CN108169496B - DHAV-3 type polypeptide indirect ELISA antibody detection kit and application thereof - Google Patents
DHAV-3 type polypeptide indirect ELISA antibody detection kit and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biological engineering, and particularly relates to a DHAV-3 type polypeptide indirect ELISA antibody detection kit for rapid detection of a DHAV-3 type (DHAV-3) antibody of duck hepatitis A virus. The indirect ELISA detection kit for duck hepatitis A virus type 3 polypeptide takes the synthesized polypeptide W7 as an envelope antigen, has high specificity to DHAV-3 antibody, good safety, does not contain irrelevant hybrid protein, is only specifically combined with DHAV-3 positive serum, does not generate cross reaction with the positive serum of other viruses, and has good antigenicity, so that the detection kit has high specificity and sensitivity.
Description
Technical Field
The invention belongs to the technical field of biological engineering, and particularly relates to a DHAV-3 type polypeptide indirect ELISA antibody detection kit for rapid detection of a DHAV-3 type (DHAV-3) antibody of duck hepatitis A virus.
Background
The duck viral hepatitis is an acute and highly lethal infectious disease which harms ducklings, has the characteristics of acute onset, short course of disease, quick transmission, high fatality rate and the like, mainly attacks the ducklings within 3 weeks, and seriously threatens the healthy development of the duck industry. The etiology of the disease is Duck Hepatitis A Virus (DHAV), and the DHAV is divided into DHAV-1, DHAV-2 and DHAV-3 because of the obvious difference between DHAV sequences and no cross reaction among different DHAV types. So far, the dominant domestic prevalence has been the DHAV-1 and DHAV-3 strains. Epidemiological studies have shown that DHAV-3 has a markedly increasing tendency to infect and become more and more harmful in recent years. With the advent of the DHAV-3 vaccine, how to evaluate the immune effect of the vaccine is urgent.
At present, the detection of DHAV antibody mainly depends on the traditional serum neutralization test, but the method has the defects of long detection period, time and labor waste, and is not beneficial to the rapid detection of batch samples; and the ELISA method established based on the whole virus limits the popularization and application of the method because DHAV purification is difficult. The gene VP1 encodes the main antigenic site and has the main type-specific neutralizing site, which is the main component determining the antigenicity of the virus. ELISA antibody detection kit developed by utilizing genetic engineering technology and aiming at DHAV-3 has been reported, but the kit selects complete VP1 protein as coating antigen, and more antigen epitopes exist on the surface of the protein, and the phenomenon of nonspecific interference may exist more or less. The synthesized specific epitope polypeptide has high antigen specificity, is an optimal envelope antigen of an ELISA detection antibody, can realize high-throughput detection of the DHAV-3 antibody, and provides technical support for comprehensive prevention and control of duck hepatitis A virus type 3.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide an indirect ELISA antibody detection kit for duck hepatitis A virus type 3 polypeptide, so as to realize the rapid detection of the antibody of duck hepatitis A virus type 3 (DHAV-3).
In order to solve the technical problem, the kit for detecting the DHAV-3 type polypeptide indirect ELISA antibody comprises an ELISA plate coated by DHAV-3 type synthetic polypeptide W7; the synthetic polypeptide W7 includes an amino acid sequence structure shown as N-SRNHRPFRFCLRLKT-CooH.
According to the DHAV-3 type polypeptide indirect ELISA antibody detection kit, the amino acid sequence structure of the synthetic polypeptide W7 is N-SRNHRPFRFCLRLKT-CooH.
The preparation method of the ELISA plate coated by the synthetic polypeptide W7 comprises the following steps: the synthetic polypeptide W7 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 ℃, blotted dry, blocked with Porcine skin Gelatin at 37 ℃ for 2 hours, washed with PBS containing 0.05% Tween-20 at pH7.4, blotted dry and stored in a bag containing a desiccant after drying.
The kit also comprises a sample diluent, a 10 multiplied concentrated washing solution, an enzyme conjugate working solution, a developing solution, a stop solution, a positive control and a negative control.
The kit for detecting the DHAV-3 type polypeptide indirect ELISA antibody comprises:
The DHAV-3 type polypeptide indirect ELISA antibody detection kit comprises:
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 DHAV-3 positive serum obtained by screening, and the OD of the positive control is650nmMore 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 control650nmLess than or equal to 0.25, and 1000U/mL streptomycin is added.
