Alkaline phosphatase developing solution and preparation method thereof

Abstract

The invention provides an alkaline phosphatase color developing solution and a preparation method thereof, and particularly relates to the technical field of immunodetection, the alkaline phosphatase color developing solution is formed by dissolving chromogen dry powder in a color developing buffer solution, and the concentration is 1-3 mg / ml; wherein the chromogen dry powder is composed of naphthol AS-MX phosphate and fast red-TR; the preparation method comprises the following steps: S1, dissolving naphthol AS-MX phosphate and fast red TR with purified water to obtain a solution; wherein the concentration of naphthol AS-MX phosphate in the solution is 0.5 to 1.5 mg / ml, and the concentration of fast red TR is 0.5 to 1.5 mg / ml; s2, freezing and storing the solution in a refrigerator, and condensing the solution into ice blocks; s3, the ice blocks are put into a freeze dryer, vacuumizing is carried out, water molecules in the ice blocks are sublimated into water vapor under the negative pressure condition, the water vapor is discharged, and chromogen dry powder can be obtained; s4, the chromogen dry powder is dissolved in the chromogenic buffer solution, and an alkaline phosphatase chromogenic solution can be obtained; the prepared developing solution has the core advantages of high sensitivity, rapid developing, multicolor compatibility, digital adaptation and the like, and an efficient and reliable tool is provided for pathological research and clinical diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to an alkaline phosphatase colorimetric solution and its preparation method. Background Technology

[0002] In immunohistochemical (IHC) staining experiments, Fast Red alkaline phosphatase chromogenic solution is a key detection tool. Its necessity lies not only in improving experimental efficiency and the reliability of results, but also in its ability to meet the urgent needs of modern pathological research and clinical diagnosis for high sensitivity, multi-target analysis, and standardized operation. DAB chromogenic reagent is currently the most commonly used chromogenic reagent in immunochemical staining techniques. During the immunoreaction, horseradish peroxidase carried by the antibody catalyzes the decomposition of DAB chromogenic reagent, resulting in a yellow-brown stained precipitate at the antigen sites in the tissue section. This stained precipitate is highly stable, insoluble in various organic solvents, and does not fade over long-term storage. Furthermore, it exhibits a very clear color difference from sections counterstained with hematoxylin, facilitating diagnosis by pathologists.

[0003] However, in some specific tumor diseases, such as melanoma and lung cancer, the staining effect of DAB chromogenic solution is not clearly distinguishable from the pigment deposition in tumor tissue, which may lead to misdiagnosis by pathologists and thus affect the formulation of the patient's treatment plan.

[0004] Fast Red offers a rapid colorimetric process, and its high contrast significantly reduces the risk of false negatives. Secondly, by optimizing its substrate formulation, Fast Red effectively suppresses interference from endogenous alkaline phosphatases (found in high concentrations in tissues such as the intestines and placenta), reducing non-specific binding and improving detection specificity. This characteristic is particularly important in the analysis of clinical samples (such as small tissue samples obtained through biopsy), preventing false positives from misleading diagnoses. Therefore, Fast Red has attracted considerable attention and is continuously being improved and developed. However, the existing Fast Red still has the following drawbacks: 1. The existing Fast Red chromogenic solution has the advantage of high contrast, but its contrast still needs to be improved when developing pigmented tumor tissues such as lung cancer and melanoma; 2. The preparation of the existing Fast Red chromogenic solution involves chemical reactions, the process is relatively complicated, and the quality is unstable; 3. Traditional chromogenic agents can show a yellow-brown coloring effect, and cannot be used in combination for the detection of the same sample. Summary of the Invention

[0005] The purpose of this invention is to provide an alkaline phosphatase chromogenic solution and its preparation method. The prepared chromogenic solution has core advantages such as high sensitivity, rapid color development, multicolor compatibility and digital adaptation, providing an efficient and reliable tool for pathological research and clinical diagnosis.

[0006] This invention provides the following technical solution: An alkaline phosphatase colorimetric solution is composed of chromogen dry powder dissolved in colorimetric buffer, with a concentration of 1-3 mg / ml; The chromogen powder consists of naphthol AS-MX phosphate and Solid Red-TR; the chromogenic buffer consists of Tris buffer with a pH of 8–9 and a molar concentration of 25–40 mM and a molar concentration of 1–10 mM MgCl2. Preferably, the chromogen dry powder is dissolved in a colorimetric buffer solution with a concentration of 2 mg / ml; wherein the chromogen dry powder is composed of 1 part by mass of naphthol AS-MX phosphate and 1 part by mass of solid red-TR.

