CN113325171A - Kit for detecting human body erythrocyte folic acid content, detection method and application - Google Patents
Kit for detecting human body erythrocyte folic acid content, detection method and application Download PDFInfo
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Abstract
The invention relates to a kit for detecting human body erythrocyte folic acid content, a detection method and application. The invention provides a kit for detecting human body erythrocyte folic acid content, which comprises a whole blood sample treatment reagent, a precipitation solution and an immunochromatography test paper card; the whole blood sample treatment reagent comprises an antioxidant, a surfactant and a reaction buffer solution, the immunochromatography test paper card comprises a supporting plate, a sample pad, a combination pad, a nitrocellulose membrane detection area and a water absorption pad are sequentially stuck on the supporting plate, and a labeled folic acid monoclonal antibody is adsorbed on the combination pad. The invention adopts the immunochromatography technology, and through screening antibodies, optimizing sample treatment reagents and the like, the provided kit can realize the effect of simply, low-cost and quickly detecting the folic acid content of the red blood cells, effectively solves the defects of complex operation or expensive instruments of the existing means, and fills the blank of measuring the folic acid content of the red blood cells by the immunochromatography method.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a kit for detecting human body erythrocyte folic acid content, a detection method and application.
Background
Folic acid is a water-soluble vitamin, is essential for the growth and reproduction of human cells, can be used for treating anemia caused by folic acid deficiency, and is also a nutrient supplement for pregnant women. Over 50% of neonatal Neural Tube Defects (NTDs) cases are associated with folate insufficiency during the early stages of pregnancy, and folate supplementation can significantly reduce the incidence of NTDs. The long-term reasonable folic acid supplementation also helps to reduce the occurrence risk of cardiovascular and cerebrovascular diseases.
Serum folic acid is considered as an index reflecting the recent folic acid nutrition status, and the detection of the serum folic acid level alone cannot distinguish the state of insufficient folic acid intake of the transient diet from the state of chronic folic acid deficiency; the level of the folic acid of the red blood cells can reflect the nutrition status of the folic acid in chronic or long-term (within 4 months), and is more suitable for evaluating the intervention effect of the folic acid.
The common methods for detecting the folic acid of the red blood cells at present comprise a microbiological method, a mass spectrometry method and a chemiluminescence method. Among them, the operation of the microbiological method and the mass spectrometry is complicated, so the chemiluminescence method is the most practical method in recent years, but the method needs a chemiluminescence analyzer, and the instrument is expensive and has high detection cost.
Disclosure of Invention
The invention aims to provide a kit for detecting human body erythrocyte folic acid content, a detection method and application, solves the defects that the detection operation of the detection kit in the prior art is complex or a detection instrument is expensive, fills the blank of measuring the erythrocyte folic acid content by an immunochromatography method, is more suitable for the requirement of people on folic acid screening, and is suitable for popularization and application in basic units or general resource areas and institutions.
The invention provides a kit for detecting human body erythrocyte folic acid content, which comprises a whole blood sample treatment reagent, a precipitation solution and an immunochromatography test paper card; the whole blood sample treatment reagent comprises an antioxidant, a surfactant and a reaction buffer solution, the immunochromatography test paper card comprises a support plate, a sample pad, a combination pad, a nitrocellulose membrane (NC membrane) detection area and a water absorption pad are sequentially stuck on the support plate, and a labeled folic acid monoclonal antibody is adsorbed on the combination pad. The whole blood sample treatment reagent adopted by the invention can completely destroy red blood cells, so that folic acid components are completely released and dissociated into monoglutamyl folic acid; the folic acid content of whole blood was measured by immunochromatography, and the folic acid content of erythrocytes was calculated by hematocrit conversion. The inventor finds that the provided kit can realize the effect of simply, low-cost and quickly detecting the folic acid content of the red blood cells by adopting the most common immunochromatography technology at present, screening antibodies, optimizing sample treatment reagents and the like, effectively solves the defects that the existing means is complex in operation and expensive in instruments, and fills the blank of measuring the folic acid content of the red blood cells by an immunochromatography method.
In addition, the kit for detecting the human body erythrocyte folic acid content according to the invention can also have the following additional technical characteristics.