The invention also discloses a using method of the DHAV-3 type polypeptide indirect ELISA antibody detection kit, which comprises the step of adding serum to be detected into the ELISA plate coated by the synthetic polypeptide W7.
The application method of the DHAV-3 type polypeptide 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 DHAV-3 type polypeptide indirect ELISA antibody detection kit in the field of DHAV-3 antibody detection.
The invention also discloses a method for rapidly detecting the DHAV-3 antibody, which comprises the step of detecting the serum to be detected by using the DHAV-3 type polypeptide indirect ELISA antibody detection kit; the judgment standard of the detection sample is as follows: when the sample to be detected is OD650nmValue and negative control OD650nmThe value ratio (P/N) is greater than or equal to 2.1, and the value of the sample OD650nm which is greater than 0.280 is judged to be positive.
The indirect ELISA detection kit for duck hepatitis A virus type 3 polypeptide takes the synthesized polypeptide W7 as an envelope antigen, has high specificity to DHAV-3 antibody, good safety, does not contain irrelevant hybrid protein, is only specifically combined with DHAV-3 positive serum, does not generate cross reaction with the positive serum of other viruses, and has good antigenicity, so that the detection kit has high specificity and sensitivity.
The indirect ELISA detection kit for duck hepatitis A virus type 3 polypeptide can evaluate the DHAV-3 antibody level, is simple and quick to operate, is suitable for detection of batch samples, and greatly improves the speed of serological diagnosis of duck hepatitis.
Detailed Description
Example 1 specificity verification of the synthetic polypeptide W7
The amino acid sequence structure of the synthesized polypeptide W7 in this example is N-SRNHRPFRFCLRLKT-CooH, and the synthesis can be performed according to the methods for polypeptide synthesis known to those skilled in the art, such as solid phase synthesis of polypeptide.
Diluting the synthesized polypeptide W7 with carbonate buffer solution respectively to protein concentration of 1 μ g/mL, coating each well with 100 μ L, placing in a wet box at 37 deg.C for 1h, standing at 4 deg.C overnight, discarding the liquid, washing with PBST for 3 times, and patting to dry; then adding sealing liquid Porcine skin Gelatin, controlling the dosage to be 300 mu L/hole, and sealing for 2h at 37 ℃; discarding the liquid, adding PBST and washing for 3 times; the chicken positive serum (DHAV-3) and the SPF chicken negative serum were subjected to 1: 100 dilution, controlling the dose to be 100 mu L/hole, and acting for 1h at 37 ℃; discard the liquid, wash 3 times with PBST; adding goat anti-chicken secondary antibody, controlling the dose at 100 μ L/hole, and acting at 37 deg.C for 30 min; discarding the liquid, PBST washing 3 times; adding substrate (TMB), controlling the dosage to be 100 μ L/hole, and keeping away from light for 15 min; adding stop solution, controlling the dosage to be 50 mu L/hole, and placing a microplate reader for reading.
Table 1 specificity verification of polypeptides
The results shown in Table 1 above show that the synthetic polypeptide W7 can only specifically react with DHAV-3 positive serum, but has no cross reaction with DHAV-1.
EXAMPLE 2 kit Components preparation
The sample diluent was 0.01M ph7.4 phosphate buffer containing 0.05% tween-20: take KH2PO40.2g，NaHPO4·12H2O2.9 g and NaCl 8g, the volume is adjusted to 1000mL, and then 0.5mL of Tween-20 is added;
the 10 x concentrated wash was 0.1M ph7.4 phosphate buffer containing 0.5% tween-20: take KH2PO42g，NaHPO4·12H2O29 g and NaCl 80g, the volume is adjusted to 1000mL, and then 5mL of Tween-20 is added;
the stop solution is a 0.31% hydrofluoric acid solution: taking 0.31mL of hydrofluoric acid, and fixing the volume of double distilled water to 100 mL;
positive control: the prepared positive serum was diluted with the sample diluent as 1: 100 dilution (OD thereof)650nmNot less than 1.0), adding 1000U/mL streptomycin, and performing sterile filtration to serve as a positive control of the kit;
negative control: negative serum (OD) obtained by screening650nmLess than or equal to 0.25), adding 1000U/mL streptomycin, sterile filtering, and using as a negative control of the kit.
