CN111735785A - Detection method for tetrahydrofolic acid production - Google Patents
Detection method for tetrahydrofolic acid production Download PDFInfo
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- CN111735785A CN111735785A CN202010633018.0A CN202010633018A CN111735785A CN 111735785 A CN111735785 A CN 111735785A CN 202010633018 A CN202010633018 A CN 202010633018A CN 111735785 A CN111735785 A CN 111735785A
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- MSTNYGQPCMXVAQ-KIYNQFGBSA-N 5,6,7,8-tetrahydrofolic acid Chemical compound N1C=2C(=O)NC(N)=NC=2NCC1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 MSTNYGQPCMXVAQ-KIYNQFGBSA-N 0.000 title claims abstract description 25
- 238000001514 detection method Methods 0.000 title claims abstract description 24
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 19
- 239000000243 solution Substances 0.000 claims abstract description 42
- 238000000034 method Methods 0.000 claims abstract description 28
- 238000007789 sealing Methods 0.000 claims abstract description 28
- 239000012468 concentrated sample Substances 0.000 claims abstract description 27
- 239000003085 diluting agent Substances 0.000 claims abstract description 27
- 239000000523 sample Substances 0.000 claims abstract description 22
- 238000012360 testing method Methods 0.000 claims abstract description 21
- 102000004190 Enzymes Human genes 0.000 claims abstract description 20
- 108090000790 Enzymes Proteins 0.000 claims abstract description 20
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 19
- 238000005406 washing Methods 0.000 claims abstract description 17
- 238000002965 ELISA Methods 0.000 claims abstract description 13
- 239000012089 stop solution Substances 0.000 claims abstract description 10
- 238000007865 diluting Methods 0.000 claims abstract description 8
- 238000002835 absorbance Methods 0.000 claims abstract description 6
- 239000000463 material Substances 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims description 16
- 238000011161 development Methods 0.000 claims description 15
- 238000004321 preservation Methods 0.000 claims description 12
- 238000004140 cleaning Methods 0.000 claims description 10
- 238000005259 measurement Methods 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 6
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 3
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 10
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 5
- 229960000304 folic acid Drugs 0.000 description 5
- 235000019152 folic acid Nutrition 0.000 description 5
- 239000011724 folic acid Substances 0.000 description 5
- 230000002596 correlated effect Effects 0.000 description 4
- 230000002950 deficient Effects 0.000 description 3
- 206010002065 Anaemia megaloblastic Diseases 0.000 description 2
- 208000000682 Megaloblastic Anemia Diseases 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000005515 coenzyme Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 231100001016 megaloblastic anemia Toxicity 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108700007696 Tetrahydrofolate Dehydrogenase Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 210000000276 neural tube Anatomy 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Engineering & Computer Science (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a detection method for tetrahydrofolic acid production, which comprises the following steps: s1: preparing a test agent and related detection materials; 1-3 parts of a test agent, an enzyme label plate with a blank hole, a standard hole and a sample hole to be tested, a sealing plate film, an FA enzyme label, a concentrated washing solution, a developing solution A, a developing solution B and a stop solution; s2: diluting a test agent to be tested into concentrated sample diluent; s3: adding a sample; adding the concentrated sample diluent in the S2 into a blank hole, a standard hole and a sample hole to be detected of the ELISA plate; s4: storing the sealing plate; sealing the ELISA plate with a sealing plate film and preserving at constant temperature; the invention obtains the concentration range more accurately by comparing the average absorbance value of the sample with the standard value through a plurality of tests, and ensures the activity of the enzyme by a plurality of times of temperature return treatment in the operation process, simulates the normal temperature of the human body, so that the detection result is closer to the reality.
Description
Technical Field
The invention particularly relates to the technical field of folic acid detection, and particularly relates to a detection method for tetrahydrofolic acid production.
Background
Tetrahydrofolic acid is a reduced folic acid, also known as coenzyme F, which is the parent compound of folic acid in the form of a coenzyme. When folic acid is deficient or some medicines inhibit folic acid reductase, folic acid can not be converted into tetrahydrofolic acid, which can affect the development and maturation of blood cells to cause megaloblastic anemia, and if tetrahydrofolic acid is deficient in organisms, various biosynthesis reactions are hindered; the higher animals are most typically shown to have affected development and maturation of red blood cells, and are susceptible to megaloblastic anemia; in pregnancy, the need is increased, which can lead to deficiency, and the badly deficient fetus can have neural tube malformation. Therefore, the production of tetrahydrofolic acid becomes very important.
