ELISA (enzyme-linked immunosorbent assay) kit for detecting canine ectopic dermatitis antibody as well as method and application thereof

Abstract

The invention relates to an ELISA kit for detecting a canine ectopic dermatitis antibody and a method and application thereof, and belongs to the technical field of animal medicine. The invention provides application of a recombinant protein Per a 10 with an amino acid sequence as shown in SEQ ID NO.1 in preparation of a reagent or a kit for detecting a canine ectopic dermatitis antibody. According to the present invention, the recombinant protein Per a 10 is adopted to establish the ELISA kit for detecting the canine ectopic dermatitis antibody, the kit has characteristics of strong specificity, high accuracy, good repeatability, simpleness and rapidness, the accuracy rate is 95.65%, and the kit is suitable for detecting canine ectopic dermatitis.

Description

Technical Field

[0001] The present invention relates to the technical field of animal medicine, in particular to an ELISA kit for detecting canine atopic dermatitis antibodies and a method and application thereof. Background Art

[0002] Like humans, dogs and cats can also develop allergies, including atopic dermatitis, food allergies, flea and insect bite allergies, and drug allergies. Atopic dermatitis is the most common, with approximately 70% of dogs clinically experiencing the disease. Canine atopic dermatitis (cAD) is a recurrent, chronic, allergic skin allergy mediated by a specific immunoglobulin E (IgE). The disease is generally hereditary, and the allergens are primarily environmental and airborne components. Canine atopic dermatitis has a defective epidermal barrier, making it easier for antigens, irritants, and microorganisms to invade, leading to the complications of several skin diseases, with the possibility of "superimposition" of itching, such as Malassezia dermatitis and pyoderma. Pruritus in dogs not only causes self-injury to the animal, but also reduces the quality of life of both the animal and the owner, and can even cause anxiety in the owner.

[0003] Currently, methods for detecting canine atopic dermatitis in dogs include exclusion testing, intradermal testing, serum IgE testing, microbial culture, PCR testing, and skin biopsy. However, these methods all have certain drawbacks, such as the time-consuming and inefficient diagnosis of exclusion testing; the complex operation of intradermal testing and the susceptibility of results to interference; the high false-positive rate of serum IgE testing; the inability of microbial culture to distinguish between secondary infection and primary causes; the high cost and low sensitivity of PCR testing; and the invasive nature of skin biopsy, which can cause complications. A simple and highly accurate method for detecting canine atopic dermatitis is urgently needed.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide an ELISA kit for detecting canine atopic dermatitis antibodies, a method and an application thereof, so as to solve the problem that the existing methods for detecting canine atopic dermatitis are complex and have low accuracy.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides use of a recombinant protein Per a 10 having an amino acid sequence as shown in SEQ ID NO. 1 in preparing a reagent or a kit for detecting canine atopic dermatitis antibodies.

[0008] The present invention provides an ELISA kit for detecting canine atopic dermatitis antibodies. The ELISA kit comprises an enzyme-labeled plate coated with a recombinant protein Per a 10, an enzyme-labeled secondary antibody, a diluent, a positive control sample, a negative control sample, a color developing solution, and a stop solution.

[0009] The amino acid sequence of the recombinant protein Per a 10 is shown in SEQ ID NO.1;

[0010] The enzyme-labeled secondary antibody is a rabbit anti-dog IgG polyclonal antibody labeled with horseradish peroxidase.

[0011] Preferably, the diluent is PBST buffer containing 0.05-0.15% BSA;

[0012] The color developing solution is TMB substrate color developing solution;

[0013] The stop solution is a 1-3 M H2SO4 aqueous solution.

[0014] Preferably, the preparation method of the ELISA plate coated with the recombinant protein Per a 10 is to mix the recombinant protein Per a 10 with a buffer to obtain a coating solution, add the coating solution to the ELISA plate, coat at 3 to 5° C. for 10 to 14 hours, discard the coating solution, add a blocking solution to the ELISA plate, let it stand at 36 to 38° C. for 1 to 3 hours, wash and dry to obtain the ELISA plate coated with the recombinant protein Per a 10.

