Walnut sensitization protein Jug r1 polyclonal antibody serum as well as preparation method and application thereof

Abstract

The invention provides a walnut sensitization protein Jugr 1 polyclonal antibody serum as well as a preparation method and application thereof, and belongs to the technical field of biomedical detection. The walnut sensitization protein Jug r 1 polyclonal antibody serum prepared from the walnut sensitization protein Jug r 1 with the amino acid sequence as shown in SEQ ID NO.1 can specifically bind and recognize the walnut sensitization protein Jug r 1, and has the advantages of high specificity, high titer, good repeatability, high precision and high sensitivity, and the preparation method is simple and easy to operate. The invention lays a foundation for screening a detection target of low-sensitization high-quality walnuts or walnut sensitization protein Jug r 1 and preparing a kit for detecting the walnut sensitization protein Jug r 1.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and in particular relates to a walnut allergenic protein Jug r 1 polyclonal antibody serum and a preparation method and application thereof. Background Art

[0002] Walnut protein is a high-quality plant protein resource, but it is also one of the world's major food allergens. Walnut allergy can affect the human respiratory tract, gastrointestinal tract and other parts, causing clinical symptoms such as nausea, lip swelling, and difficulty breathing, which seriously affects people's health, especially children. Therefore, it is of great significance to identify the allergen proteins in walnuts and conduct testing for human dietary health.

[0003] So far, the World Allergen Database (http: / / www.allergenonline.org) has collected information on six walnut allergens, namely Jugr 1, Jug n 1 (2S protein), Jug r2, Jugn 2 (7S protein), Jug r 3 (lipid transfer protein) and Jugr4 (11S globulin). Jug r 1 is a 2S seed storage protein with a molecular weight of about 16.4 kDa and is composed of 139 amino acids. Among walnut allergy patients, the proportion of allergies to the antigen Jug r 1 is large, so Jug r 1 is the main cause of walnut allergic reactions. However, there are few studies on Jug r 1 detection at home and abroad. Therefore, there is an urgent need for a reagent that can effectively detect Jug r 1 allergens.

[0004] After decades of development and application, enzyme-linked immunosorbent assay (ELISA) has become the most commonly used method for quantitative detection of food allergens. Polyclonal antibodies have the advantages of low production cost and short production cycle. However, there is no report on the establishment of an ic-ELISA method for sensitive detection of Jugr 1 antigenicity. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a walnut allergenic protein Jug r 1 polyclonal antibody serum and a preparation method and application thereof. The walnut allergenic protein Jug r 1 polyclonal antibody serum has high titer, can specifically bind to the walnut allergenic protein Jug r 1, and has high sensitivity in detecting the walnut allergenic protein Jug r 1.

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

[0007] The invention provides an application of a walnut allergenic protein Jugr 1 in preparing a polyclonal antibody against the walnut allergenic protein Jugr 1. The amino acid sequence of the walnut allergenic protein Jugr 1 is shown in SEQ ID NO.1.

[0008] The invention provides an immunogenic composition, comprising an adjuvant and the walnut allergenic protein Jug r 1.

[0009] Preferably, the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant; the concentration of the walnut allergenic protein Jug r 1 is 0.3-1.2 μg / μL; and the volume percentage of the Freund's complete adjuvant or Freund's incomplete adjuvant in the immunogenic composition is 40%-60%.

[0010] The present invention provides a method for preparing a polyclonal antiserum against walnut allergenic protein Jug r 1, comprising the following steps:

[0011] Animals are immunized with the immunogenic composition described above.

[0012] Preferably, the number of immunizations is 3 to 5 times, with an interval of 14 to 21 days between each immunization; the adjuvant in the immunogenic composition used for the first immunization is Freund's complete adjuvant, and the adjuvant in the immunogenic composition used for subsequent immunizations is Freund's incomplete adjuvant.

[0013] Preferably, the immunized animal is a mouse; the antigen content of each immunization is 40-110 μg walnut allergenic protein Jug r 1 / mouse.

[0014] The present invention provides a walnut allergenic protein Jugr 1 polyclonal antiserum prepared by the above-mentioned preparation method.

[0015] The present invention provides an application of the above-mentioned walnut allergenic protein Jug r 1 polyclonal antiserum, comprising at least one of the following:

[0016] (1) Application in the preparation of a kit for detecting walnut allergenic protein Jugr 1;

[0017] (2) Application in walnut screening or walnut allergenic protein Jug r 1 detection target screening.

