CN111426828B

CN111426828B - Duck egg yield-reduction syndrome virus antibody detection method and kit thereof

Info

Publication number: CN111426828B
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: 2023-09-22
Anticipated expiration: 2039-01-09
Also published as: CN111426828A; CN111426828A8

Abstract

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

Description

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

Technical Field

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

Background

China is a large country for raising poultry, the poultry industry rapidly develops, the annual raising amount of poultry reaches 120 hundred million feathers, the world is the first place, and China is the first large country for raising waterfowl in the world and the large country for consuming. Although the aquatic bird raising amount is increased year by year in recent years, the scale and industrialization operation level is also improved rapidly, the Chinese aquatic bird raising technology is relatively backward, especially the occurrence of aquatic bird infectious diseases, so that the elimination rate of aquatic bird raising is high, the loss of the aquatic bird industry in China is up to hundreds of billions of primordial notes each year, and the aquatic bird raising technology becomes a restriction factor which puzzles the healthy development of the aquatic bird raising industry in China.

The duck raising industry in China faces a serious threat of new infectious diseases. In 2016 years, firstly, the egg laying rate of the ducks is reduced by 20-30% due to the unknown reasons in the duckling field, and the duckling eggs are recovered after a few weeks, but the duckling eggs are difficult to recover to the egg laying rate before the disease occurrence, so that the duckling field suffers great economic loss. The main symptom of the disease is egg laying decline of the laying duck, so the disease is named as duck egg lay reduction syndrome. The duck is examined by dissection, and the liver is provided with blood stasis in the early stage of the disease, and the ovary and the oviduct are damaged to different degrees. The disease is spread continuously in China, the disease is outbreak in main egg duck production areas of China, but no detection method and kit for diagnosing the duck egg reduction syndrome exist at present, the prevention and control of the duck egg reduction syndrome are very unfavorable, and the development of an effective duck egg reduction syndrome detection kit is urgently needed.

Disclosure of Invention

The invention aims to solve the technical problem that the existing diagnosis means for duck egg yield-reducing syndrome are limited, and provides a novel duck egg yield-reducing syndrome virus antibody detection kit which is a brand-new duck egg yield-reducing syndrome virus separated from duck egg yield-reducing syndrome onset ducks.

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

in one aspect of the invention, an antibody is provided against duck egg yield reduction syndrome virus.

Preferably, the antibody is an antibody raised from an immunized animal of duck egg reduced production syndrome virus strain AH 204.

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

More preferably, the antibody consists of SEQ ID NO:2 or an active fragment thereof.

In another aspect of the invention, there is also provided a duck egg reduced production syndrome virus antibody detection kit comprising the above antibody, and/or a whole virus antigen of a duck egg reduced production syndrome virus or a major immunogenic protein antigen thereof.

The method for detecting the duck egg yield reduction syndrome virus antibody by the kit comprises the following steps: a two-way agarose diffusion assay, an indirect ELISA method, or a blocking ELISA method.

Preferably, the kit for detecting the duck egg yield-reducing syndrome virus antibody by using an indirect ELISA method or a blocking ELISA method further comprises an ELISA enzyme-labeled plate, an ELISA secondary antibody, positive control serum and negative control serum.

Preferably, the detection of duck egg yield-reducing syndrome virus antibodies by blocking ELISA method comprises the following steps:

coating ELISA enzyme label plates with purified duck egg yield reduction syndrome virus antigens;

after the serum to be tested reacts with the coating antigen, an antibody, an enzyme-labeled secondary antibody and a color development liquid are sequentially added;

reading absorbance value OD by using enzyme labeling instrument _450nm And according to the formula: blocking rate (%) = (absorbance value of negative control-absorbance value of test sample)/absorbance value of negative control x 100%, blocking rate of test serum is calculated, and detection result is obtained according to the following determination criteria:

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

Preferably, the detection of duck egg yield-reducing syndrome virus antibodies by an indirect ELISA method comprises the following steps:

coating ELISA enzyme label plates with purified duck egg yield reduction syndrome virus antigen or recombinant expressed duck egg yield reduction syndrome virus main antigen protein;

reading absorbance value OD by using enzyme labeling instrument _450nm If the OD of the serum sample to be tested _450nm Value not less than negative sample OD _450nm The mean value of the values +2 times the standard deviation is judged as positive.

In another aspect of the invention, there is also provided the use of the above antibody in the preparation of a product for diagnosing or treating duck egg dystocia syndrome.

In another aspect of the invention, the invention also provides application of the duck egg yield reduction syndrome virus antibody detection kit in preparation of products for diagnosing duck egg yield reduction syndrome.

The duck egg yield reduction syndrome virus antibody detection kit can be used for rapidly and qualitatively detecting antibodies of duck egg yield reduction syndrome viruses, and has good application prospects in diagnosis and antibody detection of duck egg yield reduction syndrome.

