CN110791479A - DEV gB protein monoclonal antibody and blocking ELISA kit for detecting DEV antibody - Google Patents

Abstract

The invention discloses a DEV gB protein monoclonal antibody and a blocking ELISA kit for detecting a DEV antibody. The gB protein with good reactionogenicity and immunogenicity is prepared by a prokaryotic expression system, and is used as an antigen to obtain a hybridoma cell strain with a microorganism preservation number of CGMCC No.17996 through screening by a cell fusion technology, wherein the secreted monoclonal antibody can react with duck virus enteritis virus natural space conformation virus protein and has strong blocking capability on DEV antibody; the invention also provides an epitope recognized by the monoclonal antibody, and the amino acid sequence of the epitope is shown in SEQ ID No. 1; the invention further uses the monoclonal antibody to construct a blocking ELISA kit for detecting the DEV antibody, and the blocking ELISA kit has the advantages of high sensitivity, good specificity, strong repeatability and the like.

Description

DEV gB protein monoclonal antibody and blocking ELISA kit for detecting DEV antibody

Technical Field

The invention relates to a monoclonal antibody and a blocking ELISA kit, in particular to a DEV gB protein monoclonal antibody and a blocking ELISA kit for detecting a DEV antibody, belonging to the field of antibody detection of duck viral enteritis virus.

Background

Duck Viral Enteritis (DVE), commonly known as Duck Plague (DP), is an acute, febrile, septic infectious disease of various anseriformes, such as ducks, geese, wild geese, and the like, caused by Duck viral enteritis virus (DEV). The viral genome encodes a dozen glycoproteins, of which the gB protein is one of the major proteins displayed on the surface of the viral envelope, and is also the major immunogenic protein. Duck viral enteritis is a herpes virus disease of duck animals, seriously harms domestic ducks and attacks various waterfowls such as wild ducks and the like. The virus identification is firstly confirmed in 1967 in the United states, virus is firstly separated from inoculated duck embryos and young ducks, then the duck embryos are cultured by primary fibroblasts, and virus identification is carried out in a neutralization test.

ELISA is used as a traditional serological diagnosis method with the widest application, has strong operability, high specificity and sensitivity and low price and cost, and is an ideal diagnosis method for monitoring the immune effect of a vaccine and infecting animals. So far, a blocking ELISA kit for detecting DEV antibodies with commercial application value is lacked.

Disclosure of Invention

One of the purposes of the invention is to provide a DEV gB protein monoclonal antibody which can react with the natural space conformation virus protein of duck viral enteritis virus and has strong blocking capability on DEV antibody;

the other purpose of the invention is to provide an epitope recognized by the DEV gB protein monoclonal antibody;

the third purpose of the invention is to construct a blocking ELISA kit for detecting DEV antibody by using the DEV gB protein monoclonal antibody;

the above object of the present invention is achieved by the following technical solutions:

the invention firstly provides a DEV gB protein monoclonal antibody which can react with the DEV natural space conformation virus protein and has strong blocking capability to the DEV antibody:

according to the invention, DEV gB protein is purified after prokaryotic expression, and the purified gB protein is subjected to WesternBlot analysis, and the result shows that the expressed protein has stronger immunogenicity. The result of the immunoreactivity IFA analysis of the gB protein shows that the expressed gB protein has good immunoreactivity.

The invention finally screens a specific hybridoma cell strain secreting the anti-DEV gB protein monoclonal antibody by using the prepared gB protein as an antigen through a cell fusion technology:

the invention mixes and emulsifies the purified DEV and DEV gB proteins with Freund's adjuvant respectively, immunizes BALB/c mice of 6 weeks, the dosage is 50 mug/mouse, the immunization is carried out for 3 times, and the immunization interval is two weeks. Mice with high titers were selected after 5 days for boosting with non-emulsified antigen and their spleens were fused with SP2/0 cells in logarithmic growth phase 3 days later. And (3) carrying out subcloning on the cells of the fusion hole for 3 times, screening by an indirect ELISA method to obtain a positive cell strain, and freezing and storing the cells after amplification culture. 4 screening by using an established indirect ELISA method to obtain 4 hybridoma cell lines capable of stably secreting anti-DEVMAb, which are respectively named as 2E3, 7E12, 9G3 and 10A9, and 7 hybridoma cell lines capable of stably secreting anti-gB protein MAb, namely 1A4, 3C2, 4H3, 3E2, 2B1, 1E8 and 2G 3. The subclass of MAb was determined using antibody subclass identification kit, and among 11 MAbs, 4 DEVMAb, 2E3 were of IgM subclass, 7E12 and 9G3 were of IgG1 subclass, and 10A9 was of IgG2b subclass; the heavy chains of 7gB protein mabs are all subclass IgG2 b. IFA detection is carried out on hybridoma cell strains capable of stably secreting anti-DEVMAb, observation is carried out under an inverted fluorescence microscope, 3MAb strains react with DEV, namely DEV MAb 2E3, 7E12 and gB protein MAb1E8, the inoculated cells emit strong green fluorescence and are concentrated on cytoplasm and cell membranes, and specific green fluorescence is not seen in a control group.

In order to screen an IgG subclass MAb which can react with a natural space conformation virus protein and has strong blocking capacity to DEV antibody from a plurality of MAbs, the MAb which is identified as positive to IFA and is identified as an IgG subclass is further screened as a MAb which is established to block an ELISA method and screen specificity; screening results show that DEV MAb 7E12 and gB protein MAb1E8 are IgG subclasses and react with natural virus proteins, and the identification result of the blocking effect of the two MAbs shows that the blocking capability of MAb1E8 is stronger than that of 7E12, so that the subsequent identification test is carried out on MAb1E 8.

Western Blot identification showed that PAb binds to denatured DEV proteins at the 30kDa, 47kDa and 120kDa bands, MAb1E8 binds to denatured DEV proteins at the 30kDa band and binds to pronucleus-expressed 51kDa proteins.

The specificity identification result of the MAb1E8 shows that the reactions of the MAb1E8 with other viruses and DEFs are negative under the condition that the positive control is established, which indicates that the MAb1E8 is specific; the ELISA identification result of the ascites titer of MAb1E8 shows that when the ascites are diluted by 10⁴Time of doubling OD_450nmThe values are all larger than 1.0, the P/N is more than or equal to 2.1 and is taken as a positive judgment standard, and the result shows that the titer of the antibody is 10⁶。

The invention further carries out epitope identification on the MAb1E 8. According to the epitope identification truncation expression schematic diagram, the gB protein is truncated by 3 rounds, and the induction expression is carried out for 6 times; through PCR, SDS-PAGE and Western Blot analysis, the size of the gB-101 amplified fragment expressed by the first truncation is 600bp, the size of the protein is 38kDa, the size of the gB-102 amplified fragment is 441bp, the size of the protein is 28kDa, and the Western Blot result shows that the MAb reacts with the second section of protein gB-102; the size of the second truncated and expressed gB-201 amplified fragment is 192bp, the size of the protein is 16kDa, the size of the gB-202 amplified fragment is 288bp, the size of the protein is 25kDa, and Western Blot results show that the MAb reacts with a second section of protein gB-202; the size of the gB-301 amplified fragment expressed by the third truncation is 171bp, the size of the protein is 15.5kDa, the size of the gB-302 amplified fragment is 153bp, the size of the protein is 15kDa, and a Western Blot result shows that the MAb reacts with both the two sections of proteins; indirect ELISA assay of mabs and synthetic overlapping polypeptides showed thatAnd (3) polypeptide reaction. Therefore, the epitope to which the MAb is directed should be the amino acid sequence of the polypeptide overlapped by the two protein segments in the third truncated expression, i.e. the amino acid sequence⁵⁷⁹RMLGDVLAVSSC⁵⁹⁰(SEQ ID No. 1). The epitope region of MAb1E8 analyzed was found to be in UL27 full-length protein 579-590 aa.

The hybridoma cell strain MAb1E8 is submitted to a patent approved organization for preservation, and the preservation number is CGMCC No. 17996; the classification nomenclature is: a DEV gB protein monoclonal antibody cell strain; the preservation time is 6 months and 19 days in 2019; the preservation unit is China general microbiological culture Collection center; the preservation address is: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.

The invention further provides a blocking ELISA detection kit for detecting DEV antibody, which comprises: DEV gB protein, coating solution, MAb1E8 antibody, enzyme-labeled secondary antibody, buffer solution, diluent, washing solution, confining solution, stop solution, positive serum control and negative serum control.