[0018] The invention provides a kit for detecting walnut allergenic protein Jug r 1, comprising the above-mentioned walnut allergenic protein Jug r 1 polyantiserum.

[0019] Preferably, the kit comprises an ELISA kit; the ELISA kit also comprises walnut allergenic protein Jugr 1 as an antigen; the antigen coating concentration is 1 μg / mL, and the dilution ratio of the walnut allergenic protein Jugr 1 polyclonal antibody serum is 1:12800.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention provides a polyantibody against walnut allergenic protein Jugr 1 and a preparation method and application thereof. The polyantibody against walnut allergenic protein Jugr 1 prepared by the present invention using the walnut allergenic protein Jugr 1 with an amino acid sequence as shown in SEQ ID NO.1 can specifically bind to and identify the walnut allergenic protein Jugr 1, has the advantages of high specificity, high titer, good repeatability, high precision and high sensitivity, and the preparation method is simple and easy to operate. The present invention lays a foundation for screening low-allergenic high-quality walnuts or walnut allergenic protein Jugr 1 detection targets and preparing a kit for detecting walnut allergenic protein Jugr 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the titer of antiserum in different groups after the last immunization;

[0023] Figure 2 The indirect ELISA diagram of the cross reaction between the antiserum of Example 1 and different plant proteins, different lowercase letters indicate significant differences (p<0.05);

[0024] Figure 3 is the ic-ELISA standard curve of different concentrations of Jugr 1 protein;

[0025] Figure 4 Fit the Logit linear regression equation. DETAILED DESCRIPTION

[0026] The invention provides an application of a walnut allergenic protein Jugr 1 in preparing a polyclonal antibody against the walnut allergenic protein Jugr 1. The amino acid sequence of the walnut allergenic protein Jugr 1 is shown in SEQ ID NO.1.

[0027] The purpose of the present invention is to prepare a walnut allergenic protein Jugr 1 polyantibody that can specifically bind to the walnut allergenic protein Jugr 1 and has a high titer. The present invention has found that by using the walnut allergenic protein Jugr 1 shown in SEQ ID NO.1 as an immunogen combined with an immune adjuvant, a walnut allergenic protein Jugr 1 polyantibody that can specifically bind to the walnut allergenic protein Jugr 1 and has a high titer can be obtained.

[0028] The present invention provides an immunogenic composition, comprising an adjuvant and the above-mentioned walnut allergenic protein Jugr 1. Immunizing animals with the above-mentioned immunogenic composition can be more conducive to obtaining walnut allergenic protein Jugr 1 polyantibodiester that specifically binds to the walnut allergenic protein Jugr 1 and has a high titer.

[0029] As a preferred embodiment, the adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant; the concentration of the walnut allergenic protein Jug r 1 is 0.3-1.2 μg / μL, more preferably 0.5-1 μg / μL, such as 0.5 or 1 μg / μL; in the immunogenic composition, the volume percentage of the Freund's complete adjuvant or Freund's incomplete adjuvant is 40%-60%, more preferably 45%-55%, such as 50%.

[0030] The present invention provides a method for preparing a polyclonal antiserum against walnut allergenic protein Jug r 1, comprising the following steps:

[0031] Animals are immunized with the immunogenic composition described above.

[0032] In the above-mentioned method for preparing polyantibody serum, the number of immunizations is preferably 3 to 5 times, such as 4 times, and the interval between each immunization is 14 to 21 days, and more preferably, the first immunization is 21 days apart, and the subsequent immunization is 14 days apart; the adjuvant in the immunogenic composition used for the first immunization is Freund's complete adjuvant, and the adjuvant in the immunogenic composition used for the subsequent immunization is Freund's incomplete adjuvant. The immunized animal is a mouse. The antigen content of each immunization is 40 to 110 μg walnut allergenic protein Jug r 1 / mouse, and more preferably 50 to 100 μg walnut allergenic protein Jug r 1 / mouse, and more preferably the antigen content used for the first immunization is 100 μg walnut allergenic protein Jug r 1 / mouse, and the antigen content used for the subsequent immunization is 50 μg walnut allergenic protein Jug r 1 / mouse. The immunization method is subcutaneous injection, and more preferably multiple subcutaneous injections at the back of the neck. The present invention does not specifically limit the sources of Freund's incomplete adjuvant and Freund's complete adjuvant, and commercially available products in the art can be used.

