Release agent for detecting folic acid content of human body and application of release agent

Abstract

The invention relates to a releasing agent for detecting the folic acid content of a human body and application of the releasing agent, namely a treating fluid R1 is a mixed solution of sodium salicylate, guanidine isothiocyanate and tris (2-formylethyl) phosphine hydrochloride, effective denaturation dissociation can be carried out on protein within the pH value range of 5.0-7.0, the mixed solution can be stably stored, mild, efficient and sufficient dissociation is realized, and the content of folic acid in the human body is detected. The folic acid detection sensitivity is improved. The releasing agent is used in a corresponding immunodetection method so as to solve the technical problems that an existing product is complex in process or incomplete in releasing, poor in sensitivity and repeatability, unstable in treating fluid and unsatisfactory in sample comparison correlation. Through magnetic particle chemiluminescence detection, folic acid and a corresponding detection reagent and a detection method, the sensitivity and repeatability of the reagent and the comparison correlation of samples are better improved, the effect is ideal, and the performance reaches the level of international products. The process is more simplified, large-batch samples can be rapidly detected at high throughput, and clinical application is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of medical in vitro immunodiagnostic reagents, in particular to a release agent for detecting human folic acid content and its application. The release agent is used for detecting folic acid content in serum or heparin plasma, and uses magnetic particle chemiluminescence technology and antigen-antibody binding technology to detect human folic acid content. Background Art

[0002] Folic acid is a water-soluble vitamin with a molecular formula of C19H19N7O6. It is named because it is very rich in green leaves. It is also known as pterylglutamic acid. It exists in several forms in nature. Its parent compound is composed of three components: pteridine, para-aminobenzoic acid and glutamic acid.

[0003] Folic acid contains one or more glutamyl groups, and most of the naturally occurring folic acid is in the form of polyglutamic acid. The biologically active form of folic acid is tetrahydrofolate. Folic acid is a yellow crystal that is slightly soluble in water, but its sodium salt is very soluble in water and insoluble in ethanol. It is easily destroyed in acidic solutions, unstable to heat, easily lost at room temperature, and easily destroyed by light. Folic acid belongs to the pteroylglutamic acid (PGA) compound and acts as a coenzyme in metabolic reactions involving the transfer of a single carbon unit from a donor to a compound acceptor. Folic acid and vitamin B12 are essential for DNA (deoxyribonucleic acid) synthesis and are required for normal red blood cell maturation. People get folic acid from their daily diet, such as fruits, green leafy vegetables, yeast, and organ meats. Folic acid is absorbed through the small intestine and stored in the liver. Gastrointestinal diseases, pregnancy, and taking certain medications, such as phenytoin (diphenylhydantoin), will lead to low folic acid intake or poor absorption, thus causing folic acid deficiency. Folic acid deficiency is associated with chronic alcoholism. Folic acid and vitamin B12 deficiency will affect DNA synthesis, resulting in megaloblastic anemia. Megaloblastic anemia is characterized by abnormal maturation of erythroid precursors in the bone marrow, the appearance of immature megaloblasts, and a shortened life span of erythrocytes. Because folate and vitamin B12 deficiencies can cause megaloblastic anemia, appropriate treatment depends on the differential diagnosis of this deficiency. Serum folate measurement can provide an early indicator of folate status in the body.

[0004] Folic acid is essential for normal DNA (deoxyribonucleic acid) synthesis and is involved in two metabolic functions that are essential for normal cell growth and DNA synthesis - the synthesis of methionine and the conversion of methylmalonyl CoA to succinyl CoA, which in turn affects the development and maturation of red blood cells. Folic acid is also an essential substance for the formation and maintenance of myelin. The human body is very precise in mobilizing the folic acid stored in the body. It reabsorbs folic acid from the ileum and then returns it to the liver, so the body excretes very little folic acid. Clinical and laboratory research results have found that B12 deficiency manifests itself in neurological diseases, decreased serum folate levels, and increased methylmalonic acid excretion. Due to folic acid deficiency, DNA synthesis is impaired, which will lead to megaloblastic anemia. The main characteristics of this anemia are abnormal maturation of erythrocyte precursors in the bone marrow, that is, