Polypeptides, kits and methods for detecting type 1 rhdv antibodies

Abstract

The application discloses a polypeptide for detecting type 1 RHDV antibody, and an amino acid sequence of the polypeptide is shown as SEQ ID NO. 1. The polypeptide is applied to preparing a kit for detecting type 1 RHDV antibody and is applied to detecting type 1 RHDV antibody. The application further discloses a kit for detecting type 1 RHDV antibody, which comprises the polypeptide for detecting type 1 RHDV antibody. The kit for detecting type 1 RHDV antibody is applied to detecting type 1 RHDV antibody. The polypeptide for detecting type 1 RHDV antibody has high specificity for type 1 RHDV antibody, is safe and has good antigenicity. The kit for detecting type 1 RHDV antibody is suitable for an indirect ELISA detection method, can evaluate type 1 RHDV antibody level, has high specificity and sensitivity and is suitable for detecting batch samples.

Description

Technical Field

[0001] This invention relates to peptides, kits, and methods for detecting type 1 RHDV antibodies, and belongs to the field of antibody detection technology. Background Technology

[0002] Rabbit viral hemorrhagic disease (RHD), also known as rabbit hemorrhagic pneumonia, rabbit hemorrhage, or rabbit plague, is an acute, septicemic, highly contagious, and fatal infectious disease in rabbits caused by rabbit hemorrhagic disease virus (RHDV). It is characterized by hemorrhage in all the body's parenchymal organs. The presence of this pathogen has been reported in many countries and regions, causing severe economic losses to the rabbit farming industry. Currently, two serotypes of RHDV with high mortality rates have been identified in China, one of which has a vaccine against RHDV serotype 1.

[0003] Currently, RHDV antibody detection in China is mainly conducted through a hemagglutination inhibition test. RHDV only agglutinates with human type O red blood cells and does not agglutinate with rabbit or other mammalian red blood cells. However, obtaining human type O blood cells for this method is difficult, and public health conditions are hard to meet. Therefore, how to evaluate the immunization efficacy of type 1 RHDV vaccine is an urgent problem to be solved.

[0004] Enzyme-linked immunosorbent assay (ELISA) is, to some extent, more sensitive and easier to standardize than hemagglutination inhibition assay. ELISA antibody detection methods for RHDV using whole RHDV virus or VP60 protein expressed through genetic engineering have been reported. However, these coating antigen proteins contain numerous epitopes, making them difficult to distinguish from type 2 RHDV antibodies in actual detection, thus unsuitable for evaluating type 1 RHDV vaccine antibodies. A single epitope peptide is the optimal coating antigen for establishing ELISA antibody detection, enabling specific detection of type 1 RHDV antibodies and providing technical support for the evaluation of rabbit hemorrhagic disease virus (RHDV) vaccines. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides a polypeptide, a kit and a method for detecting type 1 RHDV antibodies.

[0006] This invention is achieved through the following technical solution:

[0007] A polypeptide for detecting type 1 RHDV antibodies, named polypeptide E22, has the following amino acid sequence: AENSSSASVATAGIGG, as shown in SEQ ID NO.1.

[0008] The application of the peptide for detecting type 1 RHDV antibodies in the preparation of a kit for detecting type 1 RHDV antibodies, and its application in the detection of type 1 RHDV antibodies.

[0009] A kit for detecting type 1 RHDV antibodies, comprising the aforementioned polypeptide for detecting type 1 RHDV antibodies.

[0010] Furthermore, the kit includes an ELISA plate coated with peptide E22.

[0011] Furthermore, the ELISA plate coated with the peptide E22 was prepared by the following method: peptide E22 was added to the coating solution at a concentration of 10 μg / mL, and added to the ELISA reaction plate at a dose of 100 μL / well. The plate was incubated at 37°C for 2 hours, and then coated at 4°C for 10–16 hours. After drying, the plate was blocked with 1% BSA (bovine serum albumin) solution at 37°C for 2 hours. The plate was washed with PBST, dried, and then packaged in a desiccant bag for storage.