[0015] Preferably, the buffer is 0.04-0.06M carbonate buffer;

[0016] The final concentration of the recombinant protein Per a 10 in the coating solution is 0.1-0.3 μg / μL, and the volume of the coating solution added to the ELISA plate is 90-110 μL / well;

[0017] The blocking solution is a PBST buffer solution containing 0.5-1.5% BSA, and the volume of the blocking solution added to the ELISA plate is 190-210 μL / well.

[0018] The present invention provides a method for detecting canine atopic dermatitis antibodies for non-diagnostic purposes, which is performed using the ELISA kit, and the specific steps are as follows:

[0019] (1) Mix the sample to be tested with the diluent, add 90-110 μL / well to the ELISA plate coated with the recombinant protein Per a 10, and incubate at 36-38°C for 0.5-1.5 h to obtain the incubated ELISA plate;

[0020] (2) Mixing the enzyme-labeled secondary antibody with the diluent to obtain a mixed solution, adding the mixed solution to the incubated ELISA plate at a rate of 90 to 110 μL / well, incubating at 36 to 38°C for 25 to 35 minutes, washing to obtain a washed ELISA plate, adding the color developing solution to the washed ELISA plate at a rate of 90 to 110 μL / well, reacting at room temperature in the dark for 5 to 20 minutes, and obtaining a color-developed ELISA plate;

[0021] (3) Add the stop solution to the developed ELISA plate at a volume of 90 to 110 μL / well, immediately detect the OD value of the sample at 440 to 460 nm, calculate the S / P value of the sample based on the OD value of the sample, and determine whether the sample contains canine atopic dermatitis antibodies based on the S / P value of the sample;

[0022] The volume ratio of the sample to be tested and the diluent mixed in step (1) is 1:1800-2200;

[0023] The volume ratio of the enzyme-labeled secondary antibody and the diluent in step (2) is 1:8000-10000.

[0024] Preferably, the calculation formula of the S / P value in step (3) is as follows:

[0025] S / P value = (OD value of the sample to be tested - OD value of the negative control sample) / (OD value of the positive control sample - OD value of the negative control sample).

[0026] Preferably, the method for determining whether the sample to be tested contains canine atopic dermatitis antibodies in step (3) is:

[0027] When the S / P value of the sample to be tested is greater than or equal to 0.6, it is positive and the sample to be tested contains canine atopic dermatitis antibodies;

[0028] When the S / P value of the sample to be tested is less than 0.6, it is negative and the sample to be tested does not contain canine atopic dermatitis antibodies.

[0029] The present invention provides application of the ELISA kit in preparing a diagnostic product for canine atopic dermatitis.

[0030] The present invention has the following technical effects and advantages:

[0031] The present invention provides use of a recombinant protein Per a 10 having an amino acid sequence as shown in SEQ ID NO. 1 in preparing a reagent or a kit for detecting canine atopic dermatitis antibodies. An ELISA kit for detecting canine atopic dermatitis antibodies is established using the recombinant protein Per a 10. The kit has the characteristics of strong specificity, high accuracy, good repeatability, simplicity and rapidity, with an accuracy rate of 95.65%, and is suitable for detecting canine atopic dermatitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the predicted antigenic epitope map of Per a 10 protein;

[0033] Figure 2 This is the gel electrophoresis diagram of the recombinant protein Per a 10. DETAILED DESCRIPTION

[0034] The present invention provides the use of a recombinant protein Per a 10 having an amino acid sequence as shown in SEQ ID NO. 1 in preparing a reagent or a kit for detecting canine atopic dermatitis antibodies;

[0035] SEQ ID The amino acid sequence shown in NO.1 is: MFRIAIFVSVLFASCWGASLIPRRPRHDGRIVGGEPTDITNYPYQVSFEFFGSHHCGAAIISELWVVTAGHCVDGIPVSTVTFRAGTNVMEEGGTVHEAAEIIAHPNFDYLSLDCDIAVSK VTTAFTYQPSVQPVSLASADPPAGAMAVVTGWGSTSSGGALSPLLLKVSVPVVSFDACNASYSKYGGITSNMMCAGADEGGKDSCQGDSGGPLVVEKELVGIVSWGAGCGVRGYPGVYASVAVLREFVTSNTDVQ.

[0036] The present invention provides an ELISA kit for detecting canine atopic dermatitis antibodies. The ELISA kit comprises an enzyme-labeled plate coated with a recombinant protein Per a 10, an enzyme-labeled secondary antibody, a diluent, a positive control sample, a negative control sample, a color developing solution, and a stop solution.

