An antibody diluent and uses thereof

Abstract

The application discloses an antibody diluent and application thereof, and belongs to the technical field of biological detection reagents. The antibody diluent is a buffer liquid mainly comprising a stabilizer with a mass fraction of 4+ / -0.05%, a surfactant with a volume fraction of 0.05+ / -0.01% and a preservative. The preservative is composed of a broad-spectrum bacteriostatic agent and an antifungal agent, the broad-spectrum bacteriostatic agent is proclin-300 or proclin-950, and the antifungal agent is amphotericin B. The volume-mass ratio of the broad-spectrum bacteriostatic agent and the antifungal agent is 1mL:2-3mg, and the addition amount of the antifungal agent in 1L of the antibody diluent is 2-3mg. The experiment proves that the antibody diluent can increase the sensitivity of specific antigen-antibody combination, improve the antibody dilution ratio, reduce the amount of the antibody, effectively reduce the attenuation speed of the antibody titer and prolong the shelf life of the product.

Description

Technical Field

[0001] This invention relates to an antibody diluent and its application, belonging to the field of biological detection reagent technology. Background Technology

[0002] Antibodies are immunoglobulins produced by plasma cells, which differentiate from B lymphocytes, in response to antigen stimulation. These antibodies specifically bind to the corresponding antigens. Because antibodies can specifically and stably bind to specific antigens, they are widely used.

[0003] Immunological detection methods are a series of experimental methods designed based on immunological theory to measure antigens, antibodies, immune cells, and their secreted cytokines. With the interdisciplinary integration of science and technology, the scope of immunology continues to expand, and new immunological detection methods (such as immunoadsorption, immunofluorescence, immunohistochemistry, and immunoprecipitation) are constantly emerging. The application of immunological methods is also expanding, becoming not only an important method for the diagnosis of various clinical diseases but also facilitating research in numerous disciplines.

[0004] The core of immunological detection methods lies in the specific recognition and binding of antigens and antibodies. While the most crucial factor in antigen-antibody binding is the specificity of the antibody, factors such as the matching concentrations of the antigen and antibody, the reaction temperature and time, the electrolyte content of the solution, and the pH value all affect the binding. These factors necessitate the adjustment and balancing of the antibody dilution buffer.

[0005] The primary functions of antibody diluents are threefold: first, as a buffer system, providing a stable working environment for the antibody (ion concentration, pH, etc.); second, as a protective system, maintaining antibody stability over extended periods and preventing titer reduction at low concentrations; and third, as a blocking system, helping to improve antibody specificity and reduce non-specific binding. Currently, PBS or PBST are used as antibody diluents, leveraging their buffering effect to mitigate the impact of acid-base changes on experimental results. However, this method suffers from low detection intensity, poor antibody storage stability, and low detection sensitivity. While adding sodium azide as a preservative can enhance antibody storage stability, sodium azide is highly toxic and explosive, posing risks to safe experiments and management. Therefore, there is an urgent need for an antibody diluent with high storage stability and high detection intensity.

[0006] Chinese invention patent application CN113717282A, published on November 30, 2021, discloses an antibody diluent. Specifically, the diluent is composed of the following components: a buffer component selected from at least three of disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium chloride, potassium chloride, Tris, and EDTA; a protein protection component of bovine serum albumin or polyethylene glycol; a wetting component of one or more of sucrose, trehalose, and sorbitol; a freeze-proof component of glycerol; a surfactant component of one or more of Tween-20, Tween 80, and Triton X-100; and a preservative component of one or two of Proclin-300 and sodium thimerosal. This antibody diluent can maintain the stability of various antibody molecules, maximize the preservation of antibody biological activity, extend the shelf life of antibody molecules, and reduce non-specific binding of antibodies during use. However, its shelf life for antibodies still needs to be extended. Summary of the Invention

[0007] The first objective of this invention is to provide an antibody diluent that can effectively reduce the rate of antibody titer decay and extend the shelf life of the product.

[0008] The second objective of this invention is to provide the application of the above-mentioned antibody diluent in the preparation of immunological detection reagents, and to provide an immunological detection reagent that can effectively improve detection intensity, sensitivity and stability.

[0009] To achieve the above objectives, the technical solution adopted by the antibody diluent in this invention is as follows:

[0010] An antibody diluent, comprising a buffer solution mainly containing a stabilizer at a mass fraction of 4 ± 0.05%, a surfactant at a volume fraction of 0.05 ± 0.01%, and a preservative; the preservative consists of a broad-spectrum antibacterial agent and an antifungal agent, wherein the broad-spectrum antibacterial agent is proclin-300 or proclin-950, and the antifungal agent is amphotericin B; the volume-to-mass ratio of the broad-spectrum antibacterial agent to the antifungal agent is 1 mL: 2-3 mg, and the amount of the antifungal agent added per 1 L of antibody diluent is 2-3 mg.

