A method and kit for detecting a swine anti-human fepsilon ri antibody

Abstract

The application discloses a detection method and kit of pig anti-human Fc epsilon R1 antibody, and belongs to the field of detection analysis and quality control of biological products. The method is characterized in that: a solid phase carrier is coated with Fc epsilon R1 alpha protein, and is sealed with a protein-free blocking solution without serum protein; gradient-diluted standard samples and diluted to-be-detected samples are sequentially added; an anti-pig IgG secondary antibody labeled with a detectable marker is added; a substrate is added for color development, and the reaction is terminated; and then the signal value is measured; a standard curve is fitted by using a four-parameter logistic equation; and the relative content of pig anti-human Fc epsilon R1 antibody in the to-be-detected sample is calculated. The application establishes an ELISA relative quantitative detection method of pig anti-human Fc epsilon R1 antibody, and the method has good precision, accuracy, sensitivity and specificity, and can quickly and quantitatively detect the content of anti-human Fc epsilon R1 antibody in anti-human thymocyte / lymphocyte immunoglobulin products.

Description

Technical Field

[0001] This invention belongs to the field of detection, analysis and quality control of biological products, specifically relating to a method for detecting porcine anti-human FcεRI antibodies in anti-human thymocyte and / or lymphocyte porcine immunoglobulin preparations, and a kit for detecting porcine anti-human FcεRI antibodies using this method. Background Technology

[0002] Anti-thymocyte globulin (ATG) and anti-lymphocyte globulin (ALG) are potent immunosuppressive drugs widely used clinically for the prevention and treatment of organ transplant rejection, preconditioning for hematopoietic stem cell transplantation, and treatment of severe aplastic anemia. The main component of these preparations is polyclonal IgG antibody, and the key raw material for their preparation is animal immune serum (using animals such as pigs, rabbits, or horses). The immunogens used to immunize these animals are mainly human thymocytes, human T lymphocytes, or human T lymphocyte cell lines (such as Jurkat cells). Currently, the most commonly used ATG in clinical practice includes anti-human T cell porcine immunoglobulin (such as Aitege). ® Both rabbit anti-human thymocyte immunoglobulin (such as ibuprofen) and rabbit anti-human thymocyte immunoglobulin (such as ibuprofen) are mainly produced using human thymocytes as immunogens.

[0003] However, a certain number of mast cells have been shown to exist in human thymus tissue. The high-affinity IgE receptor FcεRI expressed on the surface of mast cells may act as an immunogen, leading to the production of anti-human FcεRI antibodies in immunized animals. These heterologous antibodies may also enter the final product along with immune serum or immune plasma. Since FcεRI is a key receptor mediating type I hypersensitivity reactions and is widely distributed on the surface of mast cells and basophils, when anti-human FcεRI antibodies bind to the receptors on the surface of these cells in patients, they can directly activate mast cells or basophils through a cross-linking effect, causing cell degranulation and the release of inflammatory mediators such as histamine and leukotrienes, thereby inducing acute allergic reaction symptoms. In addition, anti-FcεRI autoantibodies are also a key factor in inducing type IIb chronic spontaneous urticaria.

[0004] Several studies have reported the use of enzyme-linked immunosorbent assay (ELISA) to detect FcεRI autoantibodies. For example, the paper "Prevalence and Clinical Relevance of Anti-FcεRI Autoantibody in Crohn's Disease" (https: / / doi.org / 10.2147 / JAA.S476501) discloses an indirect ELISA method that uses human FcεRIα-coated microplates and enzyme-labeled anti-human IgG as a secondary antibody for semi-quantitative detection of IgG-type anti-FcεRI antibodies in serum. Patent application CN121079594A discloses an indirect ELISA method that involves coating human FcεRI (or its α-chain fragment / epitope) onto a solid phase, binding it to anti-FcεRI Ig in the plasma or IVIG / SCIG (intravenous immunoglobulin G / subcutaneous immunoglobulin G) fraction to form a complex, and then adding enzyme- / fluorescently labeled anti-human IgG or anti-human IgE to detect the protein colorimetric reading.