[0037] The amino acid sequence of the recombinant protein Per a 10 is shown in SEQ ID NO.1;

[0038] The enzyme-labeled secondary antibody is a rabbit anti-dog IgG polyclonal antibody labeled with horseradish peroxidase.

[0039] In the present invention, the diluent is a PBST buffer solution containing 0.05-0.15% BSA, and the content of BSA in the PBST buffer solution is preferably 0.1%;

[0040] The positive control sample is a high-temperature inactivated dilution of canine serum after immunization with canine atopic dermatitis;

[0041] The negative control sample is a high-temperature inactivated serum dilution of a dog that is not sick and not immunized with a vaccine;

[0042] The color developing solution is TMB substrate color developing solution;

[0043] The stop solution is a 1-3M H2SO4 aqueous solution, and the concentration of the H2SO4 aqueous solution is preferably 2M.

[0044] In the present invention, the preparation method of the ELISA plate coated with the recombinant protein Per a 10 is as follows: mixing the recombinant protein Per a 10 with a buffer to obtain a coating solution, adding the coating solution to the ELISA plate, coating at 3 to 5° C. for 10 to 14 hours, discarding the coating solution, adding a blocking solution to the ELISA plate, standing at 36 to 38° C. for 1 to 3 hours, washing and drying to obtain the ELISA plate coated with the recombinant protein Per a 10;

[0045] The coating temperature is preferably 4°C, and the coating time is preferably 12 hours;

[0046] The standing temperature is preferably 37° C., and the standing time is preferably 2 h.

[0047] In the present invention, the buffer solution is a 0.04-0.06M carbonate buffer solution, and the concentration of the carbonate buffer solution is preferably 0.05M;

[0048] The final concentration of the recombinant protein Per a 10 in the coating solution is 0.1-0.3 μg / μL, preferably 0.2 μg / μL, and the volume of the coating solution added to the ELISA plate is 90-110 μL / well, preferably 100 μL / well;

[0049] The blocking solution is a PBST buffer solution containing 0.5-1.5% BSA, and the volume of the blocking solution added to the ELISA plate is 190-210 μL / well, preferably 200 μL / well;

[0050] The content of BSA in the PBST buffer is preferably 1%.

[0051] The present invention provides a method for detecting canine atopic dermatitis antibodies for non-diagnostic purposes, which is performed using the ELISA kit, and the specific steps are as follows:

[0052] (1) Mix the sample to be tested with the diluent, add 90-110 μL / well to the ELISA plate coated with the recombinant protein Per a 10, and incubate at 36-38°C for 0.5-1.5 h to obtain the incubated ELISA plate;

[0053] The added amount is preferably 100 μL / well;

[0054] The incubation temperature is preferably 37°C, and the incubation time is preferably 1 hour;

[0055] (2) Mixing the enzyme-labeled secondary antibody with the diluent to obtain a mixed solution, adding the mixed solution to the incubated ELISA plate at a rate of 90 to 110 μL / well, incubating at 36 to 38°C for 25 to 35 minutes, washing to obtain a washed ELISA plate, adding the color developing solution to the washed ELISA plate at a rate of 90 to 110 μL / well, reacting at room temperature in the dark for 5 to 20 minutes, and obtaining a color-developed ELISA plate;

[0056] The amount of the mixed solution added is preferably 100 μL / well;

[0057] The incubation temperature is preferably 37°C, and the incubation time is preferably 30 minutes;

[0058] The amount of the color developing solution added is preferably 100 μL / well;

[0059] The reaction time is preferably 15 min;

[0060] (3) Add the stop solution to the developed ELISA plate at a volume of 90 to 110 μL / well, immediately detect the OD value of the sample at 440 to 460 nm, calculate the S / P value of the sample based on the OD value of the sample, and determine whether the sample contains canine atopic dermatitis antibodies based on the S / P value of the sample;

[0061] The amount of the stop solution added is preferably 100 μL / well;

[0062] The wavelength of the sample to be tested is preferably 450 nm;

[0063] In step (1), the volume ratio of the sample to be tested and the diluent is 1:1800-2200, preferably 1:2000;

[0064] The volume ratio of the enzyme-labeled secondary antibody and the diluent in step (2) is 1:8000-10000, preferably 1:9000.