The enzyme-labeled secondary antibody is goat anti-mouse IgG labeled by HRP;

the buffer solution is preferably PBS buffer solution; the coating solution is preferably 0.05mol/L carbonate buffer solution with pH value of 9.6;

the preparation of the sealing liquid is preferably as follows: 50g of skim milk was added to the PBS buffer to dissolve it sufficiently, and the volume was adjusted to 1000 mL.

The washing solution is preferably prepared by: 500mL of Tween20 was added to the PBS buffer, and the volume was adjusted to 1000 mL.

The preparation of the diluent is preferably: 10g of skim milk was added to the PBS buffer to dissolve it sufficiently, and the volume was adjusted to 1000 mL.

The preparation of the stop solution is preferably as follows: 100mL of concentrated sulfuric acid was slowly added to 80mL of deionized water to 100 mL.

For reference, the present invention provides a method for detecting DEV antibodies in a sample using the blocking ELISA test kit, comprising:

(1) diluting the purified gB protein with coating solution to 0.5. mu.g/mL, 100. mu.L per well, and coating at 4 ℃ for 12 h; washing the ELISA plate with washing solution for 3 times, and patting to dry; (2) adding 300 μ L of 5% ski mil per well, sealing at 37 deg.C for 2h, and washing the plate for 3 times; (3) centrifuging to remove impurities, reacting at room temperature for 2 hr, setting positive and negative serum control (1: 40), washing plate for 3 times with each well at 100 μ L; (4) adding MAb (1: 2000) and reacting at 37 deg.C for 1h, washing plate for 3 times, wherein each well is 100 μ L; (5) adding goat anti-mouse-HRP (1:10000) 100 μ L per well, performing reaction at 37 deg.C for 0.75h, and washing the plate for 3 times; (6) adding TMB (100 μ L per well), and developing in dark for 15 min; terminating the reaction, wherein each well contains 50 mu L of termination solution; read plate within 15 min.

364 parts of negative duck serum sample and 130 parts of positive duck serum sample are determined by adopting the blocking ELISA detection kit constructed by the invention, and one part of SPF duck serum is used as a negative control. SPSS draws an ROC curve of PI value statistical data of the positive and negative samples, and the sensitivity and the specificity are higher when the critical value is 30%. Therefore, the critical value of the detection method is 30 percent, namely the PI of the sample to be detected is more than or equal to 30 percent and is judged to be positive, otherwise, the PI is judged to be negative.

The ELISA method established by the invention is used for synchronously detecting the standard serum resisting AIV, NDV, EDSV, DTMUV, GPV and DHV-1, the reference product of the anti-DEV positive serum is used as a positive control, one part of SPF duck serum is used as a negative control, and the result shows that the detection method is specific to DEV antibody, which indicates that the blocking ELISA detection kit has good specificity.

The sensitivity of the method was tested by testing a DEV positive serum reference in an established ELISA method by diluting the positive serum 10-fold and 1280-fold maximum. Calculating the PI value of the positive serum under different dilution times, wherein the PI value decreases along with the increase of the dilution times of the serum, the detection result is positive when the serum is diluted by 80 times, the detection result is negative when the serum is diluted by 160 times, and the method can detect that the maximum dilution times of the serum are 1: 80, the blocking ELISA detection kit constructed by the invention has high sensitivity.

Respectively taking 4 plates in the same batch and 3 plates in different batches, detecting 7 positive serum samples by using the method established by the invention, determining the PI values of the same sample in the same batch and in different batches, and calculating results show that the inter-batch and intra-batch variation coefficients are less than 10 percent, wherein the intra-batch maximum difference is 6.88 percent and the inter-batch maximum difference is 8.62 percent; the test result shows that the blocking ELISA detection kit constructed by the invention has good repeatability.

And simultaneously, a commercial kit (indirect ELISA) and the blocking ELISA detection kit constructed by the invention are used for detecting 41 positive samples and 53 negative samples. The detection results of 94 samples show that the total coincidence rate of the detection kit is 84.04%, the positive coincidence rate is 82.5% and the negative coincidence rate is 85.19%.

The clinical sample detection result shows that the total positive rate of the sample antibody is not more than 20%, and the antibody positive rate of the breed ducks is obviously higher than that of the commodity ducks.

The result is combined to show that the blocking ELISA detection reagent for detecting DEV antibody provided by the invention has the advantages of high sensitivity, good specificity, strong repeatability and the like, and has good commercial application prospect.

Drawings

FIG. 1 software analysis of full-length gB protein.

FIG. 2 SDS-PAGE analysis of protein expression; m: protein Marker, 1: whole bacteria before induction; 2: performing induced whole bacteria; 3: crushing and then clearing the supernatant; 4: and precipitating after crushing.

FIG. 3 WesternBlot identification of the gB protein; m: a protein Marker; 1: duck origin positive serum.

FIG. 4 IFA analysis of immunoreactivity of gB protein; immune gB protein rabbit serum; b: non-immune rabbit serum.

FIG. 5 schematic representation of the epitope identification truncation expression.

FIG. 6 subclass identification of MAb.

FIG. 7 IFA analysis of reactivity of MAb with DEV (200 ×); a: 2E 3; b: 7E 12; c: 1E 8; d to F: and (5) negative control.

FIG. 8 WesternBlot analysis of MAb; m: a protein Marker; 1: PAb reacts with DEV; 2: MAb reacts with DEV; 2: mabs react with gB protein.

FIG. 9 specificity test of MAb.

FIG. 10 ELISA assay results for MAb1E8 titers.

FIG. 11 gB protein first truncated expression; m1: DL2000DNA Marker; m2: a protein Marker; 1.3, 5: gB-101; 2.4, 6: gB-102.

FIG. 12 second truncation of protein gB expression; m1: DL2000DNA Marker; m2: a protein Marker; 1.3, 5: gB-201; 2.4, 6: gB-202.

Fig. 13 third truncation of gB protein expressed M1: DL2000DNA Marker; m2: a protein Marker; 1.3, 5: gB-301; 2.4, 6: gB-302.

FIG. 14 MAb and polypeptide indirect ELISA assay results.

FIG. 15 MAb1E8 working concentration optimization.

FIG. 16 gB protein coating conditions were optimized.

FIG. 17 PI distribution for negative and positive serum samples.

FIG. 18 results of specific assay for blocking ELISA.

FIG. 19 shows the results of sensitivity tests of blocking ELISA.

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

Example 1 prokaryotic expression of DEV gB protein

1. Materials and methods

1.1 Strain and Experimental animals

The seed virus of the DEVCSC strain is preserved by the laboratory; SPF duck embryos were purchased from Experimental animal center of Harbin veterinary institute, national academy of agricultural sciences; new Zealand clean-grade white rabbits were purchased from Liaoning Biotechnology Ltd; the DEV positive serum reference is purchased from Chinese veterinary medicine inspection institute; the pET-32a vector plasmid was stored in this laboratory.

1.2 test methods

1.2.1 propagation of DEV

Preparing Duck Embryo Fibroblast (DEFs) from 11-day-old SPF duck embryo by conventional method, culturing the prepared primary cells in DMEM containing 5% FBS, and placing the primary cells in 5% CO₂Culturing in an incubator at 37 ℃ for 1-2 d, then carrying out passage, and after 1 passage, growing until 80% of cells are fully paved at the bottom of the bottle, thus inoculating the virus.

Inoculating virus, diluting virus suspension with serum-free DMEM 6 times, discarding cell culture supernatant, washing with PBS buffer solution for 3 times, sucking clean PBS buffer solution, adding appropriate amount of virus suspension, and placing in 5% CO bottle with the bottom of the bottle completely covered₂Adsorbing for 1-2 h in a constant temperature incubator at 37 ℃. After adsorption, the virus suspension was discarded, DMEM containing 2% FBS was added, and the culture was continued for 3 days while observing the cytopathic condition every day, and a virus-free control group was set. Harvesting viruses, inoculating DEVCSC strain viruses to DEFs after passage 1, freezing and thawing once when 90% of cells are observed to be diseased under a microscope, collecting supernatant, centrifuging at 800r/min for 8min, and removing cell debris; the cells at the bottom of the bottle are scraped off by a cell scraper, and the cells are frozen and thawed twice, centrifuged at a differential speed to remove the sediment and combined in the supernatant.