Preparing the color developing solution: weighing 200mg of Tetramethylbenzidine (TMB), dissolving with 100mL of absolute ethyl alcohol or DMSO, and then fixing the volume to 1000mL by double distilled water; 21g of citric acid (C) was weighed6H8O7·H2O), 28.2g of anhydrous disodium hydrogen phosphate (Na)2HPO4) 6.4mL of 0.75% urea hydrogen peroxide, diluting the double distilled water to 1000mL, and adjusting the pH value to 4.5-5.0; controlling the volume ratio of the two to be 1: 1.
Example 3 determination of Indirect ELISA reaction conditions
This example uses a matrix test to determine the optimal working concentration of polypeptide antigen and serum. Coating the synthetic polypeptide W7 with a coating buffer solution to obtain a coating solution with the following ratio of 1: 10,1: 20,1: 40,1: 80, coating an ELISA reaction plate, and controlling the dose to be 100 mu L/hole; positive and negative sera against DHAV-1 and DHAV-3 were prepared using sample dilutions of 1: 50,1: 100,1: 200,1: dilution series 400; performing indirect ELISA measurement; adding a developing solution for developing and adding a stop solution to stop the reaction; and the OD value at a wavelength of 650nm was measured, and the results are shown in Table 2.
Taking positive serum OD650nm1.0 or so, negative serum OD650nmAbout 0.25, and positive serum OD650nmNegative serum OD650nmNamely, the antigen concentration with the P/N value of more than 2.1 and the serum dilution are the optimal working concentration, and the result shows that the serum optimal dilution is 1: 100, the optimal coating concentration of the polypeptide is 1.0 mu g/mL.
TABLE 2 determination of optimal working concentration (OD) of polypeptide antigen and serum650nmValue)
30 parts of collected duck serum without DHAV-1 and DHAV-3 antibodies are subjected to indirect ELISA (enzyme-linked immunosorbent assay) under the optimal working conditions to determine the absorption value range of the duck serum without DHAV-1 and DHAV-3 infection
Thus, the OD of the sample to be examined is determined 650nmValue and negative OD650nmThe value ratio (P/N) is greater than or equal to 2.1, and the OD of the sample to be detected650nmValues greater than 0.280 were judged positive.
Example 4 preparation of Duck hepatitis A Virus type 3 polypeptide Indirect ELISA antibody detection reaction plate
Diluting duck hepatitis A virus type 3 synthetic polypeptide W7 to 1 μ g/mL with 0.05M carbonate buffer solution of pH9.6 as coating solution, adding into ELISA reaction plate at 100 μ L/well, acting at 37 deg.C for 2 hr, coating at 4 deg.C overnight, patting to dry, sealing with Porcine skin Gelatin37 deg.C for 2 hr, washing with PBS containing 0.05% Tween-20 pH7.4, patting to dry, packaging in a packaging bag containing desiccant, and storing for use.
Example 5 ELISA antibody detection kit
The indirect ELISA antibody detection kit for duck hepatitis A virus type 3 polypeptide comprises the following components:
ELISA strips prepared in example 4 (96 wells): 5, blocking;
10 × concentrated wash prepared in example 2: 400mL (diluted 1: 10 before);
example 2 sample dilutions were prepared: 400 mL;
enzyme conjugate working solution: goat anti-duck enzyme-labeled secondary antibody (KPL company, USA), 100 mL;
color developing solution prepared in example 2: 200 mL;
stop solution formulated in example 2: 100 mL;
positive serum control (+): 2 mL;
Negative serum control (-) formulated in example 2: 2 mL.
The using method of the ELISA antibody detection kit comprises the following steps:
(1) taking a sample diluent for serum to be detected as 1: diluting with 100, adding 100 μ L/well into antibody detection plate, setting negative serum control and positive serum control, and incubating at 37 deg.C for 45 min;
(2) discarding the liquid in the reaction well, adding 350 μ L of washing solution (PBST) into each well, washing for 3-5 times at an interval of 1min, and drying;
(3) adding 100 μ L of enzyme conjugate working solution into each well, and incubating at 37 deg.C for 45 min;
(4) repeating the step (2);
(5) adding 100 μ L of color development solution, incubating at 37 deg.C in dark for 15 min;
(6) adding 50 μ L of stop solution, measuring absorbance A value of each well at 650nm wavelength with a microplate reader, and calculating and judging the result.