[0005] However, existing ELISA detection methods are mainly limited to detecting FcεRI autoantibodies in human serum, i.e., human anti-human FcεRI antibodies. Their detection systems cannot be directly used to detect porcine anti-human FcεRI antibodies in porcine preparations such as pATG. The main reasons are as follows: (1) The enzyme-labeled secondary antibody used in existing methods is an anti-human IgG antibody. This secondary antibody cannot specifically recognize porcine IgG. ATG / ALG preparations themselves are high concentrations of heterologous immunoglobulins. If the anti-human IgG secondary antibody is used, the target signal cannot be detected, and false signals are easily generated due to cross-reaction between species. (2) ATG / ALG preparations are polyclonal antibody preparations obtained by immunization with human thymocytes or lymphocytes. In addition to the target heterologous antibody, they also contain a large number of antibodies against other human antigens. Moreover, the total protein concentration of the test sample is high (usually on the order of 50 mg / mL). In the conventional ELISA system, non-specific adsorption is easily generated, which raises the background. In particular, conventional ELISA commonly uses bovine serum albumin (BSA) or animal serum as a blocking agent, while polyclonal immunoglobulin preparations may contain antibodies that recognize albumin or other plasma proteins. These antibodies will directly bind to the blocking layer and be detected by anti-pig IgG secondary antibody, causing significant background interference, making it difficult to distinguish between negative and positive wells. The signal-to-noise ratio and sensitivity of the detection cannot meet the requirements of quality control. (3) At present, there are no porcine anti-human FcεRI monoclonal antibodies or international / national standard materials with known concentrations and traceability available for purchase. Therefore, it is impossible to establish an absolute quantitative standard curve expressed in terms of mass concentration according to conventional practices, making it difficult for this project to achieve batch-to-batch comparable quantitative detection and standardization.

[0006] Therefore, establishing a sensitive, specific, and standardized detection method for porcine anti-human FcεRI antibodies, especially for ATG / ALG formulations, is of great significance for assessing the safety of these formulations and improving their quality control system. Summary of the Invention

[0007] To address the lack of existing methods for detecting porcine anti-human FcεRI antibodies against porcine immunoglobulin preparations targeting human thymocytes and / or lymphocytes, and the problems associated with conventional ELISA systems such as background interference from blocking proteins and the lack of traceable standards for quantification, this invention aims to provide a detection method and kit for porcine anti-human FcεRI antibodies. This method enables the specific detection of porcine anti-human FcεRI antibodies against porcine immunoglobulin preparations targeting human thymocytes and / or lymphocytes, as well as porcine immune serum and / or plasma. The detection method exhibits high sensitivity, good precision, accuracy, and dilution linearity.

[0008] Specifically, the present invention adopts the following technical solution to achieve the above objectives: A method for detecting porcine anti-human FcεRI antibodies for non-disease diagnostic and therapeutic purposes, comprising the following steps: S1. Dilute the FcεRIα protein with coating buffer and add it to a solid support. Incubate the mixture to fix the FcεRIα protein onto the surface of the solid support. S2. After the incubation in step S1 is completed, wash the solid support and add the blocking solution; the blocking solution is a protein-free blocking solution that does not contain serum proteins. S3. After the incubation in step S2 is completed, wash the solid-phase carrier, add serially diluted standards and diluted test samples respectively, and incubate to allow the porcine anti-human FcεRI antibody in the sample to bind to the FcεRIα protein; the standards are anti-human thymocytes containing porcine anti-human FcεRI antibody or anti-human lymphocyte porcine immunoglobulin preparations containing porcine anti-human FcεRI antibody. S4. After the incubation in step S3 is completed, wash the solid-phase carrier, add diluted anti-pig IgG secondary antibody carrying detectable markers and incubate. S5. After the incubation in step S4 is completed, wash the solid support, add the substrate corresponding to the detectable marker for color development, add the stop solution to terminate the reaction, and measure the signal value. S6. Fit a standard curve with the relative concentration of the standard as the abscissa and the signal value corresponding to the relative concentration as the ordinate; substitute the signal value of the sample to be tested into the standard curve to obtain the relative concentration after dilution, and then multiply it by the dilution factor of the sample to be tested to obtain the relative content of porcine anti-human FcεRI antibody in the sample to be tested.

[0009] In a preferred embodiment, in step S1, the coating concentration of the FcεRIα protein is 0.75 μg / mL to 3 μg / mL.

[0010] In a preferred embodiment, in step S1, the coating buffer is a PBS buffer with a pH of 7.4.

[0011] In a preferred embodiment, in step S2, the protein-free blocking solution is prepared using PBST buffer.

[0012] In a preferred embodiment, the standard and the sample to be tested are diluted in step S3 using the protein-free blocking solution described in step S2.

[0013] In a preferred embodiment, step S3, the method for preparing the standard includes the following steps: following the methods in steps S1, S2, and S4, multiple batches of anti-human thymocyte or anti-human lymphocyte porcine immunoglobulin preparations are tested in parallel on the same ELISA plate at the same dilution factor, the batch with the highest signal value is selected as the standard, and the relative concentration of the standard is assigned to 1000 AU / mL.

