Method for detecting specific IgG antibody in SD (Sprague Dawley) rat serum

Abstract

The invention provides a method for detecting a specific IgG antibody in SD rat serum, and belongs to the technical field of biological detection. The detection method special for the SD rat serum sample is established, the operation process is standard, the result is stable and reliable, matrix interference can be effectively eliminated, and specific recognition and detection of a target antibody are achieved; the application of the method provides key technical support for preclinical efficacy and immunogenicity evaluation of related biopharmacy, significantly improves the accuracy and comparability of experimental data, and has positive significance in promoting the research and development process of drugs.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a method for detecting specific IgG antibodies in the serum of SD rats. Background Technology

[0002] Respiratory syncytial virus (RSV) is a common pathogen that causes respiratory infections, posing a significant health threat, especially to vulnerable populations such as infants and the elderly. The fusion protein (F protein) on the viral surface is a key protein for its invasion of host cells and an important target for vaccine and antibody drug development. Preclinical studies are crucial in the development of vaccines or therapeutic antibody drugs targeting RSV. Typically, researchers use model animals such as SD rats to conduct preliminary assessments of drug safety and efficacy. In these studies, detecting the production of specific antibodies against the drug (such as the RSV F protein) in animal serum is one of the core indicators for evaluating drug immunogenicity (i.e., the ability to elicit an immune response) and potential efficacy. IgG antibodies are the main type of antibody produced in humoral immune responses, and their levels directly reflect the strength of the body's immune response to a specific antigen.

[0003] Currently, enzyme-linked immunosorbent assay (ELISA) is one of the most commonly used techniques for detecting specific antibodies in serum. Its basic principle is to use antigens coated on a solid-phase support to capture the corresponding antibodies in the sample. Then, enzyme-labeled secondary antibodies bind to the captured antibodies, and finally, the enzyme catalyzes a color reaction, thereby achieving qualitative or quantitative analysis of the target antibody. However, developing a stable and reliable ELISA method is not easy. It involves many aspects, including the selection of coating antigens, optimization of reaction conditions, and methodological validation for specific sample matrices (such as sera from different species). The serum composition of SD rats differs from that of humans or other animals. Directly applying detection methods designed for other species or targets may lead to false positives or false negatives due to matrix effects and non-specific binding, failing to accurately reflect the immunogenicity of drugs. Therefore, there is an urgent need in this field for a systematically optimized and validated ELISA method specifically for detecting specific IgG antibodies against RSV-F protein in SD rat serum to meet the precision requirements of preclinical drug research. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting specific IgG antibodies in the serum of SD rats, which solves the problem in the prior art of lacking a stable and reliable enzyme-linked immunosorbent assay method specifically for detecting specific IgG antibodies in the serum of SD rats.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for detecting specific IgG antibodies in the serum of SD rats. RSV-preFProtein is used as the coating antigen, and the specific IgG antibodies in the serum of SD rats are detected by enzyme-linked immunosorbent assay (ELISA). The method includes a coating step, a washing step, a blocking step, a sample addition step, a detection working solution addition step, a color development step, a termination step, and a plate reading step. The plate reading step involves reading the absorbance value at a wavelength of 450 nm and determining the presence or concentration of the specific IgG antibody based on the absorbance value.

[0006] Preferably, in the coating step, the coating working solution is added to the ELISA plate and coated at 2-8 degrees Celsius for 16-20 hours.

[0007] Preferably, the concentration of RSV-preF Protein in the coating working solution is 0.3~0.8 micrograms per milliliter.

[0008] Preferably, the plate washing step involves washing the plate 2 to 5 times with a washing solution, wherein the washing solution is a 0.03 to 0.08% PBST solution.

[0009] Preferably, the sealing step involves incubating the sealing solution at 35-39 degrees Celsius for 100-140 minutes.

[0010] Preferably, the blocking solution is a 3-8% SM-1×PBST solution.

[0011] Preferably, the sample dilution step is included before the sample addition step, and the sample is diluted 10 times using 0.5~1.5% BSA-1 multiplied by PBST solution.

[0012] Preferably, in the sample addition step, the diluted sample is added to the ELISA plate and incubated at 35-39 degrees Celsius for 55-65 minutes; In the step of adding the detection working solution, the detection working solution is added and incubated at 35-39 degrees Celsius for 55-65 minutes. The detection working solution is prepared by diluting HRP-ProteinG by 18,000 to 22,000 times.