[0033] As a preferred embodiment, the method for preparing the walnut allergenic protein Jug r 1 polyclonal antibody serum comprises the following steps:

[0034] First immunization: 1 μg / μL of the walnut allergenic protein Jug r1 with the amino acid sequence shown in SEQ ID NO.1 was mixed with Freund's complete adjuvant at a volume ratio of 1:1 and injected subcutaneously at multiple points on the back of the neck to immunize mice. The immunization antigen content was 100 μg of walnut allergenic protein Jug r 1 per mouse.

[0035] Secondary immunization: 21 days after the first immunization, the second immunization was performed, 0.5 μg / μL of the walnut allergenic protein Jug r 1 with the amino acid sequence shown in SEQ ID NO.1 was mixed with Freund's incomplete adjuvant at a volume ratio of 1:1 and then injected subcutaneously at multiple points on the back of the neck to immunize mice, and the immunization antigen content was 50 μg of walnut allergenic protein Jug r 1 / mouse;

[0036] Booster immunization: 14 days after the second immunization, booster immunization was performed by mixing 0.5 μg / μL of the walnut allergenic protein Jug r 1 with the amino acid sequence shown in SEQ ID NO.1 with Freund's incomplete adjuvant at a volume ratio of 1:1 and then injected subcutaneously at multiple points on the back of the neck to immunize mice. The immunization antigen content was 50 μg of walnut allergenic protein Jug r 1 / mouse;

[0037] Final immunization: 14 days after the booster immunization, the final immunization was performed. 0.5 μg / μL of the walnut allergenic protein Jug r 1 with an amino acid sequence as shown in SEQID NO.1 was mixed with Freund's incomplete adjuvant in a volume ratio of 1:1 and injected subcutaneously at multiple points on the back of the neck to immunize mice. The immunization antigen content was 50 μg of walnut allergenic protein Jug r 1 / mouse. Then, 7 days after the immunization, blood was collected and centrifuged to obtain serum, namely, walnut allergenic protein Jug r 1 polyclonal antibody serum.

[0038] The present invention provides a walnut allergenic protein Jugr 1 polyclonal antiserum prepared by the above-mentioned preparation method.

[0039] The present invention provides an application of the above-mentioned walnut allergenic protein Jug r 1 polyclonal antiserum, comprising at least one of the following:

[0040] (1) Application in the preparation of a kit for detecting walnut allergenic protein Jugr 1;

[0041] (2) Application in walnut screening or walnut allergenic protein Jug r 1 detection target screening.

[0042] The invention provides a kit for detecting walnut allergenic protein Jug r 1, comprising the above-mentioned walnut allergenic protein Jug r 1 polyantiserum.

[0043] In the present invention, the kit includes an ELISA kit; the ELISA kit also includes walnut allergenic protein Jugr 1 as an antigen; the antigen coating concentration is 1 μg / mL, and the dilution ratio of the walnut allergenic protein Jugr 1 polyantibody is 1: 12800. The present invention can reduce the cost of the kit by using the above-mentioned antigen coating concentration and the dilution ratio of the walnut allergenic protein Jugr 1 polyantibody, and can also detect the walnut allergenic protein Jugr 1 specifically and with high sensitivity.

[0044] In the present invention, the ELISA kit is preferably an indirect ELISA kit, which also includes an antigen diluent (such as carbonate buffer), an antibody diluent (i.e., PBST solution), an HRP-labeled secondary antibody (such as HRP-labeled goat anti-mouse IgG), a substrate colorimetric solution (such as TMB colorimetric solution), a washing solution (PBST solution), a blocking solution (such as 5% skimmed milk powder) and a stop solution (such as 2 mol / L H2SO4).

[0045] The present invention utilizes the kit to specifically and highly sensitively identify the walnut allergenic protein Jug r 1, and has good repeatability and high precision in detecting the walnut allergenic protein Jug r 1.

