Immunohistochemistry pen liquid and preparation method thereof

Abstract

The present invention discloses an immunohistochemistry pen liquid and a preparation method thereof, and belongs to the field of immunohistochemistry technology. The solvent of the immunohistochemistry pen liquid is petroleum ether, containing phthalates with a mass volume fraction of 0.05% to 0.4%, polydimethylsiloxane with a mass volume fraction of 2% to 4%, and further containing ethanol with a volume fraction of 5% to 20% and / or ethyl acetate with a volume fraction of 5% to 20%. The immunohistochemistry pen liquid of the present invention does not contain hazardous chemicals such as butanone, cyclohexanone, acetone, and has no pungent odor, and is more green, environmentally friendly, and safe. When the immunohistochemistry pen liquid of the present invention is used to fill the immunohistochemistry pen, the pen tip does not leak liquid during use, the circle is drawn smoothly, and the handwriting is not easily interrupted due to tissue cleaning to affect the detection effect, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunohistochemistry, and in particular relates to an immunohistochemistry pen liquid and a preparation method thereof. Background Art

[0002] Immunohistochemistry utilizes the principle of antibody-antigen binding and cytochemical techniques to locate and quantify specific antigens or antibodies in tissues and cells. It is widely used in biology, tissue anatomy, and pathology. To prevent excess spillage of reagents during immunohistochemical staining, which could result in inadequate incubation and reaction between tissue and reagents, an immunohistochemistry pen is used to draw a circle around the tissue slice to prevent reagent leakage, thereby ensuring optimal staining and conserving antibodies.

[0003] The immunohistochemistry pen in the prior art has the following problems:

[0004] (1) Commercially available immunohistochemistry pens are generally expensive, have limited uses, and are costly;

[0005] (2) Most immunohistochemistry pens are prepared with volatile organic solvents and have a pungent odor;

[0006] (3) Some immunohistochemistry pens need to be left to dry after drawing circles, which takes a long time. This may cause tissue drying and background to appear;

[0007] (4) Some immunohistochemistry pen fluids tend to leak out of the covered tissue, thus affecting the tissue test results;

[0008] (5) Some immunohistochemical pen circles are prone to interruption during tissue washing, affecting the incubation of reagents and tissues, thereby affecting the test results;

[0009] (6) Some immunohistochemistry pen fluids contain butanone, cyclohexanone, acetone and other hazardous chemicals that are under national control, such as the immunohistochemistry pen disclosed in Chinese invention patent CN109100503A. Summary of the Invention

[0010] In order to solve at least one of the above technical problems, the technical solution adopted by the present invention is as follows:

[0011] The first aspect of the present invention provides an immunohistochemistry pen liquid, the solvent of which is petroleum ether, containing 0.05% to 0.4% by mass volume of phthalate and 2% to 4% by mass volume of polydimethylsiloxane.

[0012] Phthalate is a class of organic compounds with lubricating, hydrophobic, and plasticizing properties. In some embodiments of the present invention, the phthalate is selected from at least one of the group consisting of diethyl phthalate, dibutyl phthalate, dioctyl phthalate, and di-n-octyl phthalate. In some specific embodiments of the present invention, the phthalate is dibutyl phthalate.

[0013] In some embodiments of the present invention, the petroleum ether is used as a solvent. In fact, volatile organic solvents with similar properties to petroleum ether, including alcohols, ketones, ethers or alkanes (such as petroleum ether, etc.), benzenes or aromatic hydrocarbons (such as xylene, etc.), aromatic esters (such as ethyl acetate, butyl acetate, etc.), etc., can be used as solvents for the immunohistochemistry pen liquid of the present invention, mainly to make the circle dry faster. In addition, taking into account the need to avoid producing a pungent odor as much as possible and to avoid highly dangerous chemicals, petroleum ether is selected as the main solvent. Petroleum ether is a mixture of alkanes. Generally, petroleum ether with a boiling range of 60-90 degrees is selected, and the evaporation rate is more suitable.

[0014] In the present invention, polydimethylsiloxane is used as a hydrophobic agent. In some embodiments of the present invention, the viscosity of the polydimethylsiloxane is 1000 cSt to 3000 cSt.

[0015] In some specific embodiments of the present invention, the immunohistochemistry pen liquid contains 0.05% to 0.4% by mass volume of dibutyl phthalate and 0.5% to 2% by mass volume of polydimethylsiloxane with a viscosity of 1000 cSt to 3000 cSt.

