditions
The dilution and incubation conditions of the enzyme-labeled antibodies were optimized using the experimental conditions optimized in the previous step. ELISA plates were coated and grouped, and rabbit anti-bovine IgG-HRP antibodies were individually assigned to 1: 5000. 1:10000, 1: 15000. 1: 20000. 1: dilutions were serially diluted at 25000 while incubated at 37℃for 30min, 45min, 60min, 75min, respectively, at each dilution. Comparison of OD of BCoV negative and Positive serum under different dilution factors and different incubation conditions of enzyme-labeled antibody 450nm Values and P/N values, the optimal enzyme-labeled antibody dilution and incubation conditions were determined.
5. Determination of the action time of the substrate
And optimizing the action time of the substrate by using the experimental conditions optimized in the previous step. ELISA plates are coated and grouped, and TMB substrates are incubated for 5min, 8min, 10min, 13min and 15min at 37 ℃ respectively. Comparison of OD of BCoV negative and positive serum at different substrate action times 450nm The value and P/N value, the optimal action time of the substrate is determined.
6. Determination of yin-yang critical value
The OD was measured using 75 negative bovine serum as a sample using the optimized experimental conditions 450nm Value, calculate averageAnd Standard Deviation (SD), and determining the critical value of yin and yang by using a calculation formula.
7. Results and analysis
7.1 determination of conditions for coating antigen, blocking solution, primary antiserum, enzyme-labeled secondary antibody and substrate
The results showed that the optimal coating amount of the indirect ELISA antigen was determined to be 0.125 mug/mL by experiment, and the optimal dilution of serum was 1:200 (Table 1); the optimal blocking solution is 1% gelatin (Table 2), and the blocking condition is 37 ℃ for 3 hours (Table 3); the optimal incubation conditions for serum were 37℃for 30min (Table 4); the optimal dilution of the second enzyme-labeled antibody was 1:25000 (Table 5), and the optimal effect condition was 45min at 37 ℃ (Table 6); the optimal conditions for the substrate are 13min at 37℃C (Table 7).
TABLE 1 determination of optimal coating concentration of antigen and optimal dilution of serum
TABLE 2 determination of type of blocking fluid
Table 3 closed condition determination
TABLE 4 selection of optimal reaction conditions for serum
TABLE 5 determination of optimal dilution of enzyme-labeled secondary antibodies
TABLE 6 determination of optimal conditions for the action of the second enzyme-labeled antibody
TABLE 7 determination of substrate conditions
7.2 determination of the Critical value of yin-yang
Statistical analysis of OD values of 75 BCoV bovine negative serum, negative serum OD 450nm Average value of (2) Standard deviation sd=0.047, calculated according to the formula: />The negative-positive threshold was found to be 0.297. Thus, when the OD of the test serum 450nm When the value is not less than 0.297, the result is positive, and otherwise, the result is negative (see FIG. 6).
Example 3 Performance measurement of BCoV indirect ELISA antibody detection method
1. Specificity experiments
After coating the recombinant BCoV-S1 protein under optimal conditions, standard serum 1 of Bovine Viral Diarrhea (BVDV), bovine Rotavirus (BRV), bovine astrovirus (BastV), bovine parainfluenza virus (BPIV), bovine infectious rhinotracheitis virus (IBRV), bovine adenovirus (BBAV) with known antibody positivity was respectively used: 200 dilution, and simultaneously taking BCoV positive serum and negative serum as a control, carrying out specific experimental operation steps according to optimized experimental conditions, and finally according to OD 450nm Value, determining the crossover condition of the method.
The results show that the established ELISA method is used for detecting bovine viral diarrhea, bovine rotavirus, bovine astrovirus, bovine infectious rhinotracheitis virus and bovine adenovirus positive serum, and the results show that after the recombinant BCoV-S1 protein antigen reacts with different virus positive serum, OD 450nm The values are less than 0.297, and the recombinant BCoV-S1 protein antigen is not combined with positive serum of the pathogen in a non-specific way, so that the established ELISA method has better specificity. The results are shown in Table 8.
TABLE 8 specificity test
2. Sensitivity test
BCoV positive sera were diluted 1:100, 1:200, 1:400, 1:800, 1:1:600, 1:3200, 1:6400, 1:12800 and 1:25600 fold respectively, and ELISA assays were performed on the positive sera at different dilutions, respectively.