[0046] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0047] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0048] In the following examples, SPF-grade 5-6-week-old Balb / c female mice were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. (License No.: SCXK (Xiang) 2019-0004), and the laboratory unit use license number is: SYXK (Yunnan) K2020-0004. The experimental mice were fed with drinking water and feed that did not contain walnut protein; the experimental protocol was approved by the Ethics Department of the Degree Committee of Southwest Forestry University (Approval No.: SWFU-20230314) and was conducted at the Animal Training Base of the College of Biological and Food Engineering. Before the experiment began, the experimental mice were adapted to the laboratory environment for 1 week.

[0049] The main materials and reagents are shown in Table 1.

[0050] Table 1 Main materials and reagents

[0051]

[0052] Solution configuration:

[0053] (1) Carbonate buffer solution (CBS) pH 9.650 mmol / L: Add 1.59 g NaCO3 and 2.93 g NaHCO3, respectively, dissolve in ultrapure water and dilute to 1 L for preparing antigens and ELISA blocking solution.

[0054] (2) Phosphate buffer solution + Tween-20 (PBST): Dissolve the PBS freeze-dried powder package in ultrapure water and dilute to 2 L. Then add 0.05% Tween-20 as ELISA washing solution for ELISA washing and primary and secondary antibody dilution.

[0055] (3) ELISA blocking solution: CBS diluted to 5% by mass in skimmed milk powder.

[0056] (4) TMB colorimetric solution: Take a two-component TMB colorimetric kit, mix equal amounts of liquid A and liquid B, protect from light, and prepare on-site for use.

[0057] The data processing:

[0058] All experiments were repeated at least 3 times. Excel was used to process the data, and one-way ANOVA (P < 0.05) was used for statistical analysis of significant differences, and Origin 2018 was used for drawing graphs.

[0059] Example 1

[0060] The preparation of polyclonal antiserum against walnut allergenic protein Jug r 1 is as follows:

[0061] The amino acid sequence of the walnut allergenic protein Jugr 1 in this example is

[0062] AALLVALLFVANAAFRTTITTTMEIDEDIDNPRRRGEGCREQIQRQQNLN HCQYYLRQQSRSGGYDEDNQRQHFRQCCQQLSQMDEQCQCEGLRQVVRRQ QQQQGLRGEEMEEMVQSARDLPNECGISSQRCEIRRSWF(SEQID NO. 1).

[0063] Seven Balb / c female mice were immunized by multiple subcutaneous injections at the back of the neck. After 7 days of feeding without any abnormality, the experimental mice were randomly divided into an immune-positive group (a total of 5 mice, numbered "No. 1, No. 2, No. 3, No. 4, No. 5") and a control negative group (numbered "Yin 1" and "Yin 2"). The control negative group was fed normally and no subcutaneous injection was performed. The treatment method of the immune-positive group was as follows: the walnut allergenic protein Jugr 1 was diluted with 0.01mol / LpH7.4PBS and mixed with an equal volume of adjuvant (1:1), and fully mixed with a vortex mixer until emulsified, and then the immune test was performed according to the immunization scheme in Table 2. Seven days after the last immunization (56d), blood was collected from the submandibular vein, and the serum specific anti-Jug r 1 titer was detected by indirect ELISA. Depending on the titer, it was decided whether to immunize again. After successful immunization, the eyeballs were removed for large-scale blood collection (57d). The collected blood was placed at a constant temperature of 37°C for 30 minutes and kept in a refrigerator at 4°C overnight. After the serum was fully separated, it was centrifuged at 3000r / min for 5 minutes to obtain the walnut allergenic protein Jug r 1 polyantibody (antiserum), which was packaged and stored at -20°C for testing.

[0064] Table 2 Immunization schedule

[0065] frequency Interval time / d Adjuvant Dose / μL Antigen content First immunization 0 Freund's complete adjuvant 200μL / pc 100μg / each Secondary Immunization 21 Freund's incomplete adjuvant 200μL / pc 50μg / each Strengthen immunity 35 Freund's incomplete adjuvant 200μL / pc 50μg / each final immunization 49 Freund's incomplete adjuvant 200μL / pc 50μg / each

[0066] Example 2

[0067] Determination of Antiserum Titer of Example 1

[0068] The method for determining the titer of antiserum using indirect ELISA is as follows:

[0069] (1) Plate coating: dilute the walnut allergenic protein Jugr 1 shown in SEQ ID NO.1 to 10 μg / mL with CBS, 100 μL / well, and incubate at 37°C for 1 h;