[0016] Furthermore, the immunohistochemistry pen solution also contains 5% to 20% by volume of ethanol and / or 5% to 20% by volume of ethyl acetate. Ethanol and ethyl acetate are also used as solvents. When combined with petroleum ether, they can prolong the shelf life of the immunohistochemistry pen solution.

[0017] In some specific embodiments of the present invention, the solvent of the immunohistochemistry pen liquid is petroleum ether, containing 0.05% to 0.4% by mass volume fraction of dibutyl phthalate, 0.5% to 2% by mass volume fraction of polydimethylsiloxane with a viscosity of 1000 cSt to 3000 cSt, 5% to 20% by volume fraction of ethanol and 5% to 20% by volume fraction of ethyl acetate.

[0018] Furthermore, it also contains an indicator, which makes it easier to detect the consumption of the pen liquid during use and to refill it in time. In some embodiments of the present invention, the indicator is bright green, and the mass volume fraction of the bright green is 0.00625‰-0.05‰.

[0019] In some specific embodiments of the present invention, the solvent of the immunohistochemistry pen liquid is petroleum ether, containing 0.05% to 0.4% dibutyl phthalate by mass volume fraction, 0.5 to 2% polydimethylsiloxane with a viscosity of 1000 cSt to 3000 cSt by mass volume fraction, 5% to 20% ethanol by volume fraction, 5% to 20% ethyl acetate by volume fraction and 0.00625‰-0.05‰ brilliant green by mass volume fraction.

[0020] The second aspect of the present invention provides a method for preparing the immunohistochemistry pen liquid described in the first aspect of the present invention. The phthalate and the polydimethylsiloxane are first prepared into a mother liquor using the petroleum ether, and then the mother liquor is prepared according to the ratio, containing 0.05% to 0.4% phthalate by volume and 2% to 4% polydimethylsiloxane by volume, and the solvent is petroleum ether.

[0021] In some embodiments of the present invention, the method further comprises the step of preparing phthalate and polydimethylsiloxane into a mother liquor with a mass volume fraction of 10% by using petroleum ether.

[0022] In some embodiments of the present invention, the step of adding anhydrous ethanol and / or ethyl acetate is further included, so that the immunohistochemistry pen liquid also contains 5% to 20% ethanol and / or 5% to 20% ethyl acetate by volume.

[0023] In some embodiments of the present invention, the step of preparing the indicator into a stock solution with anhydrous ethanol at a weight volume fraction of 0.25%, and further adding the stock solution to the immunohistochemistry pen fluid to further contain the indicator at a weight volume fraction of 0.00625‰ to 0.05‰. In some embodiments of the present invention, the indicator is bright green.

[0024] Beneficial effects of the present invention

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The immunohistochemistry pen liquid of the present invention does not contain dangerous chemicals such as butanone, cyclohexanone, acetone, etc., and has no pungent odor, and is more green, environmentally friendly, and safe.

[0027] The immunohistochemistry pen liquid of the present invention is used to fill the immunohistochemistry pen. When in use, the pen tip does not leak liquid, circles are drawn smoothly, and the handwriting is not easily interrupted due to tissue cleaning to affect the detection effect.

[0028] After the immunohistochemistry pen liquid of the present invention is stored at room temperature for 6 months, when used, the pen tip still has the performance of no liquid leakage, smooth circle drawing, and the handwriting is not easily interrupted due to tissue cleaning to affect the detection effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The results of the immunohistochemistry pen #8 and a control pen after six months of storage at room temperature are shown. A: Immunohistochemistry pen #8 tested after six months of storage at room temperature. The test reagent was a primary antibody. The hydrophobic ring was intact, the ink was not interrupted, and there was no leakage of the incubation liquid. B: The control pen tested after six months of storage at room temperature. The test reagent was a primary antibody. The hydrophobic ring was not intact, the ink was interrupted, and there was leakage of the incubation liquid. DETAILED DESCRIPTION

