阅读说明：本技术 一种fadv a病毒间接elisa抗体检测试剂盒及其应用 (FADV A virus indirect ELISA antibody detection kit and application thereof ) 是由 高月花 于志君 东野传现 亓丽红 宋敏训 胡峰 李玉峰 于可响 于 2018-06-08 设计创作，主要内容包括：本发明属于生物工程技术领域,具体涉及一种FADV A病毒间接ELISA抗体检测试剂盒,用于FADV A病毒抗体的快速检测。本发明所述FADV A病毒间接ELISA检测试剂盒,以表达蛋白fiber2为包被抗原,由于表达的特异性蛋白对于FADV A的特异性高,安全性好,且不含无关杂蛋白,能与FADV A阳性血清进行特异结合,且不与其它病毒的阳性血清发生交叉反应,具有良好的抗原性,是理想的ELISA检测抗体的包被抗原载体,使得制得检测试剂盒具有很高的特异性和敏感性,在禽腺病毒A型的检测工作中发挥重要作用。(The invention belongs to the technical field of biological engineering, and particularly relates to an indirect ELISA antibody detection kit for FADV A, which is used for rapidly detecting FADV A antibodies. The FADV A virus indirect ELISA detection kit provided by the invention takes the expression protein fiber2 as a coating antigen, and because the expressed specific protein has high specificity to FADV A, good safety and no irrelevant hybrid protein, the kit can be specifically combined with FADV A positive serum, does not have cross reaction with positive serum of other viruses, has good antigenicity, is an ideal coating antigen carrier for ELISA detection antibodies, so that the prepared detection kit has high specificity and sensitivity, and plays an important role in detection work of avian adenovirus type A.)

1. the application of the expression protein fiber2 in preparing an FADVA virus indirect ELISA antibody detection kit, wherein the expression protein fiber2 has a nucleotide sequence shown as SEQ ID No. 1.

2. An indirect ELISA antibody detection kit for FADVA virus is characterized by comprising an ELISA plate coated with FADVA expression protein fiber 2.

3. the kit for detecting the FADVA virus indirect ELISA antibody of claim 2, wherein the ELISA plate coated with the expressed protein fiber2 is prepared by the following steps: the expressed protein fiber2 was diluted to a concentration of 1. mu.g/mL using 0.05M carbonate buffer solution at pH9.6 as a coating solution, added to an ELISA reaction plate at a dose of 100. mu.L/well, allowed to act at 37 ℃ for 2 hours, coated overnight at 4 ℃, blotted dry, blocked with a T20blocking buffer at 37 ℃ for 2 hours, washed with PBS containing Tween-20 at a concentration of 0.05% at pH7.4, blotted dry, and stored in a bag containing a desiccant, for further use.

4. The FADVA virus indirect ELISA antibody detection kit of claim 2 or 3, wherein the kit further comprises a sample diluent, a 10 x concentrated washing solution, an enzyme conjugate working solution, a color development solution, a stop solution, a positive control and a negative control.

5. The FADVA virus indirect ELISA antibody detection kit of claim 4, wherein the kit comprises:

6. The FADVA virus indirect ELISA antibody detection kit of claim 4 or 5, characterized in that:

The sample diluent is 0.01M phosphate buffer solution containing 0.05 percent of Tween-20, and the pH value is 7.4;

The 10 multiplied concentrated washing solution is 0.1M phosphate buffer solution containing 0.5 percent of Tween-20, and the pH value is 7.4;

The enzyme conjugate working solution is HRP-goat anti-chicken IgG;

The color developing liquid is prepared from the following components in a volume ratio of 1:1, a mixed solution of a Tetramethylbenzidine (TMB) solution with a concentration of 0.2mg/mL and a citric acid-phosphate buffer solution containing urea hydrogen peroxide with a concentration of 0.5 per mill;

the stop solution is a hydrofluoric acid solution with the concentration of 0.31 percent;

The positive control is FADVA positive serum obtained by screening, and OD thereof_650nmMore than or equal to 1.0, and 1000U/mL streptomycin is added;

The negative control is negative serum obtained by screening, and the OD of the negative control_650nmLess than or equal to 0.25, and 1000U/mL streptomycin is added.