As shown in the table 9 below,the results showed that when the dilution ratio of serum was 1:3200, the detection result was positive (OD 450nm > 0.297), indicating a higher sensitivity of the method.
TABLE 9 sensitivity test
3. Repeatability experiments
Selecting 6 serum samples, and carrying out in-batch repeatability test on 3 different ELISA plates coated in the same batch by using an established BCoV indirect ELISA antibody detection method; in addition, 3 ELISA plates coated in different batches were randomly selected, and the test method was used to perform batch-to-batch reproducibility test on the above 6 serum samples. Calculating their mean, standard deviation and coefficient of variation from batch to batchThe reproducibility of the detection method was evaluated. The established ELISA method was used for intra-batch and inter-batch reproducibility assays for statistical analysis.
As shown in Table 10, the results show that the maximum variation coefficient of the intra-batch repeatability test is 7.12%, the maximum variation coefficient of the intra-batch repeatability test is 6.67%, and the values are less than 10%, which indicates that the established ELISA method has good repeatability.
TABLE 10 results of repeated experiments of indirect ELISA antibody detection method for BCoV
4. Clinical application
A total of 298 clinical bovine serum samples from 3 cattle farms were collected and tested using the established BCoV indirect ELISA antibody detection method.
As shown in Table 11, the results showed that the positive detection rate of the present sample was 74.83%.
TABLE 11 sample detection results
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (9)
1. The bovine coronavirus indirect ELISA antibody detection kit is characterized by comprising BCoV-S1 protein recombinant antigen coated on an ELISA plate.
2. The bovine coronavirus indirect ELISA antibody detection kit according to claim 1, characterized in that the amino acid sequence of BCoV-S1 protein is as set forth in SEQ ID NO: 1.
3. The bovine coronavirus indirect ELISA antibody detection kit according to claim 1, characterized in that the kit further comprises: blocking solution, washing solution, positive control, negative control, enzyme-labeled secondary antibody and chromogenic solution.
4. The bovine coronavirus indirect ELISA antibody detection kit according to claim 1, wherein the preparation method of BCoV-S1 protein recombinant antigen comprises: after codon optimization of an S1 gene sequence of bovine coronavirus, adding a histidine tag at the 3' end, introducing BamHI and HindIII restriction enzyme cutting sites at the two ends of the S1 gene sequence, and transferring into a vector to construct a recombinant plasmid; and transfecting the recombinant plasmid into Sf9 insect cells, culturing to obtain virus supernatant, and separating and purifying to obtain the BCoV-S1 protein recombinant antigen.
5. The bovine coronavirus indirect ELISA antibody detection kit according to claim 1, characterized in that the optimal coating amount of BCoV-S1 protein recombinant antigen is 0.125 μg/mL; the optimal sealing conditions are as follows: sealing for 3h at 37 ℃; the enzyme-labeled secondary antibody is a rabbit anti-bovine IgG-HRP antibody, the optimal dilution is 1:25000, and the incubation time is 45min at 37 ℃; the optimal color development condition is 13min at 37 ℃.
6. Use of the detection kit according to any one of claims 1 to 5 for the identification of bovine coronavirus for non-diagnostic purposes, wherein the detection kit is used to detect a test sample by an indirect ELISA detection method to determine whether bovine coronavirus is contained in the test sample.
7. The use according to claim 6, wherein the detection of the sample to be tested by indirect ELISA comprises the following steps:
(1) Incubation of serum to be detected: performing multiple ratio dilution and incubation on a sample to be tested; then washing;
(2) And (3) incubation of enzyme-labeled secondary antibodies: diluting rabbit anti-bovine IgG-HRP antibody, and incubating; then washing;
(3) Developing a substrate: adding a color development liquid in a dark place, adding a stop solution to stop the reaction after the incubation is completed, and measuring the OD of the sample to be measured 450nm Values and P/N values to determine whether bovine coronavirus is present.
8. The use according to claim 7, wherein in steps (1) - (2), the washing solution is a PBST solution of 0.05% tween 20.
9. The use according to claim 5, wherein the criterion for determining whether bovine coronavirus is present is: critical value OD of negative and positive 450nm More than or equal to 0.297 positive, i.e. negative, OD 450nm And < 0.297 is judged negative.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117554613A (en) * | 2024-01-10 | 2024-02-13 | 深圳市绿诗源生物技术有限公司 | Colloidal gold detection test strip for bovine coronavirus antigen, and preparation method and application thereof |
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