CN112683885A - 5-methyltetrahydrofolate chemiluminescence detection kit and preparation method thereof - Google Patents
5-methyltetrahydrofolate chemiluminescence detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention provides a chemiluminescence detection kit for 5-methyltetrahydrofolate, which comprises: a first reagent, a second reagent, a third reagent, and a fourth reagent; wherein the first reagent is a 5-methyltetrahydrofolate-alkaline phosphatase conjugate, the second reagent is an anti-5-methyltetrahydrofolate antibody-magnetic microparticle working solution, the third reagent is a cleaning buffer solution, and the fourth reagent is an enzymatic luminescent substrate solution or a direct luminescent substrate solution. The kit is used for full-automatic detection of the content of 5-methyltetrahydrofolic acid in a human body by a chemiluminescence immunoassay technology.
Description
Technical Field
The invention relates to the technical field of chemiluminescence detection kits, and particularly relates to a 5-methyltetrahydrofolate chemiluminescence detection kit and a preparation method thereof.
Background
Folic acid is widely found in various foods, but natural foods contain much folic acid in the form of glutamic acid, and the content of folic acid is not high. With the progress of research, the importance of folic acid in the diet is increasingly recognized. But the actual folic acid existing in the active form in the human body is 5-methyltetrahydrofolic acid. 5-methyltetrahydrofolate is the predominant form of folate present in serum and is the only pathway for folate to participate in physiological metabolism. A paper published in PLOS ONE in 2013 analyzes the mutation rate of the folate metabolism genes of 15357 Han people in 10 regions in China. The results show that: MTHFR gene mutant TT (enzyme activity 35%) accounts for 29.8% of the total population, and the part of people cannot completely metabolize the ingested Folic Acid (Folic Acid) to convert the ingested Folic Acid into truly useful 5-methyltetrahydrofolic Acid, and even after a large amount of Folic Acid is synthesized, the Folic Acid can be insufficiently converted due to low activity of the converting enzyme. Therefore, the detection of the level of 5-methyltetrahydrofolate in human bodies can more accurately diagnose the folate deficiency and guide the scientific vitamin supplement than the detection of only folic acid.
At present, the detection method for 5-methyltetrahydrofolic acid on the market is mainly a laboratory analytical chemistry type means, such as EILSA, liquid chromatography, mass spectrometry and other methods. However, the existing 5-methyltetrahydrofolate detection methods have certain limitations, such as complex detection experiment steps, different experiment operation standards of different detection mechanisms, long time consumption, special equipment, high detection cost, incapability of realizing full-automatic high-throughput analysis and the like, and are not suitable for the determination of large-batch clinical samples. At present, 5-methyltetrahydrofolate detection reagents with good stability, high sensitivity and strong specificity, in particular to full-automatic detection reagents with good quality, are lacked in the market. The chemiluminescence method is an accurate, rapid and low-cost full-automatic detection method based on the antigen-antibody reaction principle, plays more and more important roles in the field of medical detection and clinical diagnosis in recent years, but the existing folic acid chemiluminescence detection reagent on the market aims at total folic acid rather than active folic acid. Therefore, the development of a chemiluminescence kit for specifically detecting the level of 5-methyltetrahydrofolate in a human body is particularly important for clinically diagnosing the folate deficiency and accurately treating the folate deficiency.
Disclosure of Invention
In order to solve the problems, the application provides a chemiluminescence detection kit for 5-methyltetrahydrofolate, which is used for full-automatic detection of the content of 5-methyltetrahydrofolate in a human body by a chemiluminescence immunoassay technology.
In order to achieve the above purpose, the invention provides the following technical scheme:
according to a first aspect, the present invention provides a chemiluminescent detection kit for 5-methyltetrahydrofolate, comprising: a first reagent, a second reagent, a third reagent, and a fourth reagent;
specifically, the first reagent is a 5-methyltetrahydrofolate-alkaline phosphatase connector or a 5-methyltetrahydrofolate-acridinium ester connector;
specifically, the second reagent is any one of anti-5-methyltetrahydrofolate antibody-biotin-streptavidin-magnetic microparticles, anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead working solution and anti-5-methyltetrahydrofolate antibody-carboxyl magnetic bead working solution;
specifically, the third reagent is a washing buffer;
specifically, the fourth reagent is substrate solution for enzymatic luminescence or substrate solution for direct luminescence.