[0014] In a further preferred embodiment, the standard is first diluted with a protein-free blocking solution to a relative concentration range covering 0.078 AU / mL to 10 AU / mL.

[0015] In the preferred embodiment, in step S3, normal porcine IgG is used as a negative control, and the protein-free blocking solution is used as a blank control.

[0016] In a preferred embodiment, in step S4, the anti-pig IgG secondary antibody is a goat anti-pig IgG (H+L) antibody; In a preferred embodiment, in step S4, the anti-pig IgG secondary antibody is diluted with the protein-free blocking solution at a volume ratio of 1:20000 to 1:80000.

[0017] In a preferred embodiment, in step S4, the detectable marker is horseradish peroxidase.

[0018] In a further preferred embodiment, the substrate in step S5 is TMB, and the termination solution is a 2 mol / L sulfuric acid solution.

[0019] In the preferred embodiment, the standard curve fitting in step S6 uses a four-parameter logistic regression model.

[0020] The present invention also provides a kit for detecting porcine anti-human FcεRI antibodies, wherein the kit uses the scheme described in any of the above-described embodiments to detect porcine anti-human FcεRI antibodies in the sample to be tested; the kit includes FcεRIα protein, protein-free blocking solution, standard, and anti-porcine IgG secondary antibody carrying a detectable marker.

[0021] In a preferred embodiment, the kit further includes at least one of a substrate corresponding to the detectable marker, a stop solution, a coating buffer, and a negative control; the negative control is normal porcine IgG.

[0022] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) The present invention applies the detection of anti-human FcεRI antibody to anti-human thymocyte and / or lymphocyte immunoglobulin preparations, and establishes an ELISA relative quantitative detection method for porcine anti-human FcεRI antibody.

[0023] (2) By using a protein-free blocking solution that does not contain serum proteins instead of conventional BSA blocking solution or serum blocking solution, the present invention avoids the high background interference caused by the binding of anti-albumin or / and other anti-plasma protein antibodies in polyclonal porcine immunoglobulin preparations to the blocking layer.

[0024] (3) This invention determined the optimal parameters for antigen coating concentration and enzyme-labeled secondary antibody dilution factor through checkerboard titration, and achieved relative quantification by using self-built standards in conjunction with four-parameter logistic fitting. The methodological validation results showed that the standard curve determination coefficient R 2 The coefficient of variation was 0.9993, the intra-plate coefficient of variation was 2.480%–3.155%, the inter-plate coefficient of variation was 3.758%–7.645%, the blank spike recovery rate was 90.772%–102.076%, the limit of detection was 0.079 AU / mL, the limit of quantitation was 0.156 AU / mL, and the dilution linearity coefficient of determination R was [value missing]. 2 The detection limit was 0.9998, and the results for both porcine negative plasma and human plasma were below the limit of detection, indicating that the method provided by this invention has good precision, accuracy, sensitivity and specificity.

[0025] (4) Based on the detection method established in this invention, manufacturers can quickly quantify the content of anti-human FcεRI antibodies in anti-human thymocyte or / and lymphocyte immunoglobulin products during the production process, thereby providing key data support for screening raw material immune plasma and optimizing the production process to significantly reduce the content of this harmful antibody. Attached Figure Description

[0026] Figure 1This is a standard curve obtained by plotting the relative concentration of the standard (AU / mL) on the x-axis and the absorbance value at 450nm on the y-axis in Example 4 of the present invention and fitting it with a four-parameter logistic equation. Figure 2 This is a graph showing the results of the dilution linearity verification in Example 5 of the present invention; where the horizontal axis represents the theoretical relative concentration (AU / mL), the vertical axis represents the measured relative concentration (AU / mL), and the straight line in the graph is the linear fitting curve. Figure 3 This is a graph showing the results of specificity verification in Example 5 of the present invention, where the horizontal axis represents normal porcine IgG, porcine negative plasma, and human plasma from left to right, and the vertical axis represents the absorbance value at 450 nm. Figure 4 The graph shows the relative concentration detection results of porcine anti-human FcεRI antibody in 8 batches of pATG preparations in Example 6 of this invention. The horizontal axis represents the batch number of the pATG preparation, and the vertical axis represents the concentration (AU / mL) detection value. Detailed Implementation

[0027] The following description, in conjunction with embodiments, clearly and completely describes the technical solutions of this application, so that those skilled in the art can fully understand this application. Obviously, the described embodiments are merely some preferred embodiments of this application, and not all embodiments. Any equivalent modifications or substitutions made by those skilled in the art to the following embodiments without creative effort are within the protection scope of this application.