[0011] The beneficial effects of the above technical solution are as follows: The antibody diluent of this invention is a pioneering invention. Through extensive experiments, this invention screened components such as stabilizers and preservatives in the antibody diluent, obtaining a formulation for the antibody diluent. Furthermore, experiments have demonstrated that the antibody diluent can increase the sensitivity of antigen-antibody specific binding, improve the antibody dilution ratio, and reduce the amount of antibody used; moreover, it can effectively reduce the rate of antibody titer decay and extend the shelf life of the product.

[0012] As a further improvement, the buffer solution is PBS buffer or Tris-HCl buffer.

[0013] The beneficial effects of the above technical solution are as follows: the buffer solution can provide a stable working environment for the antibody and has good compatibility with the stabilizers, surfactants, and preservatives in the antibody buffer solution. Meanwhile, PBS buffer or Tris-HCl buffer are commonly used buffer solutions in this field, with simple components and easy preparation.

[0014] As a further improvement, the buffer solution is Tris-HCl buffer; the pH of the antibody diluent is 7.20±0.1.

[0015] The beneficial effects of the above technical solution are: the Tris-HCl buffer can maintain pH stability over a long period of time, providing a stable environment for the antibody and further reducing the impact of the external environment on the antibody titer.

[0016] As a further improvement, the stabilizer is bovine serum albumin.

[0017] As a further improvement, the surfactant is one or more of Tween 20, Tween 80, and Triton X-100.

[0018] As a further improvement, the broad-spectrum antibacterial agent is proclin-300.

[0019] As a further improvement, each 1L of the antibody diluent consists of the following components: 2.5g Tris-HCl, 9g sodium chloride, 0.5mL Tween 20, 40g bovine serum albumin, 1mL proclin-300, 2.5mg amphotericin B, and the remainder is water.

[0020] The beneficial effects of the above technical solution are as follows: using the above formula to prepare antibody diluent can maintain the activity and stability of the antibody, reduce non-specific binding, reduce non-specific background staining, and improve the specificity and sensitivity of the experiment.

[0021] To achieve the above objectives, the technical solution adopted in this invention for the application of an antibody diluent in the preparation of immunological detection reagents is as follows:

[0022] Application of an antibody diluent in the preparation of immunological detection reagents.

[0023] The beneficial effects of the above technical solution are as follows: Experiments have shown that after the antibody is diluted with the above antibody diluent, even after being stored at 37°C for 35 days, there is no serious weakening of antibody titer, and it can still maintain high sensitivity in IHC, ELISA and WB detection.

[0024] As a further improvement, the immunological assays include immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Western blot.

[0025] As a further improvement, the application includes incubating the analyte after diluting the antibody with an antibody diluent. Attached Figure Description

[0026] Figure 1 The changes in OD450 of the antibody diluted with buffer 5 in Experimental Example 2 of the present invention at day 0 and day 14 (where day 0 and day 14 are represented as DAY0 and DAY14, respectively).

[0027] Figure 2 The OD450 changes of the antibody diluted with buffer 6 in Experimental Example 2 of the present invention at day 0 and day 14 (where day 0 and day 14 are represented as DAY0 and DAY14, respectively).

[0028] Figure 3 The changes in OD450 of the antibody diluted with buffer 7 in Experimental Example 2 of the present invention at day 0 and day 14 (where day 0 and day 14 are represented as DAY0 and DAY14, respectively).

[0029] Figure 4 The changes in OD450 of the antibody diluted with buffer 8 in Experimental Example 2 of the present invention at day 0 and day 14 (where day 0 and day 14 are represented as DAY0 and DAY14, respectively).

[0030] Figure 5 This is a 200× image showing the results of immunohistochemistry performed on colorectal cancer tissue immediately after the antibody was diluted with the antibody diluent from Example 1 in Experiment 4 of the present invention.

[0031] Figure 6 This is a 200× image showing the results of immunohistochemistry performed on colorectal cancer tissue immediately after the antibody was diluted with the control antibody diluent in Experiment Example 4 of the present invention.

[0032] Figure 7 The Western blot (WB) results for antibody diluted with the antibody diluent and control in Example 4 of this invention are shown below (where the lanes from left to right are Protein Marker, negative control, Example 1, and control, respectively).

[0033] Figure 8 This is a 200× image showing the results of immunohistochemistry performed on appendix tissue immediately after diluting the antibody with the antibody diluent from Example 1 in Experiment 4 of the present invention.

[0034] Figure 9This is a 200× image showing the results of immunohistochemistry performed on appendix tissue 14 days after the antibody was diluted with the antibody diluent from Example 1 in Experiment 4 of the present invention.

[0035] Figure 10 This is a 200× image showing the results of immunohistochemistry performed on appendix tissue 21 days after the antibody was diluted with the antibody diluent from Example 1 in Experiment 4 of the present invention.

[0036] Figure 11 This is a 200× image showing the results of immunohistochemistry of the appendix tissue 35 days after the antibody was diluted with the antibody diluent from Example 1 in Experiment 4 of the present invention.

[0037] Figure 12 The results of thermostability verification using direct ELISA at 7, 14, 21, and 35 days after the antibody was diluted with the antibody diluent from Example 1 in Experiment 4 of this invention are shown in the figure (OD450 value changes, where 7, 14, 21, and 35 days are represented as DAY7, DAY14, DAY21, and DAY35, respectively).

[0038] Figure 13 The results of thermal stability verification using WB at 7, 14, 21 and 35 days after the antibody was diluted with the antibody diluent of Example 1 in Experiment 4 of the present invention are shown in the figure (the changes in the grayscale analysis of the target protein are shown, where 7 days, 14 days, 21 days and 35 days are represented as DAY7, DAY14, DAY21 and DAY35, respectively). Detailed Implementation

[0039] Primary and secondary antibodies are involved in various immunological experiments, such as immunohistochemistry (IHC), Western blotting (WB), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), and immunocytochemistry (ICC). The primary antibody, commonly known as the primary antibody, specifically binds to an antigen; the secondary antibody, also known as the antibody against an antibody, binds to another antibody and is primarily used to detect the presence of that antibody. The primary antibody targets the antigen, and the secondary antibody targets the primary antibody; the principle of specific antigen-antibody binding is used to specifically detect the presence of the relevant antigen.

[0040] Antibodies used in immunological assays are stored in high-concentration stock solutions or lyophilized powders. After purchase, they are diluted to the working concentration using antibody diluents according to different testing needs before detection. Although antibodies can generally be stored at -20°C or below for a year or even longer, in practical applications, commercially available antibodies are typically stored at 4°C to avoid repeated freeze-thaw cycles. Therefore, to prevent a rapid decrease in antibody titer at 4°C or even room temperature, the selection of antibody diluents is crucial. Furthermore, in addition to slowing down the decline in antibody titer, the antibody diluent must not introduce impurities that could cause non-specific binding of the antigen and antibody.

[0041] Based on this, the present invention provides an antibody diluent and its application. Through extensive experiments, the inventors of this invention screened components such as buffers, stabilizers, and preservatives in the antibody diluent, obtaining a formulation for the antibody diluent. Furthermore, the present invention demonstrates through experiments that the antibody diluent can increase the sensitivity of antigen-antibody specific binding, improve the antibody dilution ratio, and reduce the amount of antibody used; moreover, it can effectively reduce the rate of antibody titer decay and extend the product's shelf life.

[0042] The antibody diluent provided by this invention has a wide range of applications and is suitable for antibody dilution in IHC, WB, and ELISA detection.

[0043] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0044] I. Specific embodiments of the antibody diluent of the present invention:

[0045] Example 1

[0046] The antibody diluent in this embodiment is a buffer solution mainly containing stabilizers, surfactants and preservatives, and the specific formulation is shown in Table 1.

[0047] Table 1

[0048]

[0049] The preparation process for the antibody dilution buffer is as follows:

[0050] First, dissolve the required amounts of Tris-HCl, sodium chloride, and Amphotericin B in an appropriate amount of purified water, stirring until completely dissolved. Adjust the pH to 7.20±0.1, then add BSA. Next, measure the required amounts of Tween 20 and Proclin-300 and add them to the above solution. Add purified water to the prepared volume, maintaining a stirring speed of 150-250 rpm / min to avoid foaming. The total stirring time during the preparation process should be no less than 2 hours.

[0051] II. Application of Antibody Diluent in the Preparation of Immunological Detection Reagents (Experimental Example)

[0052] This invention screens components such as buffers, stabilizers, and preservatives in antibody diluents to obtain the final formulation of the antibody diluent. The specific process is as follows:

[0053] Experimental Example 1: Buffer Screening

[0054] This experimental example screens the buffer solutions in the antibody dilution solution. The specific process is as follows:

[0055] Commonly used antibody diluents mainly consist of PBST and TBST. Prepare buffer solutions 1-4 according to the formulas in Tables 2-3, incubate at 37°C for 14 days, measure the pH changes, and select the group with the most stable pH, i.e., buffer solution 4.

[0056] Table 2

[0057]

[0058] Table 3

[0059]

[0060] Specific results: After preparation, the pH of buffer solutions 1-4 was adjusted to a uniform value of 7.20±0.05. After being placed at 37℃ for 14 days, the pH changes of buffer solutions 1-4 were detected, as shown in Table 4.

[0061] Table 4. pH changes of buffer solution 1-4

[0062]

[0063] Experimental Example 2: Screening of Stabilizers

[0064] This experimental example screens stabilizers in antibody dilution solutions. The specific process is as follows:

[0065] Commonly used protein stabilizers include BSA and skim milk. Based on the above buffer 4, different protein stabilizers are added (formulations are shown in Table 5) to prepare buffers 5-8. The antibodies are diluted with the same dilution ratio, placed at 37°C for 14 days, and their titer changes are measured using ELISA. The group with the most stable titer is selected.

[0066] Table 5

[0067]

[0068] The specific procedure for measuring changes in titer using ELISA is as follows:

[0069] Potency was determined using indirect ELISA.

[0070] Experimental materials: The antigen was ALK protein (coating amount fixed at 200 ng / well), the primary antibody was rabbit serum immunized with the above protein (diluted 1:1000 with PBS, incubated at 37℃ for 60 min), and the secondary antibody was HRP-labeled goat anti-rabbit secondary antibody (original concentration 800 μg / ml, serially diluted from 1:10000 to 1:128W using buffer 5-8, incubated at 37℃ for 30 min, with 3 replicates for each buffer). Then, the ELISA colorimetric solution was used for color development for 5 min, and the OD450 was measured using a microplate reader. The average value was plotted for comparison.

[0071] Experimental results are as follows Figures 1-4 As shown, the OD450 readings all decreased to some extent after 14 days, but the difference was the smallest in buffer 5. Therefore, buffer 5 was selected for the next experiment.

[0072] Experiment Example 3: Screening of Preservatives

[0073] This experimental example screens preservatives in antibody dilution solutions. The specific process is as follows:

[0074] Commonly used preservatives in immunoassay reagents include proclin-300 and proclin-950, but their antifungal effects are weak. Therefore, the antifungal agent "Amphotericin B" was tested. Buffer 9-12 was prepared according to the formula in Table 6 (based on buffer 5), and then the solution was placed open in a 37°C incubator to observe antibody turbidity.

[0075] Table 6

[0076]

[0077] Buffers 11 and 12 showed flocculated precipitation and off-odors by day 3, and mold appeared on the surface by day 7. Buffers 9 and 10 only showed significant flocculated precipitation and mold by day 14. This indicates that the buffers with added Amphotericin B had better overall antibacterial effects than those without. No significant difference was observed between proclin-300 and proclin-950 in this round of experiments. Therefore, considering cost and supply stability, Buffer 9 was ultimately selected as the optimal formulation.

[0078] Example 4: Application of antibody dilution buffer in the preparation of immunological detection reagents

[0079] This experimental example verifies the sensitivity and stability of the antibody dilution solution from Example 1 (i.e., the formulation of buffer 9 in Experiment 3 above). The specific procedures are as follows:

[0080] 1. Sensitivity verification:

[0081] Prepare a batch of antibody diluent according to the final Buffer 9 formulation. Prepare another batch of antibody at the same working concentration as the control (commercially available secondary antibody diluent). Verify the results using IHC, ELISA, and WB.

[0082] (1) The IHC experimental procedure is as follows:

[0083] Paraffin tissue sections were dewaxed and hydrated; antigens were retrieved under high pressure and washed; endogenous peroxidase blocking agent was added, washed after 5 min; primary antibody working solution was added, washed after 60 min; HRP-labeled secondary antibody was added, washed after 20 min; DAB chromogenic solution was added, washed after 5 min; hematoxylin was added, washed after 5 min; dehydrated, cleared, and mounted; and examined under a microscope.

[0084] Except for the secondary antibody, all other experimental materials and conditions remained completely consistent. To highlight the differences in the secondary antibody dilution solution, the secondary antibody was diluted at the recommended maximum dilution ratio of 1:5000. The results are as follows: Figure 5 and Figure 6 As shown, Figure 5 This is a staining result image of the secondary antibody working solution obtained after diluting the antibody diluent of Example 1. Figure 6 This serves as a control. In terms of staining intensity, the antibody dilution in this invention exhibits a staining intensity 10% higher than the control in the IHC experiment, and the staining background is very clean.

