Mouse IgG antibody ELISA detection kit and detection method
By screening and optimizing the ELISA detection kit composed of goat anti-mouse IgG antibodies, the cross-reaction problem in mouse IgG detection was solved, and the detection effect with high specificity and sensitivity was achieved, which was suitable for quantitative analysis of mouse IgG.
Patent Information
- Application Number
- CN202510303111.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-14
- Publication Date
- 2025-07-01
AI Technical Summary
The prior art is difficult to detect mouse IgG antibodies efficiently and specifically, and there is a cross-reaction problem, which affects the accuracy and sensitivity of the detection.
The ELISA detection kit consisting of goat anti-mouse IgG coated enzyme label plate, horseradish peroxidase-labeled goat anti-mouse secondary antibody, color development solution, sample dilution solution, termination solution, positive control and negative control was used to determine the best antibody pair through screening experiments, and the detection conditions were optimized to achieve high specificity and sensitivity detection.
A mouse IgG antibody detection method with high specificity, sensitivity and accuracy is provided, which can reliably quantify the IgG concentration in the sample, and is cheap and easy to operate.
Smart Images
Figure CN120233080A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a kit and a detection method for ELISA detection of mouse IgG antibody, belonging to the technical field of biological detection. Background Art
[0002] Immunoglobulin (Ig) refers to globulins with antibody activity or chemical structure, similar to antibody molecules. Immunoglobulin is a tetrapeptide chain structure composed of two identical light chains and two identical heavy chains connected by interchain disulfide bonds. Immunoglobulins are divided into five categories, namely immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), and immunoglobulin E (IgE).
[0003] Immunoglobulin G (IgG) is the main component of immunoglobulins in serum, accounting for about 75% of the total content of immunoglobulins in serum. It is one of the most important antibodies in the immune system. It can recognize and bind to pathogens, thereby helping the body clear infections. In immunological research, by detecting the IgG level, the immune response of the body to a certain antigen can be understood, so as to deeply explore the mechanism of immune response. In the construction of disease models, IgG detection can help evaluate the immune status of the body under disease conditions and provide a basis for the study of the pathogenesis of diseases. In addition, in drug R & D and efficacy evaluation, IgG detection can be used as an important indicator to evaluate the impact of drugs on the body's immune function and treatment effect. Therefore, by detecting the IgG level in mice, the intensity of the body's immune response to a specific antigen can be understood, which is of great significance for the study of immunology, disease models, and drug efficacy evaluation. Summary of the Invention
[0004] The purpose of the present invention is to provide a kit, a method, and an application for ELISA detection of mouse IgG antibody.
[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:
[0006] In the first aspect, the present invention provides a kit for ELISA detection of mouse IgG antibody, which includes: an enzyme-labeled plate coated with goat anti-mouse IgG, a goat anti-mouse secondary antibody labeled with horseradish peroxidase, a chromogenic solution, a sample diluent, a termination solution, a positive control, a negative control, and a standard product.
[0007] Preferably, the enzyme-labeled plate is coated with 0.5 μg / mL of goat anti-mouse IgG, the chromogenic solution is a TMB chromogenic solution, the sample diluent is a phosphate buffer solution with a pH of 7.4, and the termination solution is a 2 mol / L sulfuric acid solution.
[0008] Preferably, the positive control is purified mouse immunoglobulin, the negative control is phosphate buffer solution with pH 7.4, and the standard is purified mouse immunoglobulin.
[0009] Preferably, the method for preparing the enzyme-linked immunosorbent assay (ELISA) plate is as follows: Take goat anti-mouse IgG at 1.0 mg / mL, dilute it to 0.5 μg / mL with coating buffer and coat it in the ELISA plate, 100 μL per well. After coating at 37 °C for 1 hour, wash the plate 5 times with PBS buffer containing 0.05% Tween-20, pat it dry, then add 200 μL of PBS buffer containing 2% BSA for blocking. After blocking at 37 °C for 1 hour, wash the ELISA plate.
[0010] In a second aspect, the present invention provides the application of the kit described in the first aspect in quantitatively detecting the level of mouse IgG antibody.
