Kit for rapidly detecting peripheral T cell CD3 molecules and detection method thereof

Abstract

The invention belongs to the field of biomedical examination, and particularly provides a kit for rapidly detecting CD3 molecules of peripheral T cells and a detection method of the kit. The kit comprises a specific anti-CD3 monoclonal antibody, a second antibody, alkaline phosphatase labeled streptavidin, a buffer solution, a color developing agent and a nuclear counterstaining agent. High-sensitivity and high-specificity detection of CD3 molecules is realized through recognition of a specific anti-CD3 monoclonal antibody and cell surface CD3 molecules and application of an enzyme-linked immunosorbent assay technology. The specific detection method comprises the steps of sample treatment, capture reaction, detection reaction, color development and result analysis. The kit disclosed by the invention has the advantages of simplicity and convenience in operation, rapidness in detection, high sensitivity, good specificity and the like, is suitable for laboratory and clinical detection, and provides an efficient and reliable detection tool for immunological research, autoimmune disease diagnosis and tumor immunotherapy effect evaluation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical testing, and particularly relates to a kit for rapid detection of peripheral T cell CD3 molecules and a detection method thereof. BACKGROUND

[0002] In the immune system, CD3 molecules are key markers on the surface of T cells, which play a core role in the recognition, activation and regulation of immune responses of T cells. For example, in terms of immune response, CD3 molecules are involved in the recognition and response of T cells to antigens, and are an important basis for the normal functioning of the immune system; in the study of autoimmune diseases, abnormal expression or dysfunction of CD3 molecules is often closely related to the pathogenesis of the disease, and through the study of CD3 molecules, the pathological process of autoimmune diseases can be better understood, and new ideas and targets for diagnosis and treatment of the disease can be provided; in the field of tumor immunotherapy, CD3 molecules also play an important role. In recent years, immunotherapy based on CD3 molecules, such as CAR-T cell therapy, has achieved remarkable therapeutic effects by efficiently activating T cells to attack tumor cells, bringing new hope to tumor patients.

[0003] However, the current methods for detecting CD3 molecules have some limitations. Common detection methods include flow cytometry, immunohistochemistry, immunofluorescence staining, etc. Flow cytometry has the advantages of high sensitivity and high resolution, but its operation process is relatively complex, requires professional technicians to operate, and the detection time is relatively long, usually several hours or even longer. Moreover, the equipment cost of flow cytometry is high, and the reagent price is also high, which makes it difficult to be widely used in some primary medical institutions or laboratories with limited resources. In addition, flow cytometry can only detect single cells, and cannot detect solid tissues. Immunofluorescence staining technology uses expensive fluorescent antibodies, and is prone to fluorescence quenching, so it is not suitable for long-term storage of specimens, and therefore immunohistochemistry technology is more commonly used for pathological diagnosis in clinics. Immunohistochemistry is a commonly used method for detecting tissue sections, which can directly show the distribution of CD3 molecules in tissues. However, the general immunohistochemical detection has low sensitivity; the background around the tissue or cells presents strong color, which covers the specific staining signal, reduces the contrast and clarity of the image, and is not conducive to the observation and judgment of positive signals.

[0004] In view of the above problems existing in the existing detection methods, it is necessary to develop a rapid, accurate and simple CD3 molecule detection kit, which not only has important clinical significance, but also will have a profound impact on basic research and disease prevention and treatment. SUMMARY

[0005] In view of the problems of poor specificity, low sensitivity, and poor difference of color developing background of the existing CD3 molecule detection technology, the purpose of the present application is to provide a kit for rapidly detecting peripheral T cell CD3 molecules and a detection method thereof, which realizes high sensitivity and high specificity detection of CD3 molecules by specific antibody binding CD3 molecules and combining enzyme-linked immunosorbent assay (ELISA), and has the advantages of simple operation, rapid detection, high sensitivity, good specificity, and the like.

