CN110791479B - 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. Firstly, preparing gB protein with good reactivity and immunogenicity through a prokaryotic expression system, screening the gB protein serving as an antigen through a cell fusion technology to obtain a hybridoma cell strain with a microorganism preservation number of CGMCC No.17996, wherein the secreted monoclonal antibody can react with natural space conformational virus protein of duck viral enteritis virus and has strong blocking capability on DEV antibodies; the invention also provides an antigen epitope recognized by the monoclonal antibody, and the amino acid sequence of the antigen epitope is shown as SEQ ID No. 1; the invention further constructs a blocking ELISA kit for detecting DEV antibody by using the monoclonal 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 virus enteritis viruses.

Background

Duck Plague (DP), a common name of Duck viral enteritis (Duck virus enteritis, DVE), is an acute, febrile, and septic infectious disease of various anseriformes birds such as ducks, geese, and geese, caused by Duck viral enteritis virus (Duckenteritis virus, DEV). The viral genome encodes more than ten glycoproteins, wherein 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 viral disease of duck animals, seriously harms domestic ducks, and also affects various waterfowl such as wild ducks. In 1967, the virus was first identified by inoculating duck embryo and young duck, isolating the virus, culturing with primary fibroblasts of duck embryo, and performing 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 vaccine immune effect and infecting animals. To date, blocking ELISA kits for detecting DEV antibodies have been lacking with commercial application value.

Disclosure of Invention

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

The second object of the present invention is to provide an epitope recognized by a DEV gB protein mab;

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 a DEV natural space conformation virus protein and has strong blocking capability on a DEV antibody:

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

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

the purified DEV and DEV gB proteins are respectively mixed with Freund's adjuvant and emulsified, and 6-week-old BALB/c mice are immunized with 50 mug/mouse for 3 times at two weeks. After 5 days, the mice with high titers were boosted with non-emulsified antigen, and the spleens of the mice were fused with SP2/0 cells in the logarithmic phase after 3 days. And 3 times of subcloning are carried out on the cells in the fusion holes, positive cell strains are obtained through screening by an indirect ELISA method, and the cells are frozen after expansion culture. 4 times of screening are carried out by established indirect ELISA method to obtain 4 hybridoma cell strains capable of stably secreting anti-DEVMAb, which are respectively named as 2E3, 7E12, 9G3 and 10A9, and 7 hybridoma cell strains capable of stably secreting anti-gB protein MAb, which are 1A4, 3C2, 4H3, 3E2, 2B1, 1E8 and 2G3, are obtained. Determining subclasses of the MAbs by adopting an antibody subclass identification kit, wherein 4 strains of DEVMAb,2E3, 7E12 and 9G3 are IgM subclasses, and 10A9 is IgG2b subclasses in 11 strains of MAbs; the heavy chains of the 7 strain gB protein MAbs are all of the subclasses IgG2 b. IFA detection is carried out on hybridoma cell lines capable of stably secreting anti-DEVMAb, and when the hybridoma cell lines are observed under an inverted fluorescence microscope, 3 MAbs react with DEV, namely DEV MAb 2E3, 7E12 and gB protein MAb 1E8, and toxic-receiving cells emit strong green fluorescence and concentrate on cytoplasm and cell membranes, and a control group does not see specific green fluorescence.

To screen for IgG subclasses of mabs from the plurality of mabs that react with the native space conformational viral proteins and have strong blocking ability against DEV antibodies, mabs identified as positive for IFA and identified as IgG subclasses are further screened for specific mabs by the build block ELISA method; the screening result shows that DEV MAb 7E12 and gB protein MAb1E8 are of IgG subclass and react with natural viral proteins, and the blocking effect identification result of the two MAbs shows that the blocking capability of the MAb1E8 is stronger than that of the MAb 7E12, so that the invention carries out subsequent identification test on the MAb1E 8.

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

The specificity identification result of the MAb1E8 shows that under the condition that the positive control is established, the reactions of the MAb1E8, other viruses and DEFs are all negative, which indicates that the MAb1E8 is specific; ELISA identification results of MAb1E8 ascites titers indicate that when ascites is diluted 10 ⁴ OD at times _450nm The values are all larger than 1.0, and the result shows that the antibody titer is 10 by taking P/N not less than 2.1 as a positive judgment standard ⁶ 。

The invention further carries out epitope identification on MAb1E 8. According to the epitope identification truncated expression schematic diagram, the gB protein is truncated for 3 rounds, and the total induced expression is carried out for 6 times; the results of PCR, SDS-PAGE and Western Blot analysis show that the amplified fragment size of the gB-101 expressed by the first truncated is 600bp, the protein size is 38kDa, the amplified fragment size of the gB-102 is 441bp, and the protein size is 28kDa,Western Blot, and the MAb reacts with the second-stage protein gB-102; the result of the second truncated expression gB-201 amplified fragment size is 192bp, the protein size is 16kDa, the gB-202 amplified fragment size is 288bp, and the protein size is 25kDa,Western Blot shows that the MAb reacts with the second-stage protein gB-202; the result of the third truncated expression of gB-301 amplified fragment size of 171bp, protein size of 15.5kDa, gB-302 amplified fragment size of 153bp and protein size of 15kDa,Western Blot shows that MAb reacts with both proteins; indirect ELISA identification of MAbs with synthetic overlapping polypeptides showed that they reacted with the polypeptides. Thus, the first and second substrates are bonded together, The antigen epitope aimed by the MAb is the amino acid sequence of the polypeptide overlapped by two sections of proteins in the third truncated expression, namely ⁵⁷⁹ RMLGDVLAVSSC ⁵⁹⁰ (SEQ ID No. 1). The MAb1E8 epitope region is analyzed to be located in the UL27 full-length protein 579-590 aa.

The hybridoma cell strain MAb1E8 is submitted to a patent approval institution for preservation, and the preservation number is CGMCC No.17996; the classification nomenclature is: a DEV gB protein monoclonal antibody cell line; the preservation time is 19 days of 6 months of 2019; the preservation unit is China general microbiological culture Collection center; the deposit address is: beijing, chaoyang area, north Chen Xili No.1, 3, china academy of sciences, microbiological institute.

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

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

the buffer is preferably a PBS buffer; the coating solution is preferably a carbonate buffer solution with the pH value of 0.05mol/L and 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 to a volume of 1000mL.

The preparation of the washing liquid is preferably as follows: to the PBS buffer was added 500mL Tween 20 to a volume of 1000mL.

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

The preparation of the termination liquid is preferably as follows: 100mL of concentrated sulfuric acid was slowly added to 80mL of deionized water and the volume was fixed to 100mL.

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

(1) Diluting and purifying the gB protein to 0.5 mu g/mL by using a coating solution, wherein each hole is 100 mu L, and coating is carried out for 12 hours at 4 ℃; washing the ELISA plate with the washing liquid for 3 times, and drying; (2) mu.L of 5% ski mill was added to each well, the wells were blocked at 37℃for 2 hours, and the plates were washed 3 times; (3) Centrifuging the sample to be tested with impurities removed, reacting for 2 hours at room temperature, setting negative and positive serum control (1:40), washing the plate 3 times per 100 mu L of each hole; (4) MAb (1:2000) was added and allowed to act at 37℃for 1h, 100. Mu.L per well, and the plate washed 3 times; (5) Goat anti-mouse-HRP (1:10000) was added, 100. Mu.L per well, and the plate was washed 3 times at 37℃for 0.75 h; (6) adding TMB, and developing for 15min in a dark place with 100 mu L of TMB in each hole; stop reaction, 50 μl of stop solution per well; the plate was read within 15 min.