According to the invention, the antioxidant comprises one or more of 1% -10% ascorbic acid, 2% -8% vitamin E, 0.5% -5% Dithiothreitol (DTT) and 0.5% -10% tris (2-carboxyethyl) phosphine (TCEP).
According to the invention, the surfactant comprises one or more of 0.01-0.1% of tween-20, 0.01-0.1% of polyethylene glycol octyl phenyl ether (TritonX-100), 0.01-0.1% of saponin and 0.01-0.1% of Sodium Dodecyl Sulfate (SDS).
According to the invention, the precipitation liquid comprises a sodium hydroxide solution; wherein the concentration of the sodium hydroxide solution is 0.5-2M.
According to the invention, the labeled folic acid monoclonal antibody is a latex microsphere labeled folic acid monoclonal antibody.
According to the invention, the nitrocellulose membrane detection zone is coated with folate-BSA and goat anti-mouse.
The invention also provides a detection method of the kit for detecting the human body erythrocyte folic acid content, which comprises the steps of whole blood sample pretreatment and folic acid content determination; wherein the whole blood sample pretreatment comprises: and mixing the whole blood sample with the whole blood sample treatment reagent, incubating in a dark place, adding the precipitation solution, centrifuging, and taking the supernatant to obtain the sample to be detected. The inventor finds that the interference of impurities such as heme and the like in the whole blood sample on the immunochromatographic test paper is greatly solved by adopting the innovative whole blood sample treatment reagent and the treatment method; the detection method provided by the invention is simple, convenient and quick to operate, strong in repeatability, high in precision and low in detection cost.
In addition, the method for detecting the human body erythrocyte folic acid content by using the kit can also have the following additional technical characteristics.
According to the present invention, the ratio of the whole blood sample to the whole blood sample processing reagent by volume is 1: (3-20); incubating in dark for 60-120 min; the volume of the added precipitation solution is 1/10-1/4 of the total volume of the whole blood sample and the whole blood sample processing reagent.
According to the invention, the determination of the folic acid content comprises the steps of: dropwise adding a sample to be detected into a sample hole of a sample pad of the immunochromatography test paper card, dropwise adding an equivalent amount of dilution treatment liquid into a diluent hole in the sample pad, reacting for 10-60 min, placing the immunochromatography test paper card in an immunochromatography analyzer for detection, and calculating the folic acid detection concentration of a hemolyzed erythrocyte according to the following calculation formula to obtain the folic acid concentration of the erythrocyte: erythrocyte folate concentration = folate concentration of the erythrocyte hemolysis product/(% hematocrit) × 100.
The invention also provides application of the kit in detecting the human body erythrocyte folic acid content. The detection kit provided by the invention can meet the requirement of folic acid screening in crowds, and is suitable for popularization and application in basic level units or general resource areas and institutions.
One or more technical schemes provided by the invention at least have the following beneficial effects:
1. the invention adopts immunochromatographic test paper based on nano colored latex microspheres for detecting the folic acid of the red blood cells for the first time, and utilizes an immunochromatographic method to detect the folic acid of the red blood cells, thereby not only avoiding the defect of complicated operation of a microbiological method, but also avoiding the defect of expensive instruments required by a mass spectrometry method and a chemiluminescence method.
2. The invention adopts innovative erythrocyte treating reagent and method, greatly solves the interference of impurities such as heme and the like in a whole blood sample on the immunochromatographic test paper, overcomes the defect that the existing immunochromatographic method can only detect folic acid from serum, and fills the blank of detecting the folic acid content of erythrocytes.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic side sectional view of an immunochromatographic test strip card in example 1 of the present invention;
FIG. 2 is a schematic plan view of an immunochromatographic test strip card in example 1 of the present invention;
FIG. 3 is a graph showing the results of the quantitative curve in example 2 of the present invention;
FIG. 4 is a graph showing the results of precision analysis in example 3 of the present invention.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only examples, and the protection scope of the present invention is not limited thereby.
