Preparation method of 5-hydroxytryptamine antibody reagent

Abstract

The invention relates to the technical field of molecular detection, in particular to a preparation method of a 5-hydroxytryptamine antibody reagent, and the 5-hydroxytryptamine antibody reagent comprises the following basic components: serum, a 5-hydroxytryptamine receptor diluent, concentrated sulfuric acid, concentrated nitric acid, monoamine oxidase, glyoxylic acid and a developing solution. The basic components for forming the color developing solution are sulfuric acid, nitric acid, glyoxylic acid and ultrapure water, and the mass ratio of the sulfuric acid to the nitric acid to the glyoxylic acid is 30%; 25% of nitric acid; 25% of glyoxylic acid; and 20% of ultrapure water. The reagent has agility, can complete detection in a short time, is suitable for large-scale screening and has convenience, the reagent can observe a result without complex operation of four steps: 1, sampling, 2, centrifuging, 3, mixing of the reagent and a specimen, and 4, standing for 1-15 minutes to obtain the result, the reagent is single, and the operation is simpler and more accurate due to the fact that only a single reagent is provided; and the accuracy of double combination of shaping and quantification is higher.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular detection, in particular to a method for preparing a 5-hydroxytryptamine antibody reagent. Background Art

[0002] Serotonin is an important neurotransmitter that is widely present in mammalian tissues, especially in the central nervous system, and is involved in a variety of physiological regulatory effects;

[0003] However, in current general practice, the detection of serotonin mainly includes high-performance liquid chromatography-fluorescence detector pre-column derivatization method, enzyme-linked immunosorbent assay and liquid chromatography-mass spectrometry. High-performance liquid chromatography-fluorescence detector requires a color reaction, and the operation process is cumbersome, and it cannot accurately and intuitively quantify the content of serotonin in the tested product; enzyme-linked immunosorbent assay is complicated to operate and difficult to accurately quantify, and is not suitable for biological samples with trace content; mass spectrometry has strong specificity and high accuracy, but is expensive and the instrument operation is complicated. It is often used for the qualitative analysis of unknown samples, but for routine testing of conventional known samples, the instrument testing cost is relatively high, which is not conducive to large-scale testing by enterprises. These methods are relatively complicated, take a long time and have low accuracy. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for preparing a 5-HT antibody reagent to solve the problem that the methods proposed in the above background art are complex to operate and difficult to accurately quantify.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for preparing a 5-hydroxytryptamine antibody reagent, comprising:

[0006] The basic components of the 5-HT antibody reagent are serum, 5-HT receptor diluent, concentrated sulfuric acid, concentrated nitric acid, monoamine oxidase, glyoxylic acid and color developing solution;

[0007] The basic components of the color developing solution are sulfuric acid, nitric acid, glyoxylic acid and ultrapure water, and the mass ratio of the components is 30% sulfuric acid, 25% nitric acid, 25% glyoxylic acid and 20% ultrapure water.

[0008] Preferably, the preparation and determination process of the 5-HT antibody reagent is as follows:

[0009] ① Dilute 1 part of concentrated sulfuric acid into 4 parts with sterile water as an acidifier;

[0010] ② Dilute 1 part of concentrated nitric acid into 4 parts with sterile water as an acylating agent;

[0011] ③ Add monoamine oxidase to promote the modification of the 5-hydroxytryptamine structure and react with the acylating agent to form an acyl halide;

[0012] ④ Take two reagent bottles, add 1 ml of acidifier to each, and then add 4 × 20.06 μmol of glyoxylic acid as a color developing solution. After hydrolysis and oxidation, 5-hydroxytryptamine reacts with glyoxylic acid in a strong acid environment and is heated to form a specific fluorescent condensation product, forming an indigo analogue.

[0013] ⑤ Add 8 × 20.06 μmol 5-hydroxytryptamine to each of the two test tubes, add the color developing solution, mix well, and let it stand for a while;

[0014] ⑥ Depending on the color reaction, the original sample serum can be diluted again to make the color reaction reach the measurable range, and the sample can be quantitatively or qualitatively determined.

[0015] Preferably, the preparation process of the color developing solution is as follows:

[0016] ①Slowly add sulfuric acid and nitric acid into the glyoxylic acid solution;

[0017] ② Place the glyoxylic acid solution on a magnetic stirrer and stir at 30 rpm;

[0018] ③ Let the mixed solution stand for three days. If there are crystals, remove them and use the remaining solution as the color developing solution.

[0019] Preferably, the serum is added to a 5-hydroxytryptamine receptor diluent to enrich and collect 5-hydroxytryptamine.