[0028] The main raw materials used in the following embodiments are listed in Table 1: Table 1. Main raw material sources or components

[0029] Example 1 A method for detecting porcine anti-human FcεRIα antibody in an anti-human thymocyte immunoglobulin preparation, comprising the following steps: (1) Antigen coating: FcεRIα protein was diluted with PBS buffer to 3 μg / mL, 1.5 μg / mL and 0.75 μg / mL, and added to 96-well microplates at a volume of 50 μL per well. After sealing with a sealing film, the plates were incubated overnight (14h~18h) at 4°C.

[0030] (2) Washing and blocking: After step (1) incubation, add washing solution at a rate of 350 μL / well to wash the microplate 3 times and pat the microplate dry on absorbent paper; add protein-free blocking solution at a rate of 300 μL / well and incubate at 37°C for 1 h.

[0031] (3) Primary antibody incubation: After the incubation in step (2) is completed, wash the ELISA plate 3 times with washing buffer at a rate of 350 μL / well, and pat the ELISA plate dry on absorbent paper; add 50 μL of pATG preparation diluted (diluent is protein-free blocking buffer) to each well as a positive well, use 50 μL of normal pig IgG sample diluted (diluent is protein-free blocking buffer) to 62.5 μg / mL as a negative well, and use protein-free blocking buffer as a blank control; incubate at 37℃ for 1 h.

[0032] (4) Secondary antibody incubation: After incubation in step (3), wash the ELISA plate 5 times with washing buffer at a rate of 350 μL / well, and pat the plate dry on absorbent paper; dilute the goat anti-pig IgG (H+L)-HRP secondary antibody with protein-free blocking buffer at volume ratios of 1:80000, 1:40000, and 1:20000, respectively, add 100 μL of the diluted goat anti-pig IgG (H+L)-HRP secondary antibody to each well, cover with sealing film, and incubate at 37°C for 1 h. Those skilled in the art should understand that biotin- or alkaline phosphatase (AP) labeled secondary antibodies can also be used as the secondary antibody for incubation in this step.

[0033] (5) Color development and termination: After the incubation in step (4) is completed, add washing buffer at a rate of 350 μL / well and wash the microplate 5 times, and pat the microplate dry on absorbent paper; add 100 μL of TMB color development solution to each well and incubate at room temperature (e.g., 25°C) in the dark for 15 min; then immediately add 50 μL of stop solution (2 mol / L sulfuric acid solution) to each well and gently shake to mix.

[0034] (6) Absorbance measurement and data processing: Immediately after adding the stop solution, the absorbance (OD) of each well was measured at a wavelength of 450 nm using a microplate reader. 450nm ). Calculate the OD of positive and negative wells. 450nm The ratio of the positive to negative values ​​was used to obtain the P / N (positive / negative) ratio, and the results are shown in Table 2.

[0035] Table 2. Results of chessboard titration (P / N ratio)

[0036] The results in Table 2 show that the P / N value was highest (P / N = 4.299) when the antigen coating concentration was 1.5 μg / mL and the secondary antibody dilution was 1:20000. Considering the overall detection cost, P / N value, and maximum OD... 450nm Considering the values, the optimal concentration for antigen coating is 1.5 μg / mL, and the optimal dilution ratio for secondary antibody is 1:20000.

[0037] Example 2 Because pATG formulations are polyclonal antibody components, they may contain antibodies against albumin or other plasma proteins found in traditional blocking solutions. Therefore, protein components in BSA or serum blocking solutions may interfere with this detection. This example compares the blocking effects of BSA blocking solution and commercially available protein-free blocking solutions.

[0038] In this experiment, the ELISA plates were not coated with antigen. 300 μL / well of 5% BSA blocking solution and 300 μL / well of protein-free blocking solution were added to block the plates, and incubated at 37°C for 2 h. After blocking, the plates were washed 5 times with washing buffer and patted dry. 50 μL of serially diluted pATG reagent (batch numbers P014 and P010) was added to each well, and the plates were incubated at 37°C for 1 h. Blank control wells containing only diluent were also included. After incubation, the plates were washed 5 times with washing buffer and patted dry. 100 μL / well of goat anti-pig IgG (H+L)-HRP secondary antibody diluted 1:20000 (v / v) was added to each well, and the plates were incubated at 37°C for 1 h. Wash the microplate five times with washing buffer and blot dry. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature (e.g., 25°C) in the dark for 10 min. Then add 50 μL of stop solution to each well and immediately read the OD value at 450 nm using a microplate reader. 450nm The results are shown in Table 3.