[0012] Furthermore, the coating solution is selected from carbonate buffer, preferably a 0.05 mol / L carbonate buffer with a pH of 9.6.

[0013] Furthermore, the kit also includes sample diluent, 10× concentrated wash buffer, enzyme conjugate working solution, chromogenic solution, stop solution, positive control, and negative control.

[0014] Furthermore, the kit consists of the following components: 5 ELISA plates coated with peptide E22; 400 mL of sample dilution buffer; 400 mL of 10× concentrated wash buffer; 100 mL of enzyme conjugate working solution; 200 mL of chromogenic solution; 100 mL of stop solution; 2 mL of positive control; and 2 mL of negative control.

[0015] Furthermore, the sample diluent is PBST, i.e., 0.01 mol / L phosphate buffer at pH 7.4 containing 0.05% Tween-20.

[0016] Furthermore, the 10× concentrated washing solution is a 0.1 mol / L phosphate buffer solution with pH 7.4 containing 0.5% Tween-20.

[0017] Furthermore, the enzyme conjugate working solution is HRP-goat anti-rabbit IgG (horseradish peroxidase-labeled goat anti-rabbit IgG).

[0018] Furthermore, the colorimetric solution is a mixture of tetramethylbenzidine (TMB) solution and citrate-phosphate buffer solution in a volume ratio of 1:1. The concentration of the tetramethylbenzidine solution is 0.2 mg / mL, and the citrate-phosphate buffer solution contains hydrogen peroxide urea at a concentration of 0.5‰.

[0019] Furthermore, the terminating solution is a 0.31% hydrofluoric acid solution.

[0020] Furthermore, the positive control is RHDV-positive serum obtained through screening, whose OD... 630nm ≥1.0, and contains 1000 U / mL of penicillin and streptomycin.

[0021] Furthermore, the negative control is negative serum obtained through screening, whose OD... 630nm ≤0.243, and contains 1000 U / mL of penicillin and streptomycin.

[0022] The kit for detecting type 1 RHDV antibodies is used in the detection of type 1 RHDV antibodies.

[0023] Further, in specific applications, the method of using the kit for detecting type 1 RHDV antibodies is as follows: Dilute the serum to be tested with sample diluent at a ratio of 1:200, and then add 100 μL / well to an ELISA plate coated with peptide E22. Negative and positive controls are also included. Incubate at 37°C for 1 hour. After the reaction, discard the liquid in the reaction wells, add 350 μL of washing buffer to each well, wash 3-5 times, and blot dry. Then add 100 μL of enzyme conjugate working solution to each well and incubate at 37°C for 30 minutes. Wash 3-5 times with washing buffer and blot dry. Add 100 μL of chromogenic solution sequentially, incubate at 37°C in the dark for 15 minutes, and add 100 μL of stop solution to terminate the reaction. Measure the absorbance (A value) of each well at a wavelength of 630 nm using a microplate reader. Calculate and determine the result. The determination criterion is: when the OD value of the sample to be tested... 630nm Values ​​and negative control OD 630nm The ratio of values ​​(P / N) is greater than or equal to 2.1, and the OD of the sample to be tested is... 630nm A value greater than 0.243 is considered positive.

[0024] A method for detecting type 1 RHDV antibodies, as described above.

[0025] The polypeptide E22 of the present invention for detecting type 1 RHDV antibodies has high specificity and good safety for type 1 RHDV antibodies. It specifically binds only to type 1 RHDV positive serum and does not cross-react with type 2 RHDV positive serum or positive serum of other pathogens. It has good antigenicity, which makes the detection kit of the present invention have high specificity and sensitivity.

[0026] The kit for detecting type 1 RHDV antibodies of the present invention is suitable for indirect ELISA detection, can evaluate the level of type 1 RHDV antibodies, is suitable for batch sample detection, and is simple and fast to operate.

[0027] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0028] Figure 1 : Schematic diagram of the results of HI antibody detection.

