AU2020101144A4 - Kit for detecting anti-foot-and-mouth disease virus 3ab antibody and detection method thereby - Google Patents

Abstract

KIT FOR DETECTING ANTI-FOOT-AND-MOUTH DISEASE VIRUS 3AB ANTIBODY AND DETECTION METHOD THEREBY ABSTRACT The present invention provides a kit for detecting anti-foot-and-mouth disease virus (FMDV) 3AB antibody and a detection method thereby, and belongs to the technical field of enzyme linked immunosorbent assay (ELISA). The kit includes an ELISA plate and an enzyme-conjugated secondary antibody; the ELISA plate is coated with FMDV 3AB protein. The kit for detecting anti-FMDV 3AB antibody provided by the present invention is efficient, sensitive, specific, and reproducible; the detection method features easy operation, rapidness, and low cost; the method enables visual detection at room temperature and is suitable for popularization in clinical use, providing a reliable technical means for rapid detection of anti FMDV 3AB antibody. 1/1 FIG. 1

Description

1/1

FIG. 1

KIT FOR DETECTING ANTI-FOOT-AND-MOUTH DISEASE VIRUS 3AB ANTIBODY AND DETECTION METHOD THEREBY

TECHNICAL FIELD The present invention relates to the technical field of enzyme linked immunosorbent assay (ELISA), and in particular to a kit for detecting anti-foot-and-mouth disease virus (FMDV) 3AB antibody and a detection method thereby. BACKGROUND Foot-and-mouth disease (FMD) is an acute, febrile, highly contagious infectious disease caused by FMDV, which principally infects artiodactyls and is usually transmitted in such animals as sheep, cows, and pigs. At the onset of FMD, blisters appear in lips and hooves of susceptible animals, accompanied by such symptoms as fever and anorexia; infected animals have substantial weight loss and decrease in milk yield. FMDV tends to spread through the air, with strong infectivity and fast prevalence; susceptible animals with weak immunity may also die, causing huge economic losses to farmers and hindering the development of breeding industry seriously. So far, diagnosis of anti-FMDV 3AB antibody and evaluation of immune efficacy of vaccines are only done in laboratories, but are not suitable for primary level detection. Therefore, it is necessary to establish a faster, simpler, and more sensitive visual detection method. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. OBJECT OF THE INVENTION It is the object of the present invention to substantially overcome or at least ameliorate one or more of the disadvantages of the prior art or to provide a useful alternative. SUMMARY In one embodiment, the present invention relates to a kit for detecting anti-FMDV 3AB antibody and a detection method thereby. The kit of the present disclosure detects the anti-FMDV 3AB antibody simply, rapidly and accurately without the aid of laboratory instrument and equipment, and is more suitable for primary level detection. In one aspect, the present invention relates to a kit for detecting anti-FMDV 3AB antibody, including an ELISA plate and an enzyme-conjugated secondary antibody; the ELISA plate is coated with FMDV 3AB protein. Preferably, the FMDV 3AB protein has a coating concentration of 1-2 pg/mL.

Preferably, the kit further includes a wash buffer, a serum diluent, a chromogenic substrate solution, a stop solution, a standard positive serum, and a standard negative serum. Preferably, the wash buffer includes a phosphate buffer solution (PBS), the PBS includes 0.03-0.08 wt % Tween-20, and the PBS is at pH 7.2. Preferably, the serum diluent includes a PBS, the PBS includes skim milk powder, and the skim milk powder has a concentration of 45-55 g/L. Preferably, the chromogenic substrate solution includes a tetramethylbenzidine (TMB) solution. Preferably, the stop solution includes a sodium dodecyl sulfate (SDS) solution or a sulfuric acid solution. Preferably, the enzyme-conjugated secondary antibody includes a porcine or bovine enzyme-conjugated secondary antibody. In a further aspect, the present invention relates to a method for detecting anti-FMDV 3AB antibody with the kit according to the above technical solution for non-diagnostic purposes, including the following steps: 1) diluting a serum sample in a 1:40 volume ratio with the serum diluent, to obtain a diluted serum; 2) adding the diluted serum obtained in step 1) to wells of the ELISA plate obtained in step 1), incubating the plate at 15-30°C for 0.5-1.5 h, discarding the liquid from the wells, washing the plate with wash buffer, and pat-drying, to obtain a first pat-dried ELISA plate; 3) adding the diluted enzyme-conjugated secondary antibody to the wells of the first pat-dried ELISA plate obtained in step 2), incubating the plate at 15-30°C for 0.5-1.5 h, discarding the liquid from the wells, washing the plate with wash buffer, and pat-drying, to obtain a second pat-dried ELISA plate; and 4) adding the chromogenic substrate solution to the wells of the second pat-dried ELISA plate obtained in step 3), and developing color in the dark for 15-25 min at 15-30°C, where the anti-FMDV 3AB antibody is detected when a blue color is developed in the well, and the anti-FMDV 3AB antibody is not detected when the color is achromatic in the well. Preferably, the enzyme-conjugated secondary antibody is diluted 20,000-fold with PBS and then added into the wells. In a further aspect, the present invention relates to a kit for detecting anti-FMDV 3AB antibody, including an ELISA plate and an enzyme-conjugated secondary antibody; the ELISA plate is coated with FMDV 3AB protein. In the present invention, when an animal produces the anti-3AB antibody after FMDV infection, detection of anti-3AB antibody can achieve a detection purpose.