[0007] A method for preparing an alkaline phosphatase colorimetric solution, comprising the following steps: S1. Dissolve naphthol AS-MX phosphate and Gustorubin TR in purified water to obtain a solution; wherein the concentration of naphthol AS-MX phosphate in the solution is 0.5–1.5 mg / ml, and the concentration of Gustorubin TR is 0.5–1.5 mg / ml. S2. Then put the solution in the refrigerator and freeze it to solidify into ice cubes; S3. Place ice cubes into a freeze dryer and create a vacuum, so that the water molecules in the ice cubes sublimate into water vapor under negative pressure and are discharged, thus obtaining the color source dry powder. S4. Dissolve the chromogen powder in the chromogenic buffer to obtain the alkaline phosphatase chromogenic solution. Preferably, in step S1, a solution is obtained by dissolving naphthol AS-MX phosphate and Gustorubin TR in purified water; wherein the concentration of naphthol AS-MX phosphate in the solution is 1 mg / ml and the concentration of Gustorubin TR is 1 mg / ml. Preferably, in step S2, the solution is placed in a freezer at -85 to -75°C and frozen for 1 to 2 hours until it solidifies into ice. Preferably, in step S3, ice cubes are placed in a freeze dryer and vacuumed for 6 to 8 hours, so that water molecules in the ice cubes sublimate into water vapor under negative pressure and are discharged, thus obtaining color source dry powder. Preferably, in step S4, the chromogenic buffer dissolves the chromogen powder to obtain an alkaline phosphatase chromogenic solution with a concentration of 1–3 mg / ml.

[0008] The beneficial effects of this invention are: 1. The Fast Red alkaline phosphatase chromogenic solution provided by the present invention exhibits a bright pink color development effect, which has a very obvious contrast with the blue cell nuclei after hematoxylin counterstaining. It is particularly suitable for tumor tissues such as lung cancer and melanoma with pigment deposition. 2. Existing Fast Red alkaline phosphatase colorimetric solutions are all in highly concentrated liquid form. This invention creatively uses the "freeze-drying method" to prepare the chromogen of Fast Red alkaline phosphatase colorimetric solution, which significantly simplifies the production process. It only requires freeze-drying and then dispensing, without involving any chemical reactions. The preparation process is stable and there is almost no batch-to-batch variation. 3. Traditional chromogenic agents (such as DAB containing heavy metals) can produce a yellow-brown staining effect, while Fast Red alkaline phosphatase chromogenic solution produces a bright pink color. The two can be used together in the same sample test, such as P16 / Ki67 double staining, which can effectively reduce the number of missed diagnoses of cervical cancer, especially in cases where high-risk HPV is positive but cytology test is negative, and can provide more accurate triage management. Attached Figure Description

[0009] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings: Figure 1 This is the color development effect of the alkaline phosphatase colorimetric solution in Example 1; Figure 2 This is the color development effect of the alkaline phosphatase colorimetric solution in Example 2; Figure 3 This is the color development effect of the alkaline phosphatase colorimetric solution in Example 3; Figure 4 This describes the color development effect of the alkaline phosphatase colorimetric solution of the present invention; Figure 5 This describes the color development effect of existing alkaline phosphatase colorimetric solutions; Figure 6 This is the color development effect of the imported alkaline phosphatase colorimetric solution; Figure 7 This is a double staining pattern of P16 (brown) and Ki-67 (red) on a cervical cancer tissue section sample; Figure 8 This is a double staining pattern of P63 (brown) and CK pan (red) on a breast cancer tissue section sample; Figure 9 This is a double staining pattern of EBER (brown) and CD20 (red) on a tissue section sample of B-cell lymphoma; Figure 10 This is a color development diagram of the Fast Red alkaline phosphatase colorimetric solution prepared in Example 1; Figure 11 This is a color development diagram of the Fast Red alkaline phosphatase colorimetric solution prepared in Example 1; Figure 12 This is a color development diagram of the Fast Red alkaline phosphatase colorimetric solution prepared in Example 1. Detailed Implementation

[0010] Example 1 An alkaline phosphatase chromogenic solution is composed of a chromogen powder dissolved in a chromogenic buffer at a concentration of 1 mg / ml; wherein the chromogen powder is composed of naphthol AS-MX phosphate and Solid Red-TR; the chromogenic buffer is composed of Tris buffer with a pH of 8 and a molar concentration of 25 mM and MgCl2 with a molar concentration of 1 mM. A method for preparing an alkaline phosphatase chromogenic solution is as follows: S1, dissolve 1 mg of naphthol AS-MX phosphate and 1 mg of Solid Red TR in 2 mL of purified water to obtain a solution; wherein the concentration of naphthol AS-MX phosphate in the solution is 0.5 mg / mL and the concentration of Solid Red TR is 0.5 mg / mL; S2, then freeze the solution in a freezer at -85°C for 1 hour to allow it to solidify into ice cubes; S3, place the ice cubes in a freeze dryer and evacuate under vacuum for 6 hours, so that the water molecules in the ice cubes sublimate into water vapor under negative pressure and are discharged, thereby obtaining the chromogen powder; S4, dissolve the chromogen powder in a chromogenic buffer to obtain an alkaline phosphatase chromogenic solution with a concentration of 1 mg / mL.