[0029] Figure 2 : Schematic diagram of antibody detection results of the ELISA detection kit of the present invention. Detailed Implementation

[0030] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0031] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0032] The polypeptide E22 of the present invention is an artificially synthesized polypeptide that can be synthesized according to polypeptide synthesis methods known to those skilled in the art, such as solid-phase synthesis of polypeptides.

[0033] Screening of experimental antigen peptides

[0034] The VP60 protein of RHDV is a major structural protein of the virus. Based on the principle of linear antigenic epitopes in immunology, this invention artificially synthesized polypeptides containing 15 amino acids each, starting from the first amino acid of the VP60 protein sequence, with each polypeptide consisting of two amino acids. A total of 62 polypeptides were synthesized. ELISA screening was performed using type 1 RHDV positive serum, as detailed below:

[0035] 1. Dilute the synthetic peptide (pre-diluted with 1 mL DMSO) to 50 μg / mL with carbonate buffer, and coat each well with 100 μL. Incubate at 37°C for 1 h in a humidifier, then incubate overnight at 4°C. Wash four times with PBST. Add 300 μL of 1% BSA solution to each well. Incubate at 37°C for 2 h in a humidifier, then wash five times with PBST.

[0036] 2. Dilute type 1 RHDV positive serum (HI titer 6 log2) and SPF rabbit negative serum 1:200 with PBST, 100 μl per well. Incubate at 37℃ for 1 h. Wash 5 times with PBST.

[0037] 3. Add 100 μl of goat anti-rabbit secondary antibody to each well. Incubate at 37°C for 30 min, then wash 5 times with PBST.

[0038] 4. Add 100 μl of substrate (TMB) to each well. Incubate in the dark for 15 min.

[0039] 5. Add 100 μl of stop solution to each well. Read the values ​​using a microplate reader at OD 630 nm. Repeat each polypeptide strip in 3 wells. The results are shown in Table 1.

[0040] As shown in Table 1, the E22 polypeptide has excellent antigenicity and immunogenicity, which is significantly better than the other 61 polypeptides.

[0041] Table 1

[0042]

[0043]

[0044]

[0045] Example 1: Selection of Sealing Solution

[0046] The E22 peptide was first dissolved in DMSO, then diluted with carbonate buffer to a protein concentration of 50 μg / mL, with 100 μL per well for coating. After incubation at 37°C for 1 h in a humidified chamber, it was incubated overnight at 4°C, followed by washing four times with PBST. Blocking buffer (as shown in Table 1) was added to each well at 300 μL, and the mixture was blocked at 37°C for 2 h, followed by washing five times with PBST. Type 1 RHDV positive serum and SPF rabbit negative serum were diluted 1:200 with PBST, with 100 μL per well, and incubated at 37°C for 1 h. The mixture was washed five times with PBST. Goat anti-rabbit secondary antibody (1:2000 dilution) was added to each well at 100 μL. The mixture was incubated at 37°C for 30 min, followed by washing five times with PBST. Substrate (TMB) was added to each well at 100 μL. The mixture was incubated in the dark for 15 min. Stop solution was added to each well at 100 μL. The results were read using an OD 630 nm microplate reader. The results are shown in Table 2.

[0047] Table 2 Selection of Sealing Fluid

[0048]

[0049]

[0050] After conducting ELISA experiments (4 replicates) with 5 different blocking solutions, the 1% BSA solution showed the best results.

[0051] Example 2: Specificity verification of peptide E22

[0052] Dissolve peptide E22 in DMSO, then dilute with carbonate buffer to a protein concentration of 10 μg / mL, and coat each well with 100 μL. Incubate at 37°C for 1 h in a humidified chamber, then overnight at 4°C, and wash four times with PBST. Add blocking buffer (1% BSA solution) at 300 μL per well, with three replicates. Incubate at 37°C for 2 h in a humidified chamber, and wash five times with PBST. Dilute type 1 RHDV positive serum (HI titer 5 log2), type 2 RHDV positive serum (HI titer 5 log2), and SPF rabbit negative serum 1:200 with PBST at 1:200, 100 μL per well, and incubate at 37°C for 1 h. Wash five times with PBST. Add goat anti-rabbit secondary antibody (1:2000 dilution) at 1:2000, 100 μL per well. Incubate at 37°C for 30 min, and wash five times with PBST. Add substrate (TMB) at 100 μL per well. Incubate in the dark for 15 min. Add 100 μl of stop solution to each well. Read the values ​​using a microplate reader at OD 630 nm. Results are shown in Table 3.