The kit for detecting anti-FMDV 3AB antibody provided by the present disclosure is efficient, sensitive, specific, and reproducible; the detection method features easy operation, rapidness, and low cost; the method enables visual detection at room temperature and is suitable for popularization in clinical use, providing a reliable technical means for rapid detection of anti-FMDV 3AB antibody. Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". BRIEF DESCRIPTION OF THE DRAWINGS A preferred embodiment of the present invention will now be described by way of specific embodiments with reference to the examples and accompanying drawings in which: FIG. 1 illustrates a detection result of the kit provided by the present disclosure, where numbers, from left to right, represent as follows in sequence: 1. classical swine fever virus (CSFV)-infected serum; 2. Japanese encephalitis virus (JEV)-infected serum; 3. porcine circovirus (PCV)-infected serum; 4. porcine parvovirus (PPV)-infected serum; 5. FMDV-infected serum; 6. pseudorabies virus (PRV)-infected serum; 7. porcine reproductive and respiratory syndrome virus (prrsv)-infected serum; and 8. swine influenza virus (SIV)-infected serum. DETAILED DESCRIPTION The present disclosure relates to a kit for detecting anti-FMDV 3AB antibody, including an ELISA plate and an enzyme-conjugated secondary antibody; the ELISA plate is coated with FMDV 3AB protein. In the present disclosure, the ELISA plate is coated with FMDV 3AB protein, and the coating concentration is preferably 1-2 [g/mL. The coating method used is not particularly limited in the present disclosure, as long as a conventional coating method is used. In the present disclosure, the ELISA plate is preferably blocked with 50 g/L skim milk powder solution. In the present disclosure, the enzyme-conjugated secondary antibody preferably includes a porcine or bovine enzyme-conjugated secondary antibody; the porcine or bovine enzyme-conjugated secondary antibody is preferably a commercially available product. In the present disclosure, the kit further preferably includes a wash buffer, a serum diluent, a chromogenic substrate solution, a stop solution, a standard positive serum, and a standard negative serum. In the present disclosure, the wash buffer preferably includes a PBS, the PBS preferably includes 0.03-0.08 wt %, and more preferably 0.05 wt % Tween-20; the PBS is at pH 7.2. In the present disclosure, the serum diluent preferably includes a PBS, the PBS includes skim milk powder, and the skim milk powder preferably has a concentration of 45-55 g/L, and more preferably 50 g/L. In the present disclosure, the chromogenic substrate solution preferably includes a TMB solution; concentrations of the TMB solution are not particularly limited in the present disclosure, as long as a desired TMB concentration may be used during color development with a conventional substrate. In the present disclosure, the stop solution preferably includes an SDS solution or a sulfuric acid solution. The present disclosure has no particular limitation for concentrations of the SDS solution or the sulfuric acid solution, as long as a desired concentration may be used to stop the ELISA conventionally. In the present disclosure, the standard positive serum is preferably an FMDV-infected serum, and the standard negative serum is preferably a healthy animal serum. In examples of the present disclosure, the standard negative serum is preferably an FMDV type A negative serum; the standard positive serum is preferably an FMDV type A positive serum. The present disclosure further provides a method for detecting anti-FMDV 3AB antibody with the kit according to the above technical solution for non-diagnostic purposes, including the following steps: 1) diluting a serum sample in a 1:40 volume ratio with the serum diluent, to obtain a diluted serum; 2) adding the diluted serum obtained in step 1) to wells of the ELISA plate obtained in step 1), incubating the plate at 15-30°C for 0.5-1.5 h, discarding the liquid from the wells, washing the plate with wash buffer, and pat-drying, to obtain a first pat-dried ELISA plate; 3) adding the diluted enzyme-conjugated secondary antibody to the wells of the first pat-dried ELISA plate obtained in step 2), incubating the plate at 15-30°C for 0.5-1.5 h, discarding the liquid from the wells, washing the plate with wash buffer, and pat-drying, to obtain a second pat-dried ELISA plate; and 4) adding the chromogenic substrate solution to the wells of the second pat-dried ELISA plate obtained in step 3), and developing color in the dark for 15-25 min at 15-30°C, where the anti-FMDV 3AB antibody is detected when a blue color is developed in the well, and the anti-FMDV 3AB antibody is not detected when the color is achromatic in the well. The present disclosure dilutes the serum sample in a 1:40 volume ratio with the serum diluent, to obtain the diluted serum. The present disclosure adds the diluted serum obtained to wells of the ELISA plate, incubates the plate at 15-30°C for 