[0020] Preferably, the concentration of the concentrated sulfuric acid is a 30% V / V acid solution, and the concentration of the concentrated nitric acid is a 30% V / V acid solution.

[0021] Preferably, the monoamine oxidase is used as the oxidant, and the reagent bottle is 5000 ml.

[0022] Preferably, the test tube is 2 ml or 3 ml.

[0023] Preferably, the mixing process of the color developing solution is carried out at room temperature of 25 degrees Celsius.

[0024] Compared with existing technologies, the present invention offers the following advantages: 1. Agility: This reagent can be completed in a short time and is suitable for large-scale screening. 2. Convenience: The reagent can be used to observe results without a complex four-step process: 1. Sampling, 2. Centrifugation, 3. Mixing the reagent and specimen, and 4. Letting the reagent rest for 1-15 minutes to produce the result. 3. Single reagent: Using only a single reagent simplifies operation. 4. Accuracy: The combination of both qualitative and quantitative methods results in higher accuracy. DETAILED DESCRIPTION

[0025] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0026] An embodiment provided by the present invention: a method for preparing a 5-hydroxytryptamine antibody reagent, comprising:

[0027] The basic components of the 5-HT antibody reagent are serum, 5-HT receptor diluent, concentrated sulfuric acid, concentrated nitric acid, monoamine oxidase, glyoxylic acid and color developing solution;

[0028] The basic components of the color developing solution are sulfuric acid, nitric acid, glyoxylic acid and ultrapure water, and the mass ratio of the components is 30% sulfuric acid, 25% nitric acid, 25% glyoxylic acid and 20% ultrapure water.

[0029] Furthermore, the preparation and determination process of 5-HT antibody reagent is as follows:

[0030] ① Dilute 1 part of concentrated sulfuric acid into 4 parts with sterile water as an acidifier;

[0031] ② Dilute 1 part of concentrated nitric acid into 4 parts with sterile water as an acylating agent;

[0032] ③ Add monoamine oxidase to promote the modification of the 5-hydroxytryptamine structure and react with the acylating agent to form an acyl halide;

[0033] ④ Take two reagent bottles, add 1 ml of acidifier to each, and then add 4 × 20.06 μmol of glyoxylic acid as a color developing solution. After hydrolysis and oxidation, 5-hydroxytryptamine reacts with glyoxylic acid in a strong acid environment and is heated to form a specific fluorescent condensation product, forming an indigo analogue.

[0034] ⑤ Add 8 × 20.06 μmol 5-hydroxytryptamine to each of the two test tubes, add the color developing solution, mix well, and let it stand for a while;

[0035] ⑥ Depending on the color reaction, the original sample serum can be diluted again to make the color reaction reach the measurable range, and the sample can be quantitatively or qualitatively determined.

[0036] Furthermore, the preparation process of the color developing solution is as follows:

[0037] ①Slowly add sulfuric acid and nitric acid into the glyoxylic acid solution;

[0038] ② Place the glyoxylic acid solution on a magnetic stirrer and stir at 30 rpm;

[0039] ③ Let the mixed solution stand for three days. If there are crystals, remove them and use the remaining solution as the color developing solution.

[0040] Furthermore, the serum is added to a 5-HT receptor dilution solution to enrich and collect 5-HT. Serum is a light yellow transparent liquid separated after blood coagulation. It does not contain coagulation components such as blood cells and fibrinogen. Its main components include water, electrolytes, proteins, hormones, enzymes and metabolites.

[0041] Furthermore, the concentration of concentrated sulfuric acid is a 30% V / V acid solution, and the concentration of concentrated nitric acid is a 30% V / V acid solution. Concentrated sulfuric acid and concentrated nitric acid are both strong acids commonly used in laboratories, and are normally colorless and transparent liquids.

[0042] Furthermore, monoamine oxidase is used as an oxidant, and the reagent bottle is 5000 ml. The reagent bottle is a special container for storing chemical reagents, usually made of corrosion-resistant glass or plastic material. Its standard design includes a ground-mouth stopper, a wide-mouth / narrow-mouth bottle body and a volume scale mark.

[0043] Furthermore, the test tube is 2 ml or 3 ml. The test tube is the most basic glass instrument in chemical and biological experiments. It is usually a slender cylindrical instrument with a closed bottom and an open top. The capacity ranges from a few ml to tens of ml. It is mainly used for reactions, heating, mixing or observation of small amounts of reagents, such as acid-base neutralization, precipitation formation and other experiments.