Example 6 specificity test
And (3) cross test: the indirect ELISA detection kit established by the duck hepatitis A virus type 3 polypeptide W7 is used for respectively detecting positive serum and negative serum of duck hepatitis A virus type 1, duck hepatitis A virus type 3 and the like, duck plague, duck reovirus disease, duck newcastle disease and duck influenza, wherein each sample is repeated for 4 times, cross reactivity determination is carried out, the detection result shows that the positive serum of duck hepatitis A virus type 3 is a positive result, and the rest are negative, which shows that the detection kit has no cross reaction with the duck hepatitis A virus type 1, the duck plague, the duck reovirus disease, the duck newcastle disease, the duck influenza and the like.
Damping test: the DHAV-3 positive serum was mixed with an equal volume of DHAV-1(200 TCID)500.2ml) and DHAV-3(200 TCID)500.2ml) and duck plague virus (200 TCID)500.2ml), reacting for 30min at room temperature, and respectively carrying out inhibition test on the treated serum and the untreated serum according to the optimal reaction conditions, wherein the result shows that the DHAV-3 positive serum can only be specifically inhibited by DHAV-3 and can not be inhibited by DHAV-1 and duck plague virus.
Example 7 sensitivity test
DHAV-1 (neutralization titer 1: 186) and DHAV-3 positive serum (neutralization titer 1: 166) were made to 1: 50-1: 6400 and performing ELISA detection under the rest conditions according to the optimal reaction conditions. The results showed that the OD of the DHAV-1 and DHAV-3 positive sera was at 1:1600 when diluted650nmStill higher than the critical value of 0.278 (as shown in table 3), indicating that the ELISA detection kit has higher sensitivity.
TABLE 3 sensitivity test
EXAMPLE 8 repeatability test
5 parts of DHAV-3 positive serum and 5 parts of negative serum are detected by using the enzyme-labeled plate coated twice respectively, each sample is repeatedly detected for 5 times, and the coefficient of variation CV% (CV ═ S.D./X × 100%, S.D.: standard deviation, X: arithmetic mean) is determined. The results showed that the coefficient of variation was at most 3.27% and at least 1.19%. The 10 serum has smaller coefficient of variation and better repeatability.
Example 9 clinical applications
Clinical applications the samples examined were test samples and part of the samples to be examined. The detection is carried out by adopting a serum neutralization test and a duck hepatitis A virus type 3 polypeptide indirect ELISA antibody detection kit, the coincidence rate of the two methods is compared, and the result is shown in Table 4.
TABLE 4 comparison of ELISA test kits with neutralization assays
Note: the detected coincidence rate is (common positive number + common negative number)/total sample number multiplied by 100%
As can be seen from the data in Table 4, 10 portions of DHAV-3 positive serum were detected in the neutralization test, the positive detection rate was 33.3%, 11 portions of DHAV-3 positive serum were detected in the ELISA detection kit, the positive detection rate was 36.7%, and the compliance rate of the two methods was 96.7%. Therefore, the ELISA detection kit is suitable for clinical application.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (5)
1. A DHAV-3 type polypeptide indirect ELISA antibody detection kit is characterized by comprising an ELISA plate coated by DHAV-3 type synthetic polypeptide W7; the amino acid sequence structure of the synthetic polypeptide W7 is N-SRNHRPFRFCLRLKT-CooH.
2. The kit for detecting the DHAV-3 type polypeptide indirect ELISA antibody of claim 1, wherein the method for preparing the ELISA plate coated with the synthetic polypeptide W7 comprises the following steps: the synthetic polypeptide W7 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 ℃, blotted dry, blocked with Porcine skin Gelatin at 37 ℃ for 2 hours, washed with PBS containing 0.05% Tween-20 at pH7.4, blotted dry and stored in a bag containing a desiccant after drying.