[0065] In the present invention, the calculation formula of the S / P value in step (3) is as follows:

[0066] S / P value = (OD value of the sample to be tested - OD value of the negative control sample) / (OD value of the positive control sample - OD value of the negative control sample).

[0067] In the present invention, the method for determining whether the sample to be tested contains canine atopic dermatitis antibodies in step (3) is:

[0068] When the difference between the OD value of the positive control sample and the OD value of the negative control sample is less than 0.4, the test result is considered invalid and needs to be retested;

[0069] When the difference between the OD value of the positive control sample and the OD value of the negative control sample is greater than or equal to 0.4, the test result is considered valid and the next step of determination is carried out;

[0070] When the S / P value of the sample to be tested is greater than or equal to 0.6, it is positive and the sample to be tested contains canine atopic dermatitis antibodies;

[0071] When the S / P value of the sample to be tested is less than 0.6, it is negative and the sample to be tested does not contain canine atopic dermatitis antibodies.

[0072] The present invention provides application of the ELISA kit in preparing a diagnostic product for canine atopic dermatitis.

[0073] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0074] Example 1: Screening of antigenic proteins

[0075] The amino acid sequences of three proteins, Per a 10 (ID: A0A2P0XIE9), per a allergen (A0A2P0XJ10), and peramyosin (A0A2P0XJ16), were obtained from the uniprot database for identification, analysis, and scoring. The results are shown in Table 1.

[0076] Table 1 Identification and analysis results of different amino acid sequences

[0077]

[0078] According to Table 1, the score of per a 10 protein is the highest among the three proteins, which is 7.5975, higher than that of per aallergen protein and peramyosin protein. The epitope of Per a 10 protein was predicted using the online software https: / / www.detaibio.com / tools / index.php?r=epitope-prediction%2Findex. The results are as follows: Figure 1 As shown, according to Figure 1 It can be seen that Per a 10 protein has good antigenicity and its entire amino acid sequence can be used as an antigen for antibody detection.

[0079] Example 2: Expression and purification of recombinant protein Per a 10

[0080] 1. Expression of Per a 10 protein

[0081] The nucleotide sequence of Per a 10 protein (ID: A0A2P0XIE9) was obtained based on the amino acid sequence in the uniprot database, and then primers were designed using Primer Premier 5.0 software. PCR amplification was then performed using the primer pairs using DNA of Periplaneta americana as template DNA to obtain an amplified fragment. The amplified fragment was then cloned into the expression vector pET32a to obtain the recombinant vector pET32a. After sequencing verification, the recombinant vector pET32a was transformed into Escherichia coli BL21 (DE3) competent cells to obtain recombinant bacteria. IPTG was used as an inducer to induce gene expression in the recombinant bacteria to obtain an induced expression bacterial solution.

[0082] 2. Purification of Per a 10 Protein

[0083] The induced bacterial liquid was centrifuged at 12000 rpm for 10 min to collect the bacteria, and then the bacteria were resuspended in PBS buffer to obtain a resuspension. The resuspension was then ultrasonically treated (ultrasound 1 s, interval 1 s, time for 10 min) to lyse the bacteria to obtain a lysed bacterial liquid, which was centrifuged at 12000 rpm for 10 min to collect the supernatant, which was chromatographically purified using a nickel ion affinity chromatography column to obtain a purified recombinant protein Per a 10 protein solution, which was then subjected to gel electrophoresis. The results are shown in FIG. Figure 2 shown.

[0084] according to Figure 2 It can be seen that a clear and bright single band appeared at 26 KDa for the purified Per a 10 protein, indicating that the purified recombinant protein Per a 10 was successfully obtained. The recombinant protein Per a 10 was sequenced, and its amino acid sequence was shown in SEQ ID NO.1. The amino acid sequence described in SEQ ID NO.1 is: MFRIAIFVSVLFASCWGASLIPRRPRHDGRIVGGEPTDITNYPYQVSFEFFGSHHCGAAIISELWVVTAGHCVDGIPVSTVTFRAGTNVMEEGGTVHEAAEIIAHPNFDYLSLDCDIAVSKVTTAFTYQPSVQPVSLASADPPAGAMAVVTGWGSTSSGGALSPLLLKVSVPVVSFDACNASYSKYGGITSNMMCAGADEGGKDSCQGDSGGPLVVEKELVGIVSWGAGCGVRGYPGVYASVAVLREFVTSNTDVQ.