The invention provides a kit for detecting human body erythrocyte folic acid content, which comprises a whole blood sample treatment reagent, a precipitation solution and an immunochromatography test paper card; the whole blood sample treatment reagent comprises an antioxidant, a surfactant and a reaction buffer solution, as shown in figure 1, the immunochromatography test paper card comprises a support plate, a sample pad, a combination pad, a nitrocellulose membrane (NC membrane) detection area and a water absorption pad are sequentially stuck on the support plate, and a labeled folic acid monoclonal antibody is adsorbed on the combination pad. According to an embodiment of the present invention, in the whole blood sample processing reagent, the antioxidant comprises one or more of 1% -10% ascorbic acid, 2% -8% vitamin E, 0.5% -5% Dithiothreitol (DTT), and 0.5% -10% tris (2-carboxyethyl) phosphine (TCEP); the surfactant comprises one or more of 0.01-0.1% of tween-20, 0.01-0.1% of polyethylene glycol octyl phenyl ether (TritonX-100), 0.01-0.1% of saponin and 0.01-0.1% of Sodium Dodecyl Sulfate (SDS). It should be noted that the above antioxidant and surfactant are aqueous solutions, and the above values are mass volume ratios, for example, 1% to 10% ascorbic acid means that 100mL of aqueous solution contains 1 to 10g ascorbic acid. According to an embodiment of the invention, the precipitation solution is a 0.5-2M solution of sodium hydroxide (NaOH). The kind of the reaction buffer in the whole blood sample processing reagent of the present invention is not particularly limited. According to the embodiment of the invention, the labeled folic acid monoclonal antibody is a latex microsphere labeled folic acid monoclonal antibody; the nitrocellulose membrane detection zone was coated with folate-BSA and goat anti-mouse. The invention greatly solves the interference of impurities such as heme and the like in the whole blood sample on the immunochromatographic test paper by adopting the innovative whole blood sample processing reagent and the antibody. In some embodiments of the present invention, the sample pad is provided with a sample hole and a diluent hole for adding the sample to be tested and the diluent, respectively.
The invention also provides a detection method of the kit for detecting the human body erythrocyte folic acid content, which comprises the steps of whole blood sample pretreatment and folic acid content determination; wherein the whole blood sample pretreatment comprises: and mixing the whole blood sample with the whole blood sample treatment reagent, incubating in a dark place, adding the precipitation solution, centrifuging, and taking the supernatant to obtain the sample to be detected. In an embodiment of the invention, the ratio of the whole blood sample to the whole blood sample processing reagent is 1: (3-20); incubating in dark for 60-120 min; the volume of the added precipitation solution is 1/10-1/4 of the total volume of the whole blood sample and the whole blood sample processing reagent. In an embodiment of the invention, the determination of the folate content comprises the steps of: dropwise adding a sample to be detected into a sample hole of a sample pad of the immunochromatography test paper card, dropwise adding an equivalent amount of dilution treatment liquid into a diluent hole in the sample pad, processing the sample to be detected by the diluent on the sample pad, then moving the sample to be detected to a T line and a C line of an NC membrane through chromatography by a combination pad, placing the immunochromatography test paper card into an immunochromatography analyzer for detection after reacting for 10-60 min, and calculating the folic acid concentration of a hemolyzed product of red blood cells according to the following calculation formula: erythrocyte folate concentration = folate concentration of the erythrocyte hemolysis product/(% hematocrit) × 100. It should be noted that the results should be read within 10 minutes after the end of the reaction timing, otherwise the results are invalid and the experiment should be repeated.
In order to better understand the technical solution, the technical solution will be described in detail with reference to the drawings and the specific embodiments.
Example 1: preparation of immunochromatography test paper card in human body erythrocyte folic acid detection kit
(1) Preparation of NC Membrane detection zone
Cutting the cellulose nitrate detection membrane into strips, placing the strips on a spot spraying instrument platform, and respectively spraying folic acid-BSA and goat anti-mouse on the detection membrane to form a T line and a C line. Drying at 37 ℃ for 2 h.
(2) Preparation of the conjugate pad
And (3) taking 200 mu g of latex microspheres and 4 mu g of folic acid monoclonal antibody, carrying out one-step chemical coupling in MES buffer solution for 2h, and then suspending in PBS buffer solution to prepare the marker. Cutting the glass wool into strips, placing on a spot spraying instrument platform, spraying 90 mu L of the marker on the glass wool, and drying at 37 ℃ for 2 h.