[0011] In a third aspect, the present invention provides an ELISA detection method for mouse IgG antibody, which uses the kit described in the first aspect of the present invention for detection.
[0012] Preferably, it includes the following steps:
[0013] Dilute the sample to be detected to the fitting range with sample diluent, then add it to the ELISA plate for incubation;
[0014] After washing the ELISA plate, dilute the goat anti-mouse antibody labeled with horseradish peroxidase as the secondary antibody and add it to the ELISA plate for incubation;
[0015] After washing the plate, add TMB chromogenic solution, react for 5 - 10 min, then add the termination solution, and immediately use an ELISA reader to measure the A450 / A630 value of each well.
[0016] Preferably, the sample to be detected is diluted to the fitting range of 0 - 0.25 μg / mL.
[0017] Preferably, the dilution ratio of the goat anti-mouse antibody labeled with horseradish peroxidase is 1:10000 times.
[0018] Preferably, it also includes the establishment of a standard curve: Dilute the standard to a concentration gradient between 0 - 0.25 μg / mL with diluent, add it to the ELISA plate for detection, perform linear fitting between the measured A450 / A630 value and the concentration to obtain the standard curve.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] (1) The reagents in the detection kit of the present invention are determined through screening experiments, so as to obtain the best antibody pair for ELISA detection; therefore, the detection kit provided by the present invention has good specificity when detecting mouse IgG and does not cross-react with IgG of other species.
[0021] (2) The kit of the present invention shows excellent performance in the evaluation of sensitivity, recovery rate and stability, and can reliably quantify the IgG concentration in the sample.
[0022] (3) The kit of the present invention has the advantages of low cost, rapidity, simple and convenient operation, etc., and provides a detection method for detecting mouse IgG. Description of the Drawings
[0023] Figure 1 It is the specific result diagram of the kit;
[0024] Figure 2 It is the standard curve of the kit;
[0025] Figure 3 It is the result diagram of the stability of the coated antibody;
[0026] Figure 4 It is the result diagram of the stability of the positive standard product;
[0027] Figure 5 It is the result diagram of the real-time stability of the enzyme-labeled plate at 4°C. Detailed Embodiments
[0028] To make the present invention more obvious and understandable, the preferred embodiments are described in detail below in conjunction with the accompanying drawings.
[0029] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art; in the embodiments of the present invention, if not specifically specified, the technical means used are all conventional means well-known to those skilled in the art.
[0030] In the embodiments of the present invention, the raw materials used are all conventional commercially available products. The sources of some reagents are as follows:
[0031] Goat anti-mouse IgG: purchased from Aladdin Biology, product number: Ab138058; Rabbit anti-mouse IgG: purchased from Aladdin Biology, product number: Ab170451; Horseradish peroxidase-labeled goat anti-mouse secondary antibody: purchased from Aladdin Biology, product number: Ab179001; Horseradish peroxidase-labeled goat anti-mouse secondary antibody: purchased from Aladdin Biology, product number Ab156255; Horseradish peroxidase-labeled rabbit anti-mouse secondary antibody: purchased from Aladdin Biology, Ab176437.
[0032] Example 1: Screening of the Best Antibody Pair
[0033] Example 1 is an exploratory experiment to screen the best antibody pair. In this example, goat anti-mouse IgG (Ab138058) and rabbit anti-mouse IgG (Ab170451) were used as capture antibodies respectively. The two antibodies were diluted to 0.5 μg / mL with 0.05 mol / L carbonate buffer (pH 9.6), and 100 μL per well was added to the enzyme-linked immunosorbent assay (ELISA) plate and coated at 37 °C for 1 h. After coating, the ELISA plate was washed 3 times with 1×PBST, and 2% BSA solution was added to block the excess sites in the ELISA plate and blocked at 37 °C for 1 h. After blocking, the ELISA plate was washed 3 times with 1×PBST. Mouse IgG was diluted to 100 ng / mL and 10 ng / mL with 1×PBS respectively and added to the plate and incubated at 37 °C for 1 h. After incubation, the ELISA plate was washed 3 times with 1×PBST.
