CN111426828A

CN111426828A - Duck egg laying-reduction syndrome virus antibody detection method and kit thereof

Info

Publication number: CN111426828A
Application number: CN201910018009.8A
Authority: CN
Inventors: 李泽君; 李雪松; 宿鑫; 孙海伟
Original assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Current assignee: Shanghai Veteromaru Research Institute Caas China Animal Health And Epidemiology Center Shanghan Branch Center
Priority date: 2019-01-09
Filing date: 2019-01-09
Publication date: 2020-07-17
Anticipated expiration: 2039-01-09
Also published as: CN111426828A8; CN111426828B

Abstract

The invention discloses a duck egg laying-down syndrome virus antibody detection kit, which comprises an antibody for resisting a duck egg laying-down syndrome virus and/or a whole virus antigen of the duck egg laying-down syndrome virus or a main immunogen protein antigen thereof. The invention also discloses application of the duck egg laying-reduction syndrome virus antibody detection kit in preparation of a product for diagnosing duck egg laying-reduction syndrome. The duck egg laying-reduction syndrome virus antibody detection kit can be used for quickly, qualitatively and quantitatively detecting the antibody of the duck egg laying-reduction syndrome virus, and has good application prospects in the aspects of diagnosis and antibody detection of the duck egg laying-reduction syndrome.

Description

Duck egg laying-reduction syndrome virus antibody detection method and kit thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for detecting a duck egg laying-reduction syndrome virus antibody and a kit thereof.

Background

China is a big poultry-raising country, poultry-raising industry rapidly develops, annual feeding amount of poultry reaches 120 hundred million feathers, and the poultry occupies the first place of the world, and China is the first waterfowl breeding country and the consuming country in the world. Although the aquatic bird breeding amount is increased year by year in recent years, and the large-scale and industrialized operation level is also rapidly improved, the elimination rate of aquatic bird breeding is high due to the fact that the aquatic bird breeding technology is relatively lagged behind in China, particularly the aquatic bird infectious diseases occur, the annual loss of the aquatic bird industry in China is as high as hundreds of billions of yuan, and the aquatic bird breeding method becomes a restriction factor which troubles the healthy development of the aquatic bird breeding industry in China.

The duck breeding industry in China faces a serious threat to new infectious diseases. For 2016, egg laying rate of duck groups is reduced by 20-30% after egg laying rate reduction of unknown reasons occurs in a duck breeding farm in a continuous manner, and the duck breeding farm is difficult to recover the egg laying rate before the disease occurrence after a plurality of weeks, so that huge economic losses are caused. The main symptom of the disease is the reduction of egg laying of laying ducks, so the disease is named as duck egg drop syndrome. The caesarean examination of diseased duck shows that the liver has blood stasis-like change in the early stage of the disease, and the ovary and the oviduct have damage of different degrees. The disease is continuously spread in China, and the disease is fulminant in main laying duck production areas all over the country, but at present, a detection method and a kit for diagnosing the duck egg laying-down syndrome are not available, so that the prevention and control of the duck egg laying-down syndrome are very disadvantageous, and an effective duck egg laying-down syndrome detection kit is urgently needed to be developed.

Disclosure of Invention

The invention aims to solve the technical problem that the diagnosis means of the duck egg hypogenesis syndrome is limited at present, and provides a novel antibody detection kit for a duck egg hypogenesis syndrome virus, wherein the novel virus is a brand-new duck egg hypogenesis syndrome virus separated from ducks with duck egg hypogenesis syndrome symptoms.

In order to solve the technical problems, the invention is realized by the following technical scheme:

in one aspect of the invention, an antibody against duck egg drop-laying syndrome virus is provided.

Preferably, the antibody is an antibody produced by immunizing an animal with the strain AH204 of duck egg drop syndrome virus.

Preferably, the antibody consists of SEQ ID NO: 1 or a partial protein fragment thereof, or an antibody produced by immunizing an animal with a protein having an amino acid sequence represented by SEQ ID NO: 1 or a partial protein fragment thereof, wherein the homology of the amino acid sequence shown in the formula 1 is more than 98 percent.

More preferably, the antibody consists of SEQ ID NO: 2 or an active fragment thereof, and immunizing animals to produce antibodies.

In another aspect of the invention, the invention also provides a duck egg laying syndrome virus antibody detection kit, which comprises the antibody and/or a whole virus antigen of the duck egg laying syndrome virus or a main immunogen protein antigen thereof.

The method for detecting the duck egg laying-down syndrome virus antibody by using the kit comprises a two-way agarose diffusion test, an indirect E L ISA method or a blocking E L ISA method.

Preferably, the kit for detecting the duck egg hypogenesis syndrome virus antibody by using the indirect E L ISA method or the blocking E L ISA method further comprises an E L ISA enzyme label plate, an enzyme-labeled secondary antibody, positive control serum and negative control serum.

Preferably, the method for detecting the duck egg laying-down syndrome virus antibody by using the blocking E L ISA method comprises the following steps:

coating an E L ISA ELISA plate with the purified duck egg hypogenesis syndrome virus antigen;

after the serum to be detected and the envelope antigen act, sequentially adding an antibody, an enzyme-labeled secondary antibody and a developing solution;

reading the absorbance value OD by using an enzyme-linked immunosorbent assay_450nmAnd calculating the blocking rate of the serum to be detected according to a formula of (the absorbance value of the negative control-the absorbance value of the sample to be detected)/the absorbance value of the negative control × 100% and obtaining a detection result according to the following judgment criteria:

when the blocking rate is more than or equal to 25.4 percent, the product is positive; if the blocking rate is more than 17.6% and less than 25.4%, the detection is suspicious and needs to be repeated, and if the repeated detection result is less than 25.4%, the detection is judged to be negative; when the blocking rate is less than or equal to 17.6 percent, the product is negative.

Preferably, the detection of the duck egg laying-down syndrome virus antibody by using the indirect E L ISA method comprises the following steps:

coating an E L ISA ELISA plate with purified duck egg laying-down syndrome virus antigen or recombinant expression duck egg laying-down syndrome virus main antigen protein;

reading the absorbance value OD by using an enzyme-linked immunosorbent assay_450nmIf the OD of the serum sample to be tested is_450nmThe value is not less than the negative sample OD_450nmThe mean value of the values +2 times the standard deviation was judged to be positive.

In another aspect of the invention, the application of the antibody in preparing a product for diagnosing or treating duck egg laying-down syndrome is also provided.

In another aspect of the invention, the invention also provides application of the duck egg laying-reduction syndrome virus antibody detection kit in preparation of a product for diagnosing duck egg laying-reduction syndrome.

The duck egg laying-reduction syndrome virus antibody detection kit can be used for quickly, qualitatively and quantitatively detecting the antibody of the duck egg laying-reduction syndrome virus, and has good application prospects in the aspects of diagnosis and antibody detection of the duck egg laying-reduction syndrome.

Drawings

The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

FIG. 1 is a comparison of the homology of the amino acid sequences of the duck egg hypogenesis syndrome virus AH204 strain of the present invention and other different duck egg hypogenesis syndrome virus isolates;

FIG. 2 is a schematic view of the sample application of the two-way agarose diffusion test in example 1 of the present invention;

FIG. 3 is a schematic diagram showing the results of the two-way agarose diffusion test in example 1 of the present invention.

Detailed Description

The invention discloses a duck egg laying-reduction syndrome virus antibody detection kit which is developed on the basis of obtaining a brand new duck egg laying-reduction syndrome virus by separation in the early stage. The brand new duck egg laying reduction syndrome virus is obtained by separating tissues such as liver, spleen, lung and the like of ducks for duck egg laying reduction syndrome disease, and is preserved in China center for type culture Collection (CCTCC for short, address: preservation center of Wuchang Lodojia mountain Wuhan university in Wuhan city, Hubei province) with the preservation number of CCTCC NO: v201866, which is classified and named as Duck egg drop-laying syndrome Virus AH204 strain. Through genome sequence determination and analysis, the amino acid sequence of the Open Reading Frame (ORF) of the duck egg drop-laying syndrome virus gene sequence is shown as SEQ ID NO: 1 is shown.

The obtained genome sequence of the duck egg laying-down syndrome virus (AH204 strain) is used for designing primers, the open reading frame sequences of other duck egg laying-down syndrome viruses separated from different provinces in China are amplified, and after sequencing, the viruses have the same genome structure, the nucleotide homology of ORF genes of the viruses is 97.9-100%, and the corresponding amino acid sequences are similar to those of SEQ ID NO: 1 is also between 98-100% homologous (see figure 1).

The invention relates to a duck egg laying-reduction syndrome virus antibody detection kit, which detects a duck egg laying-reduction syndrome virus antibody by using an antibody generated by a duck egg laying-reduction syndrome virus or an antigen protein immune animal thereof through a two-way agarose diffusion test, an indirect E L ISA method or an E L ISA blocking method.

EXAMPLE 1 two-way agarose diffusion assay

1.1 preparation of agarose gel plate the agarose is melted, the melted agarose is poured into a plate on a horizontal table to prepare agarose gel with the thickness of about 3-4 mm, and holes are punched according to the required shape after cooling.

1.2 preparation of antigen, inoculating a 10-11-day-old non-immune duck embryo with a duck egg laying-reduction syndrome virus AH204 strain, continuously incubating for 6 days, harvesting the duck embryo dead after 24 hours, collecting allantoic fluid, inactivating with formaldehyde with the final concentration of 2 per mill for 24 hours, centrifuging at 4 ℃ for 60 minutes at 10000g to remove impurity proteins, and collecting supernatant. The supernatant was ultracentrifuged at 150000g for 5 hours at 4 ℃ to discard the supernatant, and the pellet was dissolved in PBS (200-fold concentrated in the original volume). Protein concentration was determined, split charged, stored at-70 ℃ and used as a two-way agarose diffusion test antigen.

1.3 Positive serum preparation

1.3.1 immunization methods with 10^4.5EID₅₀Intramuscular injection of 5 healthy non-immune ducks of 4-8 weeks old with the duck egg laying-reduction syndrome virus (AH204 strain) for 0.1ml of each duck egg, blood collection after 21 days, serum separation, uniform mixing of the serum from the 5 ducks, subpackaging and preparation of the anti-duck egg laying-reduction syndrome virus positive serum (namely the anti-duck egg laying-reduction syndrome virus antibody).

1.3.2 subpackaging and aseptically taking blood, separating serum, adding 0.01 percent of thimerosal, quantitatively subpackaging by 1 ml/tube.

1.3.3 test

1.3.3.1 Positive serum is yellow or orange yellow transparent clear liquid.

1.3.3.2 sterility test according to the appendix of the existing Chinese veterinary pharmacopoeia, and the positive serum prepared by the test is sterility.

1.3.3.3 potency determination according to Duck egg drop delivery syndrome Jones test, its antibody potency is not less than 1:2, or according to indirect E L ISA method with prokaryotic expression protein as coating antigen, its OD_450nmThe value is more than or equal to 0.412, or the OD is obtained according to the indirect E L ISA method of using the purified duck egg laying-down syndrome virus as a coating antigen_450nmThe value is more than or equal to 0.487, or the antibody blocking rate is more than or equal to 25.4 percent according to the duck egg laying-down syndrome virus blocking E L ISA antibody detection method.

1.4 negative serum preparation

1.4.1 preparation of negative serum 5 healthy non-immune duck blood of 4-8 weeks old is collected, serum is separated, serum from 5 ducks is uniformly mixed, 0.01% of thimerosal is added, quantitative subpackaging is carried out, and 1 ml/tube is used for preparing the negative serum of the duck egg laying-down syndrome virus.

1.4.2 test

1.4.2.1 the negative serum is yellow or orange-yellow transparent clear liquid.

1.4.2.2 sterility test according to the appendix of the existing Chinese veterinary pharmacopoeia, and the negative serum prepared by the test is sterility.

1.4.2.3 potency determination according to Duck egg drop delivery syndrome Jones test, its antibody is negative, or indirect E L ISA method using prokaryotic expressed protein as coating antigen, its OD_450nmThe value is less than 0.412, and the OD is obtained by using the indirect E L ISA method of using the purified duck egg drop-laying syndrome virus as a coating antigen_450nmThe value is less than 0.487, and the antibody blocking rate is less than 17.6 percent according to the detection method of duck egg laying-down syndrome virus antibody E L ISA.

1.5 adding samples as shown in figure 2, adding antigen into the middle hole, respectively adding the positive control, the negative control and the serum to be detected (according to a certain dilution multiple) of the duck egg reduced-labor syndrome virus antibody into the peripheral holes, inverting the agar plate after the liquid is soaked in the agarose, placing the agar plate in a wet box, and mutually diffusing the antigen and the antibody on the agar plate at 37 ℃.

1.6 judging that the test is established when the positive serum shows an opaque white line of precipitation in the agar and the negative serum does not show a line of precipitation. If the serum to be detected contains a specific antibody of the duck egg hypo-parturition syndrome virus, a white precipitation line appears; if the serum to be detected does not contain the specific antibody of the duck egg drop-laying syndrome virus, no white precipitation line appears. The antibody titer of the serum to be tested is the highest dilution of the serum with the precipitation line appearing in the test.

1.7 after inactivating the duck egg laying reduction syndrome virus, immunizing 10 healthy and susceptible ducks of 21 days old, collecting serum after 21 days, and performing an agar-agar amplification experiment, wherein the immunized ducks are all positive in duck egg laying reduction syndrome virus antibodies, and the antibody titer of the positive serum is the dilution multiple with the highest precipitation line in the agar-agar amplification experiment (see fig. 3).

Example 2 Indirect E L ISA method for detecting duck egg drop-off syndrome virus antibody by using prokaryotic expressed protein as envelope antigen

2.1 antigen preparation recombinant duck egg laying-down syndrome virus VP1 protein (VP1 protein amino acid sequence is shown in SEQ ID NO: 2) is expressed and purified by using a prokaryotic expression system.

2.2 determination of optimal coating concentration of antigen and optimal dilution of serum

Diluting the purified VP1 protein (1.513mg/m L) of the recombinant duck egg laying-down syndrome virus (DHSV) by a matrix titration method with 0.05 mol/L pH 9.6 carbonate buffer solution according to the concentration of 16, 8, 4, 2, 1 and 0.5ug/m L in sequence, coating E L ISA plates on each well with 100u L, and carrying out overnight at 4 ℃ to obtain positive serum and negative serum which are 1:100, 100 and,Diluting at 1:200, 1:400, 1:800 and 1:1600, coating E L ISA plate at 100u L per well, incubating at 37 ℃ for 1 hour, diluting with 1:5000 enzyme-labeled secondary antibody, coating E L ISA plate at 100u L per well, incubating at 37 ℃ for 1 hour, performing other steps according to E L ISA program, adding 100 mu L/well TMB substrate, reacting for 10min, adding 2M concentrated sulfuric acid 50 mu L/well to terminate the reaction, and measuring OD by using enzyme-labeling instrument_450nmThe value is obtained. Determination of OD_450nmValue and P/N value, Positive OD selection_450nmThe value was about 1, and the antigen coating concentration and the serum dilution factor at which the P/N value was maximal were the optimal working concentrations.

Titration results in OD_450nmThe coating dilution at a value close to 1.0 was the optimum coating concentration, at which the error was small and the reaction was sensitive, OD was measured at 2ug/m L for the antigen and 1:400 for the serum dilution_450nmThe value was closest to 1.0, thus, the optimal coating concentration for the antigen was determined to be 2ug/m L, and the optimal serum dilution was 1: 400.

TABLE 1 OD of Square matrix titration_450nmValue of

2.3 determination of optimal conditions for coating antigen

Coating the ELISA plate with the optimal antigen concentration, dividing the ELISA plate into 6 groups, and coating the group 1 at 37 ℃ for 1 h; coating at 37 ℃ for 2h in group 2; coating at 37 ℃ for 4h in group 3; group 4, coating overnight at 4 ℃; group 5 coating at 37 ℃ for 1h and overnight coating at 4 ℃; coating the 6 th group at 37 ℃ for 2h and adding the solution at 4 ℃ overnight, keeping the other conditions unchanged, and comparing OD of the negative hole, the positive hole and the control hole of each group₄₅₀The value and the P/N value determine the optimal coating condition.

The ELISA plate was coated with detection antigen 2ug/m L, and the highest P/N value of group 5 reached 6.036, indicating that coating at 37 ℃ for 1h and overnight coating at 4 ℃ were the optimal coating conditions.

TABLE 2 determination of optimal conditions for antigen coating

2.4 determination of confining liquid

To a maximum extentCoating the enzyme label plate with proper antigen concentration and optimal coating condition, washing, respectively using PBST containing 5% skimmed milk, 1% gelatin, 0.1% BSA, 10% calf serum, and commercial blocking solution, 200u L/well, keeping other conditions unchanged, selecting proper blocking solution, blocking at 37 deg.C for 2 hr, performing E L ISA detection, and determining OD_450nmSelecting the sealing liquid with the maximum P/N value as the value and the P/N value.

The P/N value when a commercial blocking liquid was used was the largest at 6.273, from which a commercial blocking liquid was selected as the blocking liquid of this example.

TABLE 3 determination of blocking solution

2.5 determination of optimal seal time

Coating the ELISA plate with the optimal antigen concentration and the coating condition, washing, adding a sealing liquid, and dividing the solution into 5 groups, sealing the 1 st group at 37 ℃ for 1h, sealing the 2 nd group at 37 ℃ for 2h, sealing the 3 rd group at 37 ℃ for 3h, sealing the 4 th group at 37 ℃ for 4h, sealing the 5 th group at room temperature for 1h, keeping the other conditions unchanged, and taking the highest P/N value as the optimal sealing time.

TABLE 4 determination of optimal seal time

2.6 selection of working concentration of enzyme-labeled Secondary antibody

Diluting HRP enzyme-labeled anti-duck secondary antibody with 1:1000, 1:2000, 1:4000, 1:8000 and 1:16000, keeping other conditions unchanged, diluting antigen and serum according to optimal dilution, and determining OD_450nmValues and P/N values.

Coating the enzyme label plate with the optimal antigen concentration and coating conditions, washing, adding a sealing solution, adding 200u L/well, analyzing the test result, and selecting the dilution of the enzyme-labeled secondary antibody as 1:2000 because the maximum P/N value is 6.544 when the enzyme-labeled secondary antibody is diluted at 1: 2000.

TABLE 5 selection of working concentration of enzyme-labeled Secondary antibody

2.7 selection of substrate action time

Coating an enzyme label plate with optimal antigen dilution, adopting optimal dilution for HRP-labeled anti-duck secondary antibody and negative and positive serum, respectively reacting at room temperature for 5, 10, 15 and 30min under constant other conditions, testing by conventional method, and determining OD_450nmValues and P/N values. As a result, the P/N value was at most 6.015 when the substrate reaction time was 10min at room temperature, and therefore the substrate reaction time was selected to be 10min at room temperature.

TABLE 6 determination of the action time of the substrate

2.8 determination of the Positive and negative cutoff value of E L ISA

Detecting 100 parts of duck egg hyposyndrome virus negative serum to be detected according to the method of E L ISA, and determining OD_450nmThe value is statistically analyzed, and the threshold OD of the positive serum is determined_450nmThe value is 0.412.

2.9 specificity test

The established indirect E L ISA method is used for respectively detecting the positive serum of duck hepatitis virus I (DHAV-I), duck hepatitis virus III (DHAV-III), duck plague virus (DEV), Duck Reovirus (DRV), novel duck goose parvovirus (NGPV) and avian influenza virus H9 (AIV), and verifying whether the indirect E L ISA method established in the experiment has cross reactivity to the positive serum of the viruses.

As a result, positive sera of DHAV-I, DHAV-III, DEV, DRV, NGPV and AIV were assayed by the established indirect E L ISA method, and OD was determined for the positive sera of the above viruses_450nmThe values are all less than the positive and negative critical value of 0.412, and are all negative, which indicates that the established indirect E L ISA method has better specificity.

TABLE 7 specificity test of Indirect E L ISA

2.10 sensitivity test

Diluting the positive serum of the duck egg laying-down syndrome virus to 1:12800 times according to the 1:100 ratio, and carrying out indirect E L ISA test.

The results showed that the test was positive after 1:1600 dilution, while the test was negative below 0.412 for the 1:3200 dilution.

TABLE 8 sensitivity test of Indirect E L ISA

2.11 in-batch repeatability test

Different E L ISA plates are coated with the recombinant antigen prepared in the same batch, 6 sera with different antibody levels are taken and are measured according to an indirect E L ISA program under the same condition, each blood sample is subjected to a 5-well parallel test, and the result is subjected to statistical analysis.

The same batch of recombinant antigens is used for carrying out repeatability detection on 3 parts of duck egg drop-laying syndrome virus positive serum and 3 parts of negative serum, the result of statistical analysis shows that the coefficient of variation is between 1.462 and 5.902 percent and is less than 10 percent, which shows that the same sample has small variation degree in the same batch of test and better repeatability.

TABLE 9 in-batch repeatability test

2.12 run-to-run repeatability test

Different E L ISA plates were coated with 3 different batches of prepared and purified recombinant proteins, 6 sera of different antibody levels were taken and tested under the same conditions according to the indirect E L ISA procedure, and the results were statistically analyzed.

The result is analyzed statistically, the coefficient of variation is between 1.548% and 6.726%, and is less than 10%, which shows that the same sample has small variation degree in different batches of antigen tests and better repeatability.

TABLE 10 repeatability tests between batches

Example 3 Indirect E L ISA method for detecting duck egg hypo-partum syndrome virus antibodies using purified duck egg hypo-partum syndrome virus as envelope antigen

3.1 preparation of antigens

Inoculating a 10-11-day-old non-immune duck embryo with a duck egg laying-reduction syndrome virus AH204 strain, continuously incubating for 6 days, harvesting the duck embryo dead after 24 hours, collecting allantoic fluid, inactivating with formaldehyde with the final concentration of 2 per mill for 24 hours, centrifuging at 4 ℃ for 60 minutes at 10000g to remove impurity proteins, collecting supernatant, ultracentrifuging at 4 ℃ for 5 hours at 150000g to remove the supernatant, dissolving the precipitate with PBS (concentrated by 200 times according to the original volume), measuring the protein concentration, subpackaging, storing at-70 ℃ and using as E L ISA coated antigen.

The purified whole virus was quantified using a protein quantification kit, and the viral protein concentration was 0.851mg/m L as a result of the assay

3.2 determination of optimal coating concentration of antigen and optimal serum dilution

Diluting the purified whole virus solution with 0.05M carbonate buffer solution with pH 9.6 of 16, 8, 4, 2, 1 and 0.5ug/M L in sequence by using a matrix titration method, coating an E L ISA plate on each well with 100u L, standing overnight at 4 ℃, diluting positive serum and negative serum from 1:100 to 1:1600 in a multiple ratio respectively, sealing the commercial confining liquid at room temperature for 1 hour, diluting the enzyme-labeled goat anti-duck secondary antibody with 1:2000, performing other steps according to an E L ISA program, finally adding 100 mu L/well TMB substrate, performing reaction for 10min, adding 2M concentrated sulfuric acid 50 mu L/well to terminate the reaction, and measuring OD (optical density) by using an enzyme-labeling instrument_450nmThe value is obtained.

The matrix titration showed that the OD of the positive serum was 1:800 at a dilution of serum and an antigen coating concentration of 2. mu.g/m L_450nmThe value can reach 1.002, and the OD of the negative serum_450nmValue of 0.207, OD of negative and positive sera_450nmValue ofThe difference was large (P/N4.84) therefore, 1:800 was chosen as the optimal serum dilution and 2 μ g/m L as the optimal coating amount of antigen.

Table 11 OD of square matrix titration_450nmValue of

3.3 determination of optimal working concentration of enzyme-labeled Secondary antibody

Using the optimal antigen coating concentration, optimal coating conditions and optimal serum dilution in the above, the HRP-labeled secondary duck antibody is diluted at 1:1000, 1:2000, 1:4000, 1:8000 and 1:16000, and indirect E L ISA measurement is performed to determine the optimal working concentration of the enzyme-labeled secondary antibody.

OD of positive and negative sera when HRP-labeled anti-duck secondary antibody was diluted 1:2000_450nmThe difference in value is greatest. Therefore, 1:2000 was determined as the optimal working concentration for the enzyme-labeled secondary antibody.

TABLE 12 selection of working concentration of enzyme-labeled Secondary antibodies

3.4 determination of optimal working time of the substrate

Coating an enzyme label plate with optimal antigen dilution, adopting optimal dilution for HRP anti-duck secondary antibody and negative and positive serum, respectively reacting at room temperature for 5, 10, 15 and 30min under constant conditions, testing by conventional method, and determining OD_450nmValues and P/N values.

As a result, the P/N value was maximum at 6.043 at a substrate reaction time of 10min at room temperature, and therefore the substrate reaction time was selected to be 10min at room temperature.