[0070] (2) Washing: discard the solution in the wells, wash the plate three times with PBST solution, 3 min each time, and pat dry;

[0071] (3) Blocking: Add 5% skim milk powder, 250 μL / well, incubate at 37°C for 1 h, wash the plate and pat dry;

[0072] (4) Adding primary antibody (antiserum): 100 μL / well, add the antiserum of Example 1 diluted with PBST at 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400 and 1:204800 in sequence, and prepare negative serum wells and blank wells at the same time, incubate at 37°C for 1 h, wash the plate and pat dry; wherein the negative serum wells are the antiserum of the control negative group prepared in Example 1, and the blank wells do not contain primary antibody and secondary antibody, and the rest of the operations are the same.

[0073] (5) Add secondary antibody (enzyme-labeled antibody): 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:2000 with PBST was added to the reaction wells, incubated at 37°C for 1 h, washed and patted dry;

[0074] (6) Add substrate colorimetric solution: 100 μL TMB colorimetric solution / well, incubate at 37°C for 15 min;

[0075] (7) Termination reaction: After color development, add 2 mol / L H2SO4 to terminate the reaction, 100 μL / well;

[0076] (8) Reading: The microplate reader reads at 450 nm and measures the OD of each detection well. 450 , the evaluation criteria are OD 阳性血清 / OD 阴性血清 ≥2.1 is positive.

[0077] Serum titer refers to the reciprocal of the maximum dilution of serum that can produce a significant and detectable reaction with the antigen in any screening test. Figure 1 .

[0078] Figure 1 The results showed that the two mouse antisera in the negative control group had an OD of 450 The value gradually tends to zero, indicating that there is no positive reaction to walnut protein. When the serum dilution factor of the immune positive group is 6400, OD 450 Still greater than 0.2, according to OD 阳性血清 / OD 阴性血清 ≥2.1, indicating that the 5 mice serum at this dilution ratio were positive, and the titer could reach 1:12800; and when the dilution ratio exceeded 1:12800, the OD of all mice in the immune positive group 450 The value began to drop sharply and gradually approached zero, and the serum dilution multiple of mouse No. 3 was still the largest at 1:204800, indicating that the immune effect of mouse No. 3 was better.

[0079] Example 3

[0080] Antiserum specificity assay

[0081] Referring to the indirect ELISA method of Example 2, the cross reaction of the antiserum prepared in Example 1 No. 3 with other common proteins was detected. The sequence of walnut allergenic protein Jug r 1 (Jug r 1), walnut protein isolate, peanut protein isolate, and soybean protein isolate with a sequence of 100 μg / mL as shown in SEQ ID NO.1 were selected as antigens for coating respectively, and the primary antibody was diluted to 1:200 times. The remaining steps were the same as the indirect ELISA method of Example 2 for titer determination of antiserum. The negative control group used the antiserum of Yin 1 as the primary antibody. Among them, walnut protein isolate (also known as crude walnut protein), peanut protein isolate (also known as peanut protein), and soybean protein isolate (also known as soybean protein) were purchased from Shanghai Yuanye Biotechnology Co., Ltd., walnut protein isolate item number: S33510, peanut protein isolate item number: S31067, and soybean protein isolate item number: S33496.

[0082] The results of cross-reactions of the antiserum prepared in Example 1 with different proteins are shown in Figure 2 .

[0083] Figure 2 The results showed that when the antiserum of Example 1 was diluted 1:200 times, the OD values ​​of peanut protein isolate and soy protein isolate as antigens were low (negative), indicating that the antiserum of the immunized mice failed to recognize peanut protein and soy protein allergens. However, the OD value of Jug r 1 as antigen was significantly higher than that of peanut protein and soy protein (p<0.05), and had no significant difference with the OD value of walnut protein isolate as antigen (p<0.05), indicating that the antiserum of the immunized mice in the experiment could only recognize walnut allergens and had no cross reaction to peanut protein and soy protein.

[0084] Example 4

[0085] Determination of the Optimal Antigen Coating Concentration and Antiserum Working Concentration

[0086] When establishing the ELISA method, the concentrations of coated antigen and antibody should be optimized to save the amount of antigen and antiserum and achieve the most suitable assay conditions. The optimal concentrations of antigen coating concentration and antiserum dilution are determined by square array titration (chessboard method).