1.2.2 extraction and PCR detection of DEV DNA

And (3) extracting the DNA of the DEVCSC strain seed virus and the cell subculture virus which are stored in a laboratory by using a virus DNA extraction kit. And (3) carrying out PCR detection by using primers in a national standard method, and sequencing a connected T vector. Using DEV CSC seed virus and cell subculture virus DNA as a template, and detecting a reaction system by PCR (polymerase chain reaction) with 50 mu L; the reaction procedure is as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 55 ℃, 30s at 72 ℃ and 25 cycles; 10min at 72 ℃.

1.2.3 coarse purification of DEV

After harvesting the virus, differential centrifugation is carried out at 4 ℃, the centrifugation is carried out once at 4000r/min and once at 8000r/min for 15min each time, and the supernatant obtained by the second centrifugation is taken and merged into the previous supernatant. Preparing 5mL of 30% (W/V) sucrose solution by using PBS buffer solution, adding the supernatant into a centrifugal tube with the bottom of the 30% (W/V) sucrose solution, centrifuging for 2h at 4 ℃ of 32000r/min, collecting the precipitate, and suspending the precipitate by using an appropriate amount of PBS buffer solution. The pellet collected after differential centrifugation was negatively stained and the virions were observed under an electron microscope.

1.2.4 sucrose discontinuous Density gradient centrifugation method for DEV purification

Preparing a sucrose discontinuous density gradient solution, sequentially adding 30%, 40%, 50% and 60% (W/V) sucrose solutions in a centrifuge tube from bottom to top, adding the collected virus suspension to the uppermost layer, centrifuging at 32000r/min for 2h at 4 ℃, collecting virus bands, diluting with 0.5mL PBS buffer solution, centrifuging at 28000r/min for 2h for desugaring, suspending the precipitate with 0.5mL PBS buffer solution, and storing in an ultra-low temperature refrigerator. And (3) measuring the concentration of the virus suspension purified by the sucrose discontinuous density gradient centrifugation method by using an ultraviolet spectrophotometer, and observing the virus suspension under an electron microscope after negative dyeing.

1.2.5DEV TCID50 determination

TCID50 was determined for seed and subculture strains of DEVCSC. Virus was cultured in DMEM basal medium 10^-2～10^-9Dilution by multiple times; discarding the cell culture solution in a 96-well plate with DEFs laid in advance, and washing the cell culture solution once by PBS; sequentially transferring the diluted virus solution into 1-8 rows, adding one column of the diluted virus solution into each row for 8 times, and repeating the steps until each hole is 100 mu L; 8-12 columns were set as normal cell controls. Lesions (CPE) are observed and recorded once every 24h after inoculation, the number of CPE holes and CPE-free holes observed each time is recorded for one week continuously, and the TCID50 of the virus is calculated according to a Reed-Muench method.

1.2.6 construction of DEV gB protein recombinant expression System

According to the full sequence of the gene (EF554401.1) of the Duck entitis virus UL27 precorsor (UL27) published by GenBank, the full-length gB protein coded by the UL27 gene contains 932 aa. Through DNASTAR Lasergene software analysis, epitope dense regions 324-628 aa are selected for expression, and the fragment of protein is good in hydrophilicity, high in antigen density and contains a loose corner structure (figure 1). The sequence primers were designed and synthesized artificially (table 1).

TABLE 1 PCR primer sequences

According to the selected target gene, a BL21 prokaryotic expression system of pET-32a-gB plasmid is constructed. The PCR reaction system for amplifying the target fragment is the same as 2.2.2; the reaction procedure is as follows: 5min at 95 ℃; 30 cycles at 95 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 3 min; 10min at 72 ℃.

And (3) recovering the target fragment by using a gel recovery kit, performing double enzyme digestion on the target fragment and the pET-32a vector, performing enzyme digestion for 6 hours in a water bath at 37 ℃, wherein the enzyme digestion system is 50 mu L.

Connecting the target fragment and the pET-32a vector at 16 ℃ in a metal bath overnight, wherein the connecting body is 10 mu L; transforming the ligation product into a TransT1 competent cell, extracting transformed bacterial plasmid, and performing double enzyme digestion identification; converting the correctly identified pET-32a-gB plasmid into BL21 competent cells, adding 10 mu L of the ligation product into the competence, carrying out ice bath for 5min at 42 ℃ for 90s, adding a culture medium, carrying out shake incubation for 1h, and coating an Amp solid LB plate; after the colony grows out, the colony is transferred to 5ml of Amp liquid LB for bacterial liquid PCR identification.

2.2.7 expression of recombinant gB protein

Inoculating a bacterial liquid with correct PCR identification into Amp liquid LB according to the ratio of 1:50, proliferating for 1h in a bacterial incubator at 37 ℃ and 220r/min until OD600 is 0.4-0.6, adding 1mM/L IPTG according to the ratio of 1:100, and carrying out induced expression for 4-5 h. Centrifuging the collected bacteria liquid at 2000r/min for half an hour, re-suspending the precipitate with PBS, then placing on ice for ultrasonic lysis, centrifuging, collecting supernatant and precipitate, and storing the sample at-20 ℃ for later use.

2.2.8 purification of recombinant gB protein

Purifying the expressed protein by adopting a nickel column affinity chromatography method. The specific operation steps are as follows:

(1) treating a sample: after ultrasonication of the protein, centrifugation at 10000g for 10min was carried out, and the pellet was retained and resuspended with a Binding buffer as the sample to be purified.

(2) Column assembling: the column and filter were rinsed with PBS buffer and 1mL of nickel chelating affinity resin was added.

(3) Column equilibration: after rinsing the column with PBS buffer, 4mL Binding buffer was added to equilibrate the column.

(4) Loading: the flow control valve was closed and 0.4mL of sample was added for each purification and allowed to stand for 5 min.

(5) Washing: the flow control valve was opened while 4mL of PBS buffer was added, and the contaminating proteins that did not bind to the nickel column were washed away.

(6) And (3) elution: the target protein is eluted by 100mM/L, 250mM/L, 500mM/L, 800mM/L and 1M/L of imidazole solution in sequence.

(7) And (3) dialysis concentration: and dialyzing the eluted protein by PBS, performing ultrafiltration concentration, preparing a sample by taking 0.01mL of protein, performing SDS-PAGE, and performing purity identification.

2.2.9 renaturation of recombinant gB protein

Renaturation of the recombinant protein is carried out according to the instructions of a GENMED protein renaturation kit.

2.2.10 Western Blot identification of recombinant gB proteins

The purified protein was subjected to SDS-PAGE, and then transferred to a PVDF membrane by a membrane transfer apparatus. And (3) taking a duck source DEV positive serum reference substance as a primary antibody and goat anti-duck IgG-HRP as a secondary antibody, performing a test, and performing DAB chemical color development.

2.2.11 IFA identification of recombinant gB proteins

2.2.11.1 preparation of gB protein polyclonal antibody

And mixing and emulsifying the purified and renatured gB protein and Freund's adjuvant, and immunizing New Zealand white rabbits by back subcutaneous multipoint injection. The total immunization is carried out for 3 times, the immunization interval is two weeks, the dose is 1 mg/mouse, blood is collected from the marginal veins 5 days after the 3 rd immunization, the serum titer is detected, blood is collected from the heart and separated for 5 days, and the non-emulsified antigen is used for boosting the immunization before the blood serum is separated.

The indirect ELISA detects the serum titer, and the specific operation steps are as follows:

(1) the purified gB protein was diluted to 5. mu.g/mL with coating solution, 100. mu.L per well, and after overnight at 4 ℃, the microplate was washed 3 times with PBST and patted dry.

(2) Add 300. mu.L of 5% skim milk to each well, block at 37 ℃ for 2h and wash the plate 3 times.

(3) Add 10 fold diluted immune rabbit serum, set up negative and blank control, each hole 100 u L, 37 degrees C were incubated for 1h, plate washing 3 times.

(4) Adding 1:10000 diluted goat anti-rabbit-HRP, 100 μ L per well, incubation at 37 deg.C for 1h, and washing plate 3 times.

(5) Adding 100 μ L of color developing solution, developing for 10min, stopping with 50 μ L of stop solutionReaction, reading OD from the enzyme standard plate within 15min_450nmThe value is determined by using P/N ≥ 2.1 as positive determination criterion.