[0034] In this example, 3 antibodies were selected as detection antibodies, namely: horseradish peroxidase-labeled goat anti-mouse secondary antibody (Ab179001), horseradish peroxidase-labeled goat anti-mouse secondary antibody (Ab156255), and horseradish peroxidase-labeled rabbit anti-mouse secondary antibody (Ab176437). The 3 detection antibodies were diluted at 1:5000, 1:10000, and 1:20000 respectively, and 100 μL per well was added to the ELISA plate and incubated at 37 °C for 0.5 h. After incubation, the ELISA plate was washed 3 times with 1×PBST. Chromogenic solution was added, and after reacting in the dark at room temperature for 8 minutes, the stop solution was added, and the OD value was read using an ELISA reader.
[0035] The results of the best antibody pair are shown in Table 1. As the concentrations of the antigen and the detection antibody gradually decreased, the Ab138058-Ab179001 antibody pair showed better performance. Therefore, this antibody pair was selected as the best antibody pair for this experiment.
[0036] Table 1 Screening results of the best antibody pair
[0037]
[0038] Example 2: Optimization of the working concentrations of the detection antibody and the capture antibody
[0039] The capture antibody, Goat Anti-Mouse IgG H&L Antibody, was diluted to three concentrations of 0.1 μg / mL, 0.5 μg / mL, and 1 μg / mL respectively with 0.05 mol / L carbonate buffer (pH 9.6), and coated on the ELISA plate at 37 °C for 1 h. After coating, the ELISA plate was washed three times with 1×PBST, and 2% BSA solution was added to block the excess sites on the ELISA plate at 37 °C for 1 h. After blocking, the ELISA plate was washed three times with 1×PBST. Mouse IgG was diluted to 100 ng / mL, 10 ng / mL, and 0 ng / mL respectively with 1×PBS and added to the plate, and incubated at 37 °C for 1 h. After incubation, the ELISA plate was washed three times with 1×PBST. The detection antibody was diluted at 1:5000, 1:10000, and 1:20000 respectively, and 100 μL of each dilution was added to the ELISA plate and incubated at 37 °C for 0.5 h. After incubation, the ELISA plate was washed three times with 1×PBST. Chromogenic solution was added, and after reacting for 8 minutes in the dark at room temperature, the stop solution was added, and the OD value was read using an ELISA reader.
[0040] The results are shown in Table 2. To save antibodies and achieve better detection effects, 0.5 μg / mL was selected as the optimal coating concentration of the capture antibody, and 1:10000 was selected as the optimal dilution factor of the detection antibody.
[0041] Table 2 Optimization of the working concentrations of the detection antibody and the capture antibody
[0042]
[0043]
[0044] Example 3 Establishment of the standard curve
[0045] This example provides a method for ELISA detection and application of mouse IgG antibody, including an ELISA plate coated with 0.5 μg / mL goat anti-mouse IgG, a goat anti-mouse secondary antibody labeled with horseradish peroxidase, a TMB chromogenic solution, a sample diluent, a stop solution, a positive control, a negative control, and a standard product. The positive control is purified mouse immunoglobulin, the negative control is phosphate buffer at pH 7.4, the sample diluent is 1×PBS buffer, the stop solution is 2 mol / L sulfuric acid solution, and the standard product is purified mouse immunoglobulin.
[0046] The preparation method of the enzyme-linked immunosorbent assay (ELISA) plate is as follows: Take goat anti-mouse IgG at 1.0 mg / mL and dilute it to 0.5 μg / mL with the coating solution to coat the ELISA plate, 100 μL per well. The ELISA plate is coated at 37 °C for 1 hour, washed 5 times with PBS buffer containing 0.05% Tween-20, patted dry, and then 200 μL of PBS buffer containing 2% BSA is added for blocking. After blocking at 37 °C for 1 hour, the ELISA plate is washed.