According to a second aspect, the present invention also provides a method for preparing a chemiluminescence detection kit for 5-methyltetrahydrofolate, comprising:
in some embodiments, an anti-5-methyltetrahydrofolate antibody-acridinium ester linker prepared by reacting an anti-5-methyltetrahydrofolate antibody with an acridinium ester is used as a first reagent;
in some embodiments, a 5-methyltetrahydrofolate-alkaline phosphatase conjugate prepared by the EDC/NHS method is used as the first reagent;
in some embodiments, an anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead working solution is prepared as a second reagent by reacting an anti-5-methyltetrahydrofolate antibody with a Tosyl magnetic bead;
in some embodiments, a 5-methyltetrahydrofolate antibody-carboxyl magnetic bead working solution is prepared as a second reagent by reacting an anti-5-methyltetrahydrofolate antibody with a carboxyl magnetic bead;
in some embodiments, anti-5-methyltetrahydrofolate antibody-biotin-streptavidin-magnetic microparticles are prepared as a second reagent by reacting an anti-5-methyltetrahydrofolate antibody with magnetic microparticles;
in some embodiments, the surfactant is mixed with phosphate buffer, tween-20 to prepare a wash buffer as the third reagent;
in some embodiments, substrate solution a and substrate solution B are combined into a fourth reagent.
Preferably, the step of preparing the first reagent comprises:
s11, dissolving 5-methyltetrahydrofolic acid in DMSO to obtain a 5-methyltetrahydrofolic acid solution;
s12, dissolving acridine ester in a DMF/DMSO solution to obtain an acridine ester solution;
s13, mixing the obtained 5-methyltetrahydrofolate solution and the acridinium ester solution, and purifying to obtain the 5-methyltetrahydrofolate-acridinium ester connector.
Preferably, the step of preparing the first reagent further comprises:
s11', dissolving 5-methyltetrahydrofolic acid in dimethyl sulfoxide, and adding EDC and NHS for activation reaction to prepare an activated 5-methyltetrahydrofolic acid solution;
s12', adding 5-methyltetrahydrofolate solution into alkaline phosphatase, activating at room temperature for 1 hour, and purifying to obtain the first reagent.
Preferably, the step of preparing the second reagent comprises:
s21, respectively dissolving the anti-5-methyltetrahydrofolate antibody and the Tosyl magnetic bead in a boric acid buffer solution to obtain a 5-methyltetrahydrofolate antibody solution and a Tosyl magnetic bead solution;
s22, mixing the 5-methyltetrahydrofolate antibody solution and the Tosyl magnetic bead solution, carrying out a blocking reaction by using a borate buffer solution, washing the Tosyl magnetic bead by using a PBS buffer solution after the reaction is finished to obtain an anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead, and preparing the anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead into the second reagent by using a Tris buffer solution.
Preferably, the step of preparing the second reagent further comprises:
s31, dissolving the 5-methyltetrahydrofolate antibody by using PBS buffer solution to prepare an anti-5-methyltetrahydrofolate antibody solution, and preparing carboxyl magnetic beads by using EDC (EDC);
s32, mixing the anti-5-methyltetrahydrofolate antibody solution and the carboxyl magnetic bead solution, carrying out a blocking reaction by using a PBS buffer solution, washing the carboxyl magnetic beads by using the PBS buffer solution after the reaction is finished to obtain anti-5-methyltetrahydrofolate antibody-carboxyl magnetic beads, and preparing the anti-5-methyltetrahydrofolate antibody-carboxyl magnetic beads into the second reagent by using a Tris buffer solution.
Preferably, the step of preparing the second reagent further comprises:
s41, preparing an anti-5-methyltetrahydrofolate antibody solution and a biotin-N-succinimidyl ester solution, mixing the anti-5-methyltetrahydrofolate antibody solution and the biotin-N-succinimidyl ester solution, reacting at room temperature for 1 hour, and then purifying to obtain a biotin-labeled anti-5-methyltetrahydrofolate antibody;
s42, resuspending the streptavidin-coated magnetic particles with PBS buffer solution, adding the biotin-labeled anti-5-methyltetrahydrofolate antibody for reaction, washing away unbound other substances to obtain anti-5-methyltetrahydrofolate antibody-biotin-streptavidin-magnetic particles, and preparing the anti-5-methyltetrahydrofolate antibody-biotin-streptavidin-magnetic particles with the PBS buffer solution to obtain the second reagent.