2.2.11.2IFA test

The specific operation steps are as follows:

(1) DEFs cell suspension is evenly paved in the six-hole cell culture plate, and the DEV is inoculated after the cells are cultured for 24 hours and just fully filled with a monolayer. (2) When the cells begin to have pathological changes after inoculation, a pipette gun sucks the culture medium in the six-hole plate completely, PBS is added for washing, the cells are discarded after being gently shaken for 5min in a horizontal shaking table for 2 times, and the PBS is discarded at the last time. (3) Adding 4% paraformaldehyde, fixing at 4 deg.C for 15min, and washing. (4) Adding 1% BSA, blocking at 37 deg.C for 30min, and washing 3. (5) Adding 0.1% TritonX-100, perforating at room temperature (20-30 deg.C) for 15min, and washing. (6) Adding rabbit polyclonal anti-positive serum diluted by 100 times with PBS, incubating for 2h at 37 ℃, and washing the same as the above steps. (7) Adding goat anti-rabbit IgG-FITC diluted by 10000 times by PBS, incubating for 1h at 37 ℃ in the dark, washing for 5 times, and finally discarding the PBS. (8) Immediately placed under an inverted fluorescence microscope for microscopic examination. The experiment was set with 3 controls, i.e. a control of non-cytotoxic cells, with no primary antibody blank and primary antibody as a non-immune rabbit serum negative control.

2.3 test results

2.3.1 Observation of infected cells

After DEFs were infected with DEV, the disease state was observed daily under a microscope. As a result, cells begin to vacuole within 24h, small vacuoles gradually increase and fuse into larger vacuoles with the passage of time, and the cells begin to become round and fall off after the vacuoles further expand to occupy more than half of the area of the cells; the number of the exfoliated cells in 72 hours is more than 90% of the total cells.

The cell is disintegrated after falling off, the cell membrane is damaged, the virus is released from the cell membrane, the virus content in the supernatant is high, and the harvested virus exists in liquid and is beneficial to next tests such as centrifugation.

Respectively extracting the genomes of the seed virus and the virus in the cell, and carrying out PCR detection by using a primer in a national standard method; the percent match between the sequencing and the reference sequence is 100%.

2.3.2 Electron microscopy of purified viruses

The virus particles are purified by adopting differential centrifugation and sucrose discontinuous density gradient centrifugation methods, the concentration of the purified virus suspension is 29.8mg/mL, and the turbidity is close to 0.0. The higher the virus purity, the greater the probability that the cell type of the secreted antibody obtained by immunizing an animal with the immunogen will meet the requirements, and the later screening process can be simplified.

Further negatively staining the virus and observing under an electron microscope, the whole virus particle is spherical, the diameter of the virus particle is about 180nm, the virus particle consists of an inner nucleocapsid and an outer cyst membrane, the shape is complete, and the sizes are relatively consistent; under the field of an electron microscope, impurities such as cell debris and the like around the virus particles purified by density gradient centrifugation are obviously less than those around the virus particles collected after differential centrifugation.

2.3.3 measurement results of TCID50

The tissue and cell toxicity TCID50 was determined by cell titration and CPE of seed and passage cells was counted (tables 2, 2). The TCID50 of DEVCSC seed toxicity calculated by Reed-Muench method is 10^-5.140.1mL, cytotoxic TCID50 of 10^-6.610.1 mL. Indicating that the virus titer increased after propagation on cells, about 10-fold higher than the seed virus, indicating that DEFs are suitable as a host for DEV parasitism, and that the virus propagated on tissues can be used in the next experiment.

TABLE 2 DEV seed toxicity CPE statistics

TABLE 3DEV cytotoxic CPE statistics

2.3.4 PCR identification of recombinant plasmids

According to antigenicity analysis, a UL27 gene partial region is selected to construct a pET-32a-gB plasmid, the plasmid is transformed into a TransT1 competent cell, the extracted plasmid is identified by double enzyme digestion, the result shows that the plasmid comprises two fragments after enzyme digestion, the sizes of the two fragments are respectively close to 915bp and 5900bp, and the result shows that the target fragment is inserted into the vector in a correct mode.

The correct pET-32a-gB plasmid identified by double enzyme digestion is transformed into BL21 competent cells, colonies are picked up and subjected to bacteria liquid PCR amplification, and the result shows that the target fragment of 1566bp is amplified from 7 selected colonies.

2.3.5 SDS-PAGE identification of protein expression of gB

Protein electrophoresis results show that after IPTG induction, gB protein expression is obviously increased compared with that before induction; bacterial pellets (inclusion bodies) and supernatants were respectively sampled for electrophoresis, the pellets contained a large amount of target protein for induction expression, while the supernatants contained almost no target protein (FIG. 2), indicating that the target protein exists in the bacteria mainly in the form of inclusion bodies. SDS-PAGE identified the purified protein to be about 51kDa, consistent with theoretical expectations.

SDS-PAGE identification of 2.3.6 gB protein purification

The samples before purification, the sample loading effluent and the samples eluted under various elution concentrations are prepared and subjected to SDS-PAGE identification, the result shows that the target protein can be eluted when the imidazole concentration is 800mM/L and 1M/L, in addition, a target band also appears in the 100mM/L elution sample, the result shows that the concentration range of protein elution is 800 mM/L-1M/L, the reason that the target band appears in the 100mM/L elution sample is probably that the sample loading concentration is too high, and a part of the target protein which is not combined flows down at the beginning of elution. The purified target band is clear and single, which shows that the purification effect is better.

SDS-PAGE identification of renaturation of 2.3.7gB proteins

After renaturation and concentration, the size of the gB protein is about 51kDa, and the concentration is 1mg/mL through an ultraviolet spectrophotometer. WesternBlot assay for the immunogenicity of 2.3.8gB proteins

And (3) taking a duck source DEV positive serum reference substance as a primary antibody, and taking goat anti-duck IgG-HRP as a secondary antibody, and performing DAB chemical color development. As a result, a clear brown band appeared at the target protein position, indicating that the protein was more immunogenic (FIG. 3).

2.3.9gB protein immunoreactivity IFA analysis

After immunizing rabbits with the gB protein for the third time, detecting the serum antibody titer by indirect ELISA to be 1:10⁶(ii) a With the positive serumIFA is carried out by taking primary goat anti-rabbit IgG-FITC as a secondary antibody, and the result shows that the inoculated cells emit strong green fluorescence and concentrate on cytoplasm and cell membranes, and specific green fluorescence is not seen in a control group (figure 4), namely antibodies combined with DEV exist in serum separated after the gB protein is immunized from rabbits, which indicates that the gB protein has good immunoreactivity.

Example 2 preparation and selection of DEV and DEV gB protein mAbs

1. Test materials

1.1 strains and cells

H5, H7 and H9 subtype Avian Influenza Virus (AIV) HI test antigens were purchased from harbividae biotech developers, and Newcastle Disease Virus (NDV), Egg Drop Syndrome Virus (EDSV), duck tembusu virus (DTMUV), and duck hepatitis i virus (DHV i) were all stored in the laboratory. SP2/0 myeloma cells were stored in the laboratory.

1.2 Experimental animals and serum samples

11-day-old SPF duck embryos were purchased from the laboratory animal center of the Harbin veterinary institute. SPF-grade female BALB/c mice at 6 weeks of age were purchased from Experimental animals technology, Inc., Viton, Beijing. 6 positive serum samples were prepared from laboratory immunized SPF ducks and identified by neutralization test; 6 negative serum samples were collected from the non-immunized SPF duck in the laboratory and identified using the neutralization test.

1.3 Primary reagents

Freund's Complete Adjuvant (FCA), Freund's incomplete adjuvant (IFCA), HAT, HT, and PEG/DMSO were all purchased from Sigma. DMEM, Fetal Bovine Serum (FBS) was purchased from GIBCO. ProteinG affinity chromatography columns were purchased from GEHealthcare. Glycine, Tris, sucrose were purchased from AMRESCO, USA; liquid paraffin was purchased from Tianjin chemical company. IRDye680LT goat anti-mouse antibody was purchased from LI-COR; goat anti-mouse IgG-HRP, IgG-FITC, goat anti-rabbit IgG-HRP, IgG-FITC, DAB color development kit were purchased from Beijing Zhonghua Jinqiao biotechnology, Inc. The SBACLonypingSystem-HRP antibody subclass identification kit was purchased from southern Biotech.

2 test method

2.1 animal immunization

After purification, DEV and DEV gB proteins were separately mixed and emulsified with Freund's adjuvant and 6-week-old BALB/c mice were immunized at a dose of 50. mu.g/mouse. The total immunization was 3 times, and the immunization interval was two weeks. The first immunization route is abdominal subcutaneous multipoint injection, and the emulsifying adjuvant is Freund's complete adjuvant; the last two immunization approaches are intraperitoneal injection, and the emulsifying adjuvant is Freund incomplete adjuvant; 5d after 3 rd immunization, cutting off the tail, collecting blood, centrifuging at 10000r/min for 10min, separating serum, and detecting the titer of serum antibody; after 5 days, mice with high titer were selected and boosted with non-emulsified antigen, and their spleens were fused with SP2/0 cells in logarithmic growth phase 3 days later.