[0013] Preferably, in the color development step, TMB substrate color development solution is added and the color development is carried out at 10-30 degrees Celsius in the dark for 3-15 minutes; The termination step involves adding a termination solution, which is a 0.5 mol / L sulfuric acid solution.

[0014] Preferably, the method further includes a step of preparing quality control samples, and the quality control samples include negative quality control samples and positive quality control samples; The negative control sample is a mixture of blank serum from SD rats, and the positive control sample includes a high-concentration positive control sample and a low-concentration positive control sample, wherein the high-concentration positive control sample has a concentration of 9000~11000 nanograms per milliliter and the low-concentration positive control sample has a concentration of 400~600 nanograms per milliliter.

[0015] The technical effects and advantages of this invention are as follows: This invention establishes a detection method specifically for serum samples from SD rats. The method has a standardized operation procedure, stable and reliable results, and can effectively eliminate matrix interference, achieving specific identification and detection of target antibodies. The application of this method provides key technical support for the preclinical efficacy and immunogenicity evaluation of related biopharmaceuticals, significantly improves the accuracy and comparability of experimental data, and has positive significance for promoting the drug development process. Detailed Implementation

[0016] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0017] Reagent preparation method in the examples: Preparation of coating solution Weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, dissolve them in 1000 mL of pure water, and mix thoroughly to obtain a 0.05 M carbonate buffer solution as the coating solution. The prepared solution should be stored in a sealed container at 2-8°C for up to 3 months.

[0018] Preparation of detergent Take 50 mL of 20×PBS buffer and add it to 950 mL of pure water. Mix well to prepare a 1×PBS working solution. Then, take 500 µL of Tween-20 and add it to 999.5 mL of 1×PBS. Mix thoroughly to obtain a 0.05% PBST washing solution. The prepared washing solution can be stored in a sealed container at 10-30°C for 7 days.

[0019] Preparation of sealing fluid Weigh 5.0 g of skim milk powder and dissolve it in 100 mL of washing solution (0.05% PBST). Mix thoroughly to obtain 5% SM-1×PBST blocking solution. The prepared blocking solution should be stored in a sealed container at 2-8°C for 7 days.

[0020] Preparation of sample diluent Weigh 1.0 g of bovine serum albumin and dissolve it in 100 mL of washing buffer (0.05% PBST). Mix thoroughly to obtain 1% BSA-1×PBST sample diluent. The prepared diluent should be stored in a sealed container at 2-8°C and has a shelf life of 7 days.

[0021] Preparation of stop solution Slowly add 27.2 mL of concentrated sulfuric acid to a certain volume of pure water, stir to mix, cool, and then add pure water to a final volume of 1000 mL. Mix thoroughly to obtain the 0.5 M sulfuric acid stop solution. The prepared stop solution can be stored in a sealed container at 10-30°C for up to 6 months.

[0022] Preparation of coating working fluid Dilute the RSV-preF Protein with coating buffer (0.05 M CBS) to a concentration of 0.5 µg / mL, and mix thoroughly to obtain the coating working solution. This working solution should be prepared fresh before use.

[0023] Preparation of working solution for testing HRP-ProteinG was diluted twice with washing buffer (0.05% PBST) to a final overall dilution factor of 20,000. After thorough mixing, the working solution was obtained. This working solution should be prepared and used immediately.

[0024] Instructions for preparing quality control samples The mixed SD rat blank serum was used as both a negative control sample and a dilution matrix for preparing positive control samples.

[0025] Example 1 Negative control sample: Mixed blank serum from SD rats was used as the negative control sample (NC).

[0026] Preparation of positive control samples The positive control samples were prepared using a serial dilution method.

[0027] The RSV-F Antibody source solution with a concentration of 1 mg / mL was diluted with mixed blank serum from SD rats to obtain intermediate solution A with a concentration of 100,000 ng / mL.

[0028] Intermediate solution A was diluted 10-fold with mixed SD rat blank serum to obtain a high-concentration positive control sample HPC with a concentration of 10,000 ng / mL.

[0029] The HPC sample was diluted 5 times to obtain a medium-concentration positive control sample MPC with a concentration of 2,000 ng / mL.

[0030] The MPC sample was diluted 4 times to obtain a low-concentration positive control sample LPC with a concentration of 500 ng / mL.