[0085] The obtained recombinant protein Per a 10 was reacted with canine serum suffering from canine atopic dermatitis. By adding horseradish peroxidase-labeled rabbit anti-canine IgG polyclonal antibody and TMB substrate colorimetric solution, it was found that the recombinant protein Per a 10 was able to react with antibodies in the serum of canines suffering from canine atopic dermatitis. The addition of TMB substrate colorimetric solution enabled color development, indicating that the recombinant protein Per a 10 can be used to detect antibodies in canine atopic dermatitis.

[0086] Example 3: Establishment of a method for detecting canine atopic dermatitis antibodies

[0087] Preparation of ELISA plates coated with recombinant protein Per a 10

[0088] The recombinant protein Per a 10 solution prepared in Example 1 was mixed with a 0.05 M carbonate buffer solution and diluted to obtain a coating solution. The coating solution was added to an ELISA plate at a rate of 100 μL / well and coated at 4°C for 12 h. The coating solution was then discarded and PBST buffer containing 1% BSA was added to the ELISA plate at a rate of 200 μL / well. The plate was allowed to stand at 37°C for 2 h, washed, and dried to obtain an ELISA plate coated with the recombinant protein Per a 10.

[0089] The detection method steps:

[0090] (1) The sample to be tested was mixed and diluted with the diluent, and added to the ELISA plate coated with the recombinant protein Per a 10 at a volume of 100 μL / well, and incubated at 37°C for 1 hour to obtain the incubated ELISA plate;

[0091] (2) Mix and dilute the enzyme-labeled secondary antibody with the diluent, add 100 μL / well of the diluted solution to the incubated ELISA plate, incubate at 37°C for 30 minutes, wash to obtain the washed ELISA plate, add the color development solution to the washed ELISA plate at 100 μL / well, react at room temperature in the dark for 15 minutes, and obtain the developed ELISA plate;

[0092] (3) Add the stop solution to the developed ELISA plate at a volume of 100 μL / well, and immediately detect the OD value of the sample at 450 nm to determine the result.

[0093] Based on the above steps, the reaction conditions of the detection method for canine atopic dermatitis antibodies were optimized.

[0094] 1. Selection of the optimal coating concentration of antigen and the optimal dilution of serum to be tested

[0095] According to the coating concentration of the recombinant protein Per a 10, the recombinant protein Per a 10 was coated with a carbonate buffer with a concentration of 0.05 M, so that the final concentrations of the recombinant protein Per a 10 in the coating solution were 0.1 μg / μL, 0.2 μg / μL, and 0.3 μg / μL, respectively. The coating solution was added to the ELISA plate at a rate of 100 μL / well, and the plate was coated at 4°C for 12 hours. The coating solution was then discarded, and PBST buffer containing 1% BSA was added to the ELISA plate at a rate of 200 μL / well. The plate was allowed to stand at 37°C for 2 hours, washed, and dried to obtain ELISA plates coated with different concentrations of the recombinant protein Per a 10. The negative control sample and the positive control sample were mixed with PBST buffer containing 0.1% BSA at a volume ratio of 1:1500, 1:2000, and 1:2500, and the OD values of the negative control sample and the positive control sample at 450 nm were measured by ELISA. The OD values of the positive control sample were calculated. 450 The OD value (P) was compared with the OD value of the positive control sample. 450 When the ratio (P / N) is the largest, it is the optimal antigen coating concentration and the optimal dilution of the serum to be tested. The results are shown in Table 1.

[0096] Table 1 Optimal coating concentration of antigen and optimal dilution of serum to be tested

[0097]

[0098] According to Table 1, when the coating concentration of the recombinant protein Per a 10 is 0.2 μg / μL and the serum dilution is 1:2000, the P / N value is the largest, which is 1.67, which is the optimal coating concentration and serum dilution.