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

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

DEV positive serum reference was tested by established ELISA, positive serum was diluted from 10 fold, with a maximum dilution of 1280 fold, and the sensitivity of the method was examined. Calculating PI value of positive serum under different dilution factors, wherein the PI value is reduced along with the increase of the dilution factor of the serum, the detection result is positive when the serum is diluted 80 times, the detection result is negative when the serum is diluted 160 times, and the maximum dilution factor of the serum can be detected by the method to be 1:80, it is demonstrated that the blocking ELISA detection kit constructed in the invention has high sensitivity.

Taking 4 plates of the same batch and 3 plates of different batches respectively, detecting 7 positive serum samples by the method provided by the invention, measuring PI values of the same sample in the same batch and different batches, and calculating the calculated results to show that the variation coefficients of the batch and the batch are smaller than 10%, wherein the maximum difference in the batch is 6.88% and the maximum difference in the batch is 8.62%; test results show that the blocking ELISA detection kit constructed by the invention has good repeatability.

41 positive samples and 53 negative samples were simultaneously detected using a commercial kit (indirect ELISA) and the blocking ELISA detection kit constructed according to the invention. The detection result of 94 samples shows that the total coincidence rate of the detection kit is 84.04 percent, the positive coincidence rate is 82.5 percent, and the negative coincidence rate is 85.19 percent.

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 breeding ducks is obviously higher than that of commercial ducks.

The results show that the blocking ELISA detection reagent for detecting DEV antibodies 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 protein expression SDS-PAGE analysis; m, protein Marker,1: whole bacteria before induction; 2: whole bacteria after induction; 3: crushing the supernatant; 4: and (5) settling after crushing.

The WesternBlot identification result of gB protein of fig. 3; m: protein markers; 1: duck source positive serum.

FIG. 4 IFA analysis of immunoreactivity of gB protein; a, immunizing gB protein rabbit serum; b: serum from non-immunized rabbits.

FIG. 5 epitope identification truncated expression schematic.

FIG. 6 subclass identification results for MAbs.

FIG. 7 IFA analysis of MAb reactivity with DEV (200X); a:2E3; b:7E12; c:1E8; D-F: negative control.

FIG. 8 WesternBlot analysis of MAb; m: protein markers; 1: PAb reacts with DEV; 2: reacting the MAb with DEV; 2: MAb reacts with gB protein.

FIG. 9 specificity test of MAbs.

FIG. 10 ELISA assay results for MAb1E8 titers.

FIG. 11 gB protein first truncated expression; m1: DL2000DNA markers; m2: protein markers; 1. 3, 5: gB-101; 2. 4, 6: gB-102.

FIG. 12 gB protein second truncated expression; m1: DL2000DNA markers; m2: protein markers; 1. 3, 5: gB-201; 2. 4, 6: gB-202.

FIG. 13 gB protein third truncation expresses M1: DL2000DNA markers; m2: protein markers; 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 are optimized.

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

FIG. 18 shows the results of a block ELISA specificity test.

FIG. 19 shows the results of a sensitivity test of blocking ELISA.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.

EXAMPLE 1 prokaryotic expression of DEV gB protein

1. Materials and methods

1.1 strains and laboratory animals

DEVCSC strain seed viruses are stored by the laboratory; SPF duck embryo is purchased from the experimental animal center of Harbin veterinary research institute of China academy of agricultural sciences; clean grade New Zealand white rabbits were purchased from Liaoning Changsheng biotechnology Co., ltd; DEV positive serum reference was purchased from China veterinary drug administration; pET-32a vector plasmid was maintained by the present laboratory.

1.2 test methods

Proliferation of 1.2.1DEV

Adopts a conventional formulaPreparation of Duck Embryo Fibroblasts (DEFs) from 11-day-old SPF duck embryo, culturing the prepared primary cells with DMEM containing 5% FBS, and placing the primary cells in 5% CO ₂ In the incubator at 37 ℃, after culturing for 1-2 d, the cells are transferred for 1 generation and then grow to 80% of the bottom of the bottle, and then the virus can be inoculated.

Inoculating virus, diluting the virus suspension with serum-free DMEM for 6 times, discarding cell culture supernatant, washing with PBS buffer solution for 3 times, sucking PBS buffer solution, adding appropriate amount of virus suspension until the bottom of cell culture bottle is completely covered, and placing in 5% CO ₂ Is adsorbed for 1 to 2 hours in a constant temperature incubator at 37 ℃. After adsorption, the virus suspension was discarded, DMEM with 2% fbs was added, and the culture was continued for 3d, during which cytopathic effect was observed daily, and a non-toxic control group was set. Harvesting virus, inoculating DEVCSC strain virus to DEFs after 1 generation transmission, freezing and thawing once when 90% of cells are observed to have lesions under a microscope, collecting supernatant, centrifuging at 800r/min for 8min, and removing cell debris; the cells were scraped off the bottom cells, frozen and thawed twice and centrifuged differentially to remove pellet and pooled in the supernatant.

1.2.2 DEV DNA extraction and PCR detection

And extracting DNA of DEVCSC strain virus and cell passage virus stored in a laboratory by using a virus DNA extraction kit. PCR detection was performed using primers in the national standard method, and sequencing was performed with the ligation T vector. Using DEV CSC seed virus and cell passage virus DNA as templates, and carrying out PCR detection on 50 mu L of a reaction system; the reaction procedure is: 94 ℃ for 5min;94 ℃ for 30s,55 ℃ for 30s,72 ℃ for 30s,25 cycles; and at 72℃for 10min.

Crude and pure 1.2.3DEV

After harvesting the virus, performing differential centrifugation at 4 ℃ for 15min each time at 4000r/min and 8000r/min, and mixing the supernatant obtained by the second centrifugation with the previous supernatant. 5mL of 30% (W/V) sucrose solution was prepared with PBS buffer, the supernatant was added to a centrifuge tube underlying the 30% (W/V) sucrose solution, centrifuged at 32000r/min at 4℃for 2h, and the pellet was collected and suspended with an appropriate amount of PBS buffer. The pellet collected after differential centrifugation was negatively stained and the virus particles were observed under electron microscopy.

1.2.4 purification of DEV by sucrose discontinuous Density gradient centrifugation

Preparing sucrose discontinuous density gradient solution, sequentially adding 30%, 40%, 50% and 60% (W/V) sucrose solution into a centrifuge tube from bottom to top, adding the collected virus suspension into the uppermost layer, centrifuging at 4deg.C for 2h at 32000r/min, collecting virus bands, diluting with 0.5mL PBS buffer, centrifuging at 28000r/min for 2h for desugaring, suspending the precipitate with 0.5mL PBS buffer, and storing in an ultralow temperature refrigerator. The concentration of the virus suspension purified by sucrose discontinuous density gradient centrifugation was determined by ultraviolet spectrophotometry and observed under electron microscope after negative staining.