(3) Assembly of test strips
A sample pad, a combination pad, an NC membrane detection area and a water absorption pad are sequentially stuck on a support plate of the test strip, and a side cut-open structure schematic diagram and a plane structure schematic diagram are respectively shown in fig. 1 and fig. 2.
Example 2: quantitative Curve preparation test
(1) The test paper card prepared in example 1 and an immunochromatography analyzer (model: KBE-6000) were used.
(2) The assembled test paper cards are removed, 8, 16, 32, 64, 128 and 256 ng/mL folic acid calibrators are used for measurement by the test paper cards, and the strength value of each calibrator is obtained.
(3) And (3) test results: as shown in FIG. 3, a scattergram was plotted with the logarithmic value of the calibrator concentration as the X-axis and the intensity value as the Y-axis, and a linear analysis was performed, R2>0.98。
Example 3: precision test
(1) The test paper card of the present invention was tested for precision using the test paper card and calibration system of example 2.
(2) Two concentration levels (80 ng/mL, 200 ng/mL) of the reference were tested, each for 10 replicates.
(3) And (3) test results: as shown in fig. 4, after repeating the measurement for the low-value reference and the high-value reference 10 times, the average value, the standard deviation and the CV were calculated, and the obtained results showed that the CV of the low-value reference was 9.7% and the CV of the high-value reference was 12.1%.
Example 4: blood sample measurement test
(1) Collecting a whole blood sample
Whole blood samples collected using EDTA, heparin-based anticoagulant blood collection tubes are recommended. Whole blood samples should be stored protected from light. The whole blood sample can be stored at 2-8 deg.C within 8 hr, and if it needs to be stored for a long time, it should be stored below-20 deg.C, and can be stable for 15 days, and can be frozen and thawed once. The erythrocyte hemolysis product can be stored at 2-8 deg.C within 3 hr, and if it needs to be stored for a long time, it should be stored below-20 deg.C, and can be stabilized for 15 days, and can be frozen and thawed once.
(2) Whole blood sample pretreatment
Adding 12ml sample releaser B (0.01M reaction buffer solution PBS solution, 10% antioxidant TCEP, 0.1% surfactant saponin) into sample releaser A (120 mg antioxidant ascorbic acid), covering the reagent bottle cap, reversing and mixing to ensure complete dissolution of the reagent to obtain the whole blood sample treatment reagent, and reversing and mixing before each use. The prepared whole blood sample processing reagent can be stable for 14 days at the temperature of 2-8 ℃. And (3) taking 100 mu L of whole blood sample, adding 300 mu L of whole blood sample treatment reagent, reversing, uniformly mixing, and standing and incubating for 60 min at 37 ℃ in a dark place. Adding 100 μ L of the precipitation solution, centrifuging at 10000rpm for 1min, and collecting the supernatant to obtain a sample to be detected.
(3) Determination of folic acid content
Taking out the immunochromatography test paper card, dripping 50 mu L of a sample to be detected into a sample hole of the immunochromatography test paper card, dripping 50 mu L of a dilution treatment solution into a dilution solution hole of the immunochromatography test paper card, starting reaction and timing for 20 min. After timing is finished, the immunochromatography test paper card is placed in a matched immunochromatography analyzer for detection, and the analyzer displays a quantitative result immediately. And reading the result within 10 min after the timing is finished, otherwise, if the result is invalid, and carrying out the experiment again. And reading out calibration information by utilizing the product calibration curve information. When the erythrocyte folic acid project is operated, the system reports that the reading is the folic acid detection concentration of an erythrocyte hemolysis product, and then the erythrocyte folic acid concentration is calculated according to the following calculation formula: erythrocyte folate concentration = folate concentration of the erythrocyte hemolysis product/(% hematocrit) × 100.
The present invention has been described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the principle of the present invention, and these changes and modifications also fall into the protection scope of the appended claims.
Claims (10)
1. A kit for detecting human body erythrocyte folic acid content is characterized in that: comprises a whole blood sample processing reagent, a precipitation solution and an immunochromatography test paper card; the whole blood sample treatment reagent comprises an antioxidant, a surfactant and a reaction buffer solution, the immunochromatography test paper card comprises a supporting plate, a sample pad, a combination pad, a nitrocellulose membrane detection area and a water absorption pad are sequentially pasted on the supporting plate, and a labeled folic acid monoclonal antibody is adsorbed on the combination pad.