[0099] 2. Selection of enzyme-labeled secondary antibody dilution

[0100] An ELISA plate coated with recombinant protein Per a 10 was prepared at a coating concentration of 0.2 μg / μL, and then the negative control sample and the positive control sample were mixed with PBST buffer containing 0.1% BSA at a volume ratio of 1:2000, and added to the ELISA plate at a volume of 100 μL / well. The plate was incubated at 37°C for 1 hour, and the enzyme-labeled secondary antibody (horseradish peroxidase-labeled rabbit anti-dog IgG polyclonal antibody) was diluted with diluent at a volume ratio of 1:8000, 1:9000, and 1:10000, and added to the incubated ELISA plate at a volume of 100 μL / well. The OD values of the negative control sample and the positive control sample at 450 nm were determined by ELISA, and the OD of the positive control sample was calculated. 450 The OD value (P) was compared with the OD value of the positive control sample. 450When the ratio (P / N) is the largest, it is the optimal enzyme-labeled secondary antibody dilution. The results are shown in Table 2.

[0101] Table 2 Results of optimal dilution of enzyme-labeled secondary antibodies

[0102]

[0103]

[0104] According to Table 2, when the volume ratio of the enzyme-labeled secondary antibody to the diluent is 1:9000, the maximum P / N ratio is 2.03, which is the optimal dilution of the enzyme-labeled secondary antibody.

[0105] 3. Determination of critical value

[0106] 12 known canine positive sera and 12 known canine negative sera were selected and tested using the optimized method for detecting canine atopic dermatitis antibodies. The OD values of the negative control sample, the positive control sample and the 12 known canine positive sera were calculated each time. 450 The S / P value was calculated and then statistical analysis was performed to find that when the difference between the OD value of the positive control sample and the OD value of the negative control sample was greater than or equal to 0.4, the S / P value of 0.6 could be used as the critical value for detection in the present application scheme. When the S / P value of the sample to be tested was greater than or equal to 0.6, it was positive; when the S / P value of the sample to be tested was less than 0.6, it was negative.

[0107] Based on the above optimization, the best method for detecting canine atopic dermatitis antibodies is:

[0108] (1) The sample to be tested was mixed with the diluent at a volume ratio of 1:2000, and added to the ELISA plate coated with the recombinant protein Per a 10 at a volume of 100 μL / well. The plate was incubated at 37°C for 1 h to obtain the incubated ELISA plate;

[0109] (2) Mix the enzyme-labeled secondary antibody and the diluent at a volume ratio of 1:9000, add 100 μL / well to the incubated ELISA plate, incubate at 37°C for 30 minutes, wash to obtain the washed ELISA plate, add the color development solution to the washed ELISA plate at a volume of 100 μL / well, react at room temperature in the dark for 15 minutes, and obtain the color-developed ELISA plate;

[0110] (3) Add the stop solution to the developed ELISA plate at a volume of 100 μL / well, immediately detect the OD value of the sample to be tested at 450 nm, and calculate the S / P value of the sample to be tested based on the OD value of the sample to be tested;

[0111] The formula for calculating the S / P value is:

[0112] S / P value = (OD value of the sample to be tested - OD value of the negative control sample) / (OD value of the positive control sample - OD value of the negative control sample).

[0113] When the difference between the OD value of the positive control sample and the OD value of the negative control sample is less than 0.4, the test result is considered invalid and needs to be retested;

[0114] When the difference between the OD value of the positive control sample and the OD value of the negative control sample is greater than or equal to 0.4, the test result is considered valid and the next step of determination is carried out;

[0115] When the S / P value of the sample to be tested is greater than or equal to 0.6, it is positive and the sample to be tested contains canine atopic dermatitis antibodies;

[0116] When the S / P value of the sample to be tested is less than 0.6, it is negative and the sample to be tested does not contain canine atopic dermatitis antibodies.

[0117] Example 4: Preparation of an ELISA kit for detecting canine atopic dermatitis antibodies

[0118] 1. Preparation of ELISA Plate Coated with Recombinant Protein Per a 10

[0119] The recombinant protein Per a 10 solution prepared in Example 1 was mixed with a 0.05 M carbonate buffer solution and diluted to obtain a coating solution. The final concentration of the recombinant protein Per a 10 in the coating solution was 0.2 μg / μL. The coating solution was added to the ELISA plate at a rate of 100 μL / well, and the plate was coated at 4°C for 12 h. The coating solution was then discarded, and PBST buffer containing 1% BSA was added to the ELISA plate at a rate of 200 μL / well. The plate was allowed to stand at 37°C for 2 h, washed, and dried to obtain an ELISA plate coated with the recombinant protein Per a 10.