1.2.5 determination of DEV TCID50

TCID50 of DEVCSC strain seed and cell passage virus were determined. Virus was subjected to 10 with DMEM basal medium ^-2 ～10 ^-9 Dilution with a multiple ratio; discarding the cell culture solution in the 96-well plate paved with DEFs in advance, and washing once with PBS; transferring diluted virus liquid into 1-8 rows in sequence, adding one column of each dilution virus liquid, repeating for 8 times, and 100 mu L of each hole; columns 8-12 were set as normal cell controls. Lesions (CPE) were recorded every 24h after virus inoculation, and the number of CPE holes and CPE-free holes observed each time were recorded separately for one week after continuous observation, and the TCID50 of the virus was calculated according to the Reed-Muench method.

1.2.6 construction of the recombinant expression System for the DEV gB protein

The full length gB protein encoded by the UL27 gene contains 932 aa according to the GenBank published full sequence of Duck enteritis virus UL pre (UL 27) gene (EF 554401.1). Through DNASTAR Lasergene software analysis, epitope dense regions 324-628 aa are selected for expression, and the protein has good hydrophilicity and higher antigen density and contains loose corner structures (figure 1). The primers were designed and synthesized (Table 1).

TABLE 1 PCR primer sequences

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

After the target fragment is recovered by using the gel recovery kit, the target fragment and the pET-32a carrier are cut by double enzyme, water bath is carried out at 37 ℃ for 6 hours, and the enzyme cutting system is 50 mu L.

Connecting the target fragment and the pET-32a carrier, and carrying out metal bath at 16 ℃ for overnight connection, wherein the connection system is 10 mu L; converting the connection product into a TransT1 competent cell, extracting the converted bacterial plasmid, and carrying out double enzyme digestion identification; transforming BL21 competent cells with the identified correct pET-32a-gB plasmid, adding 10 mu L of a connecting product into the competent cells, carrying out ice bath for 5min at 42 ℃, adding a culture medium, shaking, incubating for 1h, and coating an Amp solid LB plate; colonies were transferred to 5ml Amp liquid LB after growth, and bacterial liquid PCR was performed.

2.2.7 expression of recombinant gB proteins

And (3) inoculating the PCR identified correct bacterial liquid into Amp liquid LB according to the proportion of 1:50, proliferating for 1h in a bacterial incubator with the temperature of 37 ℃ and the speed of 220r/min until the OD600 = 0.4-0.6, adding 1mM/L of IPTG according to the proportion of 1:100, and inducing the expression for 4-5 h. Centrifuging the collected bacterial liquid at 2000r/min for half an hour, re-suspending the precipitate with PBS, then placing on ice for ultrasonic cleavage, centrifuging, collecting supernatant and precipitate, and preserving the above sample at-20deg.C for later use.

2.2.8 purification of recombinant gB proteins

And 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, 10000g was centrifuged for 10min, the pellet was left and resuspended with Binding buffer as the sample to be purified.

(2) And (3) column loading: the column and filter were rinsed with PBS buffer and 1mL of nickel chelating affinity resin was added.

(3) Balance column: after rinsing the column with PBS buffer, the column was equilibrated by addition of 4mL Binding buffer.

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

(5) Washing: the flow control valve was opened and 4mL of PBS buffer was added simultaneously to rinse out the unbound nickel column of the mixed protein.

(6) Eluting: the target protein was eluted with 100mM/L, 250mM/L, 500mM/L, 800mM/L, 1M/L of imizazole solution in this order.

(7) And (3) dialysis concentration: the eluted protein was dialyzed against PBS, concentrated by ultrafiltration, and purified by SDS-PAGE using 0.01mL of protein samples.

Renaturation of 2.2.9 recombinant gB protein

Renaturation of recombinant proteins according to the GENMED protein renaturation kit instructions.

2.2.10 Western Blot identification of recombinant gB proteins

After SDS-PAGE electrophoresis of the purified proteins, they were transferred to PVDF membrane using a transfer film apparatus. And (3) taking a duck-origin DEV positive serum reference as a primary antibody and goat anti-duck IgG-HRP as a secondary antibody, and performing test and DAB chemical color development.

IFA identification of 2.2.11 recombinant gB proteins

2.2.11.1 preparation of gB protein polyclonal antibody

Mixing the purified renatured gB protein with Freund's adjuvant, emulsifying, and immunizing New Zealand white rabbits by the subcutaneous multipoint injection at the back. The total immunization is carried out for 3 times, the immunization interval is two weeks, the dosage is 1 mg/dose, the ear margin vein blood collection is carried out 5 days after the 3 rd immunization, the serum titer is detected, the heart blood collection is carried out for 5 days, the serum is separated, and the non-lactating antigen is used for boosting the immunization before the serum is separated.

The indirect ELISA is used for detecting serum titers, and the specific operation steps are as follows:

(1) The purified gB protein was diluted with coating solution at 5. Mu.g/mL, 100. Mu.L per well, and after overnight at 4℃the ELISA plate was washed 3 times with PBST and then dried by pipetting.

(2) 300 μl of 5% skim milk was added to each well, and the plate was washed 3 times after blocking at 37deg.C for 2 h.

(3) 10-fold diluted immune rabbit serum was added, negative and blank controls were set, 100 μl per well incubated for 1h at 37 ℃, and the plates were washed 3 times.

(4) Adding 1:10000 diluted goat anti-rabbit-HRP, 100. Mu.L per well, incubated for 1h at 37℃and washed 3 times.

(5) Adding 100. Mu.L of a color development solution, developing for 10min, and stopping the reaction by using 50. Mu.L of a stopping solutionIn 15min, reading OD by enzyme label plate _450nm The value is that P/N is more than or equal to 2.1 as a positive judgment standard.

2.2.11.2IFA test

The specific operation steps are as follows:

(1) DEFs cell suspension is evenly paved in a six-hole cell culture plate, and the culture is carried out for 24 hours, and DEVs are inoculated when the cells are just full of monolayers. (2) When the cells begin to have lesions after the inoculation, a pipetting gun sucks the culture medium in the six-well plate, PBS is added for washing, the culture medium is discarded after being gently shaken for 5min by a horizontal shaking table, the process is repeated for 2 times, and the PBS is discarded for the last time. (3) Adding 4% paraformaldehyde, fixing at 4deg.C for 15min, and washing. (4) 1% BSA was added thereto, the mixture was blocked at 37℃for 30 minutes, and the mixture was washed 3 times. (5) 0.1% Triton X-100 was added, perforated at room temperature (20-30 ℃) for 15min, and the washing was performed as above. (6) The rabbit polyclonal serum diluted 100-fold with PBS was added, incubated at 37℃for 2h, and washed as above. (7) Goat anti-rabbit IgG-FITC diluted 10000 times with PBS was added, incubated at 37℃for 1h in the dark, washed 5 times, and finally without discarding PBS. (8) immediately placing under an inverted fluorescence microscope for microscopic examination. The experiment was set up with 3 controls, i.e. non-toxic cell controls, without primary antibody blank and primary antibody as non-immune rabbit seronegative controls.