[0006] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: The present application provides a kit for detecting CD3 molecules, which comprises: specific anti-CD3 monoclonal antibody, secondary antibody, alkaline phosphatase-labeled streptavidin, buffer, color developing agent, and nuclear counterstain agent; wherein the color developing agent is naphthol AS-BI salt and 4-diazobenzenamine sulfate, and the nuclear counterstain agent is safranin O.

[0007] Further, the secondary antibody is labeled with biotin.

[0008] Further, the buffer is phosphate buffer or Tris buffer, and the pH is 7.0-8.0.

[0009] Further, the color developing agent and the nuclear counterstain agent are prepared on site; wherein the color developing agent is prepared as follows: (1) Under light-proof conditions, naphthol AS-BI salt is dissolved in DMF to prepare A liquid; (2) Under normal temperature and light-proof conditions, 4-diazobenzenamine sulfate is dissolved in pure water to prepare B liquid; (3) 2-amino-2-methyl-1,3-propanediol is dissolved in pure water, and the pH is adjusted to 9.0-10.0 to obtain C liquid; (4) A liquid is added to B liquid and mixed well; C liquid is taken and added to the mixture of A liquid and B liquid, and mixed well to obtain the color developing agent.

[0010] Further, the mass / volume ratio of naphthol AS-BI salt to DMF in step (1) is 0.02-0.05 g / mL; the mass / volume ratio of 4-diazobenzenamine sulfate to pure water in step (2) is 0.01-0.04 g / mL; and the concentration of C liquid in step (3) is 0.1-0.4 g / mL.

[0011] Further, the volume ratio of A liquid, B liquid, and C liquid is 1:1: (10-20).

[0012] The nuclear counterstain agent is prepared as follows: Safranin O is dissolved in 75% ethanol, and then the safranin O ethanol solution is mixed with distilled water to obtain the nuclear counterstain agent.

[0013] Further, the mass-volume ratio of the safranin O and 75% ethanol is 0.01-0.04 g / mL; the volume ratio of the safranin O ethanol solution and distilled water is 1: (5-10).

[0014] The application further provides application of the kit in detection of peripheral T cell CD3 molecules.

[0015] The application further provides a method for detecting peripheral T cell CD3 molecules, comprising the following steps: (1) collecting a sample (blood) to be detected, pretreating the sample, and preparing a specimen slice; (2) sequentially performing specific anti-CD3 monoclonal antibody incubation (capturing CD3 molecules), secondary antibody incubation (binding with the captured CD3 molecules), alkaline phosphatase-labeled streptavidin incubation (binding with the biotin-labeled secondary antibody); (3) adopting a chromogenic agent for staining, and adopting a nuclear counterstain agent for counterstaining; the chromogenic agent is naphthol AS-BI salt and 4-diazobenzenamine sulfate, and the nuclear counterstain agent is safranin O; (4) observing the chromogenic condition of the cells; if the cells present the characteristic performance of "blue membrane red nucleus", it is indicated that the cells are T cells and normally express CD3 molecules; if the cells only present red, it is indicated that the cells do not express CD3 molecules Further, the chromogenic condition of the cells is observed by adopting an optical microscope.

[0016] The application has the following beneficial effects: (1) the application provides a kit for detecting peripheral T cell CD3 molecules, which breaks through the limitation of a traditional detection method, and is rapid, accurate, simple, high-sensitivity and specific, can accurately identify CD3 molecules, and provides an efficient and reliable detection tool for immunological basic research (such as T cell development research), autoimmune disease diagnosis and tumor immunotherapy effect evaluation. (2) the kit is rapid in detection, and the result is accurate: the kit is combined by antibody-antigen specificity and a biotin-streptavidin signal amplification system, and adopts specific staining agents and a nuclear counterstain agent, so that the cell membrane of the specifically stained cells presents blue, and the cell nucleus presents red; the characteristic performance of "blue membrane red nucleus" greatly improves the contrast between the specifically stained cells and non-specifically stained cells and the surrounding background, realizes rapid detection while ensuring high accuracy and sensitivity, and is suitable for large-scale screening. (3) low cost, easy to save: the kit has wide sample compatibility, can accurately detect cell smears, climbing sheets, tissue sections and printed sheets and other sample types, and the reagent components thereof are not only optimized and treated, can maintain stable performance at 4℃ or -20℃ for 6 months, ensure reliable detection results, and have low preparation cost, greatly reducing the detection cost, suitable for large-scale production application; (4) simple operation: the use of the kit has no complex steps, and the test can be completed by conventional operation, the technical requirements of the detection personnel are low, and it is easy to popularize. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 A positive and negative control chart for detecting whether peripheral T cells contain CD3 molecules using the kit prepared in Example 1.