0.5-1.5 h, discards the liquid from the wells, washes the plate with wash buffer, and pat-dries, to obtain the first pat-dried ELISA plate. In the present disclosure, the amount of the diluted serum added into a well of the ELISA plate is preferably 100 [L. In the present disclosure, incubation is conducted after the diluted serum is added into the wells of the ELISA plate; the incubation is conducted at 15-30°C, preferably 18-28°C, and more preferably 20-25°C; the incubation lasts for 0.5-1.5 h, preferably 1.8-1.2 h, and more preferably 1 h. In the present disclosure, the washing frequency is preferably three times. The present disclosure adds the diluted enzyme-conjugated secondary antibody to the wells of the first pat-dried ELISA plate, incubates the plate at 15-30°C for 0.5-1.5 h, discards the liquid from the wells, washes the plate with wash buffer, and pat-dries, to obtain the second pat-dried ELISA plate. In the present disclosure, the amount of the diluted enzyme-conjugated secondary antibody added into a well of the first pat-dried ELISA plate is preferably 100 L. In the present disclosure, the enzyme-conjugated secondary antibody is preferably diluted with PBS, and the dilution multiple is preferably 20,000-fold. In the present disclosure, incubation is conducted after the diluted enzyme-conjugated secondary antibody is added into the wells of the first pat-dried ELISA plate; the incubation is conducted at 15-30°C, preferably 18-28°C, and more preferably 20-25°C; the incubation lasts for 0.5-1.5 h, preferably 0.8-1.2 h, and more preferably 1 h. In the present disclosure, the washing frequency is preferably three times. The present disclosure adds the chromogenic substrate solution to the wells of the second pat-dried ELISA plate, and develops color in the dark for 15-25 min at 15-30°C; the anti-FMDV 3AB antibody is detected when a blue color is developed in the well, and the anti-FMDV 3AB antibody is not detected when the color is achromatic in the well. In the present disclosure, the amount of the chromogenic substrate solution is preferably 100 [L/well. In the present disclosure, the color development is preferably conducted at -30°C, and more preferably 18-28°C, and most preferably 20-25°C; the color development preferably lasts for 18-23 min, and more preferably 20 min. The technical solutions of the present disclosure will be described below clearly and completely with reference to the examples of the present disclosure. It is clear that the described examples are only a part of examples of the present disclosure, not all examples of the present disclosure. All other examples obtained by persons of ordinary skill in the art based on the examples of the present disclosure without creative efforts shall fall within the scope of the present disclosure. Example 1 1. Preparation of sample diluent, wash buffer, and stop solution Coating buffer was 0.05 M carbonate buffer: Na2CO3 1.59 g, NaHCO3 2.93 g, diluting to 1,000 mL with distilled water, at pH 9.6; Sample diluent was the wash buffer with 50 g/L skim milk powder. Wash buffer was 0.05% Tween-20 in 0.01 M PBS (pH 7.2), and the preparation method thereof was as follows: mixing 8.5 g of NaCl, 0.356 g of NaH2PO 4 -2H2O, and 2.772 g of NaH2PO 4 -12H20, dissolving the mixture in distilled water and diluting to 1,000 mL, and adding 0.5 mL of Tween-20 thereto, to obtain the above wash buffer. Stop solution was 1% SDS solution. 2. Determination of the optimal coating concentration of antigen and the optimal dilution of serum The result showed that, when the dilution of antigen was 2.0 pg/mL per well, a serum and an enzyme-conjugated secondary antibody were diluted 1:40 and 1:20,000, respectively. 3. Optimization of action time Optimally, the antigen was blocked for 20 min, the serum reacted for 1 h, the enzyme-conjugated secondary antibody was incubated for 40 min, and color development lasted for 20 min. 4. Determination of operation procedure Under the optimum operating conditions determined above; the determined operation procedure was as follows: using a coated and blocked ELISA plate, a serum sample was diluted 1:40 with serum diluent; the diluted serum was added to each well of the ELISA plate (100 L per well); each aliquot of the diluted serum was added to one well, and FMDV type A negative and positive sera were added to two wells, respectively (positive and negative sera were an FMDV-infected serum and a healthy animal serum, respectively, either of which is diluted 100-fold with PBS), followed by reaction for 1 h at room temperature; the liquid was discarded from the wells, and each well was washed with wash buffer three times and tapped to discard all liquid from wells; after adding 100 L of 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic substrate solution, color development was conducted in the dark for 20 min at room temperature; 100 L of SDS stop solution was added; the color was visually observed: a blue color represents a positive result, and an achromatic color represents a negative result.