2.2 cell fusion

After boosting, mice spleens were taken in a super clean bench and fused with SP2/0 cells in log phase growth. The specific operation steps are as follows:

(1) preparation of feeder layer cells: fixing a negative BALB/c mouse on a tray, wiping the abdomen of the mouse with an alcohol cotton ball, then lifting the skin of the abdomen with forceps, gradually cutting the skin to completely expose the peritoneum, sucking 5 mLHAT culture solution into the abdomen with an injector, massaging the abdomen for a plurality of times at a constant speed by using the alcohol cotton ball with moderate force, pumping back the liquid in the abdomen with the injector, supplementing the pumped back liquid to 50mL, and packaging the liquid into 5 culture plates with 96 holes approximately.

(2) Preparation of spleen cell suspension: positive BALB/c mice were fixed on a tray, their left abdomen was wiped with an alcohol cotton ball, and the spleen was removed with taking care of aseptic handling during; meanwhile, the mouse serum is reserved as a mouse polyclonal antibody (PAb); the spleen was washed with DMEM basal medium, the other tissues adhered to the spleen were removed, and then transferred to a dish containing 20ml ldmem basal medium, the spleen was punctured several tens of times with a syringe, and the spleen was ground with a syringe core on a 40 μm sieve, and the spleen cell suspension was transferred to a centrifuge tube.

(3) Cell fusion: taking SP2/0 myeloma cells with good growth state, discarding the culture medium, washing with PBS buffer solution for 3 times, adding 20mL DMEM basal medium, blowing down the cells, and transferring into a centrifuge tube containing spleen cells; horizontally centrifuging for 10min at 1000 r/min; discarding the supernatant, adding 20mL of DMEM basal medium to resuspend the cells, and horizontally centrifuging at 800r/min for 8 min; discarding the supernatant, gently mixing the cells by using a pipettor, putting the centrifuge tube into a blue-cap bottle filled with warm water of 37 ℃ to ensure the fusion efficiency, slowly adding 1mL of PEG within 1min, and standing for 1 min; then terminating the fusion, and sequentially adding 1mL, 2mL, 3mL and 4mL of DMEM basal medium within 4 min; the fused cells were plated on top of feeder cells prepared the day before.

2.3 screening of Positive hybridoma cell lines

And (3) carrying out subcloning on the cells of the fusion hole for 3 times, screening by an indirect ELISA method to obtain a positive cell strain, and freezing and storing the cells after amplification culture. Subcloning and cell cryopreservation steps are as follows:

(1) subcloning of the fused cells: performing primary subcloning, coating an enzyme-linked immunosorbent assay (ELISA) plate with 5 mu g/mL of purified DEV and DEV gB proteins, detecting a cell culture supernatant with fused cell holes by indirect ELISA, operating at 2.2.11.1, selecting a monoclonal of cells with positive holes, adjusting the cell concentration to 10/mL by cell counting, and subpackaging the monoclonal antibody into a 96-hole cell culture plate with a pre-laid feeder layer; performing secondary subcloning, detecting cell culture supernatant of each hole, selecting a monoclonal of positive hole cells, adjusting the cell concentration to 1 per hole, and subpackaging the cells into a 96-hole cell culture plate with a pre-laid feeding layer; and thirdly, subcloning, detecting the culture supernatant of each hole cell, picking out a single clone of the positive reaction hole cell, and expanding the culture for further identification.

(2) Freezing and storing hybridoma cell strains: performing cryopreservation on positive reaction cells obtained by each subcloning screening after the expanded culture; and during freezing, discarding the old solution, washing for 3 times, slightly blowing down the cells by using 5mL of DMEM basic culture medium, centrifuging for 8min at the speed of 800r/min, discarding the culture medium, adding 1mL of cell freezing solution for resuspending the cells, transferring the cell suspension into a freezing tube, putting the freezing tube into a freezing box, freezing and storing the cells in a super-low temperature refrigerator overnight, and putting the freezing tube into liquid nitrogen, so that the cells can be preserved for a long time.

2.4 subclass identification of MAb

The selected MAb subclass was determined according to the sbaclonypingsystem-HRP antibody subclass identification kit instructions.

2.5 IFA identification of MAb

IFA was performed by using MAb as primary antibody and goat anti-mouse IgG-FITC as secondary antibody, and setting 3 controls, i.e., non-toxic cell control, and non-primary antibody blank control and primary antibody as non-immune mouse negative serum control. The operation method is the same as above.

2.6 screening for MAbs with optimal blocking Rate

In order to screen an IgG subclass MAb which reacts with a native spatial conformation virus protein and has a strong blocking ability against DEV antibody from a plurality of MAbs, the MAb identified as positive in IFA and as IgG subclass in 2.4 was further screened, 6 portions of DEV negative and positive serum each stored in a laboratory were taken from an ultra-low temperature refrigerator, competition blocking ability against each serum antibody by MAb was determined by blocking ELISA, and an appropriate MAb was selected based on the values of positive serum inhibition rate (PI)/negative serum inhibition rate (PI). Wherein the positive serum PI value (PBS OD)_450nmvalue-Positive OD_450nmvalue)/PBS OD_450nmValue, negative serum PI value ═ PBSOD_450nmValue-negative OD_450nmvalue)/PBS OD_450nmThe value is obtained.

Respectively coating the ELISA plate with two antigens of purified DEV and gB proteins, performing blocking ELISA, and determining the blocking capability, coating concentration and method of each MAb, wherein the blocking method is the same as the indirect ELISA; after sealing, adding negative and positive serum, incubating at 37 ℃ for 1h, setting PBS control, washing the plate for 3 times, wherein each hole is 100 mu L; adding MAb, incubating at 37 deg.C for 1h, 100 μ L per well, and washing the plate for 3 times; adding 1:10000 diluted goat anti-mouse-HRP (horse radish peroxidase), 100 mu L of each hole, incubating for 0.75h at 37 ℃, and washing the plate for 3 times; the color development, termination and reading methods were the same as the indirect ELISA.

2.7 Western Blot identification of MAb1E8

Inoculating DEV into DEFs, collecting supernatant and bottom cells when 90% of cells are observed to be diseased under a microscope, centrifuging at a differential speed to remove precipitates, and performing ultracentrifugation at 4 ℃ for 2h at 32000 r/min; transferring the super-separation purified DEV to a PVDF membrane through SDS-PAGE; the result observation is carried out by scanning Western Blot with an infrared fluorescence imaging system by using the murine PAb and the selected MAb as primary antibodies (1:1000) and IRDye680LT goat anti-mouse antibodies as secondary antibodies (1: 10000). To identify murine PAb and reactivity of the MAb with DEV. Simultaneously, transferring the purified gB protein to a PVDF membrane through SDS-PAGE; western Blot was performed using the MAb as the primary antibody, in the same manner as above, to identify the reactivity of the MAb with the prokaryotically expressed gB protein.

2.8 specificity identification of MAb1E8

Diluting AIV (H5, H7 and H9), NDV, EDSV, DTMUV, DHV-I and DEFs by 10 times respectively to serve as antigen-coated enzyme labeling plates, and setting DEV positive control; indirect ELISA was performed with selected MAb as primary antibody. The operation method is the same as above.

2.9 preparation of ascites and purification of MAb1E8

10 weeks old mice were injected intraperitoneally with autoclaved paraffin oil at 500. mu.L/mouse, and after one week, hybridoma cells selected in 3.2.6 and SP2/0 myeloma cells were injected in the same manner, 10⁶Ascites can be collected after one week; antibodies in mouse hybridoma ascites were affinity purified using Protein G. The specific operation steps are as follows:

(1) centrifuging ascites at 10000r/min for 10min, and removing impurities; the column was assembled and equilibrated with 5ml PBS. And (2) closing the flow control valve, mixing 400 mu L of ascites with PBS (equal amount), adding the mixed solution into a column, and standing for 5min to ensure that the antibody in the ascites is fully adsorbed. (3) The flow control valve was opened and 4mL PBS was added for washing, eluting unbound hetero-proteins and loosely bound antibodies. (4) Glycine (0.1M, pH 3.0) was added for elution, and in order to keep the antibody structure stable, the liquid that flowed down was collected in a collection tube containing Tris-HCl (1M, pH 8.8), and neutralized with 0.1mL of Tris-HCl for every 1mL of antibody that was eluted.