[0030] Unless otherwise indicated, implied from the context, or customary in the art, all parts and percentages in this application are based on weight, and the test and characterization methods used are current as of the filing date of this application. Where applicable, the contents of any patents, patent applications, or publications referred to in this application are incorporated herein by reference in their entirety, and their equivalent patent families are also incorporated by reference, particularly for definitions of relevant terms in the art disclosed in such documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0031] Numerical ranges in this application are approximate values, so unless otherwise stated, they may include numerical values ​​outside the range. Numerical ranges include all numerical values ​​from the lower limit to the upper limit in increments of 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For a range comprising a numerical value less than 1 or comprising a fraction greater than 1 (e.g., 1.1, 1.5, etc.), 1 unit is appropriately considered to be 0.0001, 0.001, 0.01 or 0.1. For a range comprising a single digit less than 10 (e.g., 1 to 5), 1 unit is typically considered to be 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of the numerical values ​​between the minimum and maximum values ​​listed are considered to be clearly recorded in this application.

[0032] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, excipients or compounds unless expressly stated otherwise. In contrast, the term "essentially consisting of" excludes any other components, steps or processes from the scope of any subsequent description of the term, except those necessary for operational performance. The term "consisting of" does not include any components, steps or processes that are not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed members alone or in any combination.

[0033] 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 with reference to the embodiments.

[0034] Example

[0035] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0036] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials they cite are hereby incorporated by reference.

[0037] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0038] The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The instruments and equipment used in the following examples, unless otherwise specified, are all conventional laboratory instruments and equipment; the experimental materials used in the following examples, unless otherwise specified, are all purchased from conventional biochemical reagent stores.

[0039] Example 1 Preparation of Immunohistochemistry Pen Liquid

[0040] This embodiment provides a formulation and preparation method for an immunohistochemistry pen solution. Specifically, the immunohistochemistry pen solution contains dibutyl phthalate (DBP), polydimethylsiloxane (PDMS), ethanol, ethyl acetate, and brilliant green, using petroleum ether as the solvent.

[0041] Among them, phthalates are a class of organic compounds with lubricating, hydrophobic and plasticizing effects; polydimethylsiloxane is a hydrophobic agent with low surface tension, lubrication, and a viscosity range of 1000cSt-3000cSt; bright green is an indicator, the purpose of which is to make it easier to detect the consumption of pen liquid during use and to refill it in time.

[0042] In order to screen and optimize the best immunohistochemistry pen-in-liquid and verify the effect, the inventors prepared 15 different immunohistochemistry pen-in-liquids, the formulas of which are shown in Table 1.

[0043] Table 1 15 immunohistochemistry pen solutions

[0044]

[0045]

[0046] Before preparation, phthalate and polydimethylsiloxane were prepared into 10% stock solutions by weight to volume ratio using petroleum ether as solvent, and brilliant green was prepared into 0.25% stock solution using anhydrous ethanol.

[0047] During preparation, accurately weigh the mother liquors, anhydrous ethanol, ethyl alcohol and petroleum ether, add them into a clean sterilized container, mix thoroughly and store at room temperature for later use.

[0048] The prepared immunohistochemistry pen liquids were canned into empty immunohistochemistry pen tubes, with 3 mL of each pen liquid canned into each pen tube. The canned histochemistry pens were marked in sequence according to the immunohistochemistry pen liquid number, namely immunohistochemistry pen #1 to #15.

[0049] Implement 2 immunohistochemistry pen tests

[0050] The immunohistochemical pen #1 to #15 prepared in Example 1 was used for the incubation zone test, and a commercially available immunohistochemical pen (Sangon Biotech (Shanghai) Co., Ltd., catalog number E678004) was used as a control.

[0051] The circled incubation reagents (i.e., test reagents) are 1×PBS, endogenous peroxidase blocker (Tuling (Hangzhou) Biotechnology Co., Ltd., product number I20012C), antibody diluent (Tuling (Hangzhou) Biotechnology Co., Ltd., product number I30012A), and primary antibody product (Tuling (Hangzhou) Biotechnology Co., Ltd., product number I12192C); each immunohistochemistry pen tests 8 slides, of which every 2 slides are incubated with one test reagent (i.e., one immunohistochemistry pen tests 4 test reagents); the sample volume per slide is 0.4 mL each time.

[0052] 1× PBS formula (1 L): Measure 800 mL of purified water and pour it into a preparation container. Weigh 8.0 g of sodium chloride, 0.2 g of potassium chloride, 2.87 g of disodium hydrogen phosphate dodecahydrate, and 0.2 g of anhydrous potassium dihydrogen phosphate. After complete dissolution, adjust the pH to 7.2-7.4 and make up to 1 L.