[0011] Example 2 An alkaline phosphatase chromogenic solution is composed of a chromogen powder dissolved in a chromogenic buffer at a concentration of 2 mg / ml; wherein the chromogen powder is composed of naphthol AS-MX phosphate and Solid Red-TR; and the chromogenic buffer is composed of Tris buffer with a pH of 8.5 and a molar concentration of 35 mM and a molar concentration of 5 mM MgCl2. A method for preparing an alkaline phosphatase chromogenic solution is as follows: S1, dissolve 1 mg of naphthol AS-MX phosphate and 1 mg of Solid Red TR in 1 mL of purified water to obtain a solution; wherein the concentration of naphthol AS-MX phosphate in the solution is 1 mg / mL and the concentration of Solid Red TR is 1 mg / mL; S2, then freeze the solution in a freezer at -80℃ for 1.5 hours to allow it to solidify into ice cubes; S3, place the ice cubes in a freeze dryer and evacuate under vacuum for 7 hours, so that the water molecules in the ice cubes sublimate into water vapor under negative pressure and are discharged, thereby obtaining the chromogen powder; S4, dissolve the chromogen powder in a chromogenic buffer to obtain an alkaline phosphatase chromogenic solution with a concentration of 2 mg / mL; Using the method in Example 2, three batches were produced consecutively, and three samples were taken and recorded as batch 25JN0800301, batch 25FB0600301, and batch 25FB1900301, respectively.

[0012] Example 3 An alkaline phosphatase chromogenic solution is composed of a chromogen powder dissolved in a chromogenic buffer at a concentration of 3 mg / ml; wherein the chromogen powder is composed of naphthol AS-MX phosphate and Solid Red-TR; the chromogenic buffer is composed of Tris buffer with a pH of 9 and a molar concentration of 40 mM and MgCl2 with a molar concentration of 10 mM. A method for preparing an alkaline phosphatase chromogenic solution is as follows: S1, dissolve naphthol AS-MX phosphate and SolidRed TR in 0.7 mL of purified water to obtain a solution; wherein the concentration of naphthol AS-MX phosphate in the solution is 1.5 mg / mL and the concentration of SolidRed TR is 1.5 mg / mL; S2, then freeze the solution in a freezer at -75°C for 2 hours to allow it to solidify into ice cubes; S3, place the ice cubes in a freeze dryer and evacuate under vacuum for 8 hours, so that the water molecules in the ice cubes sublimate into water vapor under negative pressure and are discharged, thereby obtaining the chromogen powder; S4, dissolve the chromogen powder in a chromogenic buffer to obtain an alkaline phosphatase chromogenic solution with a concentration of 3 mg / mL.

[0013] According to the preparation method of Fast Red alkaline phosphatase chromogenic solution provided by this invention, based on Example 2, three batches of Fast Red alkaline phosphatase chromogenic solution were continuously produced: batch 25JN0800301, batch 25FB0600301, and batch 25FB1900301. The same tissue section samples were then used to test the batch-to-batch differences of this preparation method. The results are as follows... Figures 1 to 3 As shown, using the preparation method of the Fast Red alkaline phosphatase chromogenic solution described in this invention, three batches of Fast Red alkaline phosphatase chromogenic solution were produced consecutively with almost no batch-to-batch variation. Therefore, the preparation method of the Fast Red alkaline phosphatase chromogenic solution described in this invention has a stable preparation process.

[0014] According to the methods in Examples 1 to 3, three groups of Fast Red alkaline phosphatase chromogenic solutions were prepared. Then, the same tissue section samples were used to develop the three different concentrations of Fast Red alkaline phosphatase chromogenic solutions to investigate the optimal chromogenic concentration of this preparation method.

[0015] After the color development was complete, the samples were counterstained with hematoxylin. The color development effects of three different concentrations of FastRed alkaline phosphatase chromogenic solution were observed and compared under a microscope. Figures 10 to 12The results showed that the three different concentrations of Fast Red alkaline phosphatase chromogenic solutions exhibited strong color development, vibrant colors, clear contrast, and clean backgrounds, all meeting the diagnostic requirements of pathologists. Therefore, the preparation method described in this invention has a broader tolerance for error and can effectively avoid deviations caused by adding too much or too little reagent during the production process.