2.3 test results

2.3.1 observations of infected cells

After DEFs infection of DEV, the pathological state was observed daily under a microscope. As a result, cells begin to be vacuolated at 24 hours, small vacuoles gradually increase and merge into larger vacuoles over time, and after the vacuoles are further expanded to occupy more than half of the area of the cells, the cells begin to become round and fall off; compared with the total cells, the total cell ratio of the number of the 72h exfoliated cells is more than 90%.

After the cells fall off, the cells disintegrate, the cell membranes are broken, viruses are released from the cells, the content of viruses in the supernatant is high, and the harvested viruses exist in the liquid at the moment, so that the next test such as centrifugation is facilitated.

Extracting genome of virus in seed virus and cell respectively, and carrying out PCR detection by using primers in national standard method; the rate of sequence identity to the reference sequence was 100%.

2.3.2 Electron microscope observations of purified virus

The virus particles are purified by adopting differential centrifugation and sucrose discontinuous density gradient centrifugation, 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 type of antibody secreting cells obtained as immunogens from the immunized animal will meet the requirements, and the later screening process can be simplified.

When the virus is observed under an electron microscope after further negative infection, the whole virus particles are spherical, the diameter is about 180nm, the virus particles consist of an inner nucleocapsid and an outer envelope, the shape is complete, and the sizes are relatively consistent; and under the field of view of an electron microscope, the impurities such as cell fragments around the virus particles purified by density gradient centrifugation are obviously less than the periphery of the virus particles collected after differential centrifugation.

2.3.3TCID50 measurement results

TCID50 of tissue and cell toxins was determined by cell titration, and CPE results of seed and passage cell toxins were counted (tables 2, 2). The TCID50 of DEVCSC seed toxin is calculated to be 10 according to the Reed-Muench method ^-5.14 0.1mL, TCID50 of 10 for cytotoxicity ^-6.61 0.1mL. The virus titer is enhanced after the virus is propagated on cells, about 10 times of the seed virus, which indicates that DEFs are suitable as parasitic hosts for DEVs and the virus propagated on tissues can be used for the next experiment.

TABLE 2 statistical results of DEV seed toxicity CPE

TABLE 3 statistical results of DEV cytotoxic CPE

2.3.4 PCR identification of recombinant plasmids

According to antigenicity analysis, selecting a part region of an UL27 gene, constructing a pET-32a-gB plasmid, transforming the plasmid into a TransT1 competent cell, and identifying the extracted plasmid by double enzyme digestion, wherein the result shows that the plasmid comprises two fragments with sizes close to 915bp and 5900bp after enzyme digestion, and the result shows that the target fragment is inserted into a vector in a correct mode.

BL21 competent cells are transformed by double enzyme digestion identification of the correct pET-32a-gB plasmid, colonies are picked up, bacterial liquid PCR amplification is carried out, and the result shows that 1566bp target fragments are amplified from 7 selected colonies.

2.3.5 SDS-PAGE identification of gB protein expression

The protein electrophoresis result shows that the expression quantity of the gB protein is obviously increased after IPTG induction compared with that before induction; bacterial pellet (inclusion bodies) and supernatant were separately sampled and subjected to electrophoresis, and the pellet contained a large amount of the target protein of induced expression, while the supernatant contained almost no target protein (FIG. 2), indicating that the target protein was present mainly in the form of inclusion bodies in bacteria. SDS-PAGE identified the purified protein as approximately 51kDa, consistent with theoretical expectations.

2.3.6 SDS-PAGE identification of gB protein purification

The sample before purification, the effluent of loading and the sample eluted at each eluting concentration are subjected to SDS-PAGE identification, the result shows that the target protein can be eluted at the imidazole concentration of 800mM/L and 1M/L, and the target band appears in the 100mM/L eluting sample, the result shows that the concentration of the protein elution is in the range of 800mM/L to 1M/L, the target band appears in the 100mM/L eluting sample probably because the loading concentration is too high, and a part of unbound target protein flows down at the beginning of elution. The purified target band is clear and single, which shows that the purification effect is better.

2.3.7 SDS-PAGE identification of protein renaturation of gB

The gB protein is about 51kDa after renaturation and concentration, and the concentration is 1mg/mL by an ultraviolet spectrophotometer. 2.3.8 WesternBlot analysis of immunogenicity of gB protein

The duck DEV positive serum reference is used as a primary antibody, goat anti-duck IgG-HRP is used as a secondary antibody, and DAB is chemically developed. 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 the gB protein is immunized with the rabbit for the third time, the serum antibody effect is detected by indirect ELISA The price is 1:10 ⁶ The method comprises the steps of carrying out a first treatment on the surface of the The positive serum is used as primary anti-goat anti-rabbit IgG-FITC and is used as secondary antibody, IFA is carried out, and the result shows that the virus-inoculated cells emit strong green fluorescence, the strong green fluorescence is concentrated on cytoplasm and cell membranes, the contrast group does not see specific green fluorescence (figure 4), namely antibodies combined with DEV exist in serum separated after gB protein is immunized with rabbit, and the gB protein has good immunoreactivity.

Example 2 preparation and selection of DEV and DEV gB protein monoclonal antibodies

1. Test materials

1.1 strains and cells

H5, H7 and H9 subtype Avian Influenza Virus (AIV) HI test antigens were purchased from Harbin Visco Biotechnology development Inc., newcastle Disease Virus (NDV), egg Drop Syndrome Virus (EDSV), duck Tembusu Virus (DTMUV), duck hepatitis virus type I (DHV I) were all maintained in the laboratory. SP2/0 myeloma cells were kept by laboratory.

1.2 laboratory animals and serum samples

SPF duck embryos at day 11 were purchased from the experimental animal center of the Harbin veterinary research institute. SPF-grade female BALB/c mice at 6 weeks of age were purchased from Experimental animal technologies Inc. of Beijing veteh. 6 positive serum samples were prepared from laboratory immunized SPF ducks and identified by neutralization assays; 6 negative serum samples were collected from the laboratory from non-immunized SPF ducks and identified using a neutralization assay.

1.3 major reagents

Freund's Complete Adjuvant (FCA), freund's incomplete adjuvant (IFCA), HAT, HT and PEG/DMSO were all purchased from Sigma company. DMEM, fetal Bovine Serum (FBS) was purchased from GIBCO. ProteinG affinity chromatography columns were purchased from GEhealth care company. Glycine, tris, sucrose are all available from AMRESCO, USA; liquid paraffin was purchased from Tianda chemical company. IRDye680LT goat anti-mouse antibody was purchased from LI-COR company; goat anti-mouse IgG-HRP, igG-FITC, goat anti-rabbit IgG-HRP, igG-FITC, DAB chromogenic reagent kit were purchased from Kyoto China fir bridge biotechnology Co. SBAClotyping System-HRP antibody subclass identification kit was purchased from southern Biotech company.

