A ready-to-use PD-L1 antibody reagent for immunohistochemistry

Abstract

A ready-to-use PD-L1 antibody reagent for immunohistochemistry is prepared from pH 6.0 citric acid buffer, 5 mg / mL L-arginine, 2.5 to 5 mg / mL L-glutamic acid, and 0.02% ProClin 300. The antibody reagent of the formula can be stably stored for 3 years at 2-8°C. The preferred formula uses a small number of additives, is non-toxic, environmentally friendly, easy to use, suitable for long-term storage without affecting the staining effect. When used for immunohistochemical staining, it can omit the antigen repair operation, greatly saving working time.

Description

Technical Field

[0001] The present invention belongs to the technical field of antibody detection, and specifically relates to a ready-to-use PD-L1 antibody reagent for immunohistochemistry. Background Art

[0002] In order to preserve ready-to-use antibody reagents for a long time, preservatives are often added. Preservatives should be compatible with other components in the antibody solution system and be non-toxic or low-toxic. Commonly used preservatives in immunohistochemistry reagents include sodium azide, thimerosal, antibiotics, etc. Among them, sodium azide is similar to cyanide and is a highly toxic compound with a median lethal dose of 27 mg / kg (rat, oral). In addition to being irritating to the eyes and skin, it can also cause poisoning and death through inhalation, oral administration or skin absorption. Acute poisoning causes dizziness, headache, general weakness, decreased blood pressure, bradycardia and coma; it can explode strongly in case of high fever or severe vibration, so safety should be paid great attention when using it. Thimerosal has neurotoxicity problems because it contains organic mercury, and the safety of its use is also of great concern. The problem with antibiotics is that they are prone to drug resistance and are prone to interact with other components in the system, affecting their respective functions. In addition, the addition of sodium azide also limits the use of secondary antibodies labeled with horseradish peroxide because sodium azide is an inhibitor of horseradish peroxidase. Staff in clinical medical testing laboratories often have to deal with a large number of immunohistochemistry samples. Long-term contact with reagents containing these substances will undoubtedly put the staff in danger. In addition, the disposal of waste solutions may also cause environmental pollution.

[0003] In addition, antibodies are easily adsorbed on the tube wall during long-term storage, resulting in antibody loss. The usual practice is to add protein stabilizers such as BSA, but these stabilizers are not suitable for antibodies used in immunohistochemistry because they will compete with antibodies for binding markers. Therefore, it is necessary to develop a safe, pollution-free, easy-to-use, suitable for long-term storage, and unaffected staining ready-to-use PD-L1 antibody reagent for immunohistochemistry. Summary of the invention

[0004] The purpose of the present invention is to provide a ready-to-use PD-L1 antibody reagent for immunohistochemistry that has a small amount of additives, is non-toxic, environmentally friendly, easy to use, suitable for long-term storage without affecting the staining effect, so as to solve the problems raised in the above-mentioned background technology.

[0005] In order to achieve the above purpose, the present invention screened a series of prescriptions to determine that the prescription consists of citric acid buffer, L-arginine, L-glutamic acid, and ProClin 300. Among them, the pH of the citric acid buffer is 6.0, the concentration of L-arginine is 5 mg / mL, the content of L-glutamic acid is 2.5 to 5 mg / mL, and the content of ProClin 300 is 0.02%. The preferred prescription can be stably stored for 3 years at 2-8°C. When the preferred formula is used to stain lung cancer tissue, the staining results meet the requirements, no background staining is observed, and the antigen repair step is omitted, which greatly saves working time.

[0006] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 These are the staining effect diagrams of the preferred PD-L1 antibody reagent described in Example 6 for staining lung cancer tissue; (A) is a staining effect diagram when an antigen retrieval operation is performed, and (B) is a staining effect diagram when no antigen retrieval operation is performed. DETAILED DESCRIPTION

[0008] The following contents are used to illustrate the present invention in conjunction with the accompanying drawings, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0009] Example 1: Buffer Screening

[0010] The thermal stability (unfolding) of the PD-L1 antibody was determined by the DSC temperature curve (10-110°C). The so-called thermal unfolding is a measure for comparing various buffer systems: an increase in the Tm1 value of an antibody indicates an increase in the thermal stability of the antibody, i.e., the stability of the antibody in the corresponding buffer system is good.

[0011] Prepare PD-L1 antibody reagent at a concentration of approximately 10 mg / mL in the following buffer system without adding other components.

[0012] Table 1: Tm1 of PD-L1 antibody (10 mg / mL) in various buffer systems

[0013]

[0014]

[0015] Results: The unfolding temperature of PD-L1 antibody in various buffer systems ranged from 55.8°C to 79.5°C. From the table above, it can be seen that the thermal stability of PD-L1 antibody in 50mM sodium citrate (pH 5.0-6.5), 50mM L-histidine (pH 6.5-7.5), 50mM L-arginine (pH 6.0-7.5), and 50mM L-glycine (pH 7.0-7.5) is better than that in other buffer systems. Based on the fact that pH 6.0 citric acid buffer also has antigen retrieval function, pH 6.0 citric acid buffer is preferred for further study.

[0016] Example 2: Stirring stress test

[0017] Antibody reagents may be subjected to severe vibration during transportation, which may potentially cause protein aggregation and the formation of oligomers until visible particles. The stability of the PD-L1 antibody preparation was tested by a stirring stress test (300 rpm and 24 hours at room temperature). The oligomer content was detected by size exclusion chromatography (SEC), and the presence of visible particles was visually assessed. As a result, floccules were visible during stirring in the pH 6.0 citric acid buffer, and the oligomer content was measured to be >8%. Since L-arginine has the effect of inhibiting antibody aggregation, L-arginine was added to the buffer system. The results are shown in the following table. When 10 mg / mL L-arginine was added to the buffer system, the oligomer content was 2.3%, no particles were seen, but there was background staining; when 5 mg / mL L-arginine was added, the staining effect was qualified, no particles were seen, but 1.5% of oligomers were detected. Therefore, L-glutamic acid was continuously added to the buffer system to observe whether the oligomer content changed. The results showed that the best effect was achieved when 2.5 to 5 mg / mL L-glutamic acid was added. The oligomer content was controlled at 0.9% to 1.0%, no particles were observed, and the staining effect was also satisfactory.

[0018] Oligomer content Particles Dyeing effect 0.5mg / mL L-arginine 5.1％ visible Not evaluated 1mg / mL L-arginine 3.7％ Trace Not evaluated 5mg / mL L-arginine 1.5％ Not seen qualified 10mg / mL L-arginine 2.3％ Not seen Background 5mg / mL L-arginine + 5mg / mL L-glutamic acid 1.0％ Not seen qualified 5mg / mL L-arginine + 2.5mg / mL L-glutamic acid 0.9％ Not seen qualified 5mg / mL L-arginine + 7.5mg / mL L-glutamic acid 2.6％ Trace Background

[0019] Example 3: Accelerated stability test

[0020] 10mg / mL PD-L1 antibody reagent (pH 6.0 citric acid buffer, 5mg / mL L-arginine, 2.5mg / mL L-glutamic acid) was placed at 37°C for 0 days, 3 days, 1 week, 2 weeks, and 3 weeks, and the purity and oligomer content of PD-L1 antibody were measured. We found that the purity of PD-L1 antibody gradually decreased over time, but the oligomers did not increase, indicating that the antibody may be degraded by microorganisms during accelerated storage. So we added 0.02%, 0.03%, and 0.05% ProClin 300 to it, and placed it at 37°C again for 0 days, 3 days, 1 week, 2 weeks, and 3 weeks. As a result, the purity of PD-L1 antibody did not decrease over time, and the oligomers did not increase. Therefore, we prefer 0.02% ProClin 300 as a preservative.

[0021] Example 4: Long-term stability experiment

[0022] 10mg / mL PD-L1 antibody (pH 6.0 citrate buffer, 5mg / mL L-arginine, 2.5mg / mL L-glutamic acid, 0.02% ProClin 300) was placed at 2-8°C for 0 days, 6 months, 12 months, 24 months and 36 months. Samples were taken out at each time point to observe whether the solution was clear, whether oligomers appeared, and the purity of the PD-L1 antibody. The results showed that the PD-L1 antibody solution of the above formula can be stably stored for 36 months.

[0023] Example 5

[0024] PD-L1 antibody solutions with concentrations of approximately 1 mg / mL and 5 mg / mL were prepared in a solution system of pH 6.0 citric acid buffer, 5 mg / mL L-arginine, 2.5 mg / mL L-glutamic acid, and 0.02% ProClin300, and placed at 2-8°C for 0 days, 6 months, 12 months, 24 months, and 36 months. Samples were taken out at each time point to observe whether the solution was clear, whether oligomers appeared, and the purity of the PD-L1 antibody. The results showed that the PD-L1 antibody solution with the above formulation could be stably stored for 36 months.