[0087] This example uses the square titration method (also known as the chessboard method) to determine the optimal antigen coating concentration and antiserum dilution. Antigen dilution: The antigen Jug r 1 was diluted with CBS to 0.25, 0.5, 1, 2, 4, 8, 16, and 32 μg / mL in sequence; Antiserum dilution: The primary antibody was diluted with PBST to 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, and 1:102400; Secondary antibody dilution: The secondary antibody (HRP-labeled goat anti-mouse IgG) was diluted with PBST to 1:4000 according to the product instructions; The remaining steps were the same as the indirect ELISA method in Example 2 for the determination of the titer of the antiserum, OD 450 Antigen and antisera with values ​​of approximately 1.0 were selected as optimal working concentrations.

[0088] Table 3 Determination of Jug r 1 antigen coating and antiserum working concentration

[0089]

[0090] As shown in Table 3, when the concentration of the coating antigen Jugr 1 was 1 μg / mL, the optimal dilution ratio of the corresponding antiserum was 1:12800, and the measured OD value was around 1.0. However, based on subsequent actual operations, the optimal working conditions of Jugr 1 antigen and antiserum were finally determined to be 1 μg / mL and 1:6400, respectively.

[0091] Example 5

[0092] Establishment of ic-ELISA standard curve

[0093] (1) Plate coating: Dilute Jug r 1 to the optimal working concentration of 1 μg / mL with CBS, 100 μL / well, incubate at 37°C for 1 h, wash the plate, and pat dry;

[0094] (2) Blocking: Add 5% skim milk powder, 250 μL / well, incubate at 37°C for 1 h, wash the plate, and pat dry;

[0095] (3) Adding samples and primary antibodies: Add 50 μL of Jug r 1 standard protein solution of different concentrations (select a dilution concentration series of 0 to 512 μg / mL, dilute to 0.0078125 μg / mL by double gradient dilution method, 0 μg / mL is a non-competitive well) and the sample solution to be tested to each well, then add 50 μL of primary antibody (antiserum prepared in No. 3 of Example 1) diluted to 1:6400 with PBST, and simultaneously establish non-competitive wells (antigen-antibody binding reaction rate is 100%) and blank wells, incubate at 37°C for 1 hour, wash the plate, and pat dry. Wherein, the blank well does not contain primary antibody and secondary antibody.

[0096] (4) Add enzyme-labeled antibody (secondary antibody): add 100 μL of HRP-labeled goat anti-mouse IgG diluted 1:4000 with PBST to each well, incubate at 37°C for 1 h, wash the plate and pat dry;

[0097] (5) Add substrate colorimetric solution: 100 μL / well, incubate at 37°C for 15 min;

[0098] (6) Termination: After color development, add 2 mol / L H2SO4 to terminate the reaction, 100 μL / well;

[0099] (7) Reading: Read the sample at 450 nm using a microplate reader. Use the OD value of each concentration of Jug r 1 to convert it into (B / B0)% as the vertical coordinate, and use the logarithmic value of the corresponding concentration of Jug r 1 as the horizontal coordinate of the standard curve. Where B is the OD value of each concentration during competition. 450 Value, B0 is non-competitive OD 450 value.

[0100] The good linear relationship range of the standard curve was selected to fit the Logit (logarithmic) linear regression equation, and the linear regression equation and correlation coefficient were obtained.

[0101] The standard curve of Jug r 1ic-ELISA was drawn with the inhibition rate (B / B0)% after multiple dilution of the standard concentration of Jug r 1 as the y-axis and the logarithm of the corresponding concentration of Jug r 1 (Lg) as the horizontal coordinate.

[0102] like Figure 3 As shown, the obtained curve is in the range of 1 to 0.03125 μg / mL (corresponding to the horizontal coordinate of 0 to -1.505). The standard curve has a good linear relationship and a large slope. Therefore, the fitting of the Logit linear regression equation is selected in this part. The regression analysis results after linear regression transformation are shown in Figure 4 .

[0103] Figure 4 The results show that the adjusted linear regression equation Y is Logit ((B / B0)%), X is Lg [Jug r1], and the adjusted linear regression line equation is Y = -3.90503X-1.22183, R 2 =0.99351, which meets the standard for judging a linear relationship, so the optimal detection range is 1-0.03125 μg / mL.