[0120] 2. Preparation of dilution solution

[0121] Take 0.1 g of BSA and add PBST buffer to 100 mL to obtain a dilution solution.

[0122] 3. Preparation of Positive and Negative Control Samples

[0123] Take 1 mL of serum from a dog immunized with atopic dermatitis, sterilize it at high temperature, and then add the diluent to make the volume 10 mL to obtain a positive control sample;

[0124] Serum from a dog that does not suffer from canine atopic dermatitis and has not been immunized was obtained and sterilized at high temperature, and then a diluent was added to make the volume 10 mL to obtain a negative control sample.

[0125] 4. Preparation of Color Developing Solution

[0126] Mix 25.7 mL of 0.2 M NaHPO2 aqueous solution with 24.3 mL of 0.1 M citric acid aqueous solution, and add distilled water to 100 mL to obtain a phosphate-citrate buffer solution with a pH of 5.0;

[0127] Dimethyl sulfoxide (DMSO) and 3,3',5,5'-tetramethylbenzidine (TMB) were mixed to prepare a 1% TMB solution in DMSO;

[0128] 0.1 mL of 1% TMB dimethyl sulfoxide solution was added to 9.9 mL of pH 5.0 citric acid buffer and mixed to obtain TMB substrate color development solution.

[0129] 5. Preparation of Stop Solution

[0130] 21.7 mL of concentrated sulfuric acid (98%) was added dropwise to 178.3 mL of distilled water to obtain a 2 M H2SO4 aqueous solution, which was the stop solution.

[0131] 6. Enzyme-labeled secondary antibody

[0132] The secondary antibody was a commercially available rabbit anti-dog IgG polyclonal antibody, which was labeled with horseradish peroxidase to obtain a horseradish peroxidase-labeled rabbit anti-dog IgG polyclonal antibody.

[0133] 7. Washing liquid

[0134] The washing solution was PBST buffer.

[0135] Preparation of ELISA kit

[0136] The prepared ELISA plate coated with the recombinant protein Per a 10 was vacuum-packed, and the diluent, positive control sample, negative control sample, color development solution, stop solution, enzyme-labeled secondary antibody and washing solution were respectively placed in test tubes, labeled respectively, and finally combined into a kit to obtain an ELISA kit for detecting canine atopic dermatitis antibodies.

[0137] Example 5: Specificity Experiment

[0138] The sera of dogs with symptoms of atopic dermatitis, scale mites, Staphylococcal superficial pyoderma, and Malassezia dermatitis were used as the test sera, and the method for detecting canine atopic dermatitis antibodies in Example 3 was used for detection. The results showed that only the serum samples of dogs with atopic dermatitis tested positive, while the serum samples of the other diseases were negative, indicating that the ELISA kit prepared by the present application scheme has good specificity.

[0139] Example 6: Repeatability Experiment

[0140] Ten collected canine sera (5 positive and 5 negative) were tested using ELISA plates coated with the same batch of recombinant protein Per a 10 and a kit coated with ELISA plates coated with recombinant protein Per a 10 from different batches. Two replicates were performed for each serum sample, using the method for detecting canine atopic dermatitis antibodies described in Example 3 for inter- and intra-batch reproducibility testing. The coefficient of variation was calculated to test inter-batch reproducibility. The results are shown in Table 3.

[0141] The coefficient of variation is calculated as follows:

[0142] CV (%) = SD / X × 100%;

[0143] In the formula, SD represents standard deviation; X represents the average value of the results of two repeated tests;

[0144] Table 3 Intra-batch and inter-batch repeat test results

[0145]

[0146]

[0147] According to Table 3, the coefficients of variation between and within batches were less than 10%, indicating good experimental repeatability.

[0148] Example 7: Clinical sample detection

[0149] The kit prepared in Example 2 was used to detect 78 collected canine sera (including 24 positive sera and 54 negative sera) according to the detection method of Example 3. The test results are shown in Table 4.

[0150] Table 2 Results of 78 canine serum tests

[0151]

[0152] According to Table 4, the detection accuracy of the ELISA kit of the present application is 95.65%.

[0153] As can be seen from the above examples, the present invention provides an ELISA kit for detecting canine atopic dermatitis antibodies, as well as a method and application thereof. The ELISA kit for detecting canine atopic dermatitis antibodies was developed using the recombinant protein Per a 10 according to the present invention. The kit exhibits strong specificity, high accuracy, good reproducibility, and is simple and rapid, with an accuracy of 95.65%, making it suitable for detecting canine atopic dermatitis.