TABLE 13 selection of substrate action time

3.5 determination of negative and positive cutoff values of Indirect E L ISA

Indirect E L ISA party built as described aboveThe method is used for detecting 100 parts of negative antibody serum of the duck egg drop-laying syndrome virus stored in a laboratory. Repeat 2 wells for each sample, average the results, calculate the mean (X) and Standard Deviation (SD) of the OD450nm values of the samples, OD450nm > OD4 of the negative samples according to the statistical principle_50nmThe value X +2SD was found to be positive at a level of 99.9%.

The result shows that the average OD450nm (X) of 100 duck egg hypogenesis syndrome virus negative antibody sera subjected to indirect E L ISA is 0.317, the standard deviation SD is 0.085, according to the formula, the negative and positive critical value is X +2SD, and the experiment critical value is 0.487, namely the OD of the sample to be tested_450nmThe antibody is positive when the value is more than or equal to 0.487, and the OD_450nmValues < 0.487 are antibody negative.

3.6 specificity test

The positive sera of DHAV-I, DHAV-III, DEV, DRV, NGPV and AIV were assayed for OD using the established indirect E L ISA method_450nmValues of 0.227, 0.196, 0.309, 0.218 and 0.264, positive serum OD for the above viruses_450nmThe values are all smaller than the negative and positive critical value of 0.487, and are all negative, which indicates that the established indirect E L ISA method has better specificity.

TABLE 14 specificity test of Indirect E L ISA

3.7 sensitivity test

Diluting the positive serum of the duck egg laying-reduction syndrome virus at a 1: 100-1: 12800-fold ratio, and carrying out indirect E L ISA test.

The results showed that the test was positive after 1:3200 dilution, while the test was negative below 0.487 for the positive serum at 1:6400 dilution.

TABLE 15 sensitivity test of Indirect E L ISA

3.8 in-and between-run repeatability experiments

3 parts of duck egg drop-laying syndrome virus positive serum and 3 parts of duck egg drop-laying syndrome virus negative serum with different antibody titers are determined by the established indirect E L ISA method, the duck egg drop-laying syndrome virus positive serum and the 3 parts of duck egg drop-laying syndrome virus negative serum are added into a whole virus antigen prepared in the same batch to coat different E L ISA plates, each serum is subjected to parallel processing of 5 holes, and the OD of each serum is determined according to the OD of each serum_450nmCalculating the standard deviation of the values, and further calculating the OD of each serum_450nmThe in-plane coefficient of variation of the values. The experiment was repeated on 3 plates, according to the OD measured_450nmValue calculation of OD of each serum between plates_450nmCoefficient of variation of values (CV) batch-to-batch reproducibility test 3 different batches of purified whole virus E L ISA were tested for plate, 6 sera were tested for indirect E L ISA, and the results were statistically analyzed.

The result of the batch repeatability test shows that the coefficient of variation is 1.498 to 3.556 percent, which indicates that the same sample has small variation degree and better repeatability in the same batch test. The variation coefficient of the batch repeatability test is 2.093% -4.371%, which shows that the same sample has smaller variation degree in different batches of antigen tests and good repeatability.

TABLE 16 in-batch repeatability tests

TABLE 17 repeatability tests between batches

3.9 compliance rate test

The established indirect E L ISA test and the established Johnson amplification test (AGP) are used for comparing, 100 parts of clinical samples in the areas such as Jiangsu and Anhui are detected, the positive and negative judgment results obtained by detecting the same serum by the indirect E L ISA method are compared with the traditional Johnson amplification test, and the positive detection rates of the two are calculated.

The result is that the established indirect E L ISA method is compared with an agar diffusion test (AGP) to detect 100 clinical duck serum samples from Jiangsu, Anhui and other areas, the positive detection rate of the indirect E L ISA method is 39% (39/100), the positive detection rate of the agar diffusion test (AGP) is 18% (18/100), and the sensitivity of the established indirect E L ISA method is obviously higher than that of the agar diffusion test (AGP).

Example 4 detection method of duck egg drop-laying syndrome virus blocking E L ISA antibody

4.1 preparation of antigen A duck egg laying-reduction syndrome virus AH204 strain is inoculated to non-immune duck embryos of 10-11 days old, the duck embryos are continuously incubated for 6 days, dead duck embryos after 24 hours are harvested, allantoic fluid is collected, the duck embryos are inactivated by formaldehyde with the final concentration of 2 per mill for 24 hours, 10000g of the duck embryos are centrifuged for 60 minutes at 4 ℃ to remove impurity proteins, supernatant fluid is collected, the supernatant fluid is ultracentrifuged for 5 hours at 4 ℃, the supernatant fluid is discarded, precipitates are dissolved by PBS (concentrated by 200 times according to the original volume), the protein concentration is measured, and the duck eggs are subpackaged and stored at-70 ℃ and are used as E L ISA coated antigen.

4.2 blocking E L ISA operating program

(1) Diluting antigen with pH 9.6 carbonate buffer solution, coating reaction plate with 0.1ml per well, and standing overnight at 4 deg.C; discard coating solution, wash 3 times with 0.05% Tween-20PBS (PBST), 3 minutes each time;

(2) adding blocking solution (PBS solution containing 5% skimmed milk) 0.1ml per well, acting at 37 deg.C for 1 hr, washing with PBST, and the same method as above;

(3) adding the serum to be tested diluted by the antibody diluent and the negative and positive control serum (diluted by 10 times), 0.1ml of the serum is added into each hole, incubating for 1 hour at 37 ℃, and washing the serum, wherein the method is the same as the method;

(4) adding 0.1ml of specific antibody (diluted by 500 times) into each well, incubating for 1 hour at 37 ℃, and washing for 3 times;

(5) adding 0.1ml of enzyme-labeled secondary antibody (diluted by 2000 times) into each hole, incubating for 1 hour at 37 ℃, and washing for 3 times;

(6) adding 0.1ml of TMB substrate color development solution into each hole, and developing at room temperature in a dark place (10 minutes);

(7) adding 0.05ml stop solution into each hole, and reading the absorbance value OD by an enzyme-linked immunosorbent assay_450nmAnd calculating the blocking rate.

The blocking rate (%) - (absorbance value of negative control-absorbance value of sample to be measured)/absorbance value of negative control × 100% was 100%.

4.3 optimization of optimal antigen coating concentration and serum dilution purified antigen (1.627mg/m L) qualified for inspection is diluted by coating solution and then diluted by 2 times, the purified antigen is diluted by 16, 8, 4, 2, 1 and 0.5ug/m L in sequence, antigens with 6 dilution are respectively added into a 96-hole E L ISA ELISA plate, each dilution has 6 holes, each hole is placed at 0.1 ml.4 ℃ for 15-18 hours, the coating solution is discarded, the cleaning solution is added into the plate, the plate is discarded after 3 minutes of washing, the cleaning solution is discarded after 3 times of washing, sealing solution is added into the plate, each hole is 0.1ml, the plate is placed at 20-25 ℃ for 1 hour, the sealing solution is discarded, positive serum is diluted to 1:5, 1:10 and 1:20, the negative serum is diluted to 1:5, 1:10 and 1:20, the plate is added into the plate holes coated with different antigen concentrations, each serum is provided with 3 repeated holes, each hole is provided with 0.1, 0.1: 10 and 1:1 hour of positive serum, the plate is placed on the plate, the plate is placed under 1.1.1 hour of a 1, the plate is placed under 1.1.1, the same antigen concentration of a 1, the plate is placed under 0.1.1, the plate is placed under the same as the plate is placed under the plate, the plate is placed under the same as the plate, the plate is placed under the plate, the plate is placed under the plate_450nmThe value is obtained. OD of negative serum_450nmValue of about 1.5, negative serum OD_450nmValue and Positive serum OD_450nmThe antigen concentration at which the ratio is the maximum is determined as the optimal antigen coating concentration.

Coating the ELISA plate with detection antigen 4ug/m L as OD of negative serum_450nmValue of about 1.5, negative serum OD_450nmValue and Positive serum OD_450nmThe ratio is maximal, thus determining the antigen concentration of 4ug/m L as the optimal antigen coating concentration and the optimal dilution of serumThe number is 1: 10.

Table 18 OD of square matrix titration_450nmValue of

4.4 determination of optimal blocking solution and blocking time an ELISA plate (100. mu.l/well) is coated with an antigen with an optimal concentration determined by 2.3, the plate is placed at 4 ℃ for 15-18 hours, washed for 3 times, respectively added with 5% skim milk, 1% gelatin, 0.1% BSA, 1% bovine serum for blocking and commercialized blocking solution, and the blocking solution is blocked for 1 hour and determined according to a 2.4 operation procedure.