[0104] Example 6

[0105] Precision testing of ic-ELISA

[0106] The precision of the established ic-ELISA detection method was tested and expressed as the intra-plate coefficient of variation (CV) and inter-plate coefficient of variation, respectively.

[0107] Intraplate error: The standard sample concentration was repeated 4 times according to Example 5, and the average OD value and the standard deviation (SD) between wells were measured. The standard deviation divided by the average value was the intraplate coefficient of variation (CV), which represents the intraplate error.

[0108] Inter-plate error: The method of Example 5 was repeated three times on different ELISA plates, the OD value was measured, and the mean of the average absorbance values ​​of each concentration of the three ELISA plates was calculated, which was the mean of the average binding rate between plates; then the standard deviation between the wells of each plate was calculated and the mean of the three plates was obtained, which was the inter-plate difference; finally, the mean of the inter-well errors of the three plates was obtained, which was the total inter-plate coefficient of variation, indicating the inter-plate error.

[0109] After repeated experiments using the ic-ELISA method established in Example 5 above, the results are shown in Table 4.

[0110] Table 4 Intra-plate error and inter-plate error of ic-ELISA standard curve

[0111]

[0112] The error in the plate is expressed as the average coefficient of variation (CV) of the standard curve, where CV = SD mean / average well binding rate × 100% = 2.83 / 60.71 × 100% = 4.66%. The error between each plate is expressed as the average coefficient of variation (CV) of three determinations. From Table 4, it can be seen that the inter-plate coefficient of variation is between 2.04% and 11.85%, with an average of 5.74%. The permissible value of the intra-plate coefficient of variation of the ELISA method is usually less than 10%, and the permissible value of the inter-plate coefficient of variation is usually less than 15%. Based on the intra-plate error and inter-plate error, it can be shown that the ic-ELISA method for detecting Jugr 1 has good repeatability and high precision.

[0113] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of walnut allergenic protein Jug r 1 in the preparation of polyclonal antiserum against walnut allergenic protein Jug r 1, characterized in that: The amino acid sequence of the walnut allergenic protein Jug r 1 is shown in SEQ ID NO.

1.

2. An immunogenic composition, characterized in that The invention comprises an adjuvant and the walnut allergenic protein Jugr1 according to claim 1.

3. The immunogenic composition according to claim 2, characterized in that The adjuvant is Freund's complete adjuvant or Freund's incomplete adjuvant; the concentration of the walnut allergenic protein Jug r 1 is 0.3-1.2 μg / μL; and in the immunogenic composition, the volume percentage of the Freund's complete adjuvant or Freund's incomplete adjuvant is 40%-60%.

4. A method for preparing polyclonal antiserum against walnut allergenic protein Jugr 1, characterized in that: The following steps are involved: Immunize an animal using the immunogenic composition of claim 2 or 3.

5. The preparation method according to claim 4, characterized in that The number of immunizations is 3 to 5 times, with an interval of 14 to 21 days between each immunization; the adjuvant in the immunogenic composition used for the first immunization is Freund's complete adjuvant, and the adjuvant in the immunogenic composition used for subsequent immunizations is Freund's incomplete adjuvant.

6. The preparation method according to claim 4 or 5, characterized in that: The immunized animals are mice; the antigen content of each immunization is 40-110 μg of walnut allergenic protein Jugr 1 / mouse.

7. A polyclonal antiserum against walnut allergenic protein Jug r 1 prepared by the preparation method according to any one of claims 4 to 6.

8. The use of the polyclonal antiserum against walnut allergenic protein Jugr 1 according to claim 7, characterized in that: Include at least one of the following: (1) Application in the preparation of a kit for detecting walnut allergenic protein Jugr 1; (2) Application in walnut screening or walnut allergenic protein Jug r 1 detection target screening.

9. A kit for detecting walnut allergenic protein Jug r 1, characterized in that: The invention comprises the walnut allergenic protein Jug r 1 polyclonal antibody serum as claimed in claim 7.

10. The kit according to claim 9, characterized in that The kit comprises an ELISA kit; the ELISA kit also comprises walnut allergenic protein Jug r 1 as an antigen; the antigen coating concentration is 1 μg / mL, and the dilution ratio of the walnut allergenic protein Jug r 1 polyclonal antibody serum is 1:12800.

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