(5) And (5) carrying out SDS-PAGE electrophoresis to identify the purity of the sample.

After purification, the Protein G column was washed with 10 column volumes of PBS followed by 20% ethanol, and finally the column was stored at 4 ℃ with 5 column volumes of 20% ethanol.

2.10 ELISA identification of ascites titer by MAb1E8

Coating a gB protein with the concentration of 5 mu g/mL on an enzyme label plate, and treating MAb1E8 ascites according to the ratio of 10-10⁸10-fold dilution was performed in sequence, and the antibody titer of ascites was indirectly determined using SP2/0 ascites as a negative control. The operation method is the same as above.

2.11 epitope identification of MAb1E8

The gB protein was truncated in 3 rounds according to the epitope identification truncation expression scheme (FIG. 5), and the designed primers were artificially synthesized after 6 inducible expression (Table 4).

TABLE 4 PCR primer sequences

Constructing recombinant plasmid of 6-segment truncated protein and prokaryotic expression. The PCR amplification system is the same as above, and the respective PCR amplification procedures are as follows:

the first round of amplification procedure was 95 ℃ for 5 min; 30 cycles at 95 ℃ for 30s, 65 ℃ for 30s and 72 ℃ for 1 min; 10min at 72 ℃. The second round of amplification procedure was 95 ℃ for 5 min; 30 cycles at 95 ℃ for 30s, 65 ℃ for 30s, 72 ℃ for 40 s; 10min at 72 ℃. The third round of amplification procedure was 95 ℃ for 5 min; 30s at 95 ℃, 30s at 65 ℃, 30s at 72 ℃ and 30 cycles; 10min at 72 ℃.

A pair of truncated proteins expressed in each round were subjected to SDS-PAGE simultaneously, transferred to a PVDF membrane, and Western Blot was performed using MAb1E8 as a primary antibody and goat anti-mouse-HRP as a secondary antibody. And (3) artificially synthesizing the preliminarily identified epitope polypeptide according to the result, and identifying the reactivity of the monoclonal antibody and the polypeptide by indirect ELISA. The concentration of the synthesized polypeptide is 10mg/mL, the polypeptide is diluted to 5 mug/mL by using a coating solution, and the polypeptide is coated overnight at 4 ℃; using MAb1E8 as a primary antibody and goat anti-mouse-HRP as a secondary antibody to perform indirect ELISA; blocking and antibody incubation methods are the same as above. The gB protein coated the enzyme standard plate, MAb1E8 was primary antibody, used as a positive control, and set negative and blank controls.

3 results of the test

3.1 screening results of hybridoma cells

Screening 4 hybridoma cell lines capable of stably secreting anti-DEVMAb by using an established indirect ELISA method for 4 times to obtain 4 hybridoma cell lines which are respectively named as 2E3, 7E12, 9G3 and 10A9, and obtaining 7 hybridoma cell lines capable of stably secreting anti-gB protein MAb, namely 1A4, 3C2, 4H3, 3E2, 2B1, 1E8 and 2G 3.

3.2 identification of MAb subclasses

The subclass of MAb was determined by using antibody subclass identification kit, 2E3 in 4 DEVMAb of 11MAb was IgM subclass, 7E12 and 9G3 were IgG1 subclass, and 10A9 was IgG2b subclass; the heavy chains of 7gB protein mabs were all subclassed IgG2b (fig. 6).

3.3 IFA analysis of reactivity of MAb with DEV

After DEFs infected DEV, after cells begin to become diseased, IFA is carried out, and the cells are observed under an inverted fluorescence microscope, 3 MAbs react with DEV and respectively comprise DEV MAb 2E3, 7E12 and gB protein MAb1E8, the inoculated cells emit strong green fluorescence and concentrate on cytoplasm and cell membranes, and specific green fluorescence is not seen in a control group (figure 7).

3.4 screening MAb for establishment of blocking ELISA method

Combining the test results in 3.3 and 3.4, DEV MAb 7E12 and gB protein MAb1E8 are of IgG subclass and react with natural viral proteins, and the results of the identification of blocking effect on the above two MAbs show that MAb1E8 has stronger blocking ability than 7E12 (Table 5), so MAb1E8 can be used in subsequent tests.

TABLE 5 comparison of blocking effects of two monoclonal antibodies on 6 DEV-positive serum samples

3.5 WesternBlot analysis of MAb1E8

DEFs infected DEV were harvested and superpurified DEV was transferred to PVDF membrane for Western Blot using murine PAb and MAb1E8 as primary antibodies, respectively. At the same time, transfer purified gB protein, MAb as primary antibody, and test. The results showed that PAb binds to denatured DEV proteins at the 30kDa, 47kDa and 120kDa bands, MAb binds to denatured DEV proteins at the 30kDa band and binds to prokaryotically expressed 51kDa size proteins (FIG. 8).

3.6 identification of specificity of MAb1E8

AIV (H5, H7 and H9), NDV, EDSV, DTMUV, DHV-I and DEFs coated ELISA plates were subjected to indirect ELISA, and P/N was not less than 2.1 as a positive judgment standard. The results showed that the reaction of MAb with other viruses and DEFs was negative under the conditions that the positive control was established (fig. 9), indicating MAb specificity.

3.7 SDS-PAGE identification of ascites purification by MAb1E8

Affinity purifying MAb with Protein G, and measuring the concentration of MAb by spectrophotometry to be about 1.6 mg/mL; SDS-PAGE identified the purified MAb heavy chain at 55kDa and the light chain at about 25 kDa.

3.8 ELISA determination of antibody titer in ascites

gB protein is used to coat enzyme label plate, SP2/0 ascites is used as negative control, indirect method is used to determine the antibody titer of MAb1E8 ascites, when the ascites is diluted 10⁴Time of doubling OD_450nmThe values are all larger than 1.0 (figure 10), the P/N is larger than or equal to 2.1 and is taken as a positive judgment standard, and the result shows that the antibody titer is 10⁶(FIG. 10).

3.9 epitope identification of MAb1E8

PCR amplification, constructing a prokaryotic expression system for expressing the truncated protein, simultaneously performing SDS-PAGE on a pair of the truncated proteins expressed in each round, transferring, and performing Western Blot by using MAb1E8 as a primary antibody and goat anti-mouse-HRP as a secondary antibody. Through PCR, SDS-PAGE and Western Blot analysis, the size of the gB-101 amplified fragment expressed by the first truncation is 600bp, the size of the protein is 38kDa, the size of the gB-102 amplified fragment is 441bp, the size of the protein is 28kDa, and the Western Blot result shows that the MAb reacts with the second segment of protein gB-102 (FIG. 11); the second time of truncation expression gB-201 amplified fragment size is 192bp, protein size is 16kDa, gB-202 amplified fragment size is 288bp, protein size is 25kDa, Western Blot result shows that MAb reacts with the second section of protein gB-202 (figure 12); the size of the gB-301 amplified fragment expressed by the third truncation is 171bp, the protein size is 15.5kDa, the size of the gB-302 amplified fragment is 153bp, the protein size is 15kDa, and Western Blot results show that the MAb reacts with both the two proteins (FIG. 13).

Indirect ELISA identification of mabs and synthetic overlapping polypeptides showed that they reacted with the polypeptides (fig. 14).Therefore, the epitope to which the MAb is directed should be the amino acid sequence of the polypeptide overlapped by the two proteins in the third truncated expression, i.e. the sequence⁵⁷⁹RMLGDVLAVSSC⁵⁹⁰. The analyzed epitope region of MAb1E8 is located in UL27 full-length protein 579-590 aa.

Test example 1 establishment and application of blocking ELISA method for detecting DEV antibody

1 Material

1.1 sample sources

H5, H7 and H9 subtype Avian Influenza Virus (AIV) HI test antiserum is purchased from Harbin Vitaceae biotech development company, and positive sera of Newcastle Disease Virus (NDV), Egg Drop Syndrome Virus (EDSV), duck Tembusu virus (DTMUV), Gosling Plague Virus (GPV) and duck hepatitis virus type I (DHV I) are stored in the laboratory. DEV positive serum reference (Duck origin, neutralization potency 1: 100) was purchased from Chinese veterinary medicine institute; DEV negative serum reference (duck source, negative neutralization test) was stored in the laboratory; 130 positive serum samples and 364 negative serum samples were derived as above.

565 parts of duck-origin clinical serum sample is collected from a Shandong Weifang, 318 parts of Duck-origin clinical serum sample is collected from a Shandong Nezeo and 27 parts of Duck-origin clinical serum sample is collected from a Jilin pine source; 27 goose-derived clinical serum samples were collected from Jilin pine.