[0154] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of the recombinant protein Per a 10 having an amino acid sequence as shown in SEQ ID NO. 1 in the preparation of a reagent or kit for detecting canine atopic dermatitis antibodies.

2. An ELISA kit for detecting canine atopic dermatitis antibodies, characterized in that: The ELISA kit includes an enzyme-labeled plate coated with recombinant protein Per a 10, an enzyme-labeled secondary antibody, a diluent, a positive control sample, a negative control sample, a color development solution, and a stop solution; The amino acid sequence of the recombinant protein Per a 10 is shown in SEQ ID NO.1; The enzyme-labeled secondary antibody is a rabbit anti-dog IgG polyclonal antibody labeled with horseradish peroxidase.

3. The ELISA kit according to claim 2, wherein The diluent is a PBST buffer solution containing 0.05-0.15% BSA; The color developing solution is TMB substrate color developing solution; The stop solution is a 1-3 M H2SO4 aqueous solution.

4. The ELISA kit according to claim 2, wherein The preparation method of the ELISA plate coated with the recombinant protein Per a 10 comprises mixing the recombinant protein Per a 10 with a buffer to obtain a coating solution, adding the coating solution to the ELISA plate, coating at 3 to 5° C. for 10 to 14 hours, discarding the coating solution, adding a blocking solution to the ELISA plate, standing at 36 to 38° C. for 1 to 3 hours, washing, and drying to obtain the ELISA plate coated with the recombinant protein Per a 10.

5. The ELISA kit according to claim 4, wherein The buffer solution is 0.04-0.06M carbonate buffer solution; The final concentration of the recombinant protein Per a 10 in the coating solution is 0.1-0.3 μg / μL, and the volume of the coating solution added to the ELISA plate is 90-110 μL / well; The blocking solution is a PBST buffer solution containing 0.5-1.5% BSA, and the volume of the blocking solution added to the ELISA plate is 190-210 μL / well.

6. A method for detecting canine atopic dermatitis antibodies for non-diagnostic purposes, characterized in that: The ELISA kit according to any one of claims 2 to 5 is used for detection, and the specific steps are as follows: (1) Mix the sample to be tested with the diluent, add 90-110 μL / well to the ELISA plate coated with the recombinant protein Per a10, and incubate at 36-38°C for 0.5-1.5 h to obtain the incubated ELISA plate; (2) Mixing the enzyme-labeled secondary antibody with the diluent to obtain a mixed solution, adding the mixed solution to the incubated ELISA plate at a rate of 90 to 110 μL / well, incubating at 36 to 38°C for 25 to 35 minutes, washing to obtain a washed ELISA plate, adding the color developing solution to the washed ELISA plate at a rate of 90 to 110 μL / well, reacting at room temperature in the dark for 5 to 20 minutes, and obtaining a color-developed ELISA plate; (3) Add the stop solution to the developed ELISA plate at a volume of 90 to 110 μL / well, immediately detect the OD value of the sample at 440 to 460 nm, calculate the S / P value of the sample based on the OD value of the sample, and determine whether the sample contains canine atopic dermatitis antibodies based on the S / P value of the sample; The volume ratio of the sample to be tested and the diluent mixed in step (1) is 1:1800-2200; The volume ratio of the enzyme-labeled secondary antibody and the diluent in step (2) is 1:8000-10000.

7. The method according to claim 6, characterized in that The calculation formula of the S / P value in step (3) is as follows: S / P value = (OD value of the sample to be tested - OD value of the negative control sample) / (OD value of the positive control sample - OD value of the negative control sample).

8. The method according to claim 6, characterized in that The method for determining whether the sample to be tested contains canine atopic dermatitis antibodies in step (3) is: When the S / P value of the sample to be tested is greater than or equal to 0.6, it is positive and the sample to be tested contains canine atopic dermatitis antibodies; When the S / P value of the sample to be tested is less than 0.6, it is negative and the sample to be tested does not contain canine atopic dermatitis antibodies.

9. Use of the ELISA kit according to any one of claims 1 to 5 in the preparation of a diagnostic product for canine atopic dermatitis.

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