Preferably, the preparation method of the fourth reagent comprises the steps of:
s51, preparing a mixed solution of hydrogen peroxide and nitric acid by using a Tris-HCl solution as a substrate solution A;
s52, preparing a sodium hydroxide solution as a substrate solution B by using a Tris-HCl solution;
and S53, the substrate solution A and the substrate solution B are combined into a fourth reagent.
Preferably, the preparation method of the fourth reagent further comprises the steps of:
diluting the adamantane AMPPD substrate solution with a PBS buffer solution to prepare an enzymatic luminescent substrate solution, wherein the enzymatic luminescent substrate solution is a fourth reagent.
In some embodiments, the kit uses PBS buffer of 0.05M, pH7.4, the PBS buffer also contains 0.5% bovine serum albumin.
In some embodiments, the borate buffer used in the kit is 0.1M, pH9.5, and further comprises 2% bovine serum albumin.
The chemiluminescence detection kit for 5-methyltetrahydrofolate provided by the invention has the following beneficial effects:
5-methyltetrahydrofolate and a 5-methyltetrahydrofolate-alkaline phosphatase linker in a sample to be detected compete for binding an anti-5-methyltetrahydrofolate antibody on a magnetic particle, the anti-5-methyltetrahydrofolate antibody is bound with the anti-5-methyltetrahydrofolate-alkaline phosphatase linker and then is connected on the magnetic particle, after the reaction is completed, a magnetic bead is adsorbed by a magnetic field, unbound substances are washed away, an enzymatic chemiluminescent substrate is added, the substrate is catalytically cracked under the action of enzyme to form an unstable excited state intermediate, when the excited state intermediate returns to a ground state, photons are emitted to form a luminescence reaction, and the luminescence intensity of the reaction is measured by using a luminometer. The luminous intensity is inversely proportional to the concentration of the 5-methyltetrahydrofolate in the sample, and the concentration of the 5-methyltetrahydrofolate in the sample to be detected can be quantitatively measured and calculated by using improved four-parameter Logistic equation fitting.
Drawings
FIG. 1 is a test graph of a 5-methyltetrahydrofolate standard;
FIG. 2 shows the molecular formula of 5-methyltetrahydrofolate;
FIG. 3 is a graph showing the correlation between the results of the chemiluminescence immunoassay detection method for 5-methyltetrahydrofolate and the serum test results of the LC-MS.
Detailed Description
The invention discloses a chemiluminescence detection kit and a detection method for 5-methyltetrahydrofolate, and a person skilled in the art can realize the detection by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
The materials or reagents used in the chemiluminescence detection kit for 5-methyltetrahydrofolate provided by the invention can be purchased from the market. Wherein, the full-automatic chemiluminescence immunoassay analyzer is a Cosmei SMART 500S full-automatic chemiluminescence immunoassay analyzer.
Example one
The embodiment provides a chemiluminescent detection kit for 5-methyltetrahydrofolate, comprising: a first reagent, a second reagent, a third reagent, and a fourth reagent;
wherein the first reagent is a 5-methyltetrahydrofolate-alkaline phosphatase conjugate or a 5-methyltetrahydrofolate-acridinium ester conjugate; the second reagent is any one of anti-5-methyltetrahydrofolate antibody-biotin-streptavidin-magnetic microparticles, anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead working solution and anti-5-methyltetrahydrofolate antibody-carboxyl magnetic bead working solution; the third reagent is a washing buffer solution; the fourth reagent is substrate liquid for enzymatic luminescence or substrate liquid for direct luminescence.
Specifically, the preparation method of the first reagent comprises the following steps:
1mg of 5-methyltetrahydrofolic acid was weighed and dissolved in 1mL of dimethyl sulfoxide at room temperature to a final concentration of 1 mg/mL. 0.1mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 0.1mg of N-hydroxysuccinimide (NHS) were added to the above solutions to carry out activation reaction, and the mixture was mixed at room temperature for 20 min. After completion of the reaction, 200. mu.l of the activator was added with 1mg of alkaline phosphatase and reacted at room temperature for 60 min. Purifying the above ligation product by G-25 gel chromatography column to obtain a final product 5-methyltetrahydrofolate-alkaline phosphatase conjugate, and storing at 2-8 deg.C.