[0053] Table 3. Specificity verification of peptides

[0054]

[0055] The results shown in Table 2 indicate that peptide E22 can only specifically react with type 1 RHDV positive serum, but has no cross-reaction with type 2 RHDV positive serum.

[0056] Example 3 Reagent Kit Composition

[0057] The kit consists of the following components: 5 ELISA plates coated with peptide E22; 400 mL sample dilution buffer; 400 mL 10× concentrated wash buffer; 100 mL enzyme conjugate working solution; 200 mL chromogenic solution; 100 mL stop solution; 2 mL positive control; and 2 mL negative control.

[0058] The sample diluent was PBST, a 0.01 mol / L phosphate buffer solution with pH 7.4 containing 0.05% Tween-20. The preparation method was as follows: Take 0.2 g of KH2PO4, 2.9 g of NaHPO4·12H2O, and 8 g of NaCl, and bring the volume to 1000 mL, then add 0.5 mL of Tween-20.

[0059] The 10× concentrated washing solution is a 0.1 mol / L phosphate buffer solution with pH 7.4 containing 0.5% Tween-20. The preparation method is as follows: take 2g of KH2PO4, 29g of NaHPO4·12H2O, and 80g of NaCl, make up to 1000mL, and then add 5mL of Tween-20.

[0060] The enzyme conjugate working solution is HRP-goat anti-rabbit IgG (horseradish peroxidase-labeled goat anti-rabbit IgG).

[0061] The colorimetric solution is a mixture of tetramethylbenzidine solution and citrate-phosphate buffer solution in a volume ratio of 1:1. The concentration of the tetramethylbenzidine solution is 0.2 mg / mL, and the citrate-phosphate buffer solution contains 0.5‰ hydrogen peroxide urea. The preparation method is as follows: Weigh 200 mg of tetramethylbenzidine, dissolve it in 100 mL of anhydrous ethanol, and then make up to 1000 mL with double-distilled water; Weigh 21 g of citric acid (C6H8O7·H2O), 28.2 g of anhydrous disodium hydrogen phosphate (Na2HPO4), and 6.4 mL of 0.75% hydrogen peroxide urea, and make up to 1000 mL with double-distilled water, adjusting the pH to 5.0; control the volume ratio of the two solutions to 1:1.

[0062] The termination solution is a 0.31% hydrofluoric acid solution: take 0.31 mL of hydrofluoric acid and dilute it to 100 mL with double-distilled water.

[0063] The positive control was RHDV positive serum obtained through screening: the prepared positive serum was diluted 1:300 with sample diluent (its OD... 630nm Add 1000 U / mL of penicillin and streptomycin (≥1.0), filter aseptically, and use as a positive control for the kit.

[0064] The negative control is negative serum obtained through screening: the negative serum (OD) obtained through screening is... 630nm ≤0.25), add 1000 U / mL of penicillin and streptomycin, filter aseptically, and use as a negative control for the kit.

[0065] Example 4: Determination of Indirect ELISA Reaction Conditions

[0066] In this embodiment, a checkerboard assay was used to determine the optimal working concentrations of the peptide antigen and serum. Peptide E22 was diluted with coating buffer to concentrations of 1 μg / mL, 5 μg / mL, and 10 μg / mL, and used to coat ELISA reaction plates at 100 μL / well. Positive and negative sera against type 1 RHDV were diluted 1:100, 1:200, and 1:300, respectively, with sample dilution buffer. Indirect ELISA was performed. A chromogenic reagent was added for color development, and a stop solution was added to terminate the reaction. The OD value at a wavelength of 630 nm was measured, and the results are shown in Table 4.