[0031] Detection steps: Wash plate: Wash the plate 3 times with washing buffer (0.05% PBST), 300 μL / well, to remove residual liquid; Blocking: Add blocking solution (5% SM-1×PBST), 200 μL / well, cover with membrane, and incubate in a constant temperature incubator at 37 °C for 2 h. 20 min; Sample dilution before loading: Dilute each quality control and sample 10-fold with 1% BSA-1×PBST before loading; Washing: Wash the plate 3 times with washing buffer (0.05% PBST), 300 μL / well, to remove residual liquid; Sample addition: Following the Mapping of Samples diagram in the Watson system, add 100 μL / well of each quality control sample and the original sample to the microplate, cover with a membrane, and incubate at 37 °C for 1 h. 5 min; Washing: Wash the plate 3 times with washing buffer (0.05% PBST), 300 μL / well, to remove residual liquid; Add detection working solution: Add 100 μL / well to the microplate, cover with a membrane, and incubate in a constant temperature incubator set at 37°C for 1 h ± 5 min; Washing: Wash the plate 3 times with washing buffer (0.05% PBST), 300 μL / well, to remove residual liquid; Color development: Add 100 μL of TMB substrate color development solution to each well and develop at 10~30 °C in the dark for 3-15 min; Termination: Add 100 μL of stop solution (0.5 M H2SO4) per well to terminate the reaction; Plate reading: Use a multi-functional microplate reader (Varioskan LUX) set to a detection wavelength of 450 nm to read the OD value.

[0032] Data processing and statistical analysis The OD values ​​of the samples were detected using a Varioskan LUX multi-functional microplate reader, and the OD value data were acquired using the instrument's built-in SkanIt DDE software and imported into Watson LIMS software for data management. The acquired raw OD value data were then analyzed using Microsoft Office Excel 2010 (Microsoft Corporation).

[0033] The CF value, CP value, SIR, %CV, mean, median, median absolute deviation (MAD), standard deviation (SD), and log transformation in the experiment were all calculated and analyzed using Office Excel 2010 (Microsoft Corporation). The relevant calculation formulas are as follows: LPC / NC = The test result of each LPC sample divided by the mean of the NC test results in the same analytical batch.

[0034] HPC / NC = The test result of each HPC sample divided by the mean of the NC test results in the same analytical batch.

[0035] SIR% = (1 - OD value of sample with added antigen / OD value of sample without added antigen) × 100 Coefficient of variation percentage: %CV = SD / Mean × 100, where SD and Mean are calculated using the "STDEV" and "AVERAGE" formulas in Excel 2010, respectively.

[0036] Median performs the calculation using the "MEDIAN" formula in Excel 2010.

[0037] MAD=Median (︱X i -Median (X j First, calculate the median (X) of the data set. j Then calculate the absolute value of the deviation between each data point and its median (|X). i -Median (X j The median of the absolute values ​​of the deviations is the MAD. The Median is calculated using the "MEDIAN" formula in Excel 2010, and the absolute value is calculated using the "ABS" formula in Excel 2010.

[0038] In the experiment, the box plot method for outlier identification, the Shapiro-Wilk method for distribution identification, and the skewness coefficient calculation were all statistically analyzed using Stata software.

[0039] All the above calculation results are processed by the formula "ROUND(number, num_digits)" to retain the number of decimal places, and the results are all retained to 3 decimal places.

[0040] Experimental Example The present invention also validated the ELISA method for detecting specific IgG antibodies in the serum of SD rats to examine the sensitivity, precision, specificity, and selectivity of the bioanalytical method, and to examine whether the method can be used for the detection of specific IgG antibodies in the serum of SD rats.

[0041] The method is as follows: Sensitivity The positive control antibody (RSV-F Antibody) was serially diluted using mixed SD rat blank serum (concentrations: 5000.000, 2500.000, 1250.000, 625.000, 312.500, 156.250, 78.125, 39.063, 19.531 ng / mL; see original records for specific preparation methods) to obtain sensitivity samples (A1~A9). Each concentration was tested in duplicate (OD values ​​were averaged). The sensitivity of this method is defined as the minimum concentration of sample that yields a positive result (OD≥CP).

[0042] The acceptable sensitivity threshold is ≤500,000 ng / mL.

[0043] If none of the above-mentioned series of diluted samples has an OD lower than CP, but the concentration value meets the sensitivity acceptance standard (≤500,000 ng / mL), then the concentration report corresponding to the CP value is the lowest detectable concentration of the sample.