[0044] Furthermore, the mixing process of the color developing solution is carried out at room temperature of 25 degrees Celsius to ensure that there are no unexpected results and to ensure that there are no accidents in the mixing process of the color developing solution.

[0045] Working principle:

[0046] Step 1: Slowly add sulfuric acid and nitric acid into the glyoxylic acid solution, and place the glyoxylic acid solution on a magnetic stirrer and stir at 30 rpm.

[0047] Step 2: Let the mixed solution stand for three days. If there are crystals, remove them and use the remaining solution as the color developing solution.

[0048] Step 3: Dilute 1 part of concentrated sulfuric acid with sterile water to 4 parts as an acidifying agent, and dilute 1 part of concentrated nitric acid with sterile water to 4 parts as an acylating agent.

[0049] Step 4: Add monoamine oxidase to promote the modification of the 5-hydroxytryptamine structure and react with the acylating agent to form an acyl halide.

[0050] Step 5: Take two reagent bottles, add 1 ml of acidifier to each, and then add 4×20.06 μmol of glyoxylic acid as a color developing solution to each. After hydrolysis and oxidation, 5-hydroxytryptamine and glyoxylic acid are heated in a strong acid environment to form a specific fluorescent condensation product, forming an indigo analog.

[0051] Step 6: Add 8×20.06 μmol 5-hydroxytryptamine to each of the two test tubes, add the color developing solution, mix well, and let it stand for a while.

[0052] Step 7: Depending on the color reaction, the original sample serum can be diluted again to make the color reaction reach the measurable range, and then the sample can be quantitatively or qualitatively determined.

[0053] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

Claims

1. A method for preparing a 5-hydroxytryptamine antibody reagent, characterized in that: include, The basic components of the 5-HT antibody reagent are serum, 5-HT receptor diluent, concentrated sulfuric acid, concentrated nitric acid, monoamine oxidase, glyoxylic acid and color developing solution; The basic components of the color developing solution are sulfuric acid, nitric acid, glyoxylic acid and ultrapure water, and the mass ratio of the components is 30% sulfuric acid, 25% nitric acid, 25% glyoxylic acid and 20% ultrapure water.

2. The method for preparing a 5-hydroxytryptamine antibody reagent according to claim 1, wherein: The preparation and determination process of the 5-HT antibody reagent is as follows: ① Dilute 1 part of concentrated sulfuric acid into 4 parts with sterile water as an acidifier; ② Dilute 1 part of concentrated nitric acid into 4 parts with sterile water as an acylating agent; ③ Add monoamine oxidase to promote the modification of the 5-hydroxytryptamine structure and react with the acylating agent to form an acyl halide; ④ Take two reagent bottles, add 1 ml of acidifier to each, and then add 4 × 20.06 μmol of glyoxylic acid as a color developing solution. After hydrolysis and oxidation, 5-hydroxytryptamine reacts with glyoxylic acid in a strong acid environment and is heated to form a specific fluorescent condensation product, forming an indigo analogue. ⑤ Add 8 × 20.06 μmol 5-hydroxytryptamine to each of the two test tubes, add the color developing solution, mix well, and let it stand for a while; ⑥ Depending on the color reaction, the original sample serum can be diluted again to make the color reaction reach the measurable range, and the sample can be quantitatively or qualitatively determined.

3. The method for preparing a 5-HT antibody reagent according to claim 1, wherein: The preparation process of the color developing solution is as follows: ①Slowly add sulfuric acid and nitric acid into the glyoxylic acid solution; ② Place the glyoxylic acid solution on a magnetic stirrer and stir at 30 rpm; ③ Let the mixed solution stand for three days. If there are crystals, remove them and use the remaining solution as the color developing solution.

4. The method for preparing a 5-HT antibody reagent according to claim 1, wherein: The serum is added to a 5-hydroxytryptamine receptor diluent to enrich and collect 5-hydroxytryptamine.

5. The method for preparing a 5-hydroxytryptamine antibody reagent according to claim 2, wherein: The concentration of the concentrated sulfuric acid is 30% V / V acid solution, and the concentration of the concentrated nitric acid is 30% V / V acid solution.

6. The method for preparing a 5-HT antibody reagent according to claim 2, wherein: The monoamine oxidase is used as an oxidant, and the reagent bottle has a volume of 5000 ml.

7. The method for preparing a 5-hydroxytryptamine antibody reagent according to claim 2, wherein: The test tube is 2 ml or 3 ml.

8. The method for preparing a 5-HT antibody reagent according to claim 3, wherein: The mixing process of the color developing solution is based on the room temperature of 25 degrees Celsius.