2 test method

2.1 immunization of animals

After purification, DEV and DEV gB proteins were mixed with Freund's adjuvant and emulsified, and 6-week-old BALB/c mice were immunized at a dose of 50. Mu.g/mouse. Total immunization was 3 times, with two weeks between immunizations. The first immunization route is subcutaneous multipoint injection of abdomen, and the emulsifying adjuvant is Freund's complete adjuvant; the second immunization route is intraperitoneal injection, and the emulsifying adjuvant is Freund's incomplete adjuvant; 5d after the 3 rd immunization, tail breaking and blood sampling, centrifuging for 10min at 10000r/min, separating serum, and detecting the serum antibody titer; after 5 days, the mice with high titers were boosted with non-emulsified antigen, and the spleens of the mice were fused with SP2/0 cells in the logarithmic phase after 3 days.

2.2 cell fusion

After booster immunization, the spleens of mice were fused with SP2/0 cells in the logarithmic growth phase in a super clean bench. The specific operation steps are as follows:

(1) Preparation of feeder cells: the negative BALB/c mice are fixed on a tray, the abdomen of the mice is wiped by an alcohol cotton ball, then the abdomen skin is lifted by forceps, the skin is gradually sheared off, the peritoneum is completely exposed, 5 mLHAT culture solution is sucked into the abdominal cavity by a syringe, the abdomen is massaged for a plurality of times at a constant speed by the alcohol cotton ball, the force is moderate, the liquid in the abdominal cavity is pumped back by the syringe, the pumped back liquid is supplemented to 50mL, and the abdominal cavity is approximately divided into 5 96-hole culture plates.

(2) Preparation of spleen cell suspension: fixing the positive BALB/c mice on a tray, wiping the left abdomen with alcohol cotton ball, taking out spleen, and taking out sterile operation; meanwhile, the mouse serum is reserved as a mouse polyclonal antibody (PAb); the spleen was washed with DMEM basal medium, other tissues adhered to the spleen were removed, and then transferred to a plate containing 20mL DMEM basal medium, the spleen was pierced with a syringe under tens of spleen, the spleen was ground on a 40 μm filter screen with a syringe core, 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, flushing 3 times by using PBS buffer solution, adding 20mL of DMEM basic culture medium to blow down the cells, and transferring the cells into a centrifuge tube containing spleen cells; centrifuging at 1000r/min for 10min; the supernatant was discarded, 20mL of DMEM basal medium was added to resuspend the cells, and the cells were centrifuged at 800 r/min for 8min; discarding the supernatant, gently mixing the cells with a pipette, placing the centrifuge tube into a blue cap bottle filled with water at 37 ℃ to ensure the fusion efficiency, slowly adding 1mL of PEG within 1min, and standing for 1min; then ending the fusion, and sequentially adding 1mL, 2mL, 3mL and 4mL of DMEM basic culture medium in 4 pieces of 1min; the fused cells were plated onto feeder cells prepared the day before.

2.3 screening of positive hybridoma cell lines

And 3 times of subcloning are carried out on the cells in the fusion holes, positive cell strains are obtained through screening by an indirect ELISA method, and the cells are frozen after expansion culture. Subcloning and cell cryopreservation steps were as follows:

(1) Subcloning of fusion cells: the first subcloning, respectively coating an ELISA plate with purified DEV and DEV gB proteins of 5 mug/mL, detecting a cell culture supernatant with a fused cell hole by indirect ELISA, operating 2.2.11.1, selecting a monoclonal of positive hole cells, regulating the cell concentration to 10/mL by cell count, and sub-packaging the monoclonal into a 96-hole cell culture plate with a pre-paved feeder layer; detecting cell culture supernatant of each hole, picking monoclonal of positive hole cells, adjusting cell concentration to 1 cell/hole, and sub-packaging into 96-hole cell culture plates with feeder layers paved in advance; and (3) detecting the culture supernatant of each cell in the third subclone, picking out the monoclonal of the cells in the positive reaction well, and performing amplification culture for further identification.

(2) Cryopreservation of hybridoma cell lines: the positive reaction cells obtained by each subcloning and screening are frozen after the expansion culture; during freezing, discarding old liquid, cleaning for 3 times, lightly blowing down cells with 5mL of DMEM basic medium, centrifuging for 8min at 800r/min, discarding the medium, adding 1mL of cell freezing liquid to resuspend the cells, transferring the cell suspension into a freezing tube, putting the freezing tube into a freezing box, freezing overnight in an ultralow temperature refrigerator, and putting the freezing tube into liquid nitrogen to preserve the cells for a long time.

2.4 subclass identification of MAbs

The screened MAb subclass was assayed according to the sbaconotyping system-HRP antibody subclass identification kit instructions.

2.5 IFA identification of MAbs

And (3) taking MAb as a primary antibody and goat anti-mouse IgG-FITC as a secondary antibody, setting 3 controls at the same time, namely, a non-toxic cell control, and performing IFA without adding a blank control and taking the primary antibody as a non-immune mouse negative serum control. The operation method is the same as the above.

2.6 screening of optimal blocking Rate MAbs

To screen for IgG subclass mabs from multiple mabs that react with the native space conformational viral proteins and have strong blocking ability against DEV antibodies, the mabs identified as positive in IFA and as IgG subclass in 2.4 were further screened, 6 copies of each of the laboratory-stored DEV negative and positive serum were taken from the ultra-low temperature refrigerator, the competitive blocking ability of the MAb against each serum antibody was determined by blocking ELISA, and the appropriate MAb was selected based on the positive serum inhibition rate (PI)/negative serum inhibition rate (PI) values. Wherein positive serum PI value= (PBS OD _450nm Value-positive OD _450nm value)/PBS OD _450nm Value, negative serum PI value= (PBS OD _450nm Value-negative OD _450nm value)/PBS OD _450nm Values.

Coating ELISA plates with two antigens of purified DEV and gB proteins respectively, performing blocking ELISA, determining blocking capacity of each MAb, coating concentration and method, and blocking method in the same way as the indirect ELISA; after blocking, adding negative and positive blood serum, incubating for 1h at 37 ℃, simultaneously setting PBS control, washing the plate for 3 times at 100 mu L per hole; MAb was added and incubated at 37℃for 1h, 100. Mu.L per well, and the plates were washed 3 times; adding 1:10000 diluted goat anti-mouse-HRP, 100 μl per well, incubation at 37deg.C for 0.75h, washing the plate 3 times; the color development, termination and reading methods are the same as indirect ELISA.

2.7 Western Blot identification of MAb1E8

Inoculating DEV to DEFs, collecting supernatant and bottle bottom cells when 90% of cells are observed to have pathological changes under a microscope, centrifuging at a differential speed to remove precipitation, and performing super-separation for 2 hours at a temperature of 32000 r/min; transferring the super-separated and purified DEV to a PVDF membrane through SDS-PAGE; and respectively taking the murine PAb and one selected MAb as primary antibodies (1:1000), taking IRDye680LT goat anti-mouse antibodies as secondary antibodies (1:10000), and carrying out Western Blot to carry out result observation by scanning through an infrared fluorescence imaging system. To identify murine PAb and its reactivity with DEV. Simultaneously, transferring the purified gB protein to a PVDF membrane by SDS-PAGE; western Blot was performed using the above MAb as primary antibody, as described above, to identify the reactivity of the MAb with the prokaryotic expressed gB protein.