[0047] Using the purified mouse immunoglobulin as a reference product to prepare the standard curve: Dilute the reference product from the initial concentration of 0.25 μg / mL in the first well with the antibody diluent according to a 2-fold gradient into 10 concentrations. Add the serially diluted mouse immunoglobulin to the ELISA plate, 100 μL per well, and incubate at 37 °C for 1 h. After washing the ELISA plate, add the goat anti-mouse horseradish peroxidase labeled at 1:10000 dilution as the secondary antibody to the ELISA plate, 100 μL per well, and incubate for 0.5 h. After washing the plate, add 100 μL of TMB chromogenic solution, and after reacting for 8 min, add 50 μL of the termination solution, and immediately measure the A450 / A630 value of each well with an ELISA reader. The results are shown in Table 3 below.
[0048] Table 3 A450 / A630 values of different reference product concentrations
[0049]
[0050] The fitting curve is obtained by non-linear regression of the four-parameter Logistic curve. The results show that the reference product concentration fits well with the A value, and the R 2 value is greater than 0.99, as Figure 2 shown. Curve equation: Y = Bottom + (Top - Bottom) / (1 + 10^((LogEC50 - X)*HillSlope)), where Bottom = 0.02792, Top = 2.472, LogEC50 = 1.176, HillSlope = 1.248, EC50 = 15.00, Span = 2.444, R 2 = 0.9977.
[0051] Example 4 Methodology Verification - Specificity
[0052] The stability of the kit was detected using IgG from different species, including Horse IgG (Ab175968), Dog IgG (Ab175964), Pig IgG (Ab175962), Cat IgG (Ab175966), Bovine IgG (Ab175960), Rat IgG (Ab141774), Goat IgG (Ab170217), Rabbit IgG (Ab170215), Human IgG (Ab170213), Mouse IgG (Ab170221), Chicken IgG (Ab008417), and Sheep IgG (Ab170219). The above-mentioned IgG was diluted at 1:1000 and measured by the ELISA method in Example 3 above. The A450 / A630 value was measured using a microplate reader. The results are as Figure 1 shown. Except for a low level of cross-reactivity with rat IgG of the same subfamily, no obvious cross-reactivity was observed with IgG from other species, indicating good specificity.
[0053] Example 5 - Methodology Verification - Linearity and Detection Range
[0054] Using purified mouse immunoglobulin as a reference standard with an initial concentration of 0.25 μg / mL, it was serially diluted 2-fold into 11 concentration gradients. It was measured by the ELISA method in Example 3 above. The A450 / A630 value of each well was measured using a microplate reader. The fitting curve and the recovery rate of the samples in the curve were obtained by non-linear regression of the four-parameter Logistic curve. The results are as Figure 2 shown. Good fitting was obtained within the detection range of 0.244 - 250 ng / mL, with R 2 = 0.99.
[0055] Example 6 - Methodology Verification - Repeatability
[0056] Repeatability within and between batches: Using purified mouse immunoglobulin as a reference standard, the positive standard was diluted to 10 different concentrations using sample diluent. Six kits of the same batch were used for detection respectively, and the actual concentration and coefficient of variation were calculated by substituting into the standard curve to evaluate the repeatability within the batch of the kit. The results are shown in Table 4. The coefficient of variation within the batch of this kit was between 2.03% and 9.83%. Similarly, 10 samples with different concentrations were detected using kits from 3 batches respectively, and the actual concentration and coefficient of variation were calculated by substituting into the standard curve to evaluate the repeatability between batches of the kit. The results are shown in Table 5. The coefficient of variation between batches of this kit was between 0.95% and 7.97%. The coefficients of variation between batches and within batches of this kit were both within 10%, indicating good repeatability.
[0057] Table 4 Intra-batch Variation Repeatability Results of the Kit
[0058]
[0059]
[0060] Table 5 Inter-batch Variation Repeatability Results of the Kit
[0061]
[0062] Example 7 Methodology Verification - Accuracy
[0063] Using the purified mouse immunoglobulin as a reference product, it was diluted into 4 concentrations to prepare test samples, and measured by the above ELISA method. The average recovery rate of the actually added reference product was calculated. The formula for the sample recovery rate is: Sample recovery rate = (Actual recovery amount / Theoretical recovery amount to be recovered) × 100%. The results are shown in Table 6. The average recovery rates of the actually added reference products in the 4 test samples are all between 80% and 120%.