[0025] Embodiment 6:

[0026] The lung cancer tissue was stained using the preferred formula of the PD-L1 antibody reagent (pH 6.0 citrate buffer, 5 mg / mL L-arginine, 2.5 mg / mL L-glutamic acid, 0.02% ProClin 300). The experiment was divided into two groups, one group underwent antigen retrieval and the other group did not. The experimental steps are as follows:

[0027] 1. Dewaxing and hydration

[0028] The sections were sequentially immersed in xylene I (15 min), xylene II (15 min), and then immersed in anhydrous ethanol I (5 min) - anhydrous ethanol II (5 min) - 95% alcohol (5 min) - 80% alcohol (5 min) - 70% alcohol (5 min), and then washed with PBS for 3 times, 5 min each time.

[0029] 2. Antigen retrieval

[0030] Preheat 1x antigen retrieval solution (pH 6.0 citrate buffer) to 65°C, place tissue sections in the antigen retrieval solution, and make sure the liquid level covers the tissue. Incubate at 97°C for 20 minutes. After the incubation is complete and the temperature cools to 65°C, remove the sections and immediately wash them in PBS at room temperature for 3 times, 5 minutes each time.

[0031] 3. Inactivation

[0032] Draw a circle around the tissue with an immunohistochemistry pen, add 3% catalase blocking solution to the sliced ​​tissue to block endogenous peroxidase, incubate at room temperature for 15 minutes, and rinse with PBS three times, 5 minutes each time.

[0033] 4. Blocking and Antibody Incubation

[0034] Dry PBS with absorbent paper, add normal serum (serum from the species of the secondary antibody) on the slide, block at 37°C for 30 minutes, and wash in PBS three times, 5 minutes each time. Wipe the liquid around the slide tissue with absorbent paper, then add PD-L1 antibody reagent, and add PBS to the tissue of the control group. Then incubate in a 4°C wet box overnight. The next day, rinse the sections with PBS three times, 5 minutes each time, wipe the sections with absorbent paper, add enzyme-labeled secondary antibody, and incubate at 37°C for 30 minutes.

[0035] 5. Color rendering

[0036] Rinse the slices with PBS twice, 5 minutes each time, shake off the PBS liquid and wipe the slices dry with absorbent paper, add freshly prepared DAB color developing solution to each slice, observe under a microscope, and the positive signal is brown. Rinse the slices with PBS to stop color development.

[0037] 6. Re-staining

[0038] Re-stain with hematoxylin for about 1 minute, wash with water, differentiate with 1% hydrochloric acid alcohol, and then rinse with tap water to return to blue.

[0039] 7. Dehydration, fixation and sealing

[0040] After rinsing the slices in water for 3 minutes, place the slices in the following order: 70% alcohol-80% alcohol-90% alcohol-95% alcohol-absolute ethanol I-absolute ethanol II-xylene I-xylene II for dehydration and transparency, place in each reagent for 2 minutes, and finally air-dry the slices in a fume hood. Drop neutral gum next to the tissue and cover it with a cover glass. Lay one side flat first, then gently put down the other side to avoid bubbles. The sealed slices are placed flat in a fume hood to dry.

[0041] 8. Read the film

[0042] The dried sections can be observed or imaged under a microscope.

[0043] The results are as follows Figure 1 As shown, the staining results of the preferred formulation meet the requirements, and no background staining is observed. At the same time, the preferred formulation can also omit the antigen retrieval step.

[0044] In summary, the PD-L1 antibody reagent of the present invention can be stably stored for 3 years, and the amount of additives used is small, non-toxic, and environmentally friendly, while not affecting the staining effect of the PD-L1 antibody reagent.

Claims

1. An immunohistochemistry ready-to-use PD-L1 antibody reagent, characterized in that: The PD-L1 antibody reagent is prepared from PD-L1 antibody, citric acid buffer, L-arginine, L-glutamic acid, and ProClin 300. The pH of the citric acid buffer is 6.0, the concentration of the L-arginine is 5 mg / mL, the concentration of the L-glutamic acid is 2.5-5 mg / mL, and the content of ProClin 300 is 0.02%.

2. Use of the immunohistochemical ready-to-use PD-L1 antibody reagent as claimed in claim 1 in immunohistochemical staining of lung cancer tissue.

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