Drawings

The invention will be described in further detail with reference to the drawings and the detailed description.

FIG. 1 is a diagram showing homology between the AH204 strain of duck egg yield-reducing syndrome virus of the present invention and amino acid sequences of other isolates of different duck egg yield-reducing syndrome viruses;

FIG. 2 is a schematic illustration of the two-way agarose diffusion test loading of example 1 of the present invention;

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

The duck egg yield reduction syndrome virus used in the invention is preserved in China center for type culture collection (CCTCC for short, address: university of Wuhan, wuchang mountain and Lopa nationality, hubei province) at 11-22 days in 2018, and the preservation number is CCTCC NO: v201866, which is classified and designated as duck egg reduced production syndrome virus strain AH 204.

Detailed Description

The invention relates to a duck egg yield-reducing syndrome virus antibody detection kit, which is an antibody detection kit developed on the basis of separating and obtaining a brand new duck egg yield-reducing syndrome virus in the early stage. The novel duck egg yield reduction syndrome virus is separated from tissues such as liver, spleen and lung of duck egg yield reduction syndrome disease ducks, and is preserved in China center for type culture collection (CCTCC, address: china center for type culture Collection, wuchang mountain and Wuhan university of Wuhan, hubei province), wherein the preservation number is CCTCC NO: v201866, which is classified and designated as duck egg reduced production syndrome virus strain AH 204. Through genome sequence determination and analysis, the amino acid sequence of an Open Reading Frame (ORF) of the gene sequence of the duck egg yield-reducing syndrome virus is shown as SEQ ID NO: 1.

The genome sequence of the obtained duck egg yield-reducing syndrome virus (AH 204 strain) is utilized to design a primer, the open reading frame sequences of other duck egg yield-reducing syndrome viruses separated from different provinces in China are amplified, after sequencing, the viruses are found to have the same genome structure, the nucleotide homology of ORF genes of each virus is between 97.9 and 100 percent, and the corresponding amino acid sequence is identical with SEQ ID NO:1 is also 98-100% (see figure 1).

The duck egg yield reduction syndrome virus antibody detection kit utilizes the duck egg yield reduction syndrome virus or an antibody produced by immunization of animals with antigen proteins thereof, and detects the duck egg yield reduction syndrome virus antibody through a bidirectional agarose diffusion test, an indirect ELISA method or a blocking ELISA method.

Example 1 bidirectional agarose diffusion assay

1.1 preparation of agarose gel-forming plate melted agarose, pouring the melted agarose into a plate on a horizontal table to prepare agarose gel with a thickness of about 3-4 mm, and punching according to the required shape after cooling.

1.2 preparation of antigen duck egg production reduction syndrome virus AH204 strain is inoculated with 10-11 day old non-immune duck embryo, hatching is continued until 6 days, dead duck embryo is harvested after 24 hours, allantoic fluid is collected, formaldehyde with the final concentration of 2 per mill is used for inactivating for 24 hours, and 10000g is centrifuged at 4 ℃ for 60 minutes to remove foreign proteins, and supernatant is collected. The supernatant was ultracentrifuged at 150000g for 5 hours at 4℃and the supernatant was discarded, and the pellet was dissolved in PBS (200-fold concentrated by volume). Measuring protein concentration, packaging, and storing at-70deg.C for use as antigen for bidirectional agarose diffusion test.

1.3 preparation of Positive serum

1.3.1 immunization methods at 10 ^4.5 EID ₅₀ Intramuscular injection of duck egg-reduced syndrome virus (AH 204 strain) into 5 healthy non-immunized ducks of 4-8 weeks old, collecting blood after each of 0.1ml and 21 days, separating serum, mixing the serum from 5 ducks uniformly, and packaging to prepare duck egg-reduced syndrome virus positive serum (namely duck resisting)Egg-reduced production syndrome virus antibodies).

1.3.2 sub-packaging, aseptically taking blood, separating serum, adding 0.01% merthiolate, quantitatively packaging, and 1 ml/tube.

1.3.3 inspection

1.3.3.1 the test positive serum is yellow or orange transparent clear liquid.

1.3.3.2 aseptic inspection is carried out according to the annex of the current Chinese animal pharmacopoeia, and the positive serum prepared by detection is aseptic.

1.3.3.3 titer measurement according to Duck egg reduced production syndrome agar-agar expansion test, its antibody titer is greater than or equal to 1:2, or according to the indirect ELISA method using prokaryotic expression protein as coating antigen, its OD _450nm The value is more than or equal to 0.412; or according to an indirect ELISA method using purified duck egg yield-reducing syndrome virus as a coating antigen, its OD _450nm The value is more than or equal to 0.487; or according to the detection method of duck egg reduction syndrome virus blocking ELISA antibody, the blocking rate of the antibody is more than or equal to 25.4%.