[0064] Table 6 Accuracy Verification Results
[0065]
[0066] Example 8 Methodology Verification - Stability
[0067] In this example, the coated antibody and the positive control product were respectively subjected to 4-year simulated stability (placed at 37°C for 38 days) and 6 cycles of freeze-thaw tests. At the same time, the coated enzyme-linked immunosorbent assay (ELISA) plate was placed at 4°C for real-time stability testing. The simulated stability and freeze-thaw results of the coated antibody and the positive control product are respectively as Figure 3 and Figure 4 shown. The results show that the stabilities of these two reagents are good. In addition, the 4°C real-time stability results of the ELISA plate are as Figure 5 shown. The results show that the coated ELISA plate can be placed at 4°C for 38 days with good stability.
[0068] The above are only the preferred embodiments of the present invention, and do not impose any formal or substantial limitations on the present invention. It should be noted that for those of ordinary skill in the art, without departing from the premise of the present invention, several improvements and supplements can still be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.
Claims
1. A mouse IgG antibody ELISA detection kit, characterized in that: The kit comprises: an ELISA plate coated with goat anti-mouse IgG, a goat anti-mouse secondary antibody labeled with horseradish peroxidase, a color developing solution, a sample diluent, a stop solution, a positive control, a negative control and a standard substance.
2. The mouse IgG antibody ELISA detection kit according to claim 1, characterized in that: The ELISA plate was coated with 0.5 μg / mL goat anti-mouse IgG, the color developing solution was TMB color developing solution, the sample diluent was a phosphate buffer solution with a pH of 7.4, and the stop solution was a 2 mol / L sulfuric acid solution.
3. The mouse IgG antibody ELISA detection kit according to claim 1, characterized in that: The positive control is the mouse immunoglobulin that has been separated and purified, the negative control is a pH 7.4 phosphate buffer, and the standard is the mouse immunoglobulin that has been separated and purified.
4. The mouse IgG antibody ELISA detection kit according to claim 1, characterized in that: The preparation method of the ELISA plate is as follows: take 1.0 mg / mL of goat anti-mouse IgG, dilute it to 0.5 μg / mL with coating solution and coat it in the ELISA plate, 100 μL per well, after coating at 37°C for 1 hour, wash the plate 5 times with PBS buffer containing 0.05% Tween-20, pat dry and add 200 μL of PBS buffer containing 2% BSA for blocking, and wash the ELISA plate after blocking at 37°C for 1 hour.
5. Use of the kit according to any one of claims 1 to 4 for quantitatively detecting mouse IgG antibody levels.
6. A mouse IgG antibody ELISA detection method, characterized in that: The detection is performed using the kit according to any one of claims 1 to 4.
7. The mouse IgG antibody ELISA detection method according to claim 6, characterized in that: The following steps are involved: The sample to be tested is diluted with sample diluent to within the fitting range, and then added to the ELISA plate for incubation; After washing the ELISA plate, the horseradish peroxidase-labeled goat anti-mouse antibody was diluted and added to the ELISA plate as a secondary antibody for incubation; After washing the plate, TMB colorimetric solution was added, and after reacting for 5 to 10 minutes, the stop solution was added, and the A450 / A630 value of each well was measured using an ELISA reader.
8. The detection method according to claim 7, characterized in that: The sample to be tested is diluted to a fitting range of 0 to 0.25 μg / mL.
9. The detection method according to claim 7, characterized in that: The dilution ratio of the horseradish peroxidase-labeled goat anti-mouse antibody is 1:10000.
10. The detection method according to claim 7, characterized in that: It also includes the preparation of a standard curve: the standard is diluted with a diluent to a concentration gradient between 0 and 0.25 μg / mL, added to an ELISA plate for detection, and a linear fit is performed between the A450 / A630 value obtained by the test and the concentration to obtain a standard curve.