[0067] Collect positive serum OD 630nm Around 1.0, negative serum OD 630nm Around 0.2, and positive serum OD 630nm / negative serum OD 630nmThe optimal working concentrations were found to be antigen concentrations with a P / N ratio greater than 2.1 and serum dilutions. The results showed that the optimal serum dilution was 1:200 and the optimal peptide coating concentration was 10 μg / mL.

[0068] Table 4. Determination of Optimal Working Concentrations (OD) of Polypeptide Antigens and Serum 630nm value)

[0069]

[0070] Thirty-six collected SPF rabbit serum samples (RHDV antibody negative) were subjected to indirect ELISA under optimal working conditions to determine the absorbance range of rabbit serum in the absence of RHDV infection. Therefore, the OD of the sample to be tested was determined. 630nm Values ​​and negative OD 630nm The ratio of values ​​(P / N) is greater than or equal to 2.1, and the OD of the sample to be tested is... 630nm A value greater than 0.243 is considered positive, as shown in Table 5.

[0071] Table 5 Determination of RHDV antibody threshold values

[0072]

[0073] Example 5: Preparation of a reaction plate for indirect ELISA antibody detection of type 1 RHDV peptide.

[0074] The peptide E22 was first dissolved in DMSO, then diluted with 0.05 mol / L carbonate buffer (pH 9.6) to a protein concentration of 10 μg / mL. 100 μL was added to each well of the ELISA plate and incubated at 37°C for 2 hours, followed by coating at 4°C for 14 hours. After drying, the plate was blocked with 1% BSA solution at 37°C for 2 hours, washed five times with PBST, dried, and then packaged in a desiccant bag for storage.

[0075] Example 6: Use of the ELISA Antibody Detection Kit

[0076] The steps are as follows:

[0077] (1) Dilute the serum to be tested with sample diluent at a ratio of 1:200, add 100 μL / well to the antibody detection plate, and set up negative serum control and positive serum control at the same time. Incubate at 37°C for 1 h.

[0078] (2) Discard the liquid in the reaction wells, add 350 μL of washing buffer (PBST) to each well, wash 5 times, and pat dry;

[0079] (3) Add 100 μL of enzyme conjugate working solution to each well and incubate at 37°C for 30 min;

[0080] (4) Repeat step (2) above;

[0081] (5) Add 100 μL of colorimetric solution and incubate at 37°C in the dark for 15 min;

[0082] (6) Add 100 μL of stop solution, and use an ELISA reader to measure the absorbance A value of each well at a wavelength of 630 nm. Calculate and determine the results.

[0083] Example 7 Specificity Test

[0084] Cross-reactivity assay: The indirect ELISA antibody detection kit established using the E22 coated antigen was used to detect positive and negative sera for rabbit rotavirus, rabbit Clostridium perfringens, and rabbit Pasteurella multocida. Each sample was tested in quadruplicate for cross-reactivity assay. The results showed that only the positive serum for rabbit hemorrhagic disease type 1 virus was positive, while the rest were negative.

[0085] Example 8 Sensitivity Test

[0086] Type 1 RHDV positive serum (HI titer 7 log2) and negative serum were diluted 1:50 to 1:3200, respectively, and ELISA was performed under optimal reaction conditions. The results showed that even when the type 1 RHDV positive serum was diluted to 1:800, its OD630nm was still higher than the critical value of 0.243 and the P / N ratio was greater than 2.1 (as shown in Table 6), indicating that the ELISA kit has high sensitivity.

[0087] Table 6 Sensitivity Experiments

[0088]

[0089] Example 9 Repeatability Test

[0090] Five RHDV type 1 positive serological samples and five RHDV type 1 negative serological samples were tested using double-coated ELISA plates. Each sample was tested five times, and the coefficient of variation (CV%) was measured (CV = SD / X × 100%, where SD is the standard deviation and X is the arithmetic mean). The results showed that the maximum CV was 3.96% and the minimum was 0.46%. The CVs of all ten serological samples were relatively small, indicating good reproducibility.