3. The indirect DHAV-3 polypeptide ELISA antibody detection kit of claim 1 or 2, wherein the kit further comprises a sample diluent, a 10 x concentrated washing solution, an enzyme conjugate working solution, a color developing solution, a stop solution, a positive control and a negative control.
4. The indirect DHAV-3 polypeptide ELISA antibody detection kit of claim 3, wherein the kit comprises:
5. The kit for detecting the DHAV-3 type polypeptide indirect ELISA antibody according to claim 4, wherein:
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 x concentrated washing solution is 0.1M phosphate buffer solution containing 0.5% Tween-20, and pH 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 solution with the concentration of 0.2mg/mL and a citric acid-phosphate buffer solution containing 0.5 per thousand of urea hydrogen peroxide;
the stop solution is a hydrofluoric acid solution with the concentration of 0.31 percent;
the positive control is DHAV-3 positive serum obtained by screening, and the OD of the positive control is650nmMore 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 control650nmLess than or equal to 0.25, and adding1000U/mL of penicillin streptomycin.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127531A (en) * | 2010-12-22 | 2011-07-20 | 山东省农业科学院家禽研究所 | Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit |
| CN103235121A (en) * | 2013-03-18 | 2013-08-07 | 江苏省农业科学院 | Indirect ELISA kit for detecting porcine transfusion transmitted virus 2 (TTV2) antibody |
| CN103675275A (en) * | 2013-12-30 | 2014-03-26 | 山东滨州博莱威生物技术有限公司 | Duck flavivirus detection reagent kit |
| CN103820519A (en) * | 2014-02-14 | 2014-05-28 | 西南民族大学 | Monoclonal antibody of genetic C-type duck hepatitis A virus (DHAV) and applications thereof |
| CN104031145A (en) * | 2014-04-16 | 2014-09-10 | 西南民族大学 | Monoclonal antibody against duck hepatitis A virus type A and application thereof |
| CN104087609A (en) * | 2014-07-10 | 2014-10-08 | 山东省农业科学院家禽研究所 | General type duck hepatitis A virus antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit |
| CN104215762A (en) * | 2014-09-28 | 2014-12-17 | 武汉中博生物股份有限公司 | Indirect ELISA method and kit for detecting serum 3-type duck hepatitis virus a antibody |
-
2018
- 2018-03-14 CN CN201810211222.6A patent/CN108169496B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127531A (en) * | 2010-12-22 | 2011-07-20 | 山东省农业科学院家禽研究所 | Korean novel duck hepatitis viral antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit |
| CN103235121A (en) * | 2013-03-18 | 2013-08-07 | 江苏省农业科学院 | Indirect ELISA kit for detecting porcine transfusion transmitted virus 2 (TTV2) antibody |
| CN103675275A (en) * | 2013-12-30 | 2014-03-26 | 山东滨州博莱威生物技术有限公司 | Duck flavivirus detection reagent kit |
| CN103820519A (en) * | 2014-02-14 | 2014-05-28 | 西南民族大学 | Monoclonal antibody of genetic C-type duck hepatitis A virus (DHAV) and applications thereof |
| CN104031145A (en) * | 2014-04-16 | 2014-09-10 | 西南民族大学 | Monoclonal antibody against duck hepatitis A virus type A and application thereof |
| CN104087609A (en) * | 2014-07-10 | 2014-10-08 | 山东省农业科学院家禽研究所 | General type duck hepatitis A virus antibody ELISA (Enzyme-Linked Immunosorbent Assay) detection kit |
| CN104215762A (en) * | 2014-09-28 | 2014-12-17 | 武汉中博生物股份有限公司 | Indirect ELISA method and kit for detecting serum 3-type duck hepatitis virus a antibody |
Non-Patent Citations (3)
| Title |
|---|
| DHAV－3VP1蛋白优势抗原区的初步鉴定;崔嘉琦 等;《黑龙江畜牧兽医》;20170910(第533期);第53-57页 * |
| 基因A型和C型鸭甲型肝炎病毒卵黄抗体的研制及应用;王继练 等;《动物医学进展》;20160920;第37卷(第9期);第43-48页 * |
| 表达1型和3型鸭甲型肝炎病毒ＶＰ１基因重组新城疫病毒的构建及其免疫原性;郑文卿 等;《中国兽医学报》;20170915;第37卷(第9期);第1641-1647页 * |
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