2. The kit for detecting human body erythrocyte folate content of claim 1, which is characterized in that: the antioxidant comprises one or more of 1-10% of ascorbic acid, 2-8% of vitamin E, 0.5-5% of dithiothreitol and 0.5-10% of tri (2-carboxyethyl) phosphine.
3. The kit for detecting human body erythrocyte folate content of claim 1, which is characterized in that: the surfactant comprises one or more of 0.01-0.1% of tween-20, 0.01-0.1% of polyethylene glycol octyl phenyl ether, 0.01-0.1% of saponin and 0.01-0.1% of sodium dodecyl sulfate.
4. The kit for detecting human body erythrocyte folate content of claim 1, which is characterized in that: the precipitation solution comprises a sodium hydroxide solution; wherein the concentration of the sodium hydroxide solution is 0.5-2M.
5. The kit for detecting human body erythrocyte folate content of claim 1, which is characterized in that: the labeled folic acid monoclonal antibody is a latex microsphere labeled folic acid monoclonal antibody.
6. The kit for detecting human body erythrocyte folate content of claim 1, which is characterized in that: the nitrocellulose membrane detection zone is coated with folic acid-BSA and goat anti-mouse.
7. A method for detecting human body erythrocyte folic acid content kit according to any one of claims 1-6, which is characterized in that: the method comprises the steps of whole blood sample pretreatment and folic acid content determination; wherein the whole blood sample pretreatment comprises: and mixing the whole blood sample with the whole blood sample treatment reagent, incubating in a dark place, adding the precipitation solution, centrifuging, and taking the supernatant to obtain the sample to be detected.
8. The method for detecting the folic acid content of human erythrocytes according to claim 7, which comprises the following steps: the volume ratio of the whole blood sample to the whole blood sample processing reagent is 1: (3-20); the incubation time in dark is 60-120 min; the volume of the added precipitation solution is 1/10-1/4 of the total volume of the whole blood sample and the whole blood sample processing reagent.
9. The method for detecting the folic acid content of human erythrocytes according to claim 7, which comprises the following steps: the folic acid content determination comprises the following steps: dropwise adding the sample to be detected into a sample hole of a sample pad of the immunochromatography test paper card, dropwise adding an equivalent amount of dilution treatment liquid into a diluent hole in the sample pad, reacting for 10-60 min, placing the immunochromatography test paper card in an immunochromatography analyzer for detection, and calculating the folic acid detection concentration of the erythrocyte hemolysis product according to the following calculation formula to obtain the erythrocyte folic acid concentration: erythrocyte folate concentration = folate concentration of the erythrocyte hemolysis product/(% hematocrit) × 100.
10. Use of the kit of any one of claims 1 to 6 for detecting human red blood cell folate content.
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| CN113834944A (en) * | 2021-11-25 | 2021-12-24 | 山东子峰生物技术有限公司 | Quantum dot fluorescence detection method for folic acid in red blood cells |
| CN113933502A (en) * | 2021-10-19 | 2022-01-14 | 青岛汉唐生物科技有限公司 | Detection card and kit for quantitatively detecting folic acid by immunofluorescence chromatography |
| CN117054671A (en) * | 2023-10-10 | 2023-11-14 | 深圳市迈科龙生物技术有限公司 | Dissociation method of folic acid detection sample and folic acid detection method |
| CN117949646A (en) * | 2023-12-31 | 2024-04-30 | 深圳市新产业生物医学工程股份有限公司 | Reagent combination and method for detecting target immunosuppressant in whole blood without centrifugation |
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| CN113933502B (en) * | 2021-10-19 | 2024-02-02 | 青岛汉唐生物科技有限公司 | Detection card and kit for quantitatively detecting folic acid by immunofluorescence chromatography |
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| CN113834944B (en) * | 2021-11-25 | 2022-03-22 | 山东子峰生物技术有限公司 | Quantum dot fluorescence detection method for folic acid in red blood cells |
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| CN117949646A (en) * | 2023-12-31 | 2024-04-30 | 深圳市新产业生物医学工程股份有限公司 | Reagent combination and method for detecting target immunosuppressant in whole blood without centrifugation |
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