[0053] Soak and clean the blank slides with immunohistochemical cleaning solution, wipe off excess moisture around with dust-free paper, use the immunohistochemical pen to be tested to draw a rectangular circle (about 3.5 cm long and 2 cm wide), incubate the above-mentioned 4 test reagents separately, incubate 2 slides for each test reagent, and incubate for 45 minutes; wash the slides and incubate for another 45 minutes; wash the slides again and incubate for another 45 minutes. Observe the usage when drawing circles and the incubation status of the test reagents during the incubation process. The standards that meet the requirements are: no pungent smell, no leakage from the pen tip, smooth circle drawing, complete hydrophobic circle, no interruption of handwriting and no leakage during the incubation process. The results are shown in Table 2:

[0054] Table 2 Test results of different immunohistochemistry pens

[0055]

[0056] As shown in Table 2, the circle drawing test using immunohistochemistry pen #2 to #4, #7 to #9 and #11 to #13 all met the requirements for the four test reagents. When the immunohistochemistry pen #1 was used for the circle drawing test, when the test reagents were endogenous peroxidase blocker and antibody diluent, the pen tip occasionally leaked liquid. When the immunohistochemistry pen #5 was used for the circle drawing test, when the test reagents were 1× PBS and endogenous peroxidase blocker, the circle drawing was occasionally not smooth. When the immunohistochemistry pen #6 was used for the circle drawing test, the four test reagents met the requirements. All test reagents leaked during incubation. Using immunohistochemistry pen #10 for the circle drawing test, when the test reagent was a primary antibody product, the circle drawing was occasionally difficult. Using immunohistochemistry pen #14 for the circle drawing test, all four test reagents exhibited difficulty in drawing circles. Using immunohistochemistry pen #15 for the circle drawing test, the odor was slightly pungent, and when the test reagents were an endogenous peroxidase blocker and a primary antibody product, the pen tip occasionally leaked. Using the control immunohistochemistry pen for the circle drawing test, all had a slightly pungent odor.

[0057] Example 3 Stability test of immunohistochemistry pen stored at room temperature for 3 months

[0058] After immunohistochemical pens #2 to #5, #7 to #13, and the control immunohistochemical pen were stored at room temperature for 3 months, the circle test was performed again: the incubation reagents after the circle test (i.e., test reagents) were also 1× PBS (self-prepared), endogenous peroxidase blocker (Tuling (Hangzhou) Biotechnology Co., Ltd., Catalog No. I20012C), antibody diluent (Tuling (Hangzhou) Biotechnology Co., Ltd., Catalog No. I30012A), and primary antibody product (Tuling (Hangzhou) Biotechnology Co., Ltd., Catalog No. I12192C); each immunohistochemical pen tested 8 slides, of which 2 were incubated with 1 test reagent; the sample volume per slide was 0.4 mL each time.

[0059] Soak and clean the blank slides with immunohistochemical cleaning solution, wipe the excess water around the tissue with dust-free paper, use the immunohistochemical pen to be tested to draw a rectangular circle (about 3.5 cm long and 2 cm wide), incubate the above-mentioned 4 test reagents separately, incubate 2 slides for each test reagent, and incubate for 45 minutes; wash the slides and incubate for another 45 minutes; wash the slides again and incubate again for 45 minutes. Observe the usage when drawing circles and the incubation status of the test reagents during the incubation process. The standards that meet the requirements are: no pungent smell, no leakage from the pen tip, smooth circle drawing, complete hydrophobic circle, no interruption of handwriting and no leakage during the incubation process. The results are shown in Table 3:

[0060] Table 3 Test results of different immunohistochemistry pens after storage at room temperature for 3 months

[0061]

[0062]

[0063] Table 3 shows that, after three months of storage at room temperature, immunohistochemistry pens #2-#4 were tested for the circle drawing test, and all four test reagents met the requirements. After three months of storage at room temperature, immunohistochemistry pens #5 were tested for the circle drawing test, and the test reagents used were primary antibodies. The other three test reagents occasionally had irregular circles. After three months of storage at room temperature, immunohistochemistry pens #7-#9 were tested for the circle drawing test, and all four test reagents met the requirements. After three months of storage at room temperature, immunohistochemistry pens #10 were tested for the circle drawing test, and the test reagents used were 1× PBS. The other three test reagents occasionally had irregular circles. After three months of storage at room temperature, immunohistochemistry pens #11-#13 were tested for the circle drawing test, and all four test reagents met the requirements. The control immunohistochemistry pens, after three months of storage at room temperature, still had a slightly pungent odor.