[0044] Precision: Six sets of quality control samples were prepared using mixed SD rat blank serum to prepare the positive control antibody. Each set of samples included a negative control (NC), and positive control samples with high (HPC: 10,000.000 ng / mL), medium (MPC: 2,000.000 ng / mL), and low (LPC: 500.000 ng / mL) values. These six independently prepared quality control samples served as precision samples. Each sample was tested in duplicate (the OD value was averaged), and at least two analysts were required to complete at least three independent and valid assays over at least three days. The %CV of the OD values ​​for each precision sample was calculated for both intra-batch and inter-batch measurements.

[0045] Acceptance criteria: The average %CV of the OD values ​​of positive control samples within and between batches is ≤20%, and the average %CV of the OD values ​​of negative control samples is ≤30%, indicating that the method has good precision.

[0046] Selectivity: Select blank sera from at least 10 different sources of SD rats and at least 1 hemolysis matrix (add 2% whole blood cell lysate to the blank sera of SD rats) to prepare LPC positive control samples respectively, and investigate them simultaneously with the LPC samples prepared from the mixed blank sera of SD rats; then directly detect the above at least 10 different sources of blank sera of SD rats and at least 1 hemolysis matrix sample separately. The above samples are each set with 2 replicates (the OD value results are averaged) for detection.

[0047] Acceptance criteria: When comparing the LPC samples prepared from at least 80% of the individual sera with the samples prepared from the corresponding mixed sera, %CV ≤ 20% is satisfied and they are positive (OD ≥ CP); at least 80% of the individual serum samples are negative (OD < CP), indicating that the method has good selectivity and no matrix interference.

[0048] Specificity: Use the mixed blank sera of SD rats to prepare 1 set of quality control samples from the positive control antibody, including negative control NC, positive control samples with high (HPC: 10000.000 ng / mL), medium (MPC: 2000.000 ng / mL), and low (LPC: 500.000 ng / mL) levels. Perform sample treatment on 1 set of quality control samples with sample diluent and sample diluent containing 10.0 µg / mL antigen (RSV-preF Protein) respectively, set 2 replicates (the OD value results are averaged) within the same analysis batch for detection, and calculate the signal inhibition ratio (SIR value) between the samples without adding antigen and the samples with added antigen.

[0049] Acceptance criteria: The OD value of the positive control samples with added antigen decreases during detection, and the SIR value of the positive control samples is greater than the SIR value of the negative control samples. This indicates that the method can specifically bind to the antigen and the specificity meets the detection requirements.

[0050] The summary of the method validation is shown in Table 1 below: Table 1 Summary of Validation Results project result Sensitivity 78.125 ng / mL Precision Positive control HPC (10,000,000 ng / mL), MPC (2,000,000 ng / mL), LPC (500,000 ng / mL): Intra-assay %CV: 1.872%~4.645%; Inter-assay %CV: 9.487%~16.757%. Negative control NC: Intra-assay %CV: 2.239%~3.053%; Inter-assay %CV: 7.143%. Specificity The positive control sample with added antigen showed a decrease in OD value, and the SIR value of the positive control sample was greater than that of the negative control sample. This indicates that the method specifically binds to the antigen, and its specificity meets the detection requirements. Selective Eleven out of eleven sera from different sources (including one hemolyzed serum) were negative for individual serum samples; all LPC samples prepared from these eleven out of eleven sera (including one hemolyzed serum) were positive; compared with LPC samples prepared from mixed sera, the %CV of LPC samples prepared from eleven out of eleven individual sera ranged from 8.915% to 16.723%, satisfying the requirement of %CV ≤ 20%. The validation results met the selective acceptance criteria and showed no matrix interference. Sensitivity Detect the sensitivity samples (A1 - A9) of the positive control antibody (RSV-F Antibody) diluted to 5000.000, 2500.000, 1250.000, 625.000, 312.500, 156.250, 78.125, 39.063, 19.531 ng / mL.

[0051] Through the sensitivity validation of analysis batch Run2, the sensitivity of this method is 78.125 ng / mL, and the specific results are shown in Table 2.

[0052] Table 2 Sensitivity of Specific IgG Antibody in Serum of SD Rats Detected by ELISA

[0053] This verification was carried out by 2 analysts for 3 days with a total of 3 valid batches for precision verification. The results showed that the average %CV of OD values of positive quality control samples within and between batches was ≤ 20%, and the average %CV of OD values of negative quality control samples was ≤ 30%, indicating that the precision of this method was good. The precision results are shown in Table 3.

[0054] Table 3 Precision Results

[0055] Selectivity A total of 11 blank sera from SD rats with different sources (including 1 hemolyzed serum) were used for selectivity investigation.