2.8 Specific identification of MAb1E8

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

2.9 preparation of ascites and purification of MAb1E8

The autoclaved paraffin oil was injected into the abdominal cavity of 10-week-old mice, 500. Mu.L/mouse, and 3.2.6 selected hybridoma cells and SP2/0 myeloma cells were injected in the same manner after one week, 10 ⁶ The ascites can be collected after one week; antibodies in mouse hybridoma ascites were affinity purified with Protein G. The specific operation steps are as follows:

(1) Centrifuging the ascites 10000r/min for 10min, and removing impurities; the column was set up and equilibrated with 5mL PBS. (2) After 400 mu L of ascites is mixed with equal amount of PBS, the mixed solution is added into a column and kept stand for 5min, so that antibodies in the ascites are fully adsorbed. (3) The flow control valve was opened, 4mL PBS was added for washing, and unbound hetero-protein and unbound and unstable antibody were eluted. (4) Glycine (0.1M, pH=3.0) was added for elution, and in order to keep the antibody structure stable, the running down liquid was collected by a collection tube containing Tris-HCl (1M, pH=8.8), and 0.1mL Tris-HCl was used for neutralization per 1mL of eluted antibody collected.

(5) SDS-PAGE electrophoresis was performed to identify the purity of the samples.

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

ELISA identification of the 10MAb1E8 ascites Titer

Coating the ELISA plate with gB protein with the concentration of 5 mug/mL, and mixing MAb1E8 ascites according to the proportion of 10-10 ⁸ Sequentially performing 10-fold ratio dilution, and determining the antibody titer of the ascites by using SP2/0 ascites as a negative control and an indirect method.The operation method is the same as the above.

Epitope identification of 2.11MAb 1E8

Truncated expression schematic for epitope identification (FIG. 5) the gB protein was truncated in 3 rounds and induced for 6 total times, and the designed primers were synthesized (Table 4).

TABLE 4 PCR primer sequences

And constructing a recombinant plasmid of the 6-segment truncated protein and prokaryotic expression. The PCR amplification system was as above, and the respective PCR amplification procedure was as follows:

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

And (3) simultaneously performing SDS-PAGE on a pair of truncated proteins expressed in each round, transferring to a PVDF membrane, and performing Western Blot by taking 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 results, 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 coating liquid, and the coating is carried out at 4 ℃ for overnight; performing indirect ELISA by taking MAb1E8 as a primary antibody and goat anti-mouse-HRP as a secondary antibody; blocking and antibody incubation methods are equivalent. The gB protein coats the ELISA plate, MAb1E8 is used as a primary antibody, and is used as a positive control, and negative and blank controls are arranged.

3 test results

3.1 screening results of hybridoma cells

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

3.2 identification of MAb subclasses

Determining subclasses of the MAbs by adopting an antibody subclass identification kit, wherein 2E3 in 4 DEVMAbs in 11 MAbs is IgM subclasses, 7E12 and 9G3 are IgG1 subclasses, and 10A9 is IgG2b subclasses; the heavy chains of the 7 strain gB protein MAb are all of the IgG2b subclass (fig. 6).

3.3 IFA analysis of MAb reactivity with DEV

After DEFs infected DEVs, after cells started to appear lesions, IFA was performed, and 3 MAbs reacted with DEVs, DEV MAb 2E3, 7E12 and gB protein MAb 1E8, respectively, and the toxic cells emitted strong green fluorescence concentrated on the cytosol and cell membrane, and no specific green fluorescence was seen in the control group (FIG. 7).

3.4 screening MAbs for blocking ELISA method

Combining the test results in 3.3 and 3.4, DEV MAb 7E12 and gB protein MAb 1E8 are of the IgG subclass and react with natural viral proteins, and the blocking effect identification results of the two MAbs show that the blocking capacity of MAb 1E8 is stronger than that of 7E12 (Table 5), so that MAb 1E8 can be used for subsequent tests.

Table 5 comparison of blocking Effect of two monoclonal antibodies on 6 DEV-positive serum samples

3.5 WesternBlot analysis of MAb 1E8

Virus was harvested after DEFs infection of DEV, and after super-isolation purification, DEV was transferred to PVDF membrane, and Western Blot was performed using murine PAb and MAb 1E8 as primary antibodies, respectively. Meanwhile, the purified gB protein and MAb were transferred as primary antibodies, and experiments were performed. The results show that PAb binds to the denatured DEV proteins at 30kDa, 47kDa and 120kDa bands, MAb binds to the denatured DEV protein at 30kDa band and to the prokaryotic expressed 51kDa protein (FIG. 8).

3.6 identification of MAb 1E8 specificity

AIV (H5, H7 and H9), NDV, EDSV, DTMUV, DHV-I and DEFs are respectively coated on an ELISA plate, and indirect ELISA is carried out by taking P/N more than or equal to 2.1 as a positive judgment standard. The results showed that the MAb was negative for the reaction with other viruses and DEFs under conditions where the positive control was established (fig. 9), indicating that the MAb was specific.

3.7 SDS-PAGE identification of MAb 1E8 ascites purification

Affinity purifying MAb with Protein G, spectrophotometrically to determine its concentration to about 1.6mg/mL; SDS-PAGE identifies the purified MAb heavy chain as 55kDa and light chain as about 25kDa.

3.8 ELISA determination of antibody titers in ascites

The antibody titer of the MAb 1E8 ascites is measured by an indirect method by using the gB protein coated ELISA plate and SP2/0 ascites as a negative control, and when the ascites is diluted by 10 ⁴ OD at times _450nm The values were all greater than 1.0 (FIG. 10), and the results showed that the antibody titer was 10 with P/N.gtoreq.2.1 as a positive determination criterion ⁶ (FIG. 10).

3.9 identification of epitope of MAb 1E8

And (3) PCR amplification, constructing a prokaryotic expression system for expressing truncated proteins, carrying out SDS-PAGE on a pair of truncated proteins expressed in each round, transferring, and carrying out Western Blot by taking MAb 1E8 as a primary antibody and goat anti-mouse-HRP as a secondary antibody. The results of PCR, SDS-PAGE and Western Blot analysis show that the first truncated gB-101 amplified fragment has 600bp, the protein has 38kDa, the gB-102 amplified fragment has 441bp and the protein has 28kDa,Western Blot, and the MAb reacts with the second protein gB-102 (FIG. 11); the result of the second truncated expression of gB-201 amplified fragment size of 192bp, protein size of 16kDa, gB-202 amplified fragment size of 288bp and protein size of 25kDa,Western Blot shows that MAb reacts with the second protein gB-202 (FIG. 12); the third truncated gB-301 amplified fragment size was 171bp, protein size was 15.5kDa, gB-302 amplified fragment size was 153bp, and protein size was 15kDa,Western Blot, indicating that MAb reacted with both protein fragments (FIG. 13).