[0018] Figure 2 A positive and negative control chart for detecting whether peripheral T cells contain CD3 molecules using the kit prepared in Comparative Example 1.

[0019] Figure 3 A positive and negative control chart for detecting whether peripheral T cells contain CD3 molecules using the kit prepared in Comparative Example 2.

[0020] Figure 4 A positive and negative control chart for detecting whether peripheral T cells contain CD3 molecules using the kit prepared in Comparative Example 3.

[0021] Figure 5 A positive and negative control chart for detecting whether peripheral T cells contain CD3 molecules using the kit prepared in Comparative Example 4.

[0022] Figure 6 A positive and negative control chart for detecting whether peripheral T cells contain CD3 molecules using the kit prepared in Comparative Example 5. DETAILED DESCRIPTION

[0023] The technical solutions of the present application will be described in detail below in conjunction with examples. Obviously, the described examples are only part of the examples of the present application, not all examples. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor fall within the scope of the present application. Example 1

[0024] This embodiment provides a rapid detection kit for peripheral T cell CD3 molecules, which comprises specific antibodies (primary antibodies), secondary antibodies, alkaline phosphatase-labeled streptavidin, buffer, color developing agent and nuclear counterstain.

[0025] The primary antibody is a specific monoclonal antibody against CD3 molecules, and in this embodiment, the CD3 antibody reagent of Zhongshanjingqiao is selected and can be used directly. The secondary antibody is a biotin-labeled anti-mouse IgG, and in this embodiment, the secondary antibody is purchased from Shengong Bioengineering (Shanghai) Co., Ltd., and is diluted at a ratio of 1:100 when used. The alkaline phosphatase-labeled streptavidin in this embodiment is purchased from Shengong Bioengineering (Shanghai) Co., Ltd., and is diluted at a ratio of 1:500 when used. The buffer is phosphate buffer solution (PBS, pH 7.4).

[0026] Both the chromogenic agent and the nuclear counterstain are prepared on site, and the preparation of the chromogenic agent is as follows: (1) Preparation of A solution: 2 mg of naphthol AS-BI phosphate disodium salt hydrate is dissolved in 50 mL of DMF, and stored in the dark; (2) Preparation of B solution: 2 mg of 4-diazobenzenamine sulfate (room temperature, avoid light) is dissolved in 100 mL of Wahaha water, and stored in the dark; (3) Preparation of C solution: 2-amino-2-methyl-1,3-propanediol 105 mg is dissolved in 5 mL of pure water, and the pH is adjusted to 9.75 with 0.1 mol / L hydrochloric acid or NaOH; (4) Take A solution (20 mL) and add it to B solution (20 mL), and mix well by blowing and sucking several times; take 360 mL of C solution and add it to the above mixture, and mix well.

[0027] The nuclear counterstain is prepared as follows: Dissolve 2.5 g of ponceau O in 100 mL of 75% ethanol, and mix 10 mL of the prepared ponceau O ethanol solution with 80 mL of distilled water to obtain the nuclear counterstain. Example 2

[0028] The present embodiment provides a rapid detection kit for peripheral T cell CD3 molecules, which comprises specific antibodies (primary antibodies), secondary antibodies, alkaline phosphatase-labeled streptavidin, buffer, chromogenic agent, and nuclear counterstain.