[0039] Table 3 Comparison of blocking effects between BSA blocking solution and protein-free blocking solution

[0040] As can be seen from the results in Table 3, under conditions where the antigen was not coated, the sample wells still showed a significantly higher background signal than the blank control when using BSA blocking solution (OD at 1:50 dilution). 450nm The values ​​were 0.249 and 0.530, which were 5.1 times and 10.6 times that of the blank control, respectively. This was presumably due to antibodies in the pATG formulation that specifically or non-specifically bind to BSA. Meanwhile, the signal at various dilutions when using commercially available protein-free blocking solutions (OD at 1:50 dilution) was... 450nm The values ​​of 0.092 and 0.099 were close to the blank control levels. This demonstrates that the protein-free blocking solution used in this invention has excellent blocking effect and produces only extremely low background. Therefore, the detection method of this invention must use a protein-free blocking solution, or other validated blocking solutions that do not contain serum proteins such as BSA.

[0041] Example 3 Since there are currently no commercially available porcine anti-human FcεRI monoclonal antibodies with known actual concentrations, this embodiment screened for standards. This embodiment selected pATG formulations as standards and quantified them using relative concentration units AU / mL. To determine suitable pATG formulation batches as standards, multiple batches of pATG formulations were first tested, and then the batch with the highest detection signal value, i.e., the highest anti-human FcεRI antibody content, was selected as the standard.

[0042] The experiment was conducted according to the steps of Example 1. The sample was coated with 1.5 μg / mL FcεRIα protein, and detected using goat anti-porcine IgG (H+L)-HRP secondary antibody diluted 1:20000 (v / v). Normal porcine IgG (50 mg / mL) and 11 batches of pATG preparations (50 mg / mL) were diluted 800-fold to a total protein concentration of 62.5 μg / mL. The absorbance (OD) of each well was measured at 450 nm using a microplate reader. 450nm Each sample was tested in two replicates, and the results are shown in Table 4.

[0043] Table 4. Detection results of different batches of pATG formulations

[0044] The results in Table 4 show that the OD of batch P006 of pATG formulation... 450nm Highest value (OD) 450nm = 0.520), therefore, this batch of pATG formulation was selected as the standard in subsequent methodological validation and sample testing. Meanwhile, the experimental data in Table 4 also showed significant differences in the content of porcine anti-human FcεRI antibody in different batches of pATG formulation, while the signal (OD) of normal porcine IgG was... 450nm =0.061) is much lower than that of each batch of pATG preparation, indicating that the signal detected by the ELISA reader is indeed contributed by the anti-human FcεRI antibody produced during the immunization process.

[0045] Example 4 This embodiment uses the standard samples screened in Example 3 to construct a correlation between standard concentration and OD. 450nm The relationship curve between the values ​​was used as a standard curve. The relative concentration of the pATG formulation in batch P006 of Example 3 was assigned a value of 1000 AU / mL.

[0046] The experiment was conducted according to the steps in Example 1. The pATG formulation standard was diluted 100-fold (equivalent to a concentration of 10 AU / mL) with protein-free blocking buffer, followed by a 2-fold serial dilution to prepare a total of 8 concentration points, with a relative concentration range of 0.078 AU / mL to 10 AU / mL. Each concentration was prepared in 2 replicates. The antigen coating concentration was 1.5 μg / mL, and the secondary antibody dilution factor was 1:20000.

[0047] OD was detected 450nm After setting the values, the relative concentrations of the standards were plotted as x1 and OD. 450nm The value is y1 on the ordinate. A four-parameter logistic curve was fitted using the ELISA Calc regression / fitting software (version v0.2). The fitted equation is: y1 = 0.047 + 3.611 / [1 + (x1 / 8.819)]. -1.024 The coefficient of determination R of the fitted curve 2 =0.9993, the standard curve is as follows Figure 1 As shown in the figure. This result indicates that the standard curve established in this invention has good goodness of fit in the range of 0.078 AU / mL to 10 AU / mL, and can be used for relative quantitative detection of samples.

[0048] In summary, the specific embodiments of the present invention for detecting porcine anti-human FcεRI antibodies in anti-human thymocyte immunoglobulin preparations include the following steps: S1. Antigen coating: Dilute FcεRIα protein with PBS buffer to 0.75 μg / mL~3 μg / mL, add 50 μL to each well of a 96-well microplate, cover with sealing film and incubate overnight (14~18 h) at 4°C.

[0049] S2, Washing and Blocking: After incubation in step S1, add washing buffer at a rate of 350 μL / well and wash the microplate three times. After the last wash, pat the microplate dry on absorbent paper. Add protein-free blocking buffer at a rate of 300 μL / well and incubate at 37°C for 1 hour.