The prepared 5-methyltetrahydrofolate-alkaline phosphatase conjugate is added into PBS buffer (containing 0.5% of bovine serum albumin, 0.9% of sodium chloride, 0.1% of tween-20 and 0.05% of biological preservative Proclin 300) with the pH of 7.4 and the volume ratio of the 5-methyltetrahydrofolate-alkaline phosphatase conjugate to the PBS buffer is 1: 100-1: 20000. The volume ratio of the 5-methyltetrahydrofolate-alkaline phosphatase to the PBS buffer can also be preferably 1:200, 1:500, 1:3000, 1:5000, 1:10000, 1:15000, or more preferably 1: 2000.
The method for preparing the first reagent may also comprise the steps of:
1mg of 5-methyltetrahydrofolic acid was weighed and dissolved in 1mL of DMSO at room temperature to a final concentration of 1 mg/mL. Dissolving 0.05mg acridinium ester with 20.0uL DMF/DMSO, and mixing; taking out the dissolved 5-methyltetrahydrofolic acid to a centrifuge tube; adding the dissolved acridinium ester, oscillating and uniformly mixing, and then rotating and uniformly mixing at 37 ℃ for reaction for 1 h; 0.07mL of a reaction terminating solution (0.1 g/mL of lysine) was added thereto, and the mixture was shaken and mixed, followed by reaction at 37 ℃ for 30 min. Purifying the mixture by using a G-25 gel chromatography column to obtain a 5-methyltetrahydrofolate-acridinium ester connector, and storing at the temperature of 2-8 ℃ for later use.
The prepared 5-methyltetrahydrofolate-acridinium ester connector is added into PBS buffer (containing 0.5% of bovine serum albumin, 0.9% of sodium chloride, 0.1% of tween-20 and 0.05% of biological preservative Proclin 300) with the pH value of 7.4M, and the volume ratio of the connector to the PBS buffer is 1: 500-1: 20000. The volume ratio of the 5-methyltetrahydrofolate-acridinium ester to the PBS buffer can also be preferably 1:200, 1:500, 1:3000, 1:5000, 1:10000, 1:15000 or more preferably 1: 2000.
The preparation method of the second reagent comprises the following steps:
0.2mg of an anti-5-methyltetrahydrofolate antibody was diluted to 1mg/ml with 0.1M borate buffer, pH 9.5. Weighing 5mg of Tosyl magnetic beads, washing with 0.1M, pH9.5 borate buffer solution 3 times, resuspending the magnetic beads in 20mg/ml of mother liquor with 0.1M, pH9.5 borate buffer solution, mixing the two solutions, and adding 3M (NH)4)2SO4The solution was mixed in 400. mu.l at 37 ℃ overnight and reacted for 18 hours. After the reaction is finished, the magnetic microparticles are sealed by 0.1M borate buffer solution (containing 2% bovine serum albumin) with pH9.5, and are mixed uniformly at 37 ℃ for reaction for 4 hours. After the reaction is finished, the magnetic particles are washed for 5 times by PBS buffer solution with pH7.4, and other substances which are not combined are washed, so that the magnetic particles coated by the anti-5-methyltetrahydrofolate antibody are obtained. The anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead is prepared into 0.1-1 mg/ml working solution by using a Tris buffer solution with the pH value of 7.4, and the working solution is stored at the temperature of 2-8 ℃ for later use. The concentration of the working solution of the anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead can also be 0.2mg/ml, or 0.3mg/ml, or 0.5mg/ml, or 0.8mg/ml or more preferably 0.15 mg/ml.