[0091] Example 10 Clinical Application

[0092] Seven healthy 6-week-old rabbits were randomly selected from a rabbit farm and immunized with a commercially available type 1 rabbit hemorrhagic disease inactivated vaccine. Serum samples were collected before immunization and at 7, 14, 21, 28, 35, and 42 days post-immunization for ELISA antibody and hemagglutination inhibition (HI) antibody detection. For the HI method, 25 μl of physiological saline was added to a 96-well plate. In the first well of each row, 25 μl of post-immunization positive serum was added, followed by 25 μl of RHDV antigen diluted to 4 units. The plate was incubated at room temperature for 30 minutes, and finally, 25 μl of 1% human type O erythrocytes were added to each well. The geometric mean of the antibody levels from the seven rabbit serum samples collected was calculated. Statistical analysis showed that the correlation coefficient between the ELISA test value and the HI titer was 0.90. The results are as follows: Figure 1 , Figure 2 As shown, the detection kit of the present invention is suitable for antibody detection of rabbit hemorrhagic disease virus type 1 vaccine.

[0093] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A polypeptide for detecting type 1 RHDV antibodies, characterized in that: Its amino acid sequence is AENSSASVATAGIGG, as shown in SEQ ID NO.

1.

2. The use of the polypeptide for detecting type 1 RHDV antibodies according to claim 1 in the preparation of a kit for detecting type 1 RHDV antibodies.

3. A kit for detecting type 1 RHDV antibodies, characterized in that: Includes the polypeptide for detecting type 1 RHDV antibodies as described in claim 1.

4. The kit for detecting type 1 RHDV antibodies according to claim 3, characterized in that: The kit comprises the peptide-coated ELISA plate as described in claim 1.

5. The kit for detecting type 1 RHDV antibodies according to claim 4, characterized in that, The polypeptide-coated ELISA plate according to claim 1 is prepared by the following method: the polypeptide is added to the coating solution at a concentration of 10 μg / mL, and added to the ELISA reaction plate at a dose of 100 μL / well. The plate is incubated at 37°C for 2 hours, coated at 4°C for 10-16 hours, patted dry, blocked with 1% BSA solution at 37°C for 2 hours, washed with PBST, patted dry, and dried to obtain the final product.

6. The kit for detecting type 1 RHDV antibodies according to claim 3, characterized in that: The kit also includes sample diluent, 10× concentrated wash solution, enzyme conjugate working solution, colorimetric solution, stop solution, positive control, and negative control.

7. The kit for detecting type 1 RHDV antibodies according to claim 6, characterized in that, The kit comprises the following components: 5 ELISA plates coated with the peptides described in claim 1; 400 mL of sample dilution buffer; 400 mL of 10× concentrated wash buffer; 100 mL of enzyme conjugate working solution; 200 mL of chromogenic solution; 100 mL of stop solution; and 2 mL of positive control. Negative control, 2 mL; The coating solution was selected from a 0.05 mol / L carbonate buffer solution with a pH of 9.6; The sample diluent was PBST; The 10× concentrated washing solution is a 0.1 mol / L phosphate buffer solution with pH 7.4 containing 0.5% Tween-20; The enzyme conjugate working solution is HRP-goat anti-rabbit IgG; The colorimetric solution is a mixture of tetramethylbenzidine solution and citrate-phosphate buffer solution in a volume ratio of 1:

1. The concentration of tetramethylbenzidine solution is 0.2 mg / mL, and the citrate-phosphate buffer solution contains 0.5‰ hydrogen peroxide urea. The terminating solution is a 0.31% hydrofluoric acid solution; The positive control was RHDV-positive serum obtained through screening, whose OD... 630nm ≥1.0, and contains 1000 U / mL of penicillin and streptomycin; The negative control is negative serum obtained through screening, whose OD 630nm ≤0.243, and contains 1000 U / mL of penicillin and streptomycin.

Images