1.4 negative serum preparation

1.4.1 preparation of negative serum 5 healthy non-immune ducks blood of 4-8 weeks old are collected, serum is separated, serum from 5 ducks is uniformly mixed, 0.01% merthiolate is added, quantitative split charging is carried out, 1 ml/tube is used for preparing duck egg yield reduction syndrome virus negative serum.

1.4.2 inspection

1.4.2.1 trait test negative serum was yellow or orange-yellow clear liquid.

1.4.2.2 sterility test is carried out according to the annex of the current Chinese animal pharmacopoeia, and the negative serum prepared by detection is sterile.

1.4.2.3 titer determination according to Duck egg reduced production syndrome agar-agar amplification test, its antibody is negative, or indirect ELISA method using prokaryotic expression protein as coating antigen, its OD _450nm A value < 0.412; indirect ELISA method using purified duck egg yield-reducing syndrome virus as coating antigen, OD thereof _450nm A value < 0.487; according to ELISA detection method of duck egg reduced production syndrome virus antibody, the blocking rate of the antibody is less than 17.6%.

1.5 sample addition As shown in FIG. 2, antigen was added to the middle well, duck egg PRRSV antibody positive control, negative control and serum to be tested (at a dilution) were added to the surrounding wells, respectively, after the liquid was immersed in agarose, the agar plates were inverted, and placed in a wet box, and antigen and antibody were allowed to diffuse into each other on the agar plates at 37 ℃.

1.6 judgment the experiment was established when positive serum showed an opaque white line of sedimentation in agar, whereas negative serum did not appear. If the serum to be detected contains the duck egg production reduction syndrome virus specific antibody, a white precipitation line appears; if the serum to be detected does not contain the duck egg anti-production syndrome virus specific antibody, no white precipitation line appears. The antibody titer of the serum to be tested is the highest dilution of the serum with a precipitation line in the test.

1.7 after the duck egg reduction syndrome virus is inactivated, 10 healthy susceptible ducks of 21 days old are immunized, serum is collected after 21 days, a agar expansion experiment is carried out, all immunized ducks show positive antibody of the duck egg reduction syndrome virus, and the antibody titer of positive serum is the dilution multiple of the highest precipitation line of the agar expansion experiment (see figure 3).

Example 2 Indirect ELISA method for detecting Duck egg yield-reducing syndrome Virus antibody Using prokaryotic expressed protein as coating antigen

2.1 preparation of antigen the recombinant duck egg yield-reducing syndrome virus VP1 protein (VP 1 protein amino acid sequence is shown as 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

The purified recombinant duck egg yield-reducing syndrome virus VP1 protein (1.513 mg/mL) was diluted sequentially at 16, 8, 4, 2, 1, 0.5ug/mL with 0.05mol/L pH 9.6 carbonate buffer, 100uL per well coated ELISA plate, and overnight at 4 ℃. Positive and negative sera were diluted 1:100, 1:200, 1:400, 1:800, 1:1600, respectively, 100uL of coated ELISA plates per well, incubated for 1 hour at 37 ℃; the enzyme-labeled secondary antibody is diluted 1:5000, 100uL of each hole is coated with ELISA plate, and incubated for 1 hour at 37 ℃; other steps were performed according to ELISA procedure. Finally adding 100 mu L/hole TMB substrate, reacting for 10min, adding 2M concentrated sulfuric acid 50 mu L/hole to terminate reaction, and measuring OD with enzyme-labeled instrument _450nm Values. Determination of OD _450nm Value and P/N value, positive OD was selected _450nm The value was about 1, and the antigen coating concentration and serum dilution at the maximum P/N value were the optimal working concentrations.

Results of square titration at OD _450nm The coating dilution at a value close to 1.0 was the optimal coating concentration, at which time the error was smaller and the response was more sensitive. OD when the antigen was detected at 2ug/mL and serum dilution was 1:400 _450nm The value is closest to 1.0, and therefore the optimal coating concentration for antigen is determined to be 2ug/mL and the optimal serum dilution is 1:400.

TABLE 1 OD of matrix titration _450nm Value of

2.3 determination of optimal coating conditions for antigen

Coating the ELISA plates with the optimal antigen concentration, dividing the ELISA plates into 6 groups, and coating the ELISA plates for 1h at 37 ℃ in the 1 st group; group 2 coating for 2h at 37 ℃; group 3 coating for 4h at 37 ℃; group 4 overnight coating at 4 ℃; group 5 coating at 37 ℃ for 1h plus 4 ℃ overnight; group 6 coated for 2h at 37℃plus 4℃overnight, the other conditions were unchanged, and the OD of each group of negative wells, positive wells, control wells were compared ₄₅₀ The value and the P/N value, and the optimal coating condition is determined.

The ELISA plate was coated with 2ug/mL of the detection antigen, and the highest P/N value of group 5 was reached to 6.036, indicating that the optimal coating condition was obtained by coating at 37℃for 1h plus 4℃overnight.