7. the method for using the FADVA virus indirect ELISA antibody detection kit of any one of claims 2-6, characterized by comprising the step of adding the serum to be tested to the ELISA plate coated with the expressed protein fiber 2.

8. The use method of the FADVA virus indirect ELISA antibody detection kit of claim 7 is characterized by specifically comprising the following steps: and (3) diluting the serum to be detected with the sample diluent according to the ratio of 1: diluting at a ratio of 100, adding 100 μ L/well into the ELISA plate, setting negative control and positive control, and incubating at 37 deg.C for 45 min; after the reaction is finished, discarding the liquid in the reaction hole, adding 350 mu L of washing liquid into each hole, washing for 3-5 times, and beating to dry at intervals of 1min each time; then 100. mu.L of the enzyme conjugate working solution was added to each well and incubated at 37 ℃ for 45 min; washing with washing solution for 3-5 times at interval of 1min, and drying; and sequentially adding 100 mu L of developing solution, incubating for 15min at 37 ℃ in the dark, adding 50 mu L of stop solution to terminate the reaction, measuring the absorbance A value of each hole by using an enzyme-linked immunosorbent assay (ELIASA) at the wavelength of 650nm, and calculating and judging the result.

9. The FADVA virus indirect ELISA antibody detection kit of any one of claims 2-6, in the field of FADVA antibody detection.

10. A method for rapidly detecting FADVA virus antibodies, which is characterized by comprising the step of detecting serum to be detected by using the FADVA virus indirect ELISA antibody detection kit of any one of claims 2-6, wherein the judgment standard of a detection sample is as follows: when the sample to be detected is OD_650nmValue and negative control OD_650nmthe ratio of the values (P/N) is greater than or equal to 2.1, and the OD of the sample to be detected_650nmA value greater than 0.333 was judged to be positive.

Technical Field

The invention belongs to the technical field of biological engineering, and particularly relates to an indirect ELISA antibody detection kit for FADV A, which is used for rapidly detecting FADV A antibodies.

background

Avian adenovirus (fowladenvirus, FAdV), a conditional pathogen in poultry, does not generally exhibit clinical symptoms, but under certain conditions, particularly when infected concurrently, adenovirus can exhibit severe pathogenicity, leading to higher morbidity and mortality in poultry. Since the large-scale outbreak of the avian I group adenovirus in Shandong, Jiangsu, Hubei and other places in 2015, the avian I group adenovirus gradually spreads all over the country, and huge economic losses are caused to poultry farming in the country.

The avian adenovirus group I is mainly divided into 5 genotypes (types A-E) and 12 serotypes (types 1-7,8a,8b and 9-11), and the genotypes are commonly present in poultry bodies. The sick bird can be infected with a plurality of serotypes simultaneously, and can also discharge viruses of another serotype while having a certain serotype virus antibody. The avian adenovirus group I virus is distributed worldwide, which often causes that poultry in all age groups are susceptible to infection. Among them, FADV a type often causes the erosion of the sacs of sick birds, and most viruses replicate in the bodies of birds without causing diseases or cause mild symptoms, but when immunosuppressive diseases or mixed infection occurs, the disease deterioration is aggravated, and the health of the birds is seriously affected. At present, with the gradual development trend of industrialization, intensification and scale in poultry industry, higher requirements are put forward for prevention and control of the avian adenovirus disease, and new requirements are put forward for how to evaluate the performance and effect of immune antibodies generated by active immunity of the avian adenovirus disease.

Disclosure of Invention

therefore, the technical problem to be solved by the invention is to provide an indirect ELISA antibody detection kit for FADV A to realize rapid detection of FADV A antibody.

In order to solve the technical problem, the invention discloses application of an expression protein fiber2 in preparing an indirect ELISA antibody detection kit for FADV A virus, wherein the expression protein fiber2 has a nucleotide sequence shown as SEQ ID No. 1.

the invention also discloses a kit for detecting the FADV A indirect ELISA antibody, which comprises an ELISA plate coated with FADV A expression protein fiber 2.

Further, the preparation method of the ELISA plate coated by the expressed protein fiber2 comprises the following steps: the synthetic polypeptide W8 was diluted to a concentration of 1. mu.g/mL using 0.05M carbonate buffer pH9.6 as a coating solution, added to an ELISA reaction plate at a dose of 100. mu.L/well, allowed to act at 37 ℃ for 2 hours, coated overnight at 4 ℃, blocked with a T20blocking buffer at 37 ℃ for 2 hours after patting dry, washed with PBS containing 0.05% Tween-20 at pH7.4, patted dry and stored in a bag containing a desiccant after drying.