The method for preparing the second reagent may also comprise the steps of:
0.5mg of an anti-5-methyltetrahydrofolate antibody was diluted to 1mg/ml with 0.2M PBS buffer, pH 7.4. 1mg of biotin-N-succinimidyl ester was weighed and dissolved in 1mL of dimethyl sulfoxide at room temperature. To the anti-5-methyltetrahydrofolate antibody, 5. mu.l of 10mg/ml biotin-N-succinimidyl ester was added and the reaction was carried out at room temperature for 1 hour. After the reaction is finished, the connecting product is purified by a G-25 gel chromatography column, and the obtained final product is the biotin-labeled anti-5-methyltetrahydrofolate antibody mother liquor. The mother solution was diluted to 1ug/ml of the working solution of biotin labeling substance with 0.02M PBS buffer, pH 7.4. The concentration of the working solution of the anti-5-methyltetrahydrofolate antibody-biotin can also be 0.5ug/ml, or 0.75 ug/ml, or 1ug/ml, or 1.5ug/ml or more preferably 1 ug/ml. Washing 5mg of streptavidin-coated magnetic particles (purchased from outsourced manufacturers) for 3 times by using PBS buffer solution with the concentration of 0.02M and the pH value of 7.4, preparing streptavidin magnetic particle mother liquor with the concentration of 5mg/ml by using the PBS buffer solution, adding the biotin-labeled anti-5-methyltetrahydrofolate antibody (the mass ratio of the antibody to the magnetic beads is 1:50, or 1:100 or 1: 200) into the mother liquor, reacting for 12-18h at the temperature of 2-8 ℃, washing for 3 times, and re-suspending to 0.15mg/ml by using PBS with the concentration of 0.05M and the pH value of 7.4 to obtain second reagent working solution. The working solution concentration may also be 0.2mg/ml, or 0.3mg/ml, or 0.5mg/ml, or 0.8mg/ml or more preferably 0.15 mg/ml. The method for preparing the second reagent may also comprise the steps of:
0.2mg of an anti-5-methyltetrahydrofolate antibody was diluted to 1mg/ml with 0.1M borate buffer, pH 9.5. 5mg of Tosyl magnetic beads (purchased from outsourced) were washed 3 times with 0.1M, pH9.5 borate buffer, and the mother solution in which the magnetic beads were 20mg/ml was resuspended in 0.1M, pH9.5 borate buffer, and then the two were mixed together, and 400. mu.l of 3M (NH4)2SO4 solution was added thereto, and the mixture was mixed together overnight at 37 ℃ for 18 hours. After the reaction is finished, the magnetic microparticles are sealed by 0.1M borate buffer solution (containing 2% bovine serum albumin) with pH9.5, and are mixed uniformly at 37 ℃ for reaction for 4 hours. After the reaction is finished, the magnetic particles are washed for 5 times by PBS buffer solution with pH7.4, and other substances which are not combined are washed, so that the magnetic particles coated by the anti-5-methyltetrahydrofolate antibody are obtained. The anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead is prepared into 0.1-1 mg/ml working solution by using a Tris buffer solution with the pH value of 7.4, and the working solution is stored at the temperature of 2-8 ℃ for later use. The concentration of the working solution of the anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead can also be 0.2mg/ml, or 0.3mg/ml, or 0.5mg/ml, or 0.8mg/ml or more preferably 0.15 mg/ml. The method for preparing the second reagent may also comprise the steps of:
0.5mg of an anti-5-methyltetrahydrofolate antibody was diluted to 1mg/ml with 0.05M PBS buffer, pH7.4. 5mg of carboxyl magnetic beads (purchased from outsourced) are washed by 0.1M MES buffer solution with pH6.0 for 3 times, then the magnetic beads are resuspended to 20mg/mL, EDC (1-Ethyl-3-dimethylamino pyridine carboxylic acid) solution is added to the final concentration of 0.3mg/mL, the mixture is placed on a mixer at 2-8 ℃ and mixed evenly, after reaction for 30min, the magnetic beads are washed by 0.1M MES buffer solution with pH6.0 for 3 times, diluted antibody is added, the mixture is shaken and mixed evenly, and the mixture is placed on the mixer at 2-8 ℃ and mixed evenly for 2 hours. After the reaction is finished, the magnetic particles are washed for 3 times by 0.05M PBS buffer solution with pH7.4, then the magnetic particles are sealed by adding 0.05M PBS buffer solution with pH7.4 (containing 2 percent of bovine serum albumin), and the mixture is placed on a mixer for mixing and sealing for 4 hours at the temperature of 2-8 ℃. After the reaction is finished, the magnetic particles are washed for 3 times by PBS buffer solution with pH7.4, and other substances which are not combined are washed, so that the magnetic particles coated by the anti-5-methyltetrahydrofolate antibody are obtained. And (3) preparing the anti-5-methyltetrahydrofolate antibody-carboxyl magnetic bead microparticles into 0.1-1 mg/ml working solution by using a Tris buffer solution with the pH value of 7.4, and storing at the temperature of 2-8 ℃ for later use. The concentration of the working solution of the anti-5-methyltetrahydrofolate antibody-carboxyl magnetic bead microparticles can also be 0.2mg/ml, or 0.3mg/ml, or 0.5mg/ml, or 0.8mg/ml or more preferably 0.15 mg/ml.