TABLE 2 determination of optimal coating conditions for antigens

2.4 determination of the blocking fluid

The ELISA plate was coated with the optimal antigen concentration and optimal coating conditions, and after washing, the ELISA plate was washed with PBST containing 5% skim milk, 1% gelatin, 0.1% BSA, 10% calf serum, and commercial blocking solution, 200 uL/well, respectively, under the same conditions, and a suitable blocking solution was selected. ELISA detection is carried out after blocking for 2h at 37 ℃ to determine OD _450nm The value and P/N value, the maximum P/N value is selected as the sealing liquid.

The maximum P/N value when using a commercially available confining liquid was 6.273, and the commercially available confining liquid was selected as the confining liquid according to this example.

TABLE 3 determination of blocking solution

2.5 determination of optimal closing time

The ELISA plate was coated with the optimal antigen concentration and coating conditions, and after washing, blocking solution, 200 uL/well, was added and divided into 5 groups. Group 1, block at 37℃for 1h; group 2 was blocked at 37℃for 2h; group 3, block 3 for 3h at 37 ℃; group 4, block at 37 ℃ for 4h; the group 5 is sealed for 1h at room temperature, other conditions are unchanged, and the highest P/N value is taken as the optimal sealing time. Coating the ELISA plate under optimal antigen concentration and coating conditions, washing, adding a sealing solution, and analyzing test results, wherein the sealing solution is 200 uL/hole: the room temperature is used for sealing for 1h, and the P/N value is 6.082 at maximum, so that the room temperature is selected as the optimal sealing time for 1 h.

TABLE 4 determination of optimal closing time

2.6 selection of working concentration of enzyme-labeled secondary antibody

HRP enzyme-labeled anti-Duck secondary antibodies were diluted 1:1000, 1:2000, 1:4000, 1:8000 and 1:16000, the other conditions were unchanged, antigen and serum were diluted at the optimal dilutions, and OD was determined _450nm Values and P/N values.

Coating the ELISA plate with optimal antigen concentration and coating conditions, washing, adding a sealing solution, 200 uL/hole, and analyzing test results to obtain a sample with the concentration of 1: the P/N value at 2000 dilution is 6.544 at maximum, so that the dilution of the second enzyme-labeled antibody is 1:2000.

TABLE 5 selection of working concentration of enzyme-labeled secondary antibodies

2.7 selection of substrate action time

Coating ELISA plate with optimal antigen dilution, adopting optimal dilution of HRP marked anti-duck secondary antibody and negative and positive serum, respectively reacting substrate for 5, 10, 15, 30min at room temperature, testing by conventional method under other condition, and determining OD _450nm Values and P/N values. As a result, the P/N value was 6.015 at the maximum when the substrate reaction time was 10 minutes at room temperature, and therefore, the substrate reaction time was 10 minutes at room temperature was selected.

TABLE 6 determination of substrate action time

2.8 determination of ELISA negative-positive threshold

100 duck egg minus syndrome virus negative serum to be detected is detected according to the ELISA method established above, and OD thereof is measured _450nm Values were statistically analyzed to determine the threshold OD of positive serum _450nm The value is equal to or more than 0.412.

2.9 specificity test

Positive sera of duck hepatitis virus type I (DHAV-I), duck hepatitis virus type iii (DHAV-iii), duck plague virus (DEV), duck Reovirus (DRV), novel duck-origin goose parvovirus (NGPV) and avian influenza virus subtype H9 (AIV) were detected separately by established indirect ELISA methods, and whether the established indirect ELISA methods of the present experiment were cross-reactive with the positive sera of the above viruses was verified.

Results: the positive serum OD of the above viruses was determined by established indirect ELISA method for the positive serum of DHAV-I, DHAV-III, DEV, DRV, NGPV and AIV _450nm The values are all less than the critical value of yin and yang of 0.412, and are all negative, which indicates that the established indirect ELISA method has better specificity.

TABLE 7 specificity test of indirect ELISA

2.10 sensitivity test

And diluting duck egg yield reduction syndrome virus positive serum by 1:100, and then diluting the duck egg yield reduction syndrome virus positive serum by 1:12800 times, and performing an indirect ELISA test.

The results showed that positive was still detectable after 1:1600 dilution, while positive serum detection results were below 0.412 for 1:3200 dilution, negative.

TABLE 8 sensitivity test of indirect ELISA

2.11 within-batch repeatability test

Different ELISA plates were coated with the same batch of prepared recombinant antigen, 6 serum samples of different antibody levels were taken and assayed in the same condition by indirect ELISA procedure, 5 well replicates were run per blood sample, and the results were statistically analyzed.

The repeated detection of 3 duck egg reduction syndrome virus positive serum and 3 duck egg reduction syndrome virus negative serum is carried out by using the same batch of recombinant antigens, and the statistical analysis results show that the variation coefficient is between 1.462% and 5.902% and less than 10%, which shows that the variation degree of the same sample in the same batch of experiments is small and the repeated detection is good.