In the prior art, a plurality of detection modes of tetrahydrofolic acid exist, but the detection modes have defects, so that the detection results of the tetrahydrofolic acid have large errors, and the detection requirements of people are difficult to meet.
Disclosure of Invention
The invention aims to solve the defect of large error in tetrahydrofolic acid detection in the prior art, and provides a detection method for tetrahydrofolic acid production.
In order to achieve the purpose, the invention adopts the following technical scheme:
a detection method for tetrahydrofolic acid production comprises the following steps:
s1: preparing a test agent and related detection materials; 1-3 parts of a test agent, an enzyme label plate with a blank hole, a standard hole and a sample hole to be tested, a sealing plate film, an FA enzyme label, a concentrated washing solution, a developing solution A, a developing solution B and a stop solution;
s2: diluting a test agent to be tested into concentrated sample diluent;
s3: adding a sample; adding the concentrated sample diluent in the S2 into a blank hole, a standard hole and a sample hole to be detected of the ELISA plate;
s4: storing the sealing plate; sealing the ELISA plate with a sealing plate film and preserving at constant temperature;
s5: returning the temperature to prepare the liquid; diluting the concentrated washing liquid with distilled water for later use;
s6: washing liquor for washing plates; removing the sealing plate film, discarding liquid, spin-drying, filling the cleaning solution in S5 in each hole, standing for 20-30 seconds, discarding, repeating the operation for 3-5 times, and patting to dry;
s7: adding enzyme, and adding FA enzyme label into the standard hole and the sample hole to be detected;
s8: temperature return treatment: sealing the ELISA plate in S7 with a sealing plate film, and preserving at constant temperature;
s9: secondary cleaning; removing the sealing plate film, discarding liquid, spin-drying, filling the cleaning solution in S5 in each hole, standing for 20-30 seconds, discarding, repeating the operation for 3-5 times, and patting to dry;
s10: color development treatment: adding the color developing solution A into each hole, adding the color developing solution B, slightly vibrating, standing, and shading at constant temperature for color development;
s11: stopping, and finally adding a stopping solution to stop the reaction;
s12: the measurement was carried out by zeroing a blank well and measuring the absorbance (OD value) of each well in order at a wavelength of 450 nm.
Further, in S1, 3 parts of the test agent are taken, and in the S2 process, the test agent is diluted into a concentrated sample diluent 1, a concentrated sample diluent 2, and a concentrated sample diluent 3.
On the basis of the scheme, in the S3 process, when the concentrated sample diluent is added, the concentrated sample diluent is ensured not to touch the pore wall, and after the concentrated sample diluent is added, the elisa plate is slightly shaken.
As a further scheme of the invention, in the S4 process, the preservation temperature is controlled to be between 30 and 45 ℃, and the preservation time is controlled to be between 20 and 40 min.
Further, in the S5 process, the dilution ratio of the concentrated washing solution is 25 to 35 times.
On the basis of the scheme, in the S7, the addition content of the FA enzyme label is between 30ul and 60 ul.
As a further scheme of the invention, in the S8 process, the preservation temperature is controlled to be between 30 and 45 ℃, and the preservation time is controlled to be between 20 and 40 min.
Further, in the S10 process, the adding content of the color developing solution A and the color developing solution B is between 30ul and 60ul, the adding content of the color developing solution A and the color developing solution B is the same, and the constant-temperature shading and color developing time is between 10 and 20 min.
On the basis of the scheme, in the S11 process, the adding content of the stop solution is between 30ul and 60 ul.
As a still further aspect of the present invention, in the process of S12, the determination should be performed within 10 to 15min of the addition of the stop solution.
The invention has the beneficial effects that:
1. the concentration range is more accurate by comparing the average absorbance value of the sample with the standard value through the operation of preparing a plurality of portions of the test agent for a plurality of times of tests.
2. The activity of the enzyme is ensured through multiple times of temperature return treatment in the operation processes of S5 and S8, and the normal temperature of a human body is simulated, so that the detection result is closer to the reality.
3. By washing away unbound material after incubation during S6 and S9, the detection result is not affected by other factors and becomes more accurate.
4. By means of determination processing within 10-15min after the stop solution is added, the situation that the determination time is too long and the measurement result is inaccurate is avoided, and the accuracy of detection is improved again, wherein the higher the concentration of the sample to be detected is, the more the combination of the labeled antigen and the antibody is inhibited, and the shallower the color development is. The color depth is positively correlated with the enzyme amount and negatively correlated with the content of the substance to be detected in the sample.