[0050] S3. Sample Incubation: Using protein-free blocking buffer as a diluent, dilute the standard 100-fold first, then perform serial dilutions of 2-fold to prepare at least 8 concentration points (relative concentrations of 10 AU / mL, 5 AU / mL, 2.5 AU / mL, 1.25 AU / mL, 0.625 AU / mL, 0.3125 AU / mL, 0.156 AU / mL, and 0.078 AU / mL, respectively). Dilute the test sample to an appropriate concentration (for a formulation sample with a protein concentration of 50 mg / mL, typically dilute 400 or 800 times to a total protein concentration of 125 μg / mL or 62.5 μg / mL). Add the diluted standard and test sample to the ELISA plate, 50 μL per well, with at least two replicates for each concentration point and each sample. Use normal porcine IgG at the same concentration and amount as a negative control, and the diluent as a blank control. Cover with sealing film and incubate at 37°C for 1 hour.

[0051] S4. Secondary antibody incubation: After the incubation in step S3 is completed, add washing buffer at a rate of 350 μL / well and wash the ELISA plate 5 times. After the last wash, pat the ELISA plate dry on absorbent paper. Dilute the goat anti-pig IgG (H+L)-HRP secondary antibody with protein-free blocking buffer at a volume ratio of 1:20000~1:80000. Add 100 μL of the diluted goat anti-pig IgG (H+L)-HRP secondary antibody to each well, cover with sealing film, and incubate at 37℃ for 1 h.

[0052] S5. Color Development and Termination: After incubation in step S4, add washing buffer at a rate of 350 μL / well and wash the microplate 5 times. After the last wash, pat the microplate dry on absorbent paper. Add 100 μL of TMB color development solution to each well and incubate at room temperature (e.g., 25°C) in the dark for 10-15 minutes. Immediately afterward, add 50 μL of stop solution to each well and gently shake to mix.

[0053] S6. Absorbance Measurement and Data Processing: Immediately after adding the stop solution, measure the absorbance (OD) of each well at 450 nm using a microplate reader. 450nm The relative concentration of the standard was plotted as x1 on the x-axis, corresponding to OD. 450nm The value is the ordinate y1. A standard curve is fitted using a four-parameter logistic equation. The coefficient of determination R is required. 2 ≥0.99. The OD of the sample to be tested... 450nm Substitute the value into the standard curve to calculate the relative concentration after dilution, and then multiply it by the dilution factor of the sample to obtain the relative content (AU / mL) of porcine anti-human FcεRI antibody in the sample to be tested.

[0054] Example 5 This embodiment investigates the precision, accuracy, limit of detection, limit of quantitation, dilution linearity, and specificity of the above-described method for detecting porcine anti-human FcεRI antibodies in anti-human thymocyte immunoglobulin preparations.

[0055] 5.1 Precision Verification 5.1.1 In-plate precision Based on the experimental results in Example 3, three batches of pATG preparations (each with a protein concentration of 50 mg / mL, batch numbers P006, P011, and P010) were selected at high, medium, and low detection signal levels. These were diluted 800 times (to a protein concentration of 62.5 μg / mL) and used as test samples. Each sample was tested in six replicates on the same ELISA plate. An independent standard curve was prepared for each plate. Based on the standard curve and the sample dilution factor, the relative content of each replicate of the three batches of high, medium, and low concentration pATG preparations was calculated. The mean, standard deviation (SD), and coefficient of variation (CV) of the repeatability results were also calculated using the formula: CV = (SD / Mean) × 100%. The detection results are shown in Table 5.

[0056] Table 5. Results of In-Plate Precision Verification

[0057] As can be seen from the data in Table 5, the CVs of the detection results for high, medium and low concentration samples were 3.001%, 2.480% and 3.155%, respectively, all less than 5%, indicating that this method has good in-plate precision.

[0058] 5.1.2 Inter-plate precision The high, medium, and low concentrations of pATG formulations were tested three times on three different dates. The sample preparation method was consistent with the in-plate precision experiment in step 5.1.1. At least two replicates were set in each test plate. Then, all test results were collected, and the mean, standard deviation, and CV value of the test results of high, medium, and low concentration pATG samples on different dates were calculated. The test results are shown in Table 6.

[0059] Table 6 Results of Inter-plate Precision Verification

[0060] The experimental results in Table 6 show that the CV values ​​of the inter-plate detection results for high, medium and low concentration samples were 4.664%, 7.645% and 3.758%, respectively, all less than 10%, indicating that the method has good inter-plate precision.