TABLE 9 within-batch repeatability test

2.12 inter-batch repeatability test

Different ELISA plates were coated with 3 different batches of prepared and purified recombinant proteins, 6 serum fractions of different antibody levels were taken and tested under the same conditions by an indirect ELISA procedure and the results were statistically analyzed.

The result shows that the variation coefficient is between 1.548% and 6.726% and less than 10% through statistical analysis, so that the variation degree of the same sample in different batches of antigen tests is small, and the method has good repeatability.

TABLE 10 batch-to-batch repeatability test

Example 3 Indirect ELISA method for detecting Duck egg reduction syndrome Virus antibody Using purified Duck egg reduction syndrome Virus as coating antigen

3.1 preparation of antigen

The duck egg yield reduction syndrome virus AH204 strain is inoculated with 10-11 days old non-immune duck embryo, hatching is continued for 6 days, dead duck embryo is harvested for 24 hours, allantoic fluid is collected, formaldehyde with the final concentration of 2 per mill is used for inactivating for 24 hours, 10000g is centrifuged at 4 ℃ for 60 minutes to remove foreign proteins, and supernatant fluid is collected. The supernatant was ultracentrifuged at 150000g for 5 hours at 4℃and the supernatant was discarded, and the pellet was dissolved in PBS (200-fold concentrated by volume). Measuring protein concentration, packaging, and storing at-70deg.C for ELISA coating antigen.

The purified whole virus is quantified by using a protein quantification kit, and the concentration of the virus protein is 0.851mg/mL

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

The purified whole virus solution was diluted with 0.05M carbonate buffer at pH 9.6 in the order of 16, 8, 4, 2, 1, 0.5ug/mL, 100uL per well of coated ELISA plate, overnight at 4 ℃. The positive serum and the negative serum are diluted from 1:100 to 1:1600 respectively, the enzyme-labeled goat anti-duck secondary antibody is diluted 1:2000 after the commercial sealing liquid is sealed at room temperature for 1 hour, and other steps are carried out according to ELISA procedures. Finally adding 100 mu L/hole TMB substrate, reacting for 10min, adding 2M concentrated sulfuric acid 50 mu L/hole to terminate reaction, and measuring OD with enzyme-labeled instrument _450nm Values.

Matrix titration showed that the OD of positive serum was at a dilution of 1:800 for serum with a coating concentration of 2. Mu.g/mL for antigen _450nm The value can reach 1.002, and the OD of negative serum _450nm OD of negative and positive serum was 0.207 _450nm The values differ significantly (P/n=4.84). Thus, 1:800 was chosen as the optimal serum dilution and 2 μg/mL as the optimal coating amount of antigen.

TABLE 11 OD of matrix titration _450nm Value of

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

The HRP-labeled anti-duck secondary antibodies were diluted 1:1000, 1:2000, 1:4000, 1:8000 and 1:16000 using the optimal antigen coating concentration, optimal coating conditions and optimal serum dilution described above, and an indirect ELISA assay was performed to determine the optimal working concentration of the enzyme-labeled secondary antibodies.

OD of positive and negative sera when HRP-labeled anti-Duck secondary antibody was diluted 1:2000 _450nm The values differ maximally. Therefore, 1:2000 was determined as the optimal working concentration of the enzyme-labeled secondary antibody.

Table 12 selection of working concentration of enzyme-labeled secondary antibodies

3.4 determination of optimal working time of substrate

Coating ELISA plate with optimal antigen dilution, adopting optimal dilution of HRP anti-duck secondary antibody and negative and positive serum, respectively reacting at room temperature for 5, 10, 15, 30min under unchanged conditions, testing by conventional method, and determining OD _450nm Values and P/N values.

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

TABLE 13 selection of substrate action time

3.5 determination of the negative and Positive thresholds of indirect ELISA

The laboratory-stored 100 duck egg yield-reducing syndrome virus negative antibody serum was tested according to the established indirect ELISA method described above. 2 wells were repeated for each sample, the average was taken, the mean (X) and Standard Deviation (SD) of the OD450nm values of the samples were calculated, and the OD450nm values of the samples > OD4 of the negative samples according to statistical principles _50nm At a value of X+2SD, positive was judged at a level of 99.9%.

The results showed that 100 duck egg reduced production syndrome virus negative antibody serum was subjected to indirect ELISA with a mean OD450nm (X) of 0.317 and a standard deviation SD of 0.085 according to the formula: negative-positive threshold = x+2sd, experimental threshold 0.487. I.e. OD of sample to be measured _450nm Antibody positive, OD when the value is more than or equal to 0.487 _450nm A value < 0.487 was negative for the antibody.