Preferably, the kit further comprises a sample diluent, a 10 × concentrated washing solution, an enzyme conjugate working solution, a color development solution, a stop solution, a positive control and a negative control.

the kit for detecting the FADV A indirect ELISA antibody comprises:

In the FADV A indirect ELISA antibody detection kit:

The sample diluent is 0.01M phosphate buffer solution containing 0.05 percent of Tween-20, and the pH value is 7.4;

The 10 multiplied concentrated washing solution is 0.1M phosphate buffer solution containing 0.5 percent of Tween-20, and the pH value is 7.4;

the enzyme conjugate working solution is HRP-goat anti-duck IgG;

The color developing liquid is prepared from the following components in a volume ratio of 1:1, a mixed solution of a Tetramethylbenzidine (TMB) solution with a concentration of 0.2mg/mL and a citric acid-phosphate buffer solution containing urea hydrogen peroxide with a concentration of 0.5 per mill;

The stop solution is a hydrofluoric acid solution with the concentration of 0.31 percent;

The positive control is FADV A positive serum, the OD thereof_650nmMore than or equal to 1.0, and 1000U/mL streptomycin is added;

The negative control is negative serum obtained by screening, and the OD of the negative control_650nmLess than or equal to 0.25, and 1000U/mL streptomycin is added.

the invention also discloses a using method of the kit for detecting the FADV A indirect ELISA antibody, which comprises the step of adding the serum to be detected to the ELISA plate coated by the expressed protein fiber 2.

The use method of the FADV A indirect ELISA antibody detection kit specifically comprises the following steps: and (3) diluting the serum to be detected with the sample diluent according to the ratio of 1: diluting at a ratio of 100, adding 100 μ L/well into the ELISA plate, setting negative control and positive control, and incubating at 37 deg.C for 45 min; after the reaction is finished, discarding the liquid in the reaction hole, adding 350 mu L of washing liquid into each hole, washing for 3-5 times, and beating to dry at intervals of 1min each time; then 100. mu.L of the enzyme conjugate working solution was added to each well and incubated at 37 ℃ for 45 min; washing with washing solution for 3-5 times at interval of 1min, and drying; and sequentially adding 100 mu L of developing solution, incubating for 15min at 37 ℃ in the dark, adding 50 mu L of stop solution to terminate the reaction, measuring the absorbance A value of each hole by using an enzyme-linked immunosorbent assay (ELIASA) at the wavelength of 650nm, and calculating and judging the result.

The invention also discloses application of the FADV A indirect ELISA antibody detection kit in the field of detection of FADV A antibodies.

The invention also discloses a method for rapidly detecting the FADV A antibody, which comprises the step of detecting the serum to be detected by the FADV A indirect ELISA antibody detection kit, wherein the judgment standard of a detection sample is as follows: when the sample to be detected is OD_650nmValue and negative control OD_650nmThe ratio of the values (P/N) is greater than or equal to 2.1, and the OD of the sample to be detected_650nma value greater than 0.333 was judged to be positive.

The FADV A virus indirect ELISA detection kit takes the expression protein fiber2 as an envelope antigen, and the expression protein fiber2 is the surface protein of adenovirus, plays an important role in mediating virus infection host cells, can effectively stimulate an organism to generate humoral immunity and induce the generation of neutralizing antibodies, and has high specificity and good safety for FADV A due to the expression of specific protein, does not contain irrelevant hybrid protein, can be specifically combined with FADV A positive serum, does not have cross reaction with other virus positive serum, has good antigenicity, is an ideal envelope antigen carrier for ELISA detection antibodies, so that the prepared detection kit has high specificity and sensitivity, and plays an important role in the detection work of avian adenovirus A.

The FADV A indirect ELISA detection kit can evaluate the level of the FADV A antibody by one-time detection, so that the detection cost is reduced greatly; the kit is simple and rapid to operate, is suitable for detecting batch samples, and greatly improves the serological diagnosis speed of the avian adenovirus.

Detailed Description