[0061] 5.2 Accuracy Verification The accuracy of this detection method was verified by a blank spiked recovery experiment. 195 μL, 190 μL, and 180 μL of sample dilution buffer (i.e., protein-free blocking buffer, relative concentration of 0 AU / mL) were taken respectively, and 5 μL, 10 μL, and 20 μL of standard working solution (10 AU / mL) were added accordingly to prepare samples with low, medium, and high spiked concentrations for detection. Two replicates were performed for each spiked concentration. The remaining steps were the same as in steps S1-S6. The detection results are shown in Table 7.

[0062] Table 7. Recovery Rate of Blank Spike

[0063] The results in Table 7 show that the theoretical concentrations of the low, medium, and high spiked samples were 0.25, 0.5, and 1 AU / mL, respectively, while the measured concentrations were 0.227, 0.510, and 0.936 AU / mL, respectively. The recoveries were 90.772%, 102.076%, and 93.563%, respectively, all falling within the acceptable range of 80% to 120%, indicating that the detection method has good accuracy.

[0064] 5.3 Limit of Detection (LOD) Using normal porcine IgG (50 mg / mL, diluted at the same multiple as the test sample) as the negative sample, eight replicates were performed. The mean and standard deviation of the negative sample signal were calculated and then calculated using the following formula: LOD signal value = mean + 3 × standard deviation. The calculated signal value was substituted into the standard curve, and the resulting relative concentration was the LOD.

[0065] The test results showed that the OD of the 8 duplicate wells was... 450nm The values ​​were 0.070, 0.072, 0.069, 0.069, 0.066, 0.066, 0.064, and 0.065, respectively. The calculated average value was 0.067 and the standard deviation was 0.0028. Based on this, the signal value corresponding to LOD was calculated to be 0.076. Substituting this value into the standard curve obtained in Example 4, the limit of detection was calculated to be 0.079 AU / mL.

[0066] 5.4 Limit of Quantification (LOQ) Dilute the standard to a relative concentration of 0.156 AU / mL to prepare a low-concentration sample close to the LOD value and the lowest point of the standard curve. Repeat the test on 6 replicates. The minimum concentration that meets the requirements is LOQ, with a CV ≤ 20% and a recovery rate between 80% and 120%.

[0067] Experimental results show that the OD of the 6-well composite is... 450nmThe values ​​were 0.096, 0.094, 0.094, 0.096, 0.094, and 0.093, respectively. Substituting their average value into the standard curve, the measured relative concentration was calculated to be 0.129 AU / mL. Further calculations showed a recovery rate of 82.692% and a CV value of 2.569%, both meeting the above acceptance criteria. Therefore, the limit of quantitation was determined to be 0.156 AU / mL.

[0068] 5.5 Dilution Linearity Validation To verify whether high-concentration samples could still be accurately quantified after appropriate dilution, a linearity validation was performed. A high-concentration sample (average relative concentration of 486.975 AU / mL after three repeated assays) was first diluted 1:100 by volume, then serially diluted 2-fold until the lowest concentration approached the limit of quantitation, resulting in five concentration gradients for detection. The antigen coating concentration was 1.5 μg / mL, and the secondary antibody dilution factor was 1:20000. The remaining steps were the same as steps S1-S6. A graph was plotted with the theoretical relative concentration as the x-axis (x2) and the measured relative concentration as the y-axis (y2), and linear fitting was performed. The recovery rate for each concentration point was calculated using the following formula: Recovery rate (%) = (Measured concentration / Theoretical concentration) × 100%.

[0069] The results are as follows Figure 2 As shown, the coefficient of determination R0 for the linear fit between theoretical and measured concentrations is... 2 =0.9998, and the recoveries at the five concentration points from high to low were 108.618%, 106.771%, 99.407%, 97.424%, and 93.672%, respectively, all within the acceptable range of 80% to 120%. This indicates that the detection method of the present invention has good dilution linearity over a wide concentration range and can reliably achieve accurate quantification after appropriate dilution of high-concentration samples.

[0070] 5.6 Specificity As demonstrated in Example 2, experiments have shown that using a protein-free blocking solution achieves excellent blocking results and avoids interference from protein components in traditional blocking solutions (such as BSA or serum blocking solutions). This experiment further investigates whether complex components in plasma interfere with test results and lead to false positives by testing porcine negative plasma and human plasma samples.

[0071] In the experiment, the antigen coating concentration was 1.5 μg / mL, the secondary antibody dilution was 1:20000, and porcine negative plasma, human plasma, and normal porcine IgG samples were all diluted by the same factor (1:800 volume ratio) before detection. The remaining steps were the same as steps S1-S6. The experimental results are as follows: Figure 3 As shown, the average OD of porcine negative plasma and human plasma 450nmThe values ​​were 0.055 and 0.044, respectively, both lower than the average OD of normal pig IgG samples. 450nm The values ​​(0.057) and the converted relative concentrations are all less than the detection limit of this method, indicating that the complex components in the plasma matrix will not produce detectable false positive signals in this detection system, and this method has good specificity.