3.6 specificity test

Determination of positive serum for DHAV-I, DHAV-III, DEV, DRV, NGPV and AIV by established indirect ELISA method, OD _450nm The values are 0.227, 0.196, 0.309, 0.218, 0.264, respectively, positive serum OD against the above viruses _450nm The values are all less than the critical value of yin and yang of 0.487, and are all negative, which indicates that the established indirect ELISA method has better specificity.

TABLE 14 specificity assay of indirect ELISA

3.7 sensitivity test

And diluting duck egg yield reduction syndrome virus positive serum by 1:100-1:12800 times, and performing an indirect ELISA test.

The results showed that positive was still detectable after 1:3200 dilution, whereas positive serum detection at 1:6400 dilution was less than 0.487 negative.

TABLE 15 sensitivity test of indirect ELISA

3.8 within-and between-batch repeatability experiments

3 duck egg production reduction syndrome virus positive serum and 3 negative serum of different antibody titers were determined by established indirect ELISA methods. Different ELISA plates were coated with whole virus antigen prepared in the same batch, 5 wells were made per serum in parallel, and the OD per serum was determined _450nm Calculating standard deviation of the values, and further calculating OD of each serum _450nm Value of the intra-plate coefficient of variation. The test was repeated on 3 plates, based on the measured OD _450nm Value calculation of serum OD between plates _450nm Coefficient of Variation (CV) of the values. Batch-to-batch repeatability test: 3 different batches of purified whole virus ELISA assay plates were used to perform an indirect ELISA assay on 6 serum samples and the results were analyzed statistically.

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

TABLE 16 within-batch repeatability test

TABLE 17 batch-to-batch repeatability test

3.9 compliance test

And comparing 100 clinical samples in Jiangsu, anhui and other areas by using an established indirect ELISA test and an agar amplification test (AGP), comparing the yin-yang judgment result obtained by detecting the same serum by using an indirect ELISA method with the traditional agar amplification test, and calculating the positive detection rate of the two.

Results: 100 clinical duck serum samples from Jiangsu, anhui and other areas are detected by using the established indirect ELISA method compared with an agar diffusion test (AGP), wherein the positive detection rate of the indirect ELISA 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 ELISA method is obviously higher than that of the agar diffusion test (AGP).

Example 4 Duck egg reduction syndrome Virus blocking ELISA antibody detection method

4.1 preparation of antigen duck egg production reduction syndrome virus AH204 strain is inoculated with 10-11 day old non-immune duck embryo, hatching is continued for 6 days, dead duck embryo is harvested for 24 hours, allantoic fluid is collected, formaldehyde with the final concentration of 2 per mill is used for inactivating for 24 hours, and then the mixed protein is removed by 10000g centrifugation for 60 minutes at 4 ℃, and supernatant fluid is collected. The supernatant was ultracentrifuged at 150000g for 5 hours at 4℃and the supernatant was discarded, and the pellet was dissolved in PBS (200-fold concentrated by volume). Measuring protein concentration, packaging, and storing at-70deg.C for ELISA coating antigen.

4.2 blocking ELISA procedure

(1) Diluting the antigen with carbonate buffer solution with pH value of 9.6, coating the reaction plate, and standing overnight at 4 ℃ at 0.1ml per well; the coating solution is discarded, and washed 3 times with 0.05% Tween-20 PBS (PBST) for 3 minutes each time;

(2) Adding blocking solution (PBS solution containing 5% skimmed milk), allowing each well to act at 37deg.C for 1 hr, and washing with PBST;

(3) Adding the serum to be tested diluted by the antibody diluent, negative control serum and positive control serum (10 times dilution), incubating for 1 hour at 37 ℃ at 0.1ml per hole, and washing the mixture, wherein the method is the same as that described above;

(4) 0.1ml of specific antibody (500-fold dilution) was added to each well, and after incubation at 37℃for 1 hour, washing was performed 3 times;

(5) 0.1ml of enzyme-labeled secondary antibody (2000 times dilution) is added to each well, and after incubation for 1 hour at 37 ℃, the mixture is washed 3 times;

(6) 0.1ml TMB substrate color development liquid is added into each hole, and color development is carried out at room temperature and in dark place (10 minutes);

(7) Adding 0.05ml of stop solution into each hole, and reading absorbance value OD by using an enzyme label instrument _450nm The blocking rate was calculated.

Blocking rate (%) = (absorbance value of negative control-absorbance value of sample to be tested)/absorbance value of negative control x 100%.