[0085] (2) The ELISA experimental steps are as follows:

[0086] Prepare antigen-coated plates; incubate with primary antibody at 37°C for 60 min; incubate with secondary antibody at 37°C for 30 min; develop color for 5 min; terminate the process; read the OD450 value using an ELISA reader; analyze the data.

[0087] The antigen and primary antibody were the CD117 protein antigen (200 ng / well) used in the actual experiment for immunization, and the corresponding supernatant from rabbit spleen B cells cultured in a 96-well plate after immunization. The secondary antibody was diluted at the recommended maximum dilution ratio of 1:50000 using the antibody dilution buffer and control from Example 1. The average OD of a 96-well ELISA plate was compared. 450 The values ​​of the positive control (rabbit serum) and the negative control (corresponding secondary antibody dilutions, namely buffer 9 and control dilution) are shown in Table 7. The data show that the negative values ​​of both antibodies are well controlled, but the positive value of buffer 9 and the average value of the whole plate are higher than those of the control.

[0088] Table 7

[0089]

[0090] (3) The WB detection steps are as follows (using E. coli internal reference protein GAPDH as the detection index):

[0091] E. coli were lysed using RIPA lysis buffer; the primary antibody against GAPDH (1:200,000 dilution) was incubated at room temperature for 2 hours; the secondary antibody (diluted at the recommended maximum ratio of 1:50,000 for easy use with buffer 9 and the control) was incubated at room temperature for 1 hour; ECL staining was then performed. The results of staining with an exposure time of 1 second are shown below. Figure 7 As shown, the antibody diluent provided by this invention also outperforms the control in the WB experiment.

[0092] 2. Stability verification:

[0093] The diluted antibody from step 1 was subjected to a thermally accelerated experiment at 37°C. IHC, ELISA, and WB assays were performed on days 7, 14, 21, and 35 to detect the decrease in titer.

[0094] IHC results as follows Figures 8-11 As shown, there is a significant decrease in staining intensity only at 21 days, while there is still a relatively good staining effect at 35 days, indicating that the stability of the diluent is qualified.

[0095] The stability validation protocol for the ELISA experiment also used the indirect ELISA method, with all other conditions being exactly the same, except that the secondary antibody had different thermal stability days. The results are as follows: Figure 12 As shown in the figure. Experimental results demonstrate that the antibody exhibits excellent thermal stability over 35 days under the storage conditions of this dilution.

[0096] The results of the WB thermal stability test are displayed in grayscale levels, as follows: Figure 13 As shown, the changes over 7 days (represented by DAY7 in the figure) and 14 days (represented by DAY14 in the figure) are not significant. Although there is a relatively obvious decrease at 35 days (represented by DAY35 in the figure), the target protein can still be detected, which meets the detection requirements.

[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody diluent, characterized in that: The antibody diluent is a buffer solution mainly containing 4 ± 0.05% stabilizer, 0.05 ± 0.01% surfactant, and preservative. The preservative consists of a broad-spectrum antibacterial agent and an antifungal agent. The broad-spectrum antibacterial agent is proclin-300 or proclin-950, and the antifungal agent is amphotericin B. The volume-to-mass ratio of the broad-spectrum antibacterial agent to the antifungal agent is 1 mL: 2-3 mg, and the amount of the antifungal agent added per 1 L of antibody diluent is 2-3 mg.

2. The antibody diluent according to claim 1, characterized in that: The buffer solution is either PBS buffer or Tris-HCl buffer.

3. The antibody diluent according to claim 2, characterized in that: The buffer solution is Tris-HCl buffer; the pH of the antibody dilution solution is 7.20±0.

1.

4. The antibody diluent according to claim 2 or 3, characterized in that: The stabilizer is bovine serum albumin.

5. The antibody diluent according to claim 4, characterized in that: The surfactant is one or more of Tween 20, Tween 80, and Triton X-100.

6. The antibody diluent according to claim 5, characterized in that: The broad-spectrum antibacterial agent is proclin-300.

7. The antibody diluent according to claim 6, characterized in that: Each 1L of the antibody diluent consists of the following components: 2.5g Tris-HCl, 9g sodium chloride, 0.5mL Tween 20, 40g bovine serum albumin, 1mL proclin-300, 2.5mg amphotericin B, and the remainder is water.

8. The use of the antibody diluent as described in any one of claims 1 to 7 in the preparation of immunological detection reagents.

9. The application of the antibody diluent according to claim 8 in the preparation of immunological detection reagents, characterized in that: The immunological tests include immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Western blot.

10. The use of the antibody diluent according to claim 8 or 9 in the preparation of immunological detection reagents, characterized in that: The application includes incubating the analyte after diluting the antibody with an antibody diluent.

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