[0016] Three identical tumor tissue section samples were prepared and subjected to the first antibody staining according to the conventional immunostaining procedure. Then, the samples were developed using the preparation method described in Example 2 of this invention, as well as the Fast Red alkaline phosphatase chromogenic solution prepared by conventional methods and imported.

[0017] After the color development was complete, the samples were counterstained with hematoxylin, and the color development effects of the three Fast Red alkaline phosphatase solutions were observed and compared under a microscope. Figures 4 to 6 The results showed that the Fast Red alkaline phosphatase prepared using the method described in this invention exhibited a clearer contrast and more vibrant pink color, better meeting the diagnostic requirements of pathologists. Therefore, the preparation method described in this invention can obtain a Fast Red alkaline phosphatase chromogenic solution with superior staining effects, surpassing traditional preparation methods and imported Fast Red alkaline phosphatase chromogenic solutions of the same type.

[0018] Prepare three different tumor tissue section samples. Perform the first antibody staining according to the standard immunostaining procedure. After the DAB staining is completed, perform the second antibody staining according to the standard immunostaining procedure. Then, use the alkaline phosphatase staining solution described in this invention for staining.

[0019] After the color development was completed, the above samples were counterstained with hematoxylin. The dual color development effect of using DAB colorimetric solution and the alkaline phosphatase colorimetric solution described in this invention was observed under a microscope. Figures 7 to 9 .

[0020] The results showed that the brown and pink staining of P16 antibody and Ki-67 antibody on cervical cancer tissue sections, P63 antibody and CK pan antibody on breast cancer tissue sections, and EBER probe and CD20 antibody on B-cell lymphoma tissue sections exhibited clear contrast and vibrant colors, which met the diagnostic requirements of pathologists.

[0021] Therefore, the alkaline phosphatase chromogenic solution described in this invention can be used in conjunction with DAB chromogenic solution to achieve staining of two different antibodies on a single tissue section, effectively saving sample and staining time, and providing pathologists with more accurate diagnostic information.

[0022] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An alkaline phosphatase chromogenic solution, composed of chromogen powder dissolved in chromogenic buffer, with a concentration of 1-3 mg / ml; in, The chromogen powder consists of naphthol AS-MX phosphate and Solid Red-TR; the colorimetric buffer consists of Tris buffer with a pH of 8-9 and a molar concentration of 25-40 mM and MgCl2 with a molar concentration of 1-10 mM.

2. The alkaline phosphatase colorimetric solution according to claim 1, characterized in that: The solution consists of chromogen powder dissolved in a colorimetric buffer with a concentration of 2 mg / ml; wherein the chromogen powder is composed of 1 part by mass of naphthol AS-MX phosphate and 1 part by mass of Solid Red TR.

3. The alkaline phosphatase colorimetric solution according to claim 1 is prepared by the following method: S1. Dissolve naphthol AS-MX phosphate and Gustorubin TR in purified water to obtain a solution; wherein the concentration of naphthol AS-MX phosphate in the solution is 0.5-1.5 mg / ml and the concentration of Gustorubin TR is 0.5-1.5 mg / ml; S2. Then put the solution in the refrigerator and freeze it to solidify into ice cubes; S3. Place ice cubes into a freeze dryer and create a vacuum, so that the water molecules in the ice cubes sublimate into water vapor under negative pressure and are discharged, thus obtaining the color source dry powder. S4. Dissolve the chromogen powder in the chromogenic buffer to obtain the alkaline phosphatase chromogenic solution.

4. The method for preparing an alkaline phosphatase colorimetric solution according to claim 3, characterized in that: In step S1, naphthol AS-MX phosphate and Gustocris TR are dissolved in purified water to obtain a solution; wherein the concentration of naphthol AS-MX phosphate in the solution is 1 mg / ml and the concentration of Gustocris TR is 1 mg / ml.

5. The method for preparing an alkaline phosphatase colorimetric solution according to claim 3, characterized in that: In step S2, the solution is placed in a freezer at -85 to -75°C and frozen for 1 to 2 hours until it solidifies into ice.

6. The method for preparing an alkaline phosphatase colorimetric solution according to claim 3, characterized in that: In step S3, ice cubes are placed in a freeze dryer and vacuumed for 6-8 hours, allowing water molecules in the ice cubes to sublimate into water vapor under negative pressure and be discharged, thus obtaining the colorant dry powder.

7. The method for preparing an alkaline phosphatase colorimetric solution according to claim 3, characterized in that: In step S4, the chromogenic powder is dissolved in the chromogenic buffer to obtain an alkaline phosphatase chromogenic solution with a concentration of 1–3 mg / ml.

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