4.3 optimization of optimal antigen coating concentration and serum dilution purified antigen (1.627 mg/mL) that was checked for eligibility was diluted with coating solution and then subjected to 2-fold ratio dilution. The dilution was sequentially carried out at 16, 8, 4, 2, 1, 0.5ug/mL, and a total of 6 dilutions of antigen were added to 96-well ELISA plates, each dilution was 6 wells, and each well was 0.1mL. Placing for 15-18 hours at the temperature of 4 ℃; removing the coating liquid, adding the washing liquid, standing for 3 minutes, removing the washing liquid, washing for 3 times, and removing the washing liquid; adding sealing liquid, placing for 1 hour at 20-25 ℃ at 0.1ml of each hole, and discarding the sealing liquid. Positive serum was diluted 1:5, 1:10 and 1:20, negative serum was diluted 10-fold, and each serum was added to enzyme-labeled wells coated with different antigen concentrations, 3 duplicate wells were set up at the same antigen concentration for each serum, and 0.1ml per well. After the serum is added, the mixture is placed at 37 ℃ for 1 hour, serum diluent is discarded, and the mixture is washed for 3 times; discarding the washing solution, adding specific antibody 1 diluted 1:500, and standing at 37 ℃ for 1 hour at 0.1ml per well, and washing for 3 times; the washes were discarded, goat anti-mouse IgG enzyme-labeled secondary antibody (0.8 mg/ml) was added at 1:2000 dilution, and the mixture was left at 37℃for 1 hour in each well at 0.1ml, and after washing 3 times as above, the washes were discarded, TMB substrate solution was added, and the mixture was left at 0.1ml in each well, and left at room temperature in a dark place for 10 minutes, and 0.05ml of stop solution was added in each well. Determination of OD of each well on an ELISA apparatus _450nm Values. OD when negative serum _450nm About 1.5, negative serum OD _450nm Value and positive serum OD _450nm The antigen concentration at which the ratio is maximum is determined as the optimal antigen coating concentration.

Coating ELISA plate with detection antigen 4ug/mL, and detecting OD of negative serum _450nm About 1.5, negative serum OD _450nm Value and positive serum OD _450nm The ratio was maximized, thus determining an antigen concentration of 4ug/mL as the optimal antigen coating concentration, and an optimal dilution of serum of 1:10.

Table 18 OD for square titration _450nm Value of

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

The optimal sealing liquid determined by the test is commercial sealing liquid, and the sealing condition is that sealing is carried out for 1 hour at 37 ℃.

TABLE 19 determination of blocking solution

4.5 determination of optimal dilutions and working time of specific antibodies the elisa plate was coated with antigen at the determined optimal concentration and blocked with the determined blocking solution and conditions. The positive serum and the negative serum are taken to act according to the determined dilutions and time, the specific antibodies are respectively diluted by 1:250, 1:500, 1:1000, 1:2000 and 1:4000, 0.1ml is added to each well, the mixture is incubated for 1 hour at 37 ℃, and the optimal dilution of the specific antibodies is determined according to the operation procedure.

The assay determines that the specific antibody used was diluted 1:1000 fold for a 1 hour working time.

TABLE 20 determination of optimal dilutions of specific antibodies

4.6 determining the optimal working concentration and working time of the enzyme-labeled antibody, coating the enzyme-labeled plate according to the determined optimal concentration of antigen, sealing by using the determined sealing liquid and conditions, performing serum dilution and working time according to the determined conditions, performing specific antibody dilution according to the determined conditions, respectively performing 1:1000, 1:2000, 1:4000 and 1:8000 times dilution on the goat anti-mouse enzyme-labeled antibody, and determining the optimal working concentration and working time of the enzyme-labeled antibody according to the operation procedure at 37 ℃ for 1 hour.

The assay determines that the enzyme-labeled antibody used is diluted 1:2000 times at 37℃for 1 hour.

Table 21 determination of optimal dilutions of enzyme-labeled antibodies

4.7 determining the color development time of the substrate, coating the ELISA plate according to the determined antigen with the optimal concentration, sealing with the determined sealing liquid and conditions, performing serum dilution and action time according to the determined conditions, performing specific antibody according to the determined conditions, adding the substrate TMB into the ELISA plate, performing light-shielding color development at room temperature, and determining the optimal color development time according to the operation procedures, wherein the color development time is 5, 10, 15 and 20 minutes respectively.

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

TABLE 22 determination of substrate development time

4.8 threshold determination 200 negative sera from the clinic were tested after the blocking ELISA method was established, blocking rate was calculated and the threshold of detection was determined. Critical value = negative sample mean inhibition +2 or 3 standard deviations.

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

4.9 specificity test

Positive sera of duck hepatitis virus type I (DHAV-I), duck hepatitis virus type iii (DHAV-iii), duck plague virus (DEV), duck Reovirus (DRV), novel duck-origin goose parvovirus (NGPV) and avian influenza virus subtype H9 (AIV) were detected separately by established blocking ELISA methods, and whether the ELISA methods were cross-reactive with the positive sera of the above viruses was verified.

Results: determination of positive serum for DHAV-I, DEV, DRV, NGPV and AIV by established indirect ELISA method, OD thereof _450nm The values are respectively 4.4%, 0.9%, 5.8%, 7.1% and 1.2%, and the positive serum blocking rate of the viruses is less than 17.6% of the critical value of yin and yang, which indicates that the established blocking ELISA method has better specificity.