The preparation method of the third reagent comprises the following steps:
weighing Na2HPO4•12H2O 4.37g、NaH2PO4•2H21.22g of O, 9g of NaCI, 5g of Tween-20,
Putting 0.5g of Proclin300 into a beaker, adding 0.8L of purified water, uniformly mixing, adjusting the pH to 7.5, finally fixing the volume to 1L, and storing at 2-8 ℃ for later use.
The preparation method of the fourth reagent comprises the following steps:
a certain amount of adamantane AMPPD substrate solution was measured and diluted 10-fold with 0.02M, pH-7.0 PBS buffer solution to be used as an enzymatic luminescent substrate solution.
The method for preparing the fourth reagent further comprises the steps of:
preparing substrate solution A by using 0.01mol/LpH7.5 Tris-Hl solution to prepare hydrogen peroxide with the concentration of 0.01mol/L and nitric acid solution with the concentration of 0.2 mol/L; 0.01mol/L Tris-HCl solution with pH7.5 is used to prepare a solution containing 0.1g/L sodium hydroxide to prepare a substrate solution B.
Example two
The embodiment provides a method for detecting the content of 5-methyltetrahydrofolate in a sample by using a 5-methyltetrahydrofolate chemiluminescence detection kit, which comprises the following specific steps:
adding 50 mul of sample or standard substance to be detected, 50 mul of first reagent and 50 mul of second reagent into a reaction hole for reaction, incubating for 5min at 37 ℃, taking 300 mul of third reagent for magnetic separation and cleaning for 3 times after the reaction is finished, adding 100 mul of fourth reagent after the cleaning is finished, and loading the fourth reagent on a SMART 500S full-automatic chemiluminescence immunoassay analyzer for detection.
Establishment of a standard curve: 5-methyltetrahydrofolate standard (NIST 1950, concentration of 12.36 +/-0.32 ng/ml) is prepared into standard working solution of 0, 1, 2.5, 5.0, 10.0 and 12.36ng/ml by adopting commercial human serum matrix, and the 5-methyltetrahydrofolate detection reagent is adopted for detection. The results of the standard curve test are shown in FIG. 1.
The basic performance of the reagents was tested:
(1) accuracy evaluation:
the deviation between the mean of the NIST 19503, 3 measurements and the target value was measured after scaling the reagents according to the standard curve, as shown in table 1:
TABLE 1 WHO Standard NIST1950 test results for 5-methyltetrahydrofolate
From the results in table 1, it can be seen that: the 5-methyltetrahydrofolic acid in the international standard NIST1950 is measured by adopting the 5-methyltetrahydrofolic acid detection reagent, the deviation is within +/-5 percent, and the measurement accuracy is higher.
(2) And (3) precision evaluation:
the 5-methyltetrahydrofolic acid pure product is diluted by commercial human serum to final concentrations of 5.0 ng/ml and 10.0ng/ml respectively, so as to form low-concentration and high-concentration repetitive samples. The measurement was repeated 10 times on the above-mentioned low and high concentration repetitive samples using the above-mentioned chemiluminescence immunoassay detection method for 5-methyltetrahydrofolate and the calibration standard curve of fig. 1, and the average value and the coefficient of variation were calculated.
TABLE 2 results of precision measurements
From the results in table 2, it can be seen that: the 5-methyltetrahydrofolic acid detection reagent is adopted to determine the variation coefficients of 5-methyltetrahydrofolic acid in different concentration levels to be less than 6 percent, and the precision is higher.