[0072] Example 6 Following the methods described in steps S1-S6 of Example 4, two replicate tests were performed on eight production batches (P010-P017) of pATG finished product (i.e., pATG formulation) from 2025, conducted on different dates. In step S1, the antigen coating concentration was 1.5 μg / mL; in step S2, the secondary antibody dilution was 1:20000; each sample was tested in duplicate, with samples diluted 1:400 by volume. An independent standard curve was prepared for each plate. The test results are as follows: Figure 4 As shown, from Figure 4 As can be seen above, the highest relative concentration of porcine anti-human FcεRIα antibody among the eight batches was 465.016 AU / mL, and the lowest was 200.892 AU / mL.

[0073] The embodiments described above are merely preferred embodiments of this application and are not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by anyone skilled in the art. Any simple equivalent changes and modifications made based on the scope of protection claimed in this application and the content of the specification should be included within the scope of protection of this application.

Claims

1. A method for detecting porcine anti-human FcεRI antibodies for non-disease diagnosis and treatment purposes, characterized in that, Includes the following steps: S1. Dilute the FcεRIα protein with coating buffer and add it to a solid support. Incubate the mixture to fix the FcεRIα protein onto the surface of the solid support. S2. After the incubation in step S1 is completed, wash the solid support and add the blocking solution; the blocking solution is a protein-free blocking solution that does not contain serum proteins. After incubation in steps S3 and S2, the solid support is washed, and then serially diluted standards and diluted test samples are added respectively. The samples are then incubated to allow the porcine anti-human FcεRI antibody in the sample to bind to the FcεRIα protein. The standards are anti-human thymocyte or anti-human lymphocyte porcine immunoglobulin preparations containing porcine anti-human FcεRI antibody. S4. After the incubation in step S3 is completed, wash the solid-phase carrier, add diluted anti-pig IgG secondary antibody carrying detectable markers and incubate. S5. After the incubation in step S4 is completed, wash the solid support, add the substrate corresponding to the detectable marker for color development, add the stop solution to terminate the reaction, and measure the signal value. S6. Fit a standard curve with the relative concentration of the standard as the abscissa and the signal value corresponding to the relative concentration as the ordinate; substitute the signal value of the sample to be tested into the standard curve to obtain the relative concentration after dilution, and then multiply it by the dilution factor of the sample to be tested to obtain the relative content of porcine anti-human FcεRI antibody in the sample to be tested.

2. The method according to claim 1, characterized in that, In step S1, the coating concentration of the FcεRIα protein is 0.75 μg / mL to 3 μg / mL; Or / and, in step S1, the coating buffer is a PBS buffer with a pH of 7.4; Or / and, in step S2, the protein-free blocking solution is prepared using PBST buffer.

3. The method according to claim 1, characterized in that, In step S3, both the standard and the sample to be tested are diluted using the protein-free blocking solution described in step S2.

4. The method according to claim 1, characterized in that, In step S3, the screening method for the standard includes the following steps: following the method in steps S1 to S5, on the same ELISA plate, multiple batches of anti-human thymocyte or anti-human lymphocyte porcine immunoglobulin preparations are tested in parallel at the same dilution factor in step S3, and the sample of the batch with the highest signal value is selected as the standard, and the relative concentration of the standard is assigned to 1000 AU / mL.

5. The method according to claim 4, characterized in that, When using the standard, it should first be diluted with protein-free blocking buffer to a relative concentration range of 0.078 AU / mL to 10 AU / mL.

6. The method according to claim 1, characterized in that, In step S3, normal porcine IgG was used as a negative control, and the protein-free blocking solution was used as a blank control.

7. The method according to claim 1, characterized in that, In step S4, the anti-pig IgG secondary antibody is a goat anti-pig IgG (H+L) antibody; Or / and, in step S4, the anti-pig IgG secondary antibody is diluted with the protein-free blocking solution at a volume ratio of 1:20000 to 1:80000.

8. The method according to claim 1, characterized in that, In step S4, the detectable marker is horseradish peroxidase.

9. A kit for detecting porcine anti-human FcεRI antibodies using the method described in any one of claims 1 to 8, characterized in that, This includes FcεRIα protein, protein-free blocking solution, standards, and anti-pig IgG secondary antibody carrying detectable markers.

10. The reagent kit according to claim 9, characterized in that, It includes at least one of a substrate corresponding to the detectable marker, a stop solution, a coating buffer, and a negative control; the negative control is normal porcine IgG.

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