Indirect ELISA identification of MAbs with synthetic overlapping polypeptides showed that they reacted with polypeptides (FIG. 14). Therefore, the antigen epitope aimed by the MAb should be the amino acid sequence of the polypeptide overlapped by the two proteins in the third truncated expression, namely ⁵⁷⁹ RMLGDVLAVSSC ⁵⁹⁰ . The MAb 1E8 epitope region is analyzed to be located in the 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 Source

H5, H7 and H9 subtype Avian Influenza Virus (AIV) HI test antisera were purchased from Harbin Vicat Biotechnology development Inc., and positive serum was all maintained in the laboratory for Newcastle Disease Virus (NDV), egg Drop Syndrome Virus (EDSV), duck Tembusu virus (DTMUV), gosling Plague Virus (GPV), duck hepatitis virus type I (DHV I). DEV positive serum reference (Duck source, neutralization titers 1:100) were purchased from China veterinary drug administration; DEV negative serum references (Duck source, negative for neutralization test) were kept by laboratory; 130 positive serum samples and 364 negative serum samples were sourced as above.

565 parts of duck-origin clinical serum samples were collected from Shandong's back-office, 318 parts from Shandong's lotus, 27 parts from Ji Linsong stock; 27 goose-derived clinical serum samples were collected from Ji Linsong stock.

1.2 Main reagents and kit

Goat anti-mouse IgG-HRP and TMB color development solutions were both purchased from sigma company; tween20 was purchased from BioSharp company; PBS buffer (dry powder) was purchased from beijing laser technologies limited; the domestic duck plague virus ELISA antibody detection kit (TSZ brand) was purchased from Shanghai bang Jing Shengwu reagent Co. The preparation of the important reagents is as follows:

(1) PBS buffer: according to the instructions, with deionized water dissolved 50X PBS buffer (dry powder), volume to 1000mL, after autoclaving, can be stored at room temperature.

(2) Coating solution (0.05 mol/L carbonate buffer at ph=9.6): 1.50g of anhydrous sodium carbonate and 2.98g of sodium bicarbonate are diluted by deionized water, the volume is fixed to 1000mL, and the solution can be stored at room temperature after high-pressure sterilization.

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

(4) Washing liquid: 500mL of Tween 20 is added into the PBS buffer solution, the volume is fixed to 1000mL, and the mixture is prepared for use.

(5) Dilution liquid: 10g of skim milk is added into PBS buffer solution to be fully dissolved, and the volume is fixed to 1000mL, and the mixture is prepared for use.

(6) Stop solution: 100mL of concentrated sulfuric acid was slowly added to 80mL of deionized water and the volume was fixed to 100mL.

2 test method

2.1 determining working concentration

Taking DEV negative serum reference as negative control, diluting DEV positive serum reference by 1.25-10 mug/mL for 2 times, diluting antigen coating concentration by 20-160 mug/mL for 2 times, blocking ELISA, and selecting a negative value of 0.8-1.2 by a square titration method, wherein the concentration of the coated antigen and the positive serum reference is comprehensively lower, and the condition corresponding to a larger pore of an inhibition rate (PI) value is the working concentration of the coated antigen and the positive serum reference; and fixing the concentration of the coated antigen and the positive serum reference, and performing subsequent experiments according to a conventional method, wherein the working concentration of the target antigen is selected as the condition corresponding to the hole with PI of more than 40% and MAb 1E8 with low concentration.

2.2 determination of gB protein coating conditions

4 different coating conditions were set at 37℃for 1h,37℃for 2h, and then at 4℃for 12h,4℃for 12h after 2h with a protein coating plate of 0.5. Mu.g/mL. And (3) carrying out subsequent experiments 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 the gB protein with optimal conditions, four blocking fluid species were set: 5% BSA,5% skimmill, 0.5% gelatin (gelatin), 5% FBS. At 37 ℃, each sealing liquid is sealed for 2 hours, and the sealing liquid is added longitudinally, wherein 4 sealing liquids are parallel to each other. And (3) carrying out subsequent experiments according to a conventional method, integrating the PI value and the price factor of the coating liquid, and selecting the sealing liquid with economy and good effect.

2.4 determining the incubation conditions of the samples to be examined

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

2.5 determination of MAb incubation time

And incubating the MAb 1E8 with the working concentration for 0.5h, 1h, 1.5h and 2h respectively at 37 ℃. And (3) carrying out subsequent experiments 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

Working concentration goat anti-mouse IgG-HRP antibody was incubated for 30min, 45min, 1h at 37 ℃. And (3) carrying out subsequent experiments according to a conventional method, and selecting the condition with large PI value and less time consumption as the optimal incubation time.

2.7 determination of color development time

Setting 4 color development times at 37 ℃ for 5min, 10min, 15min and 20min in sequence. And (3) carrying out subsequent experiments 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 decision criteria

Taking out the negative and positive serum samples determined by neutralization test from the ultra-low temperature refrigerator, and determining OD of 364 negative duck serum samples and 130 positive duck serum samples _450nm Values, negative control with DEV negative serum reference. According to the formula PI value= (negative control OD _450nm Value-serum OD to be tested _450nm Value /) negative control OD _450nm Calculating a value, namely using GraphPad Prism 6 as a scatter distribution map, drawing an ROC curve by using IBM SPSS Statistics software, and selecting a PI value corresponding to a point with proper sensitivity and specificity as a critical value.

2.9 specificity test

The ELISA plate is coated with the determined antigen concentration, and 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, DEV positive serum reference with the optimal concentration is used as positive control, and DEV negative serum reference is used as negative control. Under the optimal conditions, incubating standard serum and negative and positive control serum, 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 DEV antibody.

2.10 sensitivity test

Coating an ELISA plate with the antigen with a determined concentration, and mixing DEV positive serum reference with the antigen according to the weight ratio of 1:10 to 1:1280 sequentially carrying out 2-time ratio dilution, incubating diluted DEV positive serum under the optimal condition, adding MAb for incubation after plate washing, incubating enzyme-labeled secondary antibodies after plate washing, setting DEV negative serum reference as negative control, calculating PI value of positive serum under each dilution, determining the maximum dilution which can detect positive serum, and verifying the sensitivity of the method.

2.11 repeatability test

2.11.1 in-batch repeat

Taking out 4 plates coated in the same batch, taking 7 positive samples stored in a laboratory, coating the ELISA plates with determined antigen concentration, incubating the positive serum samples under the optimal condition, and setting negative control on each plate; and (3) manually washing the plate, adding MAb for incubation, fully washing the plate, incubating the enzyme-labeled secondary antibody, measuring the PI value of the same sample in the same batch, and calculating the variation coefficient.

2.11.2 inter-batch repetition

Taking out 4 plates coated in different batches, taking 7 positive samples stored in a laboratory, coating an ELISA plate with determined antigen concentration, and incubating the negative and positive serum samples under the optimal condition, wherein each plate is provided with a negative control; and (3) manually washing the plate, adding MAb for incubation, fully washing the plate, incubating the enzyme-labeled secondary antibody, measuring PI values of the same sample in different batches, and calculating the variation coefficient.

2.12 compliance test

From the positive and negative serum samples stored in the laboratory, 41 DEV positive serum samples and 53 DEV negative serum samples determined by a neutralization test are randomly extracted, the serum samples are taken out from an ultralow temperature refrigerator, detection is carried out according to an indirect ELISA method described in a commercial reagent kit specification, meanwhile, a blocking ELISA method established by the research is used for carrying out the test on a 96-hole ELISA plate, and the coincidence rate of the results of the two is analyzed.