The best sealing liquid determined by the test is commercial sealing liquid, and the sealing condition is sealing for 1 hour at 37 ℃.

TABLE 19 determination of confining liquids

4.5 determination of optimal dilution and working time of specific antibody according to the determined antigen coated ELISA plate with optimal concentration, and sealing with determined sealing liquid and conditions. Taking positive serum and negative serum to act according to determined dilution and time, respectively diluting specific antibody at 1:250, 1:500, 1:1000, 1:2000 and 1:4000, adding 0.1ml into each hole, incubating for 1 hour at 37 ℃, and determining the optimal dilution of the specific antibody according to the operation program.

The test determined that the specific antibody used was diluted 1:1000 fold at working concentration for 1 hour.

TABLE 20 determination of optimal dilution of specific antibodies

4.6 determination of optimal working concentration and working time of the enzyme-labeled antibody, namely coating the enzyme-labeled plate with the determined antigen with the optimal concentration, sealing with the determined sealing solution and conditions, diluting the serum according to the determined dilution and the action time, diluting the specific antibody according to the determined conditions, diluting the goat anti-mouse enzyme-labeled antibody by 1:1000, 1:2000, 1:4000 and 1:8000 times respectively, diluting at 37 ℃ for 1 hour, and determining the optimal working concentration and the working time of the enzyme-labeled antibody according to the operation program.

The test determines that the working concentration of the used enzyme-labeled antibody is 1:2000 times diluted, the working condition is 37 ℃, and the incubation is carried out for 1 hour.

TABLE 21 determination of the optimal dilution of enzyme-labeled antibodies

4.7 determination of substrate color development time according to the determined antigen coating enzyme label plate with the optimum concentration, sealing with the determined sealing liquid and conditions, determining the serum dilution and action time according to the determined conditions, performing specific antibody according to the determined conditions, performing enzyme-labeled antibody according to the determined conditions, adding a substrate TMB, performing color development at room temperature in a dark place, wherein the color development time is respectively 5, 10, 15 and 20 minutes, and determining the optimum color development time according to the operation program.

The substrate development time determined in this experiment was 10 minutes at room temperature.

TABLE 22 determination of the development time of the substrate

4.8 cut-off value determination after the establishment of the blocking E L ISA method, 200 clinically derived negative sera were tested, the blocking rate was calculated, and the cut-off value of the test was determined as the mean inhibition rate of negative samples + 2-fold or 3-fold standard deviation.

The detection results show that 200 clinical duck egg laying-reduction syndrome virus negative serum samples show that the average blocking rate of the duck negative serum is 2.0 percent, the standard deviation is 7.8 percent, and the optimal critical value of the blocking rate is the standard deviation (17.6 percent and 25.4 percent) of the critical value which is the average inhibition rate plus 2 times or 3 times of the negative sample. According to the detection result, the determination standard of the duck egg drop-laying syndrome virus serum antibody is determined as follows: when the blocking rate is more than or equal to 25.4 percent, the product is positive; the product is suspicious when the blocking rate is more than 17.6% and less than 25.4%, and the product is judged to be negative when the retest rate is still less than 25.4%; when the blocking rate is less than or equal to 17.6 percent, the product is negative.

4.9 specificity assay

The established E L ISA blocking method is used for respectively detecting positive sera of duck hepatitis virus I (DHAV-I), duck hepatitis virus III (DHAV-III), duck plague virus (DEV), Duck Reovirus (DRV), novel duck goose parvovirus (NGPV) and avian influenza virus subtype H9 (AIV), and verifying whether the E L ISA method has cross reactivity to the positive sera of the viruses.

As a result, positive sera of DHAV-I, DEV, DRV, NGPV and AIV were assayed for OD by the established indirect E L ISA method_450nmThe values are 4.4%, 0.9%, -5.8%, 7.1% and 1.2%, respectively, and the positive serum blocking rate of the viruses is less than the negative and positive critical value of 17.6%, which indicates that the established method for blocking the E L ISA has better specificity.

Table 23 specificity test for blocking E L ISA

4.10 sensitivity test

Diluting the positive serum of the duck egg laying-reduction syndrome virus at a ratio of 1: 10-1: 1280, and performing a blocking E L ISA test.

The results show that the serum was still detectable as positive after dilution 1:320, while the blocking rate of the serum was negative below 17.6% when the serum was positive at dilution 1: 640.

TABLE 24 susceptibility test to block E L ISA

4.11 in-and between-run repeatability tests

The method comprises the steps of coating different E L ISA plates with duck egg laying-down syndrome virus antigens prepared in the same batch, taking 3 parts of positive serum and 3 parts of negative serum of different antibody levels of the duck egg laying-down syndrome virus, determining the positive serum and the negative serum according to an indirect E L ISA program in the same condition, performing a 5-hole parallel test on each blood sample, performing statistical analysis on the result, coating different E L ISA plates with viruses prepared and purified in 3 different batches, taking 6 parts of the serum, determining the serum according to a blocking E L ISA program in the same condition, and performing statistical analysis on the result.

The result is that the antigen of the same batch is used for carrying out repeated detection on 3 parts of duck egg drop-laying syndrome virus positive serum and 3 parts of duck egg drop-laying syndrome virus negative serum, the result is analyzed by statistics, the coefficient of variation is between 0.907 and 3.656 percent and is less than 10 percent, which shows that the same sample has small variation degree in the same batch of tests and better repeatability, the repeated tests among E L ISA plate batches are prepared by 3 different batches, the coefficient of variation is between 1.318 and 5.158 percent, which shows that the same sample has small variation degree in the antigen tests of different batches of the antigen and has better repeatability.

TABLE 25 in-batch repeatability tests

TABLE 26 repeatability tests between batches

4.12 compliance test

The established blocking E L ISA test and the established Johnson amplification test (AGP) are used for comparing, 100 parts of clinical samples in the areas of Jiangsu, Anhui and the like are detected, the positive and negative judgment results obtained by detecting the same serum by using the blocking E L ISA method are compared with the traditional Johnson amplification test, and the positive detection rate and the coincidence rate of the two are calculated.

The result is that the established E L ISA blocking method is compared with an agar diffusion test (AGP) to detect 100 clinical duck serum samples from Jiangsu, Anhui and other areas, the positive detection rate of the E L ISA blocking method is 37 percent (37/100), the positive detection rate of the Johnson amplification test (AGP) is 18 percent (18/100), and the sensitivity of the established E L ISA blocking method is obviously higher than that of the Johnson amplification test (AGP).

Table 27 compliance testing

The results in the block E L ISA test and the agar diffusion test (AGP) parallel test of the same value.

The above-mentioned embodiments only express the embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Shanghai animal doctor institute of Chinese academy of agricultural sciences (Shanghai center of Chinese centers of animal health and epidemiology)

<120> duck egg laying reduction syndrome virus antibody detection method and kit thereof

<160>2

<170>PatentIn version 3.3

<210>1

<211>2767

<212>PRT

<213> Duck egg drop syndrome virus (Duck egg-drop syndrome virus)

<400>1

Met Asp Thr Leu Thr Gln Pro Ile Lys Lys Ile Ala Gly Glu Val Glu

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Ile Ser Ala Ala Thr His Gln Gly Glu Gln Ala Ala Ser Ile Glu Thr

35 40 45

Gly Gln Pro Thr Glu Val Thr Asp Val His Thr Asp Gly Ser Thr Asp

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Asp Met Leu Ser Cys Ser Met Ser Val Asp Phe Tyr Lys Glu Asn Phe

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Thr Lys Leu Val His Leu Ser Thr Phe Gln Trp Asn Thr Thr Asn Gly

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Ile Gly His Arg Leu Asp Gly Arg Trp Leu Pro Asn Val Phe Phe Gln

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Asn Phe Met Pro Thr Gln Ser Ala Ser Gln Leu Phe Ser Tyr Leu Arg

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Cys Gly Tyr His Phe Arg Met Leu Val Asn Ala Ser Pro Gly Val Leu

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Gly Ser Leu Ile Cys Val Tyr Ile Pro Gly Gly Tyr Cys Lys Thr Phe

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Asp Ser Ser Phe Pro Arg Asp Phe Lys Ser Val Leu Ser Leu Pro His

165 170 175

Thr Ile Leu Asp Val Arg Cys Ser Asn Gln Ala Asp Leu Val Val Pro

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Tyr Ala Asn Tyr Thr Asn Tyr Val His Tyr Thr Lys Thr Gly Pro Leu