(3) And (3) specific evaluation:
using the chemiluminescence immunoassay detection method of 5-methyltetrahydrofolate and the calibration standard curve of FIG. 1, 750ng/mL tetrahydrofolate, 750ng/mL folic acid, 750ng/mL dihydrofolate and 5000ng/mL vitamin B12 pure products were measured, and the cross-reactivity ratios were calculated as follows.
Table 3 results of analogue cross-reaction testing
From the results in Table 3, it can be seen that: when the 5-methyltetrahydrofolic acid detection reagent is used for detecting higher-concentration analogs such as tetrahydrofolic acid, dihydrofolic acid, folic acid and VB12, the cross reaction rate is lower than 1 percent, and the specificity is higher.
(4) Comparison with serum sample test results of liquid phase secondary mass spectrometry (LC-MS/MS):
the mass spectrometry method is a universal analysis method with high specificity, high sensitivity and high accuracy, and can accurately measure the content of substances in a sample. Now, 52 clinical serum samples are synchronously tested by using a liquid phase secondary mass spectrometry method and the 5-methyltetrahydrofolate chemiluminescence immunoassay detection method, and the test results of the two detection methods are subjected to correlation comparative analysis.
For sexual analysis, the data determined is shown in Table 4 and for comparative analysis of the correlation is shown in FIG. 3.
TABLE 45 comparison of Methyltetrahydrofolate chemiluminescence to LC-MS/MS methodology
From the results of table 4 and fig. 3, it can be seen that: the result of a methodology comparison between 52 serum samples measured by the 5-methyltetrahydrofolate detection reagent and a liquid-phase secondary mass spectrometry shows that the correlation coefficient R is greater than 0.9, and the correlation is high.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (11)
1. A chemiluminescence detection kit for 5-methyltetrahydrofolate is characterized by comprising: a first reagent, a second reagent, a third reagent, and a fourth reagent;
the first reagent is a 5-methyltetrahydrofolate-alkaline phosphatase connector or a 5-methyltetrahydrofolate-acridinium ester connector;
the second reagent is any one of anti-5-methyltetrahydrofolate antibody-biotin-streptavidin-magnetic microparticles, anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead working solution and anti-5-methyltetrahydrofolate antibody-carboxyl magnetic bead working solution;
the third reagent is a washing buffer solution;
the fourth reagent is substrate liquid for enzymatic luminescence or substrate liquid for direct luminescence.
2. The preparation method of the chemiluminescence detection kit for 5-methyltetrahydrofolate is characterized by comprising the following steps:
the method comprises the following steps of (1) preparing an anti-5-methyltetrahydrofolate antibody-acridinium ester connector serving as a first reagent through a reaction of the anti-5-methyltetrahydrofolate antibody and acridinium ester;
5-methyltetrahydrofolate-alkaline phosphatase conjugate prepared by a mixed anhydride method as a first reagent;
the working solution of the anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead prepared by the reaction of the anti-5-methyltetrahydrofolate antibody and the Tosyl magnetic bead is used as a second reagent;
5-methyltetrahydrofolate antibody-carboxyl magnetic bead working solution prepared by reacting an anti-5-methyltetrahydrofolate antibody with carboxyl magnetic beads is used as a second reagent;
the anti-5-methyltetrahydrofolate antibody-biotin-streptavidin-magnetic microparticles prepared by reacting the anti-5-methyltetrahydrofolate antibody with the magnetic microparticles are used as a second reagent;
a cleaning buffer solution prepared by mixing a surfactant with phosphate buffer solution and Tween-20 is used as a third reagent;
the substrate solution A and the substrate solution B are combined to form a fourth reagent.
3. The method for preparing a chemiluminescent detection kit for 5-methyltetrahydrofolate according to claim 2, wherein the step of preparing the first reagent comprises:
s11, dissolving 5-methyltetrahydrofolic acid in DMSO to obtain a 5-methyltetrahydrofolic acid solution;
s12, dissolving acridine ester in a DMF/DMSO solution to obtain an acridine ester solution;
s13, mixing the 5-methyltetrahydrofolate solution and the acridinium ester solution, and purifying to obtain the 5-methyltetrahydrofolate-acridinium ester connector.