[0029] The primary antibody is a specific monoclonal antibody against CD3 molecules, and in this embodiment, the CD3 antibody reagent of Zhongshanjingqiao is selected and can be used directly. The secondary antibody is a biotin-labeled anti-mouse IgG, and in this embodiment, the secondary antibody is purchased from Shengong Bioengineering (Shanghai) Co., Ltd., and is diluted at a ratio of 1:100 when used. Alkaline phosphatase-labeled streptavidin was purchased from Shanghai Yingayuan Bioengineering Co., Ltd. and was diluted at a ratio of 1:500 when used. The buffer was phosphate buffered saline (PBS, pH 7.5).

[0030] Both the chromogenic agent and the nuclear counterstain were prepared immediately before use. The specific preparation of the chromogenic agent was as follows: (1) Preparation of A solution: 1 mg of naphthol AS-BI phosphate disodium salt hydrate was dissolved in 50 mL of DMF, and the solution was stored in the dark; (2) Preparation of B solution: 1 mg of 4-diazobenzenamine sulfate was dissolved in 100 mL of Wahaha water at room temperature and in the dark, and the solution was stored in the dark; (3) Preparation of C solution: 52.5 mg of 2-amino-2-methyl-1,3-propanediol was dissolved in 5 mL of pure water, and the pH was adjusted to 9.8 with 0.1 mol / L hydrochloric acid or NaOH; (4) A solution (20 mL) was added to B solution (20 mL), and the mixture was mixed thoroughly by blowing and sucking several times. 200 mL of C solution was added to the above mixture and mixed thoroughly.

[0031] The nuclear counterstain was prepared as follows: 1.0 g of ponceau O was dissolved in 100 mL of 75% ethanol, and 10 mL of the prepared ponceau O ethanol solution was mixed with 50 mL of distilled water to obtain the nuclear counterstain. Example 3

[0032] The present example provides a rapid detection kit for peripheral T cell CD3 molecules, which comprises specific antibodies (primary antibodies), secondary antibodies, alkaline phosphatase-labeled streptavidin, buffer, chromogenic agent, and nuclear counterstain.

[0033] The primary antibody is a specific monoclonal antibody against CD3 molecules. In the present example, the primary antibody is a CD3 antibody reagent from Zhongshanjingqiao, which can be used directly. The secondary antibody is a biotin-labeled anti-mouse IgG. In the present example, the secondary antibody was purchased from Shanghai Yingayuan Bioengineering Co., Ltd. and was diluted at a ratio of 1:100 when used. Alkaline phosphatase-labeled streptavidin was purchased from Shanghai Yingayuan Bioengineering Co., Ltd. and was diluted at a ratio of 1:500 when used. The buffer was phosphate buffered saline (PBS, pH 7.4).

[0034] Both the chromogenic agent and the nuclear counterstain were prepared immediately before use. The specific preparation of the chromogenic agent was as follows: (1) Preparation of A liquid: 2.5 mg of naphthol AS-BI phosphate disodium salt hydrate was dissolved in 50 mL of DMF, and stored in the dark; (2) Preparation of B liquid: 4 mg of 4-diazoaniline sulfate salt (room temperature, dark) was dissolved in 100 mL of Wahaha water, and stored in the dark; (3) Preparation of C liquid: 2-amino-2-methyl-1, 3-propanediol 210 mg was dissolved in 5 mL of pure water, and the pH was adjusted to 9.6 with 0.1 mol / L hydrochloric acid or NaOH; (4) Take A liquid (20 mL) and add it to B liquid (20 mL), and mix well by blowing and sucking several times; take 400 mL of C liquid and add it to the above mixture, and mix well.

[0035] The nuclear counterstain was prepared as follows: Take 4.0 g of safranin O and dissolve it in 100 mL of 75% ethanol. Take 10 mL of the prepared safranin O ethanol solution and mix it with 100 mL of distilled water to obtain the nuclear counterstain.