1.2 Main reagents and kits

Goat anti-mouse IgG-HRP and TMB developing solutions were purchased from sigma; tween20 was purchased from BioSharp corporation; PBS buffer (dry powder) was purchased from beijing lygbuck technologies ltd; a duck plague virus ELISA antibody detection kit (TSZ brand) produced in China is purchased from Shanghai Pongjing biological reagent Co. The formulation of the important agents is as follows:

(1) PBS buffer: according to the instruction, 50 XPBS buffer solution (dry powder) is dissolved by deionized water, the volume is adjusted to 1000mL, and the solution can be stored at room temperature after autoclaving.

(2) Coating solution (carbonate buffer with pH 9.6 at 0.05 mol/L): diluting anhydrous sodium carbonate 1.50g and sodium bicarbonate 2.98g with deionized water, diluting to 1000mL, autoclaving, and storing at room temperature.

(3) Sealing liquid: 50g of skim milk is added into the PBS buffer solution to be fully dissolved, and the volume is determined to be 1000mL, so that the composition is prepared for use.

(4) Washing liquid: 500mL of Tween20 is added into the PBS buffer solution, the volume is adjusted to 1000mL, and the Tween is prepared for use.

(5) Diluting liquid: 10g of skim milk is added into the PBS buffer solution to be fully dissolved, and the volume is determined to be 1000mL, so that the composition is prepared for use.

(6) Stopping liquid: 100mL of concentrated sulfuric acid was slowly added to 80mL of deionized water to 100 mL.

2 test method

2.1 determination of working concentration

Taking a DEV negative serum reference substance as a negative control, diluting the DEV positive serum reference substance by 2 times from 1.25-10 mu g/mL, diluting the antigen coating concentration by 2 times from 20-160 mu g/mL, blocking ELISA, selecting a negative value of 0.8-1.2 by a square matrix titration method, comprehensively lowering the concentrations of the coating antigen and the positive serum reference substance, and taking the conditions corresponding to the holes with larger inhibition ratio (PI) values as the working concentrations of the coating antigen and the positive serum reference substance; and then fixing the concentrations of the coated antigen and the positive serum reference substance, carrying out subsequent tests according to a conventional method, and selecting the working concentration corresponding to the hole with PI being more than 40% and MAb1E8 being low.

2.2 determination of gB protein coating conditions

Coating the plate with 0.5 μ g/mL protein, setting 4 different coating conditions, 37 deg.C, 1h, 37 deg.C, 2h, 4 deg.C, 12 h. And performing subsequent tests according to a conventional method, and selecting the condition with large PI value and less time consumption as the optimal coating condition.

2.3 determination of the type of blocking fluid

After coating gB protein in optimal conditions, four sealant fluid categories were set: 5% BSA, 5% skimmilk (skim milk), 0.5% gelatin (gelatin), 5% FBS. At 37 ℃, each confining liquid was confined for 2h, and confining liquids were added longitudinally, 4 in each group being parallel. And performing subsequent tests according to a conventional method, integrating the PI value and the coating solution price factors, and selecting the sealing solution which is economical and practical and has better effect.

2.4 determining incubation conditions of the test sample

3 different incubation conditions were set, 37 ℃, 0.5h, 37 ℃, 1h, room temperature, 2 h. And performing subsequent tests according to a conventional method, and selecting the condition corresponding to the larger PI value as the incubation condition of the sample to be detected.

2.5 determination of MAb incubation time

And incubating the MAb1E8 with the working concentration for 0.5h, 1h, 1.5h and 2h respectively at 37 ℃. And performing subsequent tests according to a conventional method, and selecting the time with large PI value and less time consumption as the optimal incubation time of the MAb.

2.6 determination of goat anti-mouse IgG-HRP incubation time

And incubating goat anti-mouse IgG-HRP antibody with the working concentration for 30min, 45min and 1h respectively at 37 ℃. And performing subsequent experiments by a conventional method, and selecting the condition with large PI value and less time consumption as the optimal incubation time.

2.7 determination of the color development time

Setting 4 color development times at 37 ℃ for 5min, 10min, 15min and 20min in sequence. And (4) carrying out subsequent tests according to a conventional method, and selecting the condition with large PI value and less time consumption as the optimal color development time.

2.8 determining the criteria

Taking out the negative and positive serum samples determined by the neutralization test from the ultralow temperature refrigerator, and determining the OD of 364 parts of negative duck serum samples and 130 parts of positive duck serum samples_450nmValues were negative control with DEV negative serum reference. PI value (negative control OD) according to formula_450nmValue-measured serum OD_450nmValue /) negative control OD_450nmValue calculation, GraphPad Prism6 is used as a scatter plot, IBM SPSS Statistics software is used for drawing an ROC curve, and PI values corresponding to points with proper sensitivity and specificity are selected as critical values.

2.9 specificity test

The enzyme label plate is coated by a determined antigen concentration, the standard serum of influenza virus (AIV), Newcastle Disease Virus (NDV), Egg Drop Syndrome Virus (EDSV), tembusu virus (DTMUV), Gosling Plague Virus (GPV) and duck hepatitis I virus (DHV I) is synchronously detected, the DEV positive serum reference substance with the optimal concentration is used as a positive control, and the DEV negative serum reference substance is used as a negative control. Incubating standard serum and negative and positive control serum under the optimal condition, manually washing the plate, adding MAb for incubation, fully washing the plate, incubating goat anti-mouse-HRP, and measuring the absorbance of each hole to judge the specificity of the detection method for the DEV antibody.

2.10 sensitivity test

Coating the ELISA plate with antigen with determined concentration, and mixing DEV positive serum reference substances according to the ratio of 1: 10-1: 1280 sequentially diluting by 2 times, incubating diluted DEV positive serum under the optimal condition, washing plate, adding MAb, incubating, washing plate sufficiently, incubating enzyme-labeled secondary antibody, setting DEV negative serum reference as negative control, calculating PI value of positive serum under each dilution, determining the maximum dilution capable of detecting positive serum, and verifying the sensitivity of the method.

2.11 repeatability test

2.11.1 Intra-batch repeat

Taking out 4 coated plates in the same batch, taking 7 positive samples stored in a laboratory, coating the ELISA plates with a determined antigen concentration, incubating the positive serum samples under the optimal condition, and setting negative control on each plate; adding MAb for incubation after manually washing the plate, incubating the enzyme-labeled secondary antibody after fully washing the plate, measuring the PI value of the same sample in the same batch, and calculating the coefficient of variation.

2.11.2 batch to batch repetition

Taking out 4 plates coated in different batches, taking 7 positive samples stored in a laboratory, coating the ELISA plates with a determined antigen concentration, incubating the positive and negative serum samples under an optimal condition, and setting negative contrast for each plate; adding MAb for incubation after manually washing the plate, incubating enzyme-labeled secondary antibody after fully washing the plate, measuring PI values of the same sample in different batches, and calculating the coefficient of variation.

2.12 compliance test

41 DEV positive serum samples and 53 DEV negative serum samples determined by a neutralization test are randomly extracted from negative and positive serum samples stored in a laboratory, the serum samples are taken out from an ultra-low temperature refrigerator and detected according to an indirect ELISA method described in a commercial kit specification, and simultaneously, a blocking ELISA method established by the research is used for testing on a 96-hole ELISA plate and analyzing the coincidence rate of the results of the two.

2.13 clinical sample testing

Sampling is carried out on the Shandong Weifang and the farms of the Weize and Jilinsong respectively, and 937 clinical serum samples are collected. The Weifang is used for collecting 565 parts of the breeding duck serum, 318 parts of the commercial duck serum, 27 parts of the commercial duck serum and 27 parts of the commercial goose serum. Coating an enzyme label plate with a determined antigen concentration, incubating clinical serum samples under an optimal condition, adding MAb for incubation after washing the plate, incubating enzyme-labeled secondary antibody after fully washing the plate, measuring the absorbance of each hole, taking a DEV negative serum reference substance as a negative control, and calculating and counting the antibody positive rate of each batch of samples.