195 200 205

Glu Asp Gly Ala Met Val Cys Val Tyr Val Phe Ala Lys Met Glu Val

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Gly Ser Gly Phe Ser Gly Glu Ile Asp Val Ser Leu Tyr Gly Glu Leu

225 230 235 240

Ile Glu Ala Asp Phe Gln Ala Pro Arg Pro Ile Asn Gln Gly Arg Pro

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Lys Arg Arg Gln Val Lys Pro Pro Pro Lys Pro Pro Val Lys His His

260 265 270

Thr Met Val Asp Gly Ala Pro Gly Cys Cys Asn Leu Ser Asn Val Glu

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Ser Thr Gly Thr Ser Glu Ser Leu Ala Leu Val Gly Glu Ser Thr Ala

290 295 300

Ile Asp Tyr Ala Thr Ala Gly Cys Ser Ser Asn Ile Asn Asp Phe Ile

305 310 315 320

Glu Ile Met Arg Lys Trp Val Ile Ile Asp Gln Gly Val Trp Glu Asn

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Thr Val Gly Arg Gly Ser Glu Ile Thr Ala Leu Asn Leu Gln Pro Tyr

340 345 350

Arg Tyr Gly Asn Met Gly Leu Ile Leu Gly Cys Phe Gln Phe Phe Arg

355 360 365

Gly Ser Phe Glu Ile Lys Val Leu Thr Tyr Ala Ser Pro Leu Ala Thr

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Ala Arg Tyr Gln Ile Thr Trp Phe Pro Glu Tyr Tyr Glu Thr Val Gly

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Ile Asp Lys Gln Arg Asn Gly Val Tyr Leu Thr Ala Asp Ile Gly Cys

405 410 415

Glu Ser Gly Thr Leu Val Leu Pro Phe Thr Ser Ser Thr Trp Arg Arg

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Gln Cys Asp Gln Pro Tyr Gly Arg Ile Thr Met Ser Cys Ile Asn Lys

435 440 445

Ile Ala Tyr Asn Thr Thr Ala Pro Asn Lys Val Ser Tyr Lys Ile Leu

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Met Arg Val Gly Pro Asp Phe Gln Val Phe Cys Pro Arg Leu Pro Val

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Leu Ser Ile Gln Gly Leu Gly Asp Gly Ser Ser Asp Pro Val Leu Phe

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Ile Asn Tyr Glu Val Asp His Ile Pro Ile Gln Ser Gln Ser His Ser

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Asn Val Asn Ala Leu Leu Gly Arg Ile Gln His Tyr Gly Lys Phe Thr

515 520 525

Leu Thr Ala Ala Thr Ile Asn Gln Ala Glu Ile Thr Leu Val Asn Lys

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Arg Pro Tyr Lys Ile Leu Glu Thr Val Ala Tyr Trp Ser Gly Glu Leu

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Leu Phe Ser Ile Leu Asn His Cys Pro Ser Pro Leu Tyr Phe Ala His

565 570 575

Lys Tyr Thr Ser Tyr Asn Phe Thr Asn Gln Glu Asp Met Met Ala His

580 585 590

Gly Val Ile Leu Ile Pro Ala Asn Gly Met Lys Thr Ile Asn Ile Pro

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Phe Tyr Ser Asp Thr Pro Leu Arg Arg Thr Ile Asp Asn Phe Gly Arg

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Ile Ala Leu Leu Ser Lys Glu Ala Gly Gln Val Glu Val Asn Ile Ala

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Phe Arg Lys Pro Ser Phe Phe Phe Pro Ile Pro Cys Thr Ser Thr Ala

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Gln Ala Thr Ser Tyr Val Lys Asp Leu Thr Ile Asp Gly Asp Val Glu

660 665 670

Ser Asn Pro Gly Pro Gln Tyr Val Arg Gln Arg Ile Asp Leu Gly Glu

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Glu Tyr Ile Gln Tyr Glu Phe Lys Lys Trp Arg Gly Leu Leu Val Thr

690 695 700

Asn Lys Val Leu Val Trp Ser Leu His Lys Gly Pro Tyr Pro Ser Pro

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Ala Ser Phe Thr Cys Tyr Glu Gln Thr Lys Gln Thr Leu Trp Lys Lys

725 730 735

Lys Met Phe Ile Glu Trp Glu Leu Asp Tyr Asn Gly Val Thr Tyr Trp

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Ser Arg Gln Glu Ile Glu Arg Lys Trp Asn Phe Gln Lys Thr Cys Arg

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Glu Val Glu Gly Ser Tyr Val Lys Asp Leu Thr Glu Glu Gly Ile Glu

770 775 780

Pro Asn Pro Gly Pro Ala Asp Phe Ser Ser Phe Ser Ser Gly Phe Ser

785 790 795 800

Ser Val Thr Ser Arg Ala Tyr Gly Glu Asn Phe Thr His Tyr Trp Gln

805 810 815

Asn Asn Asn Tyr Thr Asn Gly Tyr Tyr Thr Thr Asn Leu Thr Thr Val

820 825 830

His Ala Lys Ser Asn Gly Ile Pro Ser Pro Pro Val Phe Thr Ala Lys

835 840 845

Val Thr Thr Thr Ala Ser Trp Phe Thr Val Lys Val Arg Val Val Val

850 855 860

Ser Ile Arg Thr His Gln Gly Trp Lys Met Phe Arg Ala Lys Phe Lys

865 870 875 880

Leu Asn Arg Met Thr Phe Ala Arg Trp Asn Pro Ile Glu Phe Gln Glu

885 890 895

Thr Leu Pro Glu Ile Val Ala Tyr Ser Thr Val Thr Asn Ser Gly Thr

900 905 910

Arg Gly Ser Gln Trp Thr Val Ile Arg Ser Thr Glu Val Ala Gly Ser

915 920 925

Ala Val Ser Leu Ser Lys Thr Trp Asp Arg Ile Asp Val Arg Val Tyr

930 935 940

Pro Thr Gln His Thr Gln Pro Tyr Leu Leu Lys Ile Gly Asn Ala Ala

945 950 955 960

Trp Val Arg Asp Leu Thr Glu Asp Gly Asp Val Glu Glu Asn Pro Gly

965 970 975

Pro Thr Gln Trp Leu Cys Asp Arg Asp Met Ile His Ala Arg Asp Gly

980 985 990

Trp Met Met Val Asp Thr Tyr Ile Leu Val Lys Lys Asn Gly Asp Arg

995 1000 1005

Met Arg Met Val Gly Pro Leu Ile Ser Val Ile Lys Thr Lys Leu

1010 1015 1020

Asn Asp Gly Arg Ser Gln Val Glu Tyr Arg Trp Arg Arg Ser Leu

1025 1030 1035

Ile His Ser Pro Glu Ser Val Val Tyr Asn Cys His Glu Ser Cys

1040 1045 1050

Trp Asn Arg Asp Leu Thr Ile Asp Gly Asp Val Glu Leu Asn Pro

1055 1060 1065

Gly Pro Arg Asp Pro Leu Pro Cys Ser Thr Val Glu Arg Lys Glu

1070 1075 1080

Trp Glu His Asn Gly Val Leu Tyr Tyr Tyr Thr Lys Tyr Thr Lys

1085 1090 1095

Ser Phe Arg Trp Phe Gly His Val Tyr Glu Gly Asn Val Ser Leu

1100 1105 1110

Val His Val Met Glu Val His Pro Asp Asn Arg Arg Lys Asp Thr

1115 1120 1125

Phe Lys Leu Lys Asp Glu Asn Gly Gln Ile Tyr Asp Trp Val Phe

1130 1135 1140

Lys Cys His Glu Lys Cys Trp Gln Lys Asp Pro Thr Gln Asp Gly

1145 1150 1155

Asp Val Glu Gln Asn Pro Gly Pro Tyr Leu Glu Ile Thr Thr Trp

1160 1165 1170

Arg Val Gly Asn Val His Ile Thr Glu His Cys Tyr Asp Gly Gly

1175 1180 1185

Leu Ile Ile His Gln Thr Phe Ile Asn Trp Ser Asn Gly Ala Lys

1190 1195 1200

Lys Glu Val Phe Ile Val Glu Asp Arg Cys Tyr Glu Phe Lys Cys

1205 1210 1215

His Glu His Cys Trp Val Arg Asp Leu Thr Met Asp Gly Asp Val

1220 1225 1230

Glu Glu Asn Pro Gly Pro Trp Ser Pro Asp Thr Lys Thr Ile Met

1235 1240 1245

Val Leu Gly Ala Thr Gly Ser Gly Lys Ser Tyr Ala Ala Asn Lys

1250 1255 1260

Ile Leu Gly Lys Glu Ala Phe Ile His Lys Leu Ser Thr Lys Ser

1265 1270 1275

Val Thr Tyr Cys Asp Gln Ala Ile Thr His Gly Asn Leu Thr Val

1280 1285 1290

Ile Asp Thr Ala Pro Leu Pro Lys Leu Thr Asp Phe Val Ser Gly

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Phe Val Tyr Val His Lys Ala Gly Arg Phe Asn Ala Glu Glu Lys