[0064] Example 4 Stability test of immunohistochemistry pen stored at room temperature for 6 months

[0065] After 6 months of storage at room temperature, immunohistochemistry pens #2-#5, #7-#13, and the control immunohistochemistry pen were circle-tested again: the incubation reagents (i.e., test reagents) after circle-testing were also 1× PBS, endogenous peroxidase blocker, antibody diluent, and primary antibody product; each immunohistochemistry pen tested 8 slides, of which 2 were incubated with 1 test reagent each; the sample volume per slide was 0.4 mL each time.

[0066] Soak and clean the blank slides with immunohistochemical cleaning solution, wipe the excess water around the tissue with dust-free paper, use the immunohistochemical pen to be tested to draw a rectangular circle (about 3.5 cm long and 2 cm wide), incubate the above-mentioned 4 test reagents separately, incubate 2 slides for each test reagent, and incubate for 45 minutes; wash the slides and incubate for another 45 minutes; wash the slides again and incubate again for 45 minutes. Observe the usage when drawing circles and the incubation status of the reagents during the incubation process of the test reagents. The standards that meet the requirements are: no pungent smell, no leakage from the pen tip, smooth circle drawing, complete hydrophobic circle, no interruption of handwriting and no leakage during the incubation process. The results are shown in Table 4:

[0067] Table 4 Test results of different immunohistochemistry pens after storage at room temperature for 6 months

[0068]

[0069]

[0070] As shown in Table 4, immunohistochemistry pen #7 to #9 and #11 to #13 were stored at room temperature for 6 months before the circle drawing test was conducted, and the four test reagents still met the requirements; immunohistochemistry pen #2 was stored at room temperature for 6 months before the circle drawing test, and when the test reagent was 1×PBS, it met the requirements, but when the test reagent was endogenous peroxidase blocker, antibody diluent and primary antibody product, occasional circle drawing was not smooth; immunohistochemistry pen #10 was stored at room temperature for 6 months before the circle drawing test, and for the four test reagents, occasional circle drawing was not smooth; the control immunohistochemistry pen was stored at room temperature for 6 months before the circle drawing test, and there was still a slightly pungent odor, and when the test reagent was a primary antibody product, there was incubation leakage. Among them, the circle drawing test results of immunohistochemistry pen #8 and the control immunohistochemistry pen after 6 months of storage at room temperature are as follows. Figure 1 shown.

[0071] The above results show that immunohistochemistry pens #7 to #9 and #11 to #13 still have excellent performance after being stored at room temperature for 6 months.

[0072] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An immunohistochemistry pen liquid, characterized in that: The solvent is petroleum ether, containing 0.05% to 0.2% by mass volume fraction of dibutyl phthalate and 2% to 4% by mass volume fraction of polydimethylsiloxane, wherein the viscosity of the polydimethylsiloxane is 1000 cSt to 3000 cSt.

2. The immunohistochemistry pen liquid according to claim 1, characterized in that It also contains 5% to 20% by volume of ethanol and / or 5% to 20% by volume of ethyl acetate.

3. The immunohistochemistry pen liquid according to claim 1, characterized in that Also contains an indicator.

4. The immunohistochemistry pen liquid according to claim 3, characterized in that The indicator is brilliant green, and the mass volume fraction of the brilliant green is 0.00625‰-0.05‰.

5. The method for preparing the immunohistochemistry pen liquid according to claim 1, characterized in that: First, the dibutyl phthalate and the polydimethylsiloxane are prepared into a mother liquor using the petroleum ether, and then the immunohistochemistry pen liquid is prepared according to the ratio.

6. The preparation method according to claim 5, characterized in that The method further includes adding anhydrous ethanol and / or ethyl acetate, so that the immunohistochemistry pen liquid contains 5% to 20% ethanol and / or 5% to 20% ethyl acetate by volume.

7. The preparation method according to claim 5 or 6, characterized in that: The method further includes the step of adding an indicator so that the immunohistochemistry pen liquid contains the indicator at a mass volume fraction of 0.00625‰-0.05‰.

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