[0056] The results showed that 11 / 11 (including 1 hemolyzed serum) blank serum samples from SD rats with different sources were negative (meeting OD < CP); meanwhile, 11 / 11 (including 1 hemolyzed serum) LPC samples prepared from individual sera were all positive (meeting OD ≥ CP), and compared with the LPC samples prepared from mixed sera, the %CV of OD values was between 8.915% and 16.723%, meeting %CV ≤ 20%.

[0057] The verification results met the selectivity acceptance criteria, indicating that the method had good selectivity and no matrix interference.

[0058] The specific results are shown in Table 4.

[0059] Table 4 Selectivity of Specific IgG Antibody in Serum of SD Rats Detected by ELISA

[0060] Note: "*" Calculate the %CV of the OD value of the selective sample compared with LPC (Mean) , and LPC (Mean) is the average value of the OD values of 2 sets of corresponding concentration LPC samples prepared with mixed blank serum in this analysis batch; SE-LPC11 and SE-NC11 are prepared with hemolyzed serum; " / " no relevant information.

[0061] Specificity The results showed that the OD value of the detection result of the sample added with antigen decreased significantly, and the SIR value of the positive quality control sample was greater than that of the negative quality control sample, indicating that the method could specifically bind to the antigen, and the specificity met the detection requirements.

[0062] The verification results met the specificity acceptance criteria, indicating that the method had good specificity. The specific results are shown in Table 5.

[0063] Table 5. Specificity of ELISA detection of specific IgG antibodies in SD rat serum

[0064] As demonstrated by the above embodiments, this invention provides a standardized and reliable ELISA detection method. This method successfully detected specific IgG antibodies in the serum of SD rats. The entire detection system exhibited good precision and repeatability, effectively distinguishing between positive and negative results, and proving its specific recognition ability for the target antibody. Furthermore, the method demonstrated good stability under different conditions and met the required anti-interference capabilities, confirming its applicability for the analysis of real samples and providing an effective analytical tool for preclinical research of related biological products.

[0065] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting specific IgG antibodies in the serum of SD rats, characterized in that, The RSV-preF Protein was used as the coating antigen to detect specific IgG antibodies in the serum of SD rats by enzyme-linked immunosorbent assay. The method included coating, washing, blocking, sample addition, addition of detection working solution, color development, termination, and plate reading. The plate reading step involved reading the absorbance value at a wavelength of 450 nm and determining the presence or concentration of specific IgG antibodies based on the absorbance value.

2. The method as described in claim 1, characterized in that, In the coating step, the coating working solution is added to the ELISA plate and coated at 2-8 degrees Celsius for 16-20 hours.

3. The method as described in claim 2, characterized in that, The concentration of RSV-preF Protein in the coating working solution is 0.3~0.8 micrograms per milliliter.

4. The method as described in claim 1, characterized in that, The plate washing step involves washing the plate 2 to 5 times with a washing solution, which is a 0.03 to 0.08% PBST solution.

5. The method as described in claim 1, characterized in that, The sealing step involves incubating the solution at 35-39 degrees Celsius for 100-140 minutes.

6. The method as described in claim 5, characterized in that, The blocking solution is a 3-8% SM-1×PBST solution.

7. The method as described in claim 1, characterized in that, The sample addition step includes a sample dilution step, in which the sample is diluted 10 times using 0.5-1.5% BSA-1 multiplied by PBST solution.

8. The method as described in claim 1, characterized in that, In the sample addition step, the diluted sample is added to the ELISA plate and incubated at 35-39 degrees Celsius for 55-65 minutes. In the step of adding the detection working solution, the detection working solution is added and incubated at 35-39 degrees Celsius for 55-65 minutes. The detection working solution is prepared by diluting HRP-ProteinG by 18,000 to 22,000 times.

9. The method as described in claim 1, characterized in that, In the color development step, TMB substrate color development solution is added and the color development is carried out at 10-30 degrees Celsius in the dark for 3-15 minutes. The termination step involves adding a termination solution, which is a 0.5 mol / L sulfuric acid solution.

10. The method as described in claim 1, characterized in that, The method further includes a step of preparing quality control samples, and the quality control samples include negative quality control samples and positive quality control samples; The negative control sample is a mixture of blank serum from SD rats, and the positive control sample includes a high-concentration positive control sample and a low-concentration positive control sample, wherein the high-concentration positive control sample has a concentration of 9000~11000 nanograms per milliliter and the low-concentration positive control sample has a concentration of 400~600 nanograms per milliliter.

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