4. The method for preparing a chemiluminescent detection kit for 5-methyltetrahydrofolate according to claim 2, wherein the step of preparing the first reagent further comprises:
s11', dissolving 5-methyltetrahydrofolic acid in dimethyl sulfoxide, and adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) for activation reaction to prepare an activated 5-methyltetrahydrofolic acid solution;
s12', adding 5-methyltetrahydrofolate solution into alkaline phosphatase, activating at room temperature for 1 hour, and purifying to obtain the first reagent.
5. The method for preparing a chemiluminescent detection kit for 5-methyltetrahydrofolate according to claim 2, wherein the step of preparing the second reagent comprises:
s21, respectively dissolving the anti-5-methyltetrahydrofolate antibody and the Tosyl magnetic bead in a boric acid buffer solution to obtain a 5-methyltetrahydrofolate antibody solution and a Tosyl magnetic bead solution;
s22, mixing the 5-methyltetrahydrofolate antibody solution and the Tosyl magnetic bead solution, carrying out a blocking reaction by using a borate buffer solution, washing the Tosyl magnetic bead by using a PBS buffer solution after the reaction is finished to obtain an anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead, and preparing the anti-5-methyltetrahydrofolate antibody-Tosyl magnetic bead into the second reagent by using a Tris buffer solution.
6. The method for preparing a chemiluminescent detection kit for 5-methyltetrahydrofolate according to claim 2, wherein the step of preparing the second reagent further comprises:
s31, dissolving the 5-methyltetrahydrofolate antibody by using PBS buffer solution to prepare an anti-5-methyltetrahydrofolate antibody solution, and preparing carboxyl magnetic beads by using EDC (EDC);
s32, mixing the anti-5-methyltetrahydrofolate antibody solution and the carboxyl magnetic bead solution, carrying out a blocking reaction by using a PBS buffer solution, washing the carboxyl magnetic beads by using the PBS buffer solution after the reaction is finished to obtain anti-5-methyltetrahydrofolate antibody-carboxyl magnetic beads, and preparing the anti-5-methyltetrahydrofolate antibody-carboxyl magnetic beads into the second reagent by using a Tris buffer solution.
7. The method for preparing a chemiluminescent detection kit for 5-methyltetrahydrofolate according to claim 2, wherein the step of preparing the second reagent further comprises:
s41, preparing an anti-5-methyltetrahydrofolate antibody solution and a biotin-N-succinimidyl ester solution, mixing the anti-5-methyltetrahydrofolate antibody solution and the biotin-N-succinimidyl ester solution, reacting at room temperature for 1 hour, and then purifying to obtain a biotin-labeled anti-5-methyltetrahydrofolate antibody;
s42, resuspending the streptavidin-coated magnetic particles with PBS buffer solution, adding the biotin-labeled anti-5-methyltetrahydrofolate antibody for reaction, washing away unbound other substances to obtain anti-5-methyltetrahydrofolate antibody-biotin-streptavidin-magnetic particles, and preparing the anti-5-methyltetrahydrofolate antibody-biotin-streptavidin-magnetic particles with the PBS buffer solution to obtain the second reagent.
8. The method for preparing a chemiluminescence detection kit according to claim 2, wherein the method for preparing the fourth reagent comprises the steps of:
s51, preparing a mixed solution of hydrogen peroxide and nitric acid by using a Tris-HCl solution as a substrate solution A;
s52, preparing a sodium hydroxide solution as a substrate solution B by using a Tris-HCl solution;
and S53, the substrate solution A and the substrate solution B are combined into a fourth reagent.
9. The method for preparing a chemiluminescent detection kit for 5-methyltetrahydrofolate according to claim 2, wherein the method for preparing the fourth reagent further comprises the steps of:
diluting the adamantane AMPPD substrate solution with a PBS buffer solution to prepare an enzymatic luminescent substrate solution, wherein the enzymatic luminescent substrate solution is a fourth reagent.
10. The method for preparing a 5-methyltetrahydrofolate chemiluminescence detection kit according to claim 5 or 6, wherein the PBS buffer is 0.05M PBS buffer, pH7.4, and further comprises 0.5% bovine serum albumin.
11. The method for preparing a 5-methyltetrahydrofolate chemiluminescence detection kit according to claim 5, wherein the borate buffer is 0.1M, ph9.5, and further comprises 2% bovine serum albumin.
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