[0036] Example 4 Detection of CD3 molecules This example uses the kit prepared in Example 1 to detect CD3 molecules in peripheral T cells, which includes the following steps: 1. Sample preparation Collect the sample to be tested (such as human peripheral blood mononuclear cells) to make a slide, and the specific steps are as follows: (1) Isolation of peripheral blood mononuclear cells (PBMCs): (1-1) Plastic soft test tube marker pen labeling: 1, 2, 3, 4; (1-2) Add heparin sodium: use a syringe to draw out and add to the plastic test tube, 3-4 drops per tube; (1-3) Draw blood: 4 mL of blood per portion, diluted 1-fold with PBS, 4 mL of blood + 4 mL of PBS (PBS can be a little more); each portion of blood is about 4 mL; (1-4) Diluted blood is introduced into the upper layer of the separation medium: the diluted blood is slowly adhered and added to ensure that the diluted blood is in the upper layer of the separation medium, and the layers are clear; 2 mL of separation medium is added to each centrifuge tube; (1-5) Centrifugation: first centrifuge at 2000 rpm for 20 min; then, the cloud-like mononuclear cells are sucked into a new centrifuge tube, and PBS is added to 10 mL, and centrifuged at 1500 rpm for 10 min.

[0037] (2) Slide pretreatment: choose a slide with a anti-falling agent, i.e. an adhesive slide, and mark it with a marker pen.

[0038] (3) Smear: two. After centrifugation, the supernatant was discarded, about 300-500 mL liquid was reserved, mixed with a glass pipette, 30 mL (one drop) of cell liquid was sucked to the circle, and whether there were cells was observed with (x10) objective for 3-5 min. After 5 min, the supernatant was absorbed by filter paper. The liquid was dried by a hair dryer. The sample was placed at room temperature for 2 h, or at 4°C overnight, or at 37°C for 30 min.

[0039] 2. Immunohistochemical experiment (1) Fixation: select a slide, add 50 mL of fixing solution to each membrane, and place the slide at room temperature for 2-3 min. Then, rinse the slide with PBS, and wipe off the PBS solution on the back of the slide and outside the circle; (2) Add primary antibody: add 30 mL of anti-CD3 monoclonal antibody to each membrane, and place the slide in a wet box (containing water) at 4°C overnight; (3) Rinse the slide with PBS for 3 times, add an appropriate amount of biotin-labeled anti-mouse IgG (secondary antibody, 1:100) (30 mL), and incubate at 37°C for 60 min; (4) Rinse the slide with PBS for 3 times, add an appropriate amount of alkaline phosphatase-labeled streptavidin S-A / AP (30 mL), and incubate at 37°C for 60 min; (5) Rinse the slide with PBS for 3 times, add 50-80 mL of color developing agent, and develop color at 37°C for 30 min in the dark; (6) Rinse the slide with PBS for 3 times, add a nuclear counterstain, and counterstain the nucleus at room temperature for 20-30 min.

[0040] 3. Observation of color developing result Place the slide after nuclear counterstaining under an oil immersion lens to observe. If the cells show the characteristic performance of "blue membrane red nucleus", it indicates that the lymphocyte surface expresses CD3 molecules, which is positive. Otherwise, if the whole cell nucleus shows red state, it indicates that the cell does not express CD3 molecules, which is negative. The results of this example are shown in Figure 1 , the cell membrane of positive cells is blue, and the cell nucleus is red. This characteristic performance of "blue membrane red nucleus" has bright color development, and the boundary with negative cells and background is clear, effectively solving the misjudgment and omission problem caused by background interference in the traditional method, and improving the high sensitivity and high specificity.

[0041] The kit prepared in Examples 2 and 3 was used to detect CD3 molecules in peripheral T cells, and the same detection results as Figure 1 were obtained.

[0042] The kit prepared in Example 1 was stored at 4°C or -20°C for 6 months, and the above detection was repeated, and the color development result was unchanged.

[0043] Comparative Example 1 Preparation of the detection kit The reagents in the kit in the present comparative example are the same as those in Example 1, except that the chromogenic reagent and the nuclear counterstain are different.