Drawings
FIG. 1 is a schematic flow chart of a detection method for tetrahydrofolic acid production according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are exemplary only for the purpose of explaining the present patent and are not to be construed as limiting the present patent.
In the description of this patent, it is noted that unless otherwise specifically stated or limited, the terms "mounted," "connected," and "disposed" are to be construed broadly and can include, for example, fixedly connected, disposed, detachably connected, disposed, or integrally connected and disposed. The specific meaning of the above terms in this patent may be understood by those of ordinary skill in the art as appropriate.
Referring to fig. 1, a detection method for tetrahydrofolic acid production, the method comprising the steps of:
s1: preparing a test agent and related detection materials; 1-3 parts of a test agent, in the embodiment, 3 parts of the test agent are taken, the elisa plate is provided with a blank hole, a standard hole and a sample hole to be tested, the sealing plate film, the FA elisa, the concentrated cleaning solution, a developing solution A and a developing solution B, in the embodiment, the developing solution A and the developing solution B are H2O2, TMB and a stop solution;
s2: diluting a test agent to be tested into concentrated sample diluent; respectively diluting 3 parts of a to-be-tested agent into a concentrated sample diluent 1, a concentrated sample diluent 2 and a concentrated sample diluent 3; the concentration range is more accurate by comparing the average absorbance value of the sample with a standard value in multiple tests;
s3: adding a sample; adding the concentrated sample diluent in the S2 into a blank hole, a standard hole and a sample hole to be detected of the ELISA plate; when the concentrated sample diluent is added, the concentrated sample diluent is ensured not to touch the hole wall, after the concentrated sample diluent is added, the ELISA plate is slightly shaken to prevent the concentrated sample diluent with overlarge force from splashing,
s4: storing the sealing plate; sealing the ELISA plate with a sealing plate film and preserving at constant temperature; the preservation temperature is controlled to be 30-45 ℃, specifically 36 ℃ in the embodiment, and the preservation time is controlled to be 20-40min, specifically 32min in the embodiment; in addition, multiple times of temperature return treatment in the operation process ensure the activity of enzyme, simulate the lowest normal temperature of a human body and enable the detection result to be closer to the reality;
s5: returning the temperature to prepare the liquid; diluting the concentrated washing liquid with distilled water for later use; the specific dilution factor of the concentrated washing solution in this example was 30 times;
s6: washing liquor for washing plates; removing the sealing plate film, discarding liquid, spin-drying, filling the cleaning solution in S5 in each hole, standing for 25 seconds, discarding, repeating the operation for 3-5 times, and patting to dry;
s7: adding enzyme, and adding FA enzyme label into the standard hole and the sample hole to be detected; in the embodiment, the adding content of the FA enzyme label is 50 ul; to serve a marking function;
s8: temperature return treatment: sealing the ELISA plate in S7 with a sealing plate film, and preserving at constant temperature; the preservation temperature is controlled to be 30-45 ℃, specifically 36 ℃ in the embodiment, and the preservation time is controlled to be 20-40min, specifically 32min in the embodiment;
s9: secondary cleaning; removing the sealing plate film, discarding liquid, spin-drying, filling the cleaning solution in S5 in each hole, standing for 20-30 seconds, discarding, repeating the operation for 3-5 times, and patting to dry; after incubation, the unbound material is removed by washing;
s10: color development treatment: adding the color developing solution A into each hole, adding the color developing solution B, slightly vibrating, standing, and shading at constant temperature for color development; the adding contents of the developing solution A and the developing solution B are both 30ul-60ul, the adding contents of the developing solution A and the developing solution B are both 50ul, the constant-temperature shading and developing time is 10-20min, specifically 15min, and standing for developing;
s11: stopping, finally adding a stopping solution to stop the reaction, wherein the blue color is immediately changed into yellow; TMB is converted to blue by the catalysis of peroxidase and to the final yellow by the action of an acid.
S12: and (3) performing measurement, namely performing zero adjustment on blank holes, and sequentially measuring the absorbance (OD value) of each hole at the wavelength of 450nm, wherein the measurement is performed within 10-15min after adding the stop solution, so that the longer the measurement time is, the inaccurate measurement result is obtained, the higher the concentration of the sample to be measured is, the more the combination of the labeled antigen and the antibody is inhibited, and the shallower the color development is. The color depth is positively correlated with the enzyme amount and negatively correlated with the content of the substance to be detected in the sample.