3 results of the test

3.1 determination of optimum working concentration

And (3) establishing a blocking ELISA method by taking gB protein as a coating antigen and MAb1E8 as a competitive antibody through preliminary combination. The PI value is larger at 0.5 mu g/mL of antigen, and the OD of negative control_450nmThe value is in the range of 0.8-1.2, the sensitivity of the experiment is higher, so the optimal coating amount of the antigen is determined to be 0.5 mug/mL, and the working concentration of the DEV positive serum reference is 1:40 (FIG. 15 and Table 6); fixing the concentration of the envelope antigen and the positive serum, calculating the PI value of the positive serum detected by the MAb1E8 under different dilution times, wherein the PI value is reduced along with the increase of the dilution times of the MAb1E8, when the dilution times are 2000 times, the PI value is larger, the PI value is almost the same as that when the dilution times are 1000 times, and compared with the dilution times of 1000 times, the antibody is saved, and on the basis, the working concentration of the MAb1E8 is determined to be 1: 2000 (fig. 15).

TABLE 6 working concentration optimization of coated gB protein and DEV Standard Positive antisera

3.2 optimization of the coating conditions for gB protein

From 4 different coating times and temperatures, the optimal coating condition was selected as the one with a larger PI value and less time consumption, and the gB protein coating condition was determined to be 4 ℃ incubation for 12h (FIG. 16).

3.3 selection of the type of blocking solution

The blocking effect of 4 different blocking solutions is tested, the material with the best effect is selected, and the result shows that the blocking effect of 5% skimmik is better, and the material cost is more economical and practical, so that 5% skimmik is selected as the blocking solution for blocking ELISA.

3.4 optimization of incubation conditions for test samples

Setting 3 different conditions, selecting the condition with the best effect as the incubation condition of the detected sample, and determining that the incubation condition of the detected sample is room temperature incubation for 2h because the result shows that the PI value of the detected sample is larger under the room temperature (25 ℃) incubation condition for 2 h.

3.5 optimization of MAb incubation time

Setting 4 different times, and selecting the MAb incubation time with the best effect from the different times, wherein the result shows that the MAb is incubated for 1h at 37 ℃, the PI value is larger, and therefore, the MAb incubation time is determined to be 1 h.

3.6 optimization of goat anti-mouse IgG-HRP incubation time

3 different times are set, the goat anti-mouse IgG-HRP incubation time with the best effect is selected, and the result shows that the goat anti-mouse IgG-HRP antibody is incubated for 45min at 37 ℃, the PI value is larger, and therefore, the incubation time is determined to be 45 min.

3.7 optimization of the development time

4 different times are set, the color development time with the best effect is selected, and the result shows that the color development effect hardly changes along with the time after the color development is carried out for 15min at 37 ℃, so that the color development time is determined to be 15 min.

In summary, by determining the optimal working concentration and optimizing the reaction conditions, the optimal operation flow is established as follows:

(1) diluting the purified gB protein with coating solution to 0.5. mu.g/mL, 100. mu.L per well, and coating at 4 ℃ for 12 h; washing the ELISA plate with the washing solution for 3 times, and patting to dry.

(2) Add 300. mu.L of 5% ski mil to each well, block for 2h at 37 ℃ and wash the plate 3 times.

(3) Adding the sample to be tested, centrifuging at low speed to remove impurities, reacting at room temperature for 2h, setting negative and positive serum contrast (1: 40), washing plate for 3 times, and washing 100 μ L per well.

(4) MAb (1: 2000) was added and the plates were washed 3 times at 100. mu.L per well for 1h at 37 ℃.

(5) Goat anti-mouse-HRP (1:10000) was added to 100. mu.L/well and the plate was washed 3 times at 37 ℃ for 0.75 h.

(6) Adding TMB (100 μ L per well), and developing in dark for 15 min; terminating the reaction, wherein each well contains 50 mu L of termination solution; read plate within 15 min.

3.8 establishment of the criteria

364 negative duck serum samples and 130 positive duck serum samples were determined by established methods, with one SPF duck serum as a negative control. SPSS plots the ROC curve of the PI value statistics for positive and negative samples, and it can be seen that the sensitivity and specificity of the method are high at a cut-off value of 30% (Table 7). Therefore, the critical value of the method is 30 percent, namely the PI of the sample to be detected is more than or equal to 30 percent and is judged to be positive, otherwise, the PI is judged to be negative.

TABLE 7 statistics of negative samples PI

Further plotting the scattered point distribution chart of the PI value statistical result of 494 negative and positive serum samples (FIG. 17), the result shows that the PI of most negative samples is distributed below 30%, and the PI of positive samples is distributed above 30%. The main purpose of the test is to establish a detection method for eliminating antibody positive individuals, and as can be seen from the results, a higher negative rate on the population level can be ensured by taking 30% as a critical value.

3.9 results of specificity test

The standard sera against AIV, NDV, EDSV, DTMUV, GPV and DHV-1 were synchronously detected by the established ELISA method, the DEV-positive serum reference was used as a positive control, and one SPF duck serum was used as a negative control, showing that the method is specific for DEV antibodies (fig. 18).

3.10 results of sensitivity test

The sensitivity of the method was tested by testing a DEV positive serum reference in an established ELISA method by diluting the positive serum 10-fold and 1280-fold maximum. Calculating the PI value of the positive serum under different dilution times, wherein the PI value decreases along with the increase of the dilution times of the serum, the detection result is positive when the serum is diluted by 80 times, the detection result is negative when the serum is diluted by 160 times, and the method can detect that the maximum dilution times of the serum are 1: 80 (fig. 19).

3.11 repeatability test results

Taking 4 plates of the same batch and 3 plates of different batches respectively, detecting 7 positive serum samples by the established method, determining the PI values of the same sample in the same batch and different batches, and calculating results show that the variation coefficients between batches and within batches are less than 10%, wherein the maximum difference between batches is 6.88%, and the maximum difference between batches is 8.62% (tables 8 and 9).

TABLE 8 results of in-batch variation detection

TABLE 9 results of inter-batch Difference detection

3.12 compliance rate test results

The detection method adopted by the commercial kit is indirect ELISA, and the kit and the blocking ELISA method established in the research are used for detecting 41 positive samples and 53 negative samples. The detection results of 94 samples show that the total coincidence rate is 84.04%, the positive coincidence rate is 82.5% and the negative coincidence rate is 85.19%.

3.13 clinical sample test results

4 samples were collected at 4 sampling sites, and 937 clinical samples. The results showed that the Shandong breed ducks had a positive rate of 23%, the Shandong commodity ducks had a positive rate of 1.25%, and no antibody positive individuals were detected from the Songyuan commodity ducks and geese (Table 10). From the detection result, the total positive rate of the sample antibody is not more than 20%, and the positive rate of the antibody of the breed duck is obviously higher than that of the commercial duck.

TABLE 10 test results of clinical samples

SEQUENCE LISTING

<110> Harbin veterinary institute of Chinese academy of agricultural sciences (Harbin center of Chinese center of animal health and epidemiology)

<120> DEV gB protein monoclonal antibody and blocking ELISA kit for detecting DEV antibody

<130>HLJ-2001-190409A

<160>1

<170>PatentIn version 3.5

<210>1

<211>12

<212>PRT

<213>Duckenteritis virus

<400>1

Arg Met Leu Gly Asp Val Leu Ala Val Ser Ser Cys

1 5 10

Claims (10)

1. A hybridoma cell strain secreting monoclonal antibody of duck viral enteritis virus gB protein is characterized in that the microorganism preservation number is CGMCC No. 17996.

2. A monoclonal antibody secreted by the hybridoma cell line of claim 1.

3. The use of the hybridoma cell strain of claim 1 in the preparation of a reagent or a medicament for diagnosing or preventing duck viral enteritis virus.

4. The use of the monoclonal antibody of claim 2 in the preparation of a reagent or a medicament for diagnosing or preventing duck viral enteritis virus.

5. The epitope recognized by the monoclonal antibody of claim 2, wherein the amino acid sequence of the epitope is represented by SEQ ID No. 1.

6. Use of the epitope of claim 5 in the preparation of a reagent or a medicament for diagnosing or preventing duck viral enteritis virus.

7. A blocking ELISA detection kit for detecting DEV antibodies comprising: DEV gB protein, coating solution, primary antibody, enzyme-labeled secondary antibody, buffer solution, diluent, washing solution, confining solution, stop solution, positive serum control and negative serum control; wherein said primary antibody is the monoclonal antibody of claim 2.

8. The blocking ELISA detection kit of claim 7, wherein the enzyme-labeled secondary antibody is HRP-labeled goat anti-mouse IgG.

9. The blocking ELISA detection kit of claim 7, wherein the buffer is PBS buffer.

10. The blocking ELISA test kit according to claim 7, wherein the coating solution is 0.05mol/L carbonate buffer at pH 9.6.

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