Table 23 specificity test of blocking ELISA

4.10 sensitivity test

And diluting duck egg yield reduction syndrome virus positive serum by a ratio of 1:10-1:1280, and performing a blocking ELISA test.

The results showed that positive was still detectable after 1:320 dilution, whereas positive serum with 1:640 dilution was negative with a blocking rate of less than 17.6%.

TABLE 24 sensitivity test of blocking ELISA

4.11 within-and inter-batch repeatability test

Different ELISA plates are coated with the same batch of prepared duck egg production reduction syndrome virus antigens, 3 parts of positive serum and 3 parts of negative serum with different antibody levels of the duck egg production reduction syndrome virus are taken, the positive serum and the 3 parts of negative serum are measured according to an indirect ELISA program under the same condition, 5-hole parallel tests are carried out on each blood sample, and the results are statistically analyzed. Different ELISA plates were coated with 3 different batches of prepared and purified virus, 6 parts of the above serum were taken and assayed under the same conditions by blocking ELISA procedure and the results were statistically analyzed.

Results: the repeated detection of 3 duck egg reduction syndrome virus positive serum and 3 duck egg reduction syndrome virus negative serum is carried out by using the same batch of antigens, and the statistical analysis results show that the variation coefficient is between 0.907% and 3.656% and less than 10%, which shows that the variation degree of the same sample in the same batch of experiments is small and the repeated detection is good. The ELISA plate batch-to-batch repeatability test is prepared by 3 different batches, and the variation coefficient is 1.318% -5.158%, which shows that the variation degree of the same sample in the antigen tests of different batches is small, and the ELISA plate batch-to-batch repeatability test has good repeatability.

Table 25 in-batch repeatability test

Table 26 inter-lot repeatability test

4.12 compliance test

100 clinical samples in Jiangsu, anhui and other areas are detected by comparing the established blocking ELISA test with an agar amplification test (AGP), and the positive detection rate and the coincidence rate of the two are calculated by comparing the yin-yang judgment result obtained by detecting the same serum by the blocking ELISA method with the traditional agar amplification test.

Results: 100 clinical duck serum samples from Jiangsu, anhui and other areas are detected by using the established blocking ELISA method compared with an agar diffusion test (AGP), wherein the positive detection rate of the blocking ELISA method is 37 percent (37/100), the positive detection rate of the agar diffusion test (AGP) is 18 percent (18/100), and the sensitivity of the established blocking ELISA is obviously higher than that of the agar diffusion test (AGP).

Table 27 compliance test

* Refer to the same values as the parallel assay results in blocking ELISA experiments and agar diffusion experiments (AGP).

The foregoing examples merely illustrate embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Sequence list

<110> Shanghai veterinary institute of agricultural sciences of China (center for sea division of China center for animal health and epidemiology)

<120> duck egg yield 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 yield-reducing 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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Glu Thr Ile Glu Lys Thr Thr Glu Ala Ala Thr Leu Ile Ser Asp Thr

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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

115 120 125

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

130 135 140

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

145 150 155 160

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

180 185 190

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

210 215 220

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

245 250 255

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

275 280 285

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

325 330 335

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

370 375 380

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

385 390 395 400

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

420 425 430

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

450 455 460

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

465 470 475 480

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

485 490 495

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

500 505 510

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

530 535 540

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

545 550 555 560

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

625 630 635 640

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

645 650 655

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

675 680 685

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

740 745 750

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

755 760 765

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

1295 1300 1305

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

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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

1415 1420 1425

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 Asp Leu Asp Phe Asn

1460 1465 1470

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

1490 1495 1500

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

1595 1600 1605

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

1700 1705 1710

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

1715 1720 1725

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

1745 1750 1755

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

1790 1795 1800

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 His Thr 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 Val Arg 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 yield-reducing 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

Tyr Asn 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 yield-reducing syndrome virus, which comprises the amino acid sequence of CCTCC NO: antibodies raised by animals immunized with duck egg production-reducing syndrome virus strain AH204 of V201866.

2. A duck egg reduced production syndrome virus antibody detection kit comprising the antibody of claim 1, and/or a whole virus antigen of a duck egg reduced production syndrome virus or a major immunogenic protein antigen thereof.

3. The duck egg reduction syndrome virus antibody detection kit as claimed in claim 2, wherein the kit further comprises an ELISA enzyme-labeled plate, an ELISA secondary antibody, a chromogenic solution, positive control serum and negative control serum.

4. Use of the antibody of claim 1 for the preparation of a product for diagnosing or treating duck egg reduction syndrome.

5. Use of the duck egg yield reduction syndrome virus antibody detection kit according to claim 2 or 3 for preparing a product for diagnosing duck egg yield reduction syndrome.