1310 1315 1320

Ala Tyr Leu Asp Leu Leu Asp Lys Met Leu Pro Asn Trp Gln Ala

1325 1330 1335

His Ala Val Leu Leu Val Pro Gln Arg Glu Val Gln Lys Val Tyr

1340 1345 1350

Val Glu Asp Tyr Ile Lys Asn His Lys Glu Leu Ser Gly Leu Ala

1355 1360 1365

Phe Lys Met Met Gly Arg Ile Thr Asp Ser Tyr Glu Ile Ala Lys

1370 1375 1380

Lys Met Ile Cys Glu Cys Leu Pro Ser Pro Tyr Phe Ser His His

1385 1390 1395

Tyr Lys Leu Val Tyr Lys Asn Arg Gly Ala Tyr Arg His Tyr Gly

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Val Leu Ser His Gly Arg Val Phe His Leu Asn Thr Ala Asp Ile

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Leu Lys Ser Ala Leu Ser Gly Ser Ala Ser Val Gln Val Asp His

1430 1435 1440

Asn Pro Gln Glu Trp Ile Lys Ala Glu Glu Asn Gly Tyr Arg Ser

1445 1450 1455

Ala Leu Tyr Leu Val Asn Ala Gly Ala Ile AspLeu Asp Phe Asn

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Phe Asp Ser Asn Cys Glu Thr Trp Ala Lys Thr Ile Leu Gly Ser

1475 1480 1485

Asp Gln Ala Cys Gln Gly His Arg Leu Lys Trp Cys Leu Thr Leu

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Ala Ala Ala Ala Ala Phe Met Phe Ser Gly Val His Ile Glu Asp

1505 1510 1515

Gln Ser Pro Gly Leu Phe Ser Lys Ile Ile Thr Ser Ile Ser Gly

1520 1525 1530

His Phe Tyr Lys Asn Leu Glu Cys Val Val Ile Lys Thr Val Ile

1535 1540 1545

Arg Thr Val Cys Arg Ile Leu Cys Tyr Leu Ile Leu Tyr Cys His

1550 1555 1560

Ser Pro Asn Leu Leu Thr Thr Gly Val Ile Ile Ala Leu Ile Ser

1565 1570 1575

Met Asp Val Thr Ser Ile Glu Ile Asp Ala Arg Val Lys Ala Ala

1580 1585 1590

Cys Glu Ser Leu Ala Asn Gly Glu Phe Ala Gln Phe Cys Ser Asp

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Ile Ile Asp Leu Thr Gly Asp Pro Asp Tyr Val Asp Leu Lys Ser

1610 1615 1620

His Ile Pro Gln Phe Thr Asn Lys Asn Tyr Thr Leu Gln Arg Leu

1625 1630 1635

Gln Gln Glu Ala Ile His Gly Gln Ile Glu Met Glu Lys Lys Met

1640 1645 1650

Gly Pro Leu Ser Ile Asn Glu His Pro Asp Asn Cys Asp Cys Tyr

1655 1660 1665

Leu Cys Ser Asp Leu Lys Thr Cys Ser Gly Lys Glu Asp Cys Tyr

1670 1675 1680

Cys Pro Lys Cys Arg Lys Pro His Asn Gln Gly Pro Lys Ser Phe

1685 1690 1695

Asn Asp Trp Thr Thr Ala Ala Lys Asn Val Lys Trp Trp Ile Glu

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Ser Leu Met Lys Cys Phe Glu Trp Leu Arg Asp Lys Ile Phe Pro

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Gln Asp Ala Ala Lys Lys Ile Ala Glu Leu Glu Leu Arg Ser Ala

1730 1735 1740

Glu Ile Ala Thr Val Met Ala Leu Ala Asp Glu His Ile Cys Lys

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Cys Arg Thr Asn Lys Asn Tyr Val Leu His Lys Asp Thr Pro Lys

1760 1765 1770

Lys His Ala Ala Leu Val Asp Arg Leu Leu Ser Phe His Ile Asp

1775 1780 1785

Glu Leu Pro Ser Gln Leu Ser His Leu Gln Gln Lys Leu Asn Asn

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Leu Leu Thr Arg Leu Gln Asn Ile Asn Ile Glu Pro Pro Leu Gln