2.13 clinical sample detection

Sampling is carried out on the farm of the Shandong Weifang, the lotus and Ji Linsong original farms, and 937 clinical blood serum samples are collected in total. Wherein the Weifang collects 565 parts of duck serum, the lotus is 318 parts of commercial duck serum, the Pingyuan is 27 parts of commercial duck serum and 27 parts of commercial goose serum. Coating an ELISA plate with determined antigen concentration, incubating clinical serum samples under the optimal condition, adding MAb for incubation after plate washing, incubating an ELISA secondary antibody after plate washing is performed fully, measuring the absorbance of each hole, taking DEV negative serum reference as negative control, and calculating and counting the antibody positive rate of each batch of samples.

3 test results

3.1 determination of optimal working concentration

And (3) using the gB protein as a coating antigen, using MAb1E8 as a competitive antibody, and establishing a blocking ELISA method by preliminary combination. PI value is larger at antigen 0.5 mug/mL, negative control OD _450nm The values were in the range of 0.8-1.2, the sensitivity of the experiment was high, so that the optimal coating amount of antigen was determined to be 0.5. Mu.g/mL, and the working concentration of DEV positive serum reference was 1:40 (FIG. 15 and Table 6); fixing the concentration of the coating antigen and the positive serum, calculating PI values of MAb1E8 detected positive serum under different dilution factors, wherein the PI values are reduced along with the increase of the dilution factor of the MAb1E8, when the dilution factor is 2000 times, the PI values are larger and are almost the same as those when the dilution factor is 1000 times, and compared with the dilution factor of 1000 times, the antibodies are saved, and determining that the working concentration of the MAb1E8 is 1:2000 (FIG. 15).

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

3.2 optimization of protein coating conditions

From 4 different coating times and temperatures, the conditions with greater PI and less time consuming were selected as optimal coating conditions for a 0.5 μg/mL gB protein coated plate, and therefore, the gB protein coated conditions were determined to be 4 ℃ for 12h incubation (fig. 16).

3.3 selection of types of blocking fluid

The blocking effect of 4 different blocking solutions was tested, from which the best material was selected, and the results showed that 5% of the shimmill had better blocking effect and was more economical from the standpoint of raw material cost, so 5% of the shimmill was chosen as blocking solution for blocking ELISA.

3.4 optimization of incubation conditions for samples to be examined

3 different conditions are set, and the condition with the best effect is selected as the incubation condition of the detected sample, and the result shows that the PI value of the detected sample is larger under the condition of incubation for 2 hours at room temperature (25 ℃), so that the incubation condition of the detected sample is determined to be the incubation condition for 2 hours at room temperature.

3.5 optimization of MAb incubation time

The time of MAb incubation with the best effect was selected from among 4 different times, and the results showed that MAb incubation was performed at 37℃for 1h and PI was large, thus determining the time of MAb incubation for 1h.

Optimization of goat anti-mouse IgG-HRP incubation time

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

3.7 optimization of color development time

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

In summary, by determination of the optimal working concentration and optimization of the reaction conditions, the optimal operation flow is established as follows:

(1) Diluting and purifying the gB protein to 0.5 mu g/mL by using a coating solution, wherein each hole is 100 mu L, and coating is carried out for 12 hours at 4 ℃; washing the ELISA plate with the washing solution for 3 times, and drying.

(2) mu.L of 5% ski mill was added to each well, the wells were blocked at 37℃for 2 hours, and the plates were washed 3 times.

(3) The sample to be tested, from which impurities were removed by low-speed centrifugation, was added and allowed to act at room temperature for 2 hours, while negative-positive serum controls (1:40) were set, 100. Mu.L per well, and plates were washed 3 times.

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

(5) Goat anti-mouse-HRP (1:10000) was added and 100. Mu.L per well was allowed to act at 37℃for 0.75h and the plates were washed 3 times.

(6) Adding TMB, and developing for 15min in a dark place with 100 mu L of TMB in each hole; stop reaction, 50 μl of stop solution per well; the plate was read within 15 min.

3.8 establishment of decision criteria

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

TABLE 7 statistical results of negative samples PI

Further plotting scatter distribution map of PI value statistics results of 494 negative and positive serum samples (fig. 17), the results show that most negative samples PI are distributed below 30% and positive samples are distributed above 30%. The main purpose of the test is to establish a detection method for eliminating antibody-positive individuals, and the result shows that 30% is taken as a critical value to ensure higher negative rate on the population level.

3.9 specificity test results

Standard sera against AIV, NDV, EDSV, DTMUV, GPV and DHV-1 were synchronously tested by established ELISA methods, with anti-DEV positive serum reference as positive control and one SPF duck serum as negative control, and the results showed that the method was specific for DEV antibodies (fig. 18).

3.10 sensitivity test results

DEV positive serum reference was tested by established ELISA, positive serum was diluted from 10 fold, with a maximum dilution of 1280 fold, and the sensitivity of the method was examined. Calculating PI value of positive serum under different dilution factors, wherein the PI value is reduced along with the increase of the dilution factor of the serum, the detection result is positive when the serum is diluted 80 times, the detection result is negative when the serum is diluted 160 times, and the maximum dilution factor of the serum can be detected by the method to be 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 using an established method, measuring PI values of the same sample in the same batch and different batches, and calculating results show that the inter-batch and intra-batch variation coefficients are smaller than 10%, wherein the maximum intra-batch difference is 6.88% and the maximum inter-batch difference is 8.62% (tables 8 and 9).

TABLE 8 within-batch Difference detection results

TABLE 9 results of batch-to-batch variation detection

3.12 results of compliance test

The detection method adopted by the commercialized kit is indirect ELISA, and 41 positive samples and 53 negative samples are detected by the kit and the blocking ELISA method established by the research. The test results of 94 samples showed a total compliance of 84.04%, a positive compliance of 82.5% and a negative compliance of 85.19%.

3.13 clinical sample detection results

4 samples were collected for a total of 4 sampling sites, and 937 clinical samples. The results showed that the Shandong ducks had a positive rate of 23%, the Shandong commercial ducks had a positive rate of 1.25%, and the pine commercial ducks and geese did not detect antibody-positive individuals (Table 10). From the detection result, the total positive rate of the sample antibody is not more than 20%, and the antibody positive rate of the breeding ducks is obviously higher than that of commercial ducks.

TABLE 10 detection results of clinical samples

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SEQUENCE LISTING

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

<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 a duck virus enteritis virus gB protein monoclonal antibody 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. Use of the hybridoma cell line of claim 1 in the preparation of a reagent or a medicament for diagnosing or preventing duck viral enteritis virus.

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

5. The epitope peptide recognized by the monoclonal antibody according to claim 2, wherein the amino acid sequence of the epitope peptide is shown in SEQ ID No. 1.

6. The use of the epitope peptide according to claim 5 for preparing a reagent or a medicament for diagnosing or preventing duck viral enteritis virus.

7. A blocking ELISA detection kit to detect DEV antibodies comprising: DEV gB protein, coating liquid, primary antibody, enzyme-labeled secondary antibody, buffer solution, diluent, washing liquid, sealing liquid, stop solution, positive serum control and negative serum control; 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 of claim 7, characterized in that the coating solution is a carbonate buffer at ph=9.6 of 0.05 mol/L.

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