Example 2 Specificity test for the kit: The method established in Example 1 was used to test a known group of positive serum samples of SIV, CSFV, JEV, PCV, PPV, PRV, PRRSV, and FMDV 3AB. A negative result displayed an achromatic color, and a positive result displayed a blue color. The result is shown in FIG. 1. As shown in FIG. 1, use of the kit provided by the present disclosure can detect FMD accurately, has no cross reaction with other viruses, and has specificity. It can be concluded from the above examples that: the kit for detecting anti-FMDV 3AB antibody provided by the present disclosure is efficient, sensitive, specific, and reproducible; the detection method features easy operation, rapidness, and low cost; the method enables visual detection at room temperature and is suitable for popularization in clinical use, providing a reliable technical means for rapid detection of anti- FMDV 3AB antibody. The foregoing descriptions are merely preferred examples of the present invention, it should be noted that various modifications and variations can be made by those skilled in the art without departing from the principles of the present invention and are within the scope of the invention.

Claims (5)

What is claimed is:

1. A kit for detecting anti-foot-and-mouth disease virus (FMDV) 3AB antibody, wherein the kit comprises an ELISA plate and an enzyme-conjugated secondary antibody; and the ELISA plate is coated with FMDV 3AB protein.

2. The kit according to claim 1, wherein the FMDV 3AB protein has a coating concentration of 1-2 pg/mL.

3. The kit according to claim 1 or 2, wherein the kit further comprises a wash buffer, a serum diluent, a chromogenic substrate solution, a stop solution, a standard positive serum, and a standard negative serum; wherein the wash buffer comprises a phosphate buffer solution (PBS), the PBS comprises 0.03-0.08 wt % Tween-20, and the PBS is at pH 7.2; wherein the serum diluent comprises a PBS, the PBS comprises skim milk powder, and the skim milk powder has a concentration of 45-55 g/L; wherein the chromogenic substrate solution comprises a tetramethylbenzidine (TMB) solution; wherein the stop solution comprises a sodium dodecyl sulfate (SDS) solution or a sulfuric acid solution.

4. The kit according to claim 1, wherein the enzyme-conjugated secondary antibody comprises a porcine or bovine enzyme-conjugated secondary antibody.

5. A method for detecting anti-FMDV 3AB antibody with the kit according to any one of claims 1 to 4 for non-diagnostic purposes, comprising the following steps: 1) diluting a serum sample in a 1:40 volume ratio with the serum diluent, to obtain a diluted serum; 2) adding the diluted serum obtained in step 1) to wells of the ELISA plate obtained in step 1), incubating the plate at 15-30°C for 0.5-1.5 h, discarding the liquid from the wells, washing the plate with wash buffer, and pat-drying, to obtain a first pat-dried ELISA plate; 3) adding the diluted enzyme-conjugated secondary antibody to the wells of the first pat-dried ELISA plate obtained in step 2), incubating the plate at 15-30°C for 0.5-1.5 h, discarding the liquid from the wells, washing the plate with wash buffer, and pat-drying, to obtain a second pat-dried ELISA plate; and

4) adding the chromogenic substrate solution to the wells of the second pat-dried ELISA plate obtained in step 3), and developing color in the dark for 15-25 min at 15-30°C, wherein the anti-FMDV 3AB antibody is detected when a blue color is developed in the well, and the anti-FMDV 3AB antibody is not detected when the color is achromatic in the well.