1805 1810 1815

Tyr Ala His Arg Val Glu Pro Leu Gly Ile Trp Ile Gln Gly Ala

1820 1825 1830

Pro Gly Cys Gly Lys Ser Phe Leu Ser His Tyr Ile Val Lys Glu

1835 1840 1845

Leu Gln Lys Arg Tyr Gly Trp Glu Pro Tyr Ser His Pro Ile Gly

1850 1855 1860

Ser Glu His Met Asp Gly Tyr Thr Asp Gln Glu Ile His Ile Phe

1865 1870 1875

Asp Asp Leu Gly Gln Asn Arg Glu Glu Glu Asp Val Gly Leu Met

1880 1885 1890

Cys Asn Leu Ile Ser Ser Val Pro Phe Ile Val Pro Lys Ala Ala

1895 1900 1905

Leu Glu Ser Lys Gly Cys Gln Tyr Asn Gly Lys Val Val Ile Ala

1910 1915 1920

Thr Thr Asn Lys Arg Asp Phe Thr Thr Asn Lys Leu Leu Asp Ser

1925 1930 1935

Gly Ala Leu Gln Arg Arg Phe Pro Ile Ile Leu Glu Ile Arg Pro

1940 1945 1950

Arg Glu Lys Tyr Arg Arg Asp Asp Ala Cys Lys Trp Ser Lys Phe

1955 1960 1965

Asn Ala Val Asn Ala Thr Gly Asp Gly Ser Leu Met Arg Gly Glu

1970 1975 1980

Cys Trp Glu Ile Asn Val Asp Ala Arg Asn Thr Leu Arg Thr Ser

1985 1990 1995

Glu His Trp Gln His Leu Asn Pro Gln Asn Leu Met Asp Glu Ile

2000 2005 2010

Phe Gln Glu Ile Asp Ser Arg Leu Lys Val Cys Asn Phe Met Asn

2015 2020 2025

Gln Gly Lys Cys Arg Ile Thr Leu Asp Ser Asp Glu Pro Asp Met

2030 2035 2040

Leu Ser Asp Met Phe Pro Glu Pro Pro Lys Asn Lys Glu Lys Phe

2045 2050 2055

Val Gln Tyr Val Ser Ser Ala Ile Gly Ser Phe Lys Glu Phe Val

2060 2065 2070

Asp Arg Asn Arg Thr Trp Phe Val Ala Ala Gly Ala Leu Gly Thr

2075 2080 2085

Ile Ile Ser Leu Ala Ser Ile Thr Ile Pro Tyr Val Lys Lys Trp

2090 2095 2100

Met Ala Lys Asp Ala Thr Glu Glu Glu Asn Phe Tyr Gly Gly Lys

2105 2110 2115

Val Gly Pro Leu Arg Leu Lys Asp Tyr Lys Leu Pro Leu His Asn

2120 2125 2130

Gln Gly Pro Leu Asp Met Lys Ser Ile Ser Lys Leu Leu Val Thr

2135 2140 2145

Ile Glu Asp Glu Asp Gly Asp Leu Ala Thr Gly Leu Ala Ile Gly

2150 2155 2160

Asp Lys Thr Val Val Thr Phe Gly His Glu Asn Phe Lys Lys Val

2165 2170 2175

Val Cys Phe Arg Asp Thr Glu Val Asn Trp Glu Met Val Asn Ser

2180 2185 2190

Thr Gln Ile Thr Ile Asn Gly Asp Ser Met Asp Leu Arg Gln Tyr

2195 2200 2205

Asp Val Lys Ser Asp Ile Gln Phe Lys Ser Val Asn His Lys Ile

2210 2215 2220

Tyr Gly Glu Asp Tyr His Gly Asp Gly Tyr Leu Val Trp Lys Glu

2225 2230 2235

Met Lys His Tyr Leu Tyr Ile Pro Val Thr Asn Ile Arg Pro Thr

2240 2245 2250

Ser Thr Ile Ile Thr Gln Gln Gly Thr Thr Thr Gln HisThr Tyr

2255 2260 2265

Ser Tyr Val Gly Lys Thr Trp Arg Gly Leu Cys Gly Ala Leu Leu

2270 2275 2280

Val Gly Val Val Asn Gly Asn Pro Lys Ile Leu Gly Ile His Val

2285 2290 2295

Ala Gly Asn Lys Ser Leu Gly Met Ala Ala Arg Leu Phe Pro Met

2300 2305 2310

Phe Asn Gln Gly Lys Ala Lys Val Val Gly Pro Asn Pro Thr Pro

2315 2320 2325

Tyr Tyr Gln Pro Arg Lys Thr Lys Tyr Glu Pro Ser Pro Val Gln

2330 2335 2340

Gln Asp Glu Pro Thr Phe Gly Pro Ala Val Leu Ser Asn Lys Asp

2345 2350 2355

Lys Arg Leu Glu Val Gln Ile Glu Asp Ile Thr Lys His Ala Ala

2360 2365 2370

Gln Lys Tyr Ile Gly Asn His Phe Asp Pro Pro Arg Gly Ala Phe

2375 2380 2385

Gln Met Ala Lys Ser His Val Thr Gln Leu Leu Ser Gln Val Leu

2390 2395 2400

Glu Val Glu Asp Asn Met Ser Phe Glu Gln Ala Val Thr Ser Asp

2405 2410 2415

Val Leu Pro Ile Asp Trp Gln Thr Ser Ser Gly Leu Lys Tyr Ile

2420 2425 2430

Gly Phe Ser Lys Lys Gln Leu Val Gln Met Glu Ser Phe Lys Ala

2435 2440 2445

Asp Val Leu Lys Ile Leu Glu Gly Gly Glu Thr Phe Phe Thr Cys

2450 2455 2460

Tyr Leu Lys Asp Glu Leu Arg Pro Asn Asp Lys Val Ala Ile Gly

2465 2470 2475

Lys Thr Arg Ala Ile Glu Ala Gly Asn Phe Asp Tyr Val Ile Ala

2480 2485 2490

Trp Arg Met Val Met Gly Arg Leu Thr Ala Arg Leu Phe Asn Asp

2495 2500 2505

Phe Asp Arg Ile Thr Gly Phe Ala Pro Gly Leu Asn Pro Tyr Val

2510 2515 2520

Tyr Trp Asp Ser Met Met Glu Asn Val Lys Glu Ser Val Ile Gly

2525 2530 2535

Leu Asp Phe Lys Asn Tyr Asp Gly Ser Leu Ser Pro Gln Val Met

2540 2545 2550

Glu Ala Ala Val Glu Val Leu Ala Cys Phe His Lys Gln Pro Glu

2555 2560 2565

Leu Val Lys Leu Ile His Tyr Pro Thr Ile Tyr Ser Thr Asn Leu

2570 2575 2580

Val Ser Asp Glu Lys Trp Phe Val Glu Gly Gly Met Cys Ser Gly

2585 2590 2595

Ser Pro Cys Thr Thr Val Leu Asn Thr Ile Val Asn Leu Ile Val

2600 2605 2610

Asn Tyr Thr Val Met Phe Asp Tyr Gly Tyr Ser Pro Ser Glu Leu

2615 2620 2625

Tyr Ile Ile Gly Tyr Gly Asp Asp Thr Val Ile Ser Ala Asp Arg

2630 2635 2640

Lys Val Ala Ile Ser Asp Ile Ala Ser Lys Tyr Lys Lys Tyr Phe

2645 2650 2655

Gly Met Asn Val Thr Ser Ala Ala Lys Thr Asp Gln Ile Gly Trp

2660 2665 2670

Gln Pro Lys Glu Lys Leu Glu Phe Leu Lys Arg Ser Thr Ala Leu

2675 2680 2685

Phe Pro His Thr Thr Lys Ile Val Gly Lys Leu Asp Leu Lys Asn

2690 2695 2700

Met Val Gly His Leu Asp Trp Thr Ser Gly Thr Phe Gln Glu Gln

2705 2710 2715

Leu Asn Ser Phe Tyr Leu Glu Leu Val Leu His Gly Gln Glu Ile

2720 2725 2730

Tyr Asp Lys ValArg Asn Tyr Asn Gln Lys Lys Ala Pro Ser Tyr

2735 2740 2745

Asn His Leu Ser Phe Gly Ala Ala Tyr Glu Met Met Lys Thr Ile

2750 2755 2760

Cys Leu Val Tyr

2765

<210>2

<211>173

<212>PRT

<213> Duck egg drop syndrome virus (Duck egg-drop syndrome virus)

<400>2

Gly Leu Gly Asp Gly Ser Ser Asp Pro Val Leu Phe Ile Asn Tyr Glu

1 5 10 15

Val Asp His Ile Pro Ile Gln Ser Gln Ser His Ser Asn Val Asn Ala

20 25 30

Leu Leu Gly Arg Ile Gln His Tyr Gly Lys Phe Thr Leu Thr Ala Ala

35 40 45

Thr Ile Asn Gln Ala Glu Ile Thr Leu Val Asn Lys Arg Pro Tyr Lys

50 55 60

Ile Leu Glu Thr Val Ala Tyr Trp Ser Gly Glu Leu Leu Phe Ser Ile

65 70 75 80

Leu Asn His Cys Pro Ser Pro Leu Tyr Phe Ala His Lys Tyr Thr Ser

85 90 95

TyrAsn Phe Thr Asn Gln Glu Asp Met Met Ala His Gly Val Ile Leu

100 105 110

Ile Pro Ala Asn Gly Met Lys Thr Ile Asn Ile Pro Phe Tyr Ser Asp

115 120 125

Thr Pro Leu Arg Arg Thr Ile Asp Asn Phe Gly Arg Ile Ala Leu Leu

130 135 140

Ser Lys Glu Ala Gly Gln Val Glu Val Asn Ile Ala Phe Arg Lys Pro

145 150 155 160

Ser Phe Phe Phe Pro Ile Pro Cys Thr Ser Thr Ala Gln

165 170

Claims

1. An antibody against duck egg drop-laying syndrome virus.

2. The antibody of claim 1, wherein said antibody is an antibody produced by immunizing an animal with strain AH204 of duck egg drop delivery syndrome virus.

3. The antibody of claim 1, wherein said antibody consists of SEQ ID NO: 1 or a partial protein fragment thereof, and immunizing animals to produce antibodies.

4. The antibody of claim 1, wherein said antibody consists of a heavy chain variable region of SEQ ID NO: 1 or a partial protein fragment thereof, wherein the homology of the amino acid sequence shown in the formula 1 is more than 98 percent.

5. The antibody of claim 3, wherein said antibody consists of SEQ ID NO: 2 or an active fragment thereof, and immunizing animals to produce antibodies.

6. A duck egg drop syndrome virus antibody detection kit comprising the antibody of any one of claims 1-5, and/or a whole virus antigen of duck egg drop syndrome virus or a major immunogenic protein antigen thereof.

7. The duck egg hypogenesis syndrome virus antibody detection kit according to claim 6, wherein the method for detecting duck egg hypogenesis syndrome virus antibody by using the kit comprises a two-way agarose diffusion test, an indirect E L ISA method, or a blocking E L ISA method.

8. The detection kit for duck egg hypogenesis syndrome virus antibody according to claim 7, wherein the kit for detecting duck egg hypogenesis syndrome virus antibody by using indirect E L ISA method or blocking E L ISA method further comprises an E L ISA enzyme label plate, enzyme-labeled secondary antibody, developing solution, positive control serum and negative control serum.

9. The duck egg hypopartum syndrome virus antibody detection kit according to claim 8, wherein the detection of duck egg hypopartum syndrome virus antibodies by the method of blocking E L ISA comprises the following steps:

10. The duck egg hypogenesis syndrome virus antibody detection kit according to claim 8, wherein the detection of duck egg hypogenesis syndrome virus antibodies by using indirect E L ISA method comprises the following steps:

11. Use of an antibody according to any one of claims 1 to 5 for the manufacture of a product for the diagnosis or treatment of duck egg drop-in syndrome.

12. Use of the duck egg hypopartus syndrome virus antibody detection kit of any one of claims 6-10 in the preparation of a product for diagnosing duck egg hypopartus syndrome.