In the description of this patent, it is to be understood that the terms "center," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like are used in the orientations and positional relationships indicated in the drawings for the convenience of describing the patent and for the simplicity of description, and are not intended to indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the patent.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A detection method for tetrahydrofolic acid production is characterized by comprising the following steps:
s1: preparing a test agent and related detection materials; 1-3 parts of a test agent, an enzyme label plate with a blank hole, a standard hole and a sample hole to be tested, a sealing plate film, an FA enzyme label, a concentrated washing solution, a developing solution A, a developing solution B and a stop solution;
s2: diluting a test agent to be tested into concentrated sample diluent;
s3: adding a sample; adding the concentrated sample diluent in the S2 into a blank hole, a standard hole and a sample hole to be detected of the ELISA plate;
s4: storing the sealing plate; sealing the ELISA plate with a sealing plate film and preserving at constant temperature;
s5: returning the temperature to prepare the liquid; diluting the concentrated washing liquid with distilled water for later use;
s6: washing liquor for washing plates; removing the sealing plate film, discarding liquid, spin-drying, filling the cleaning solution in S5 in each hole, standing for 20-30 seconds, discarding, repeating the operation for 3-5 times, and patting to dry;
s7: adding enzyme, and adding FA enzyme label into the standard hole and the sample hole to be detected;
s8: temperature return treatment: sealing the ELISA plate in S7 with a sealing plate film, and preserving at constant temperature;
s9: secondary cleaning; removing the sealing plate film, discarding liquid, spin-drying, filling the cleaning solution in S5 in each hole, standing for 20-30 seconds, discarding, repeating the operation for 3-5 times, and patting to dry;
s10: color development treatment: adding the color developing solution A into each hole, adding the color developing solution B, slightly vibrating, standing, and shading at constant temperature for color development;
s11: stopping, and finally adding a stopping solution to stop the reaction;
s12: the measurement was carried out by zeroing a blank well and measuring the absorbance (OD value) of each well in order at a wavelength of 450 nm.
2. The method for detecting the production of tetrahydrofolic acid according to claim 1, characterized in that: in the step S1, 3 parts of the test agent are taken, and in the step S2, the test agent is diluted into a concentrated sample diluent 1, a concentrated sample diluent 2 and a concentrated sample diluent 3.
3. The method for detecting the production of tetrahydrofolic acid according to claim 1, characterized in that: and in the S3 process, when the concentrated sample diluent is added, the concentrated sample diluent is ensured not to touch the hole wall, and after the concentrated sample diluent is added, the ELISA plate is slightly shaken.
4. The method for detecting the production of tetrahydrofolic acid according to claim 1, characterized in that: in the S4 process, the preservation temperature is controlled to be 30-45 ℃, and the preservation time is controlled to be 20-40 min.
5. The method for detecting the production of tetrahydrofolic acid according to claim 1, characterized in that: in the S5 process, the dilution multiple of the concentrated washing solution is 25-35 times.
6. The method for detecting the production of tetrahydrofolic acid according to claim 1, characterized in that: in the S7, the addition content of the FA enzyme label is between 30ul and 60 ul.
7. The method for detecting the production of tetrahydrofolic acid according to claim 1, characterized in that: in the S8 process, the preservation temperature is controlled to be 30-45 ℃, and the preservation time is controlled to be 20-40 min.
8. The method for detecting the production of tetrahydrofolic acid according to claim 1, characterized in that: in the S10 process, the adding content of the color development liquid A and the color development liquid B is between 30ul and 60ul, the adding content of the color development liquid A and the color development liquid B is the same, and the constant-temperature shading and color development time is between 10 and 20 min.
9. The method for detecting the production of tetrahydrofolic acid according to claim 1, characterized in that: in the S11 process, the addition content of the stop solution is between 30ul and 60 ul.
10. The method for detecting the production of tetrahydrofolic acid according to claim 1, characterized in that: in the process of S12, the determination should be carried out within 10-15min of adding the stop solution.
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2020
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CN1621808A (en) * | 2004-12-10 | 2005-06-01 | 张家港市国泰华荣化工新材料有限公司 | Method for measuring lithium salt in lithium ion battery electrolyte |
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CN103777021A (en) * | 2012-10-18 | 2014-05-07 | 辽宁成大生物股份有限公司 | Rabies vaccine glycoprotein content detection method |
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