[0044] Specifically, in the present comparative example, the chromogenic reagent is prepared as follows (freshly prepared): (1) Preparation of A solution: 2 mg of naphthol AS-BI phosphate disodium salt hydrate is dissolved in 50 μL of DMF, and stored in the dark; (2) Preparation of B solution: 40 mg of hexaazotetrazolium formazan (diazonium salt), 0.2 mL of concentrated hydrochloric acid, and 0.8 mL of DMF are uniformly mixed to obtain a stock solution 1; 4% sodium nitrite (dissolved in water) is used as a stock solution 2; the stock solution 1 and the stock solution 2 are mixed in equal volumes under low temperature conditions, and stored in the dark;

[0045] (3) Preparation of C solution: 105 mg of 2-amino-2-methyl-1,3-propanediol is dissolved in 5 mL of pure water, and the pH is adjusted to 9.75 with 0.1 mol / L hydrochloric acid or NaOH;

[0046] (4) A solution (20 μL) is added to B solution (20 μL), and after being mixed well by blowing and sucking several times, 360 μL of buffer C solution is added to the mixture, and the mixture is mixed well.

[0047] The nuclear counterstain is Mayer hematoxylin stain.

[0048] 2. Detection of CD3 molecules The detection steps of CD3 molecules in the present comparative example are the same as those in Example 2.

[0049] 3. Analysis of the chromogenic results The slide after nuclear counterstaining is observed under an oil immersion lens. If the cells show the characteristic performance of "red membrane and blue nucleus", the lymphocyte surface expresses CD3 molecules, which is positive; otherwise, if the entire cell nucleus shows blue, the cell does not express CD3 molecules, which is negative. The results of the present comparative example are shown in Figure 2 It can be seen that the entire cell is red, the blue color is not obvious, the positive signal and the negative signal are mixed, and the contrast is very low, making it difficult to effectively observe.

[0050] Comparative Example 2 The present comparative example provides a kit, and the components in the kit are the same as those in Example 1, except that the configuration ratio of the nuclear counterstain is different from that in Example 1.

[0051] Specifically, in the present comparative example, the nuclear counterstain is prepared as follows (freshly prepared): 1. The nuclear counterstain is prepared as follows: Eosin 1 g was dissolved in 100 mL of water to obtain the nuclear counterstain.

[0052] The results of the present comparative example are shown in Figure 3 As shown, the positive cells showed a characteristic "blue membrane red nucleus" coloration, but the coloration was very dim, and the standard blue and red colors expected in the desired effect were not present. The red coloration of the negative cells was a dark purple color, which was not intuitive and had poor visual effects and low key feature recognition.

[0053] Comparative Example 3 The present comparative example provides a kit, the components of which are the same as in Example 1, except that the configuration ratio of the nuclear counterstain is different from that of Example 1.

[0054] Specifically, in the present comparative example, the nuclear counterstain is configured as follows (freshly prepared for immediate use): 1. The nuclear counterstain is prepared as follows: 2.5 g of eosin O was dissolved in 100 mL of 95% ethanol, and 10 mL of the prepared eosin O ethanol solution was mixed with 80 mL of distilled water to obtain the nuclear counterstain.

[0055] The results of the present comparative example are shown in Figure 4 As shown, the coloration of the positive and negative cells was very bright, but the positive and negative cells were both strongly colored, which caused the key "blue membrane red nucleus" feature of the positive cells to be unclear, and the positive signal was almost submerged, making it impossible to accurately distinguish between positive and negative, and providing no reliable basis for accurate judgment of the results.

[0056] Comparative Example 4 The present comparative example provides a kit, the components of which are the same as in Example 1, except that the configuration ratio of the nuclear counterstain is different from that of Example 1.

[0057] Specifically, in the present comparative example, the nuclear counterstain is configured as follows (freshly prepared for immediate use): 1. The nuclear counterstain is prepared as follows: 0.25 g of eosin O was dissolved in 100 mL of 75% ethanol, and 10 mL of the prepared eosin O ethanol solution was mixed with 80 mL of distilled water to obtain the nuclear counterstain.

[0058] The results of the present comparative example are shown in Figure 5 As shown, the overall coloration intensity was insufficient, the "red nucleus" of the positive cells and the negative cells were both light pink, the contrast between the positive and negative cells was not so strong, the visual effect was poor, and it was difficult to observe intuitively and effectively.

[0059] Comparative Example 5 The present comparative example improves a kit, and the configuration ratio of the components in the kit is different from that of the nuclear counterstain except that the configuration ratio of the reagent and the staining agent is the same as that of example 1.

[0060] Specifically, in the present comparative example, the nuclear counterstain is configured as follows (fresh preparation): 1. The nuclear counterstain is configured as follows: 1. The nuclear counterstain is configured as follows: Take 25 g safranin O and dissolve it in 100 mL of 75% ethanol, take 10 mL of the above prepared safranin O ethanol solution and mix it with 80 mL of distilled water, and then the nuclear counterstain is obtained.

[0061] The results of the present comparative example are shown in Figure 6 As the concentration is too high, the red nucleus of the positive cells presents black purple color, and the blue film of the positive cells cannot be accurately displayed, so that the characteristic of the positive cells "blue film red nucleus" is not clearly displayed, and the color of the negative cells presents blue due to the too high concentration, and the background appears a large number of blue attachments due to the too high concentration, so that the background is messy, and the color contrast between the positive and negative cells is not obvious, which directly affects the color development effect and the visual effect.

[0062] The above only describes the preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A kit for detecting peripheral T cell CD3 molecules, characterized by, The kit comprises: specific anti-CD3 monoclonal antibody, secondary antibody, alkaline phosphatase-labeled streptavidin, buffer, color developing agent, and nuclear counterstain agent; wherein the color developing agent is naphthol AS-BI salt and 4-diazo benzidine sulfate, and the nuclear counterstain agent is safranin O.

2. The kit of claim 1, wherein The secondary antibody is labeled with biotin.

3. The kit of claim 1, wherein The buffer is phosphate buffer or Tris buffer, and the pH is 7.0-8.

0.

4. The kit of claim 1, wherein The color developing agent is prepared as follows: (1) Dissolve naphthol AS-BI salt in DMF under light-proof condition to prepare solution A; (2) Dissolve 4-diazo benzidine sulfate in pure water under light-proof condition at room temperature to prepare solution B; (3) Dissolve 2-amino-2-methyl-1,3-propanediol in pure water and adjust the pH to 9.0-10.0 to obtain solution C; (4) Add solution A to solution B and mix well; Draw solution C and add it to the mixture of solution A and solution B and mix well to obtain the color developing agent.

5. The kit according to claim 4, characterized in that, In step (1), the mass-volume ratio of naphthol AS-BI salt to DMF is 0.02-0.05 g / mL; in step (2), the mass-volume ratio of 4-diazo benzidine sulfate to pure water is 0.01-0.04 g / mL; and in step (3), the concentration of solution C is 0.1-0.4 g / mL.

6. The kit of claim 4, wherein The volume ratio of solution A, solution B, and solution C is 1:1: (10-20).

7. The kit of claim 1, wherein The nuclear counterstain agent is prepared as follows: Dissolve safranin O in 75% ethanol, and then mix the safranin O ethanol solution with distilled water to obtain the nuclear counterstain agent.

8. The kit of claim 1, wherein The mass-volume ratio of safranin O to 75% ethanol is 0.01-0.04 g / mL, and the volume ratio of the safranin O ethanol solution to distilled water is 1: (5-10).

9. The kit of claim 1 for detecting CD3 molecules in peripheral T cells.

10. A method of detecting peripheral T cell CD3 molecules, characterized by, The kit comprises the following steps: (1) Collect the sample to be tested, pretreat the sample, and prepare a specimen slice; (2) Incubate the specimen slice with specific anti-CD3 monoclonal antibody, secondary antibody, and alkaline phosphatase-labeled streptavidin in sequence; (3) Stain the specimen slice with the color developing agent and counterstain it with the nuclear counterstain agent; the color developing agent is naphthol AS-BI salt and 4-diazo benzidine sulfate, and the nuclear counterstain agent is safranin O; (4) Observe the color development of the cells; if the cells show the characteristic performance of "blue membrane and red nucleus", it indicates that the cells are T cells and normally express CD3 molecules; if the cells only show red color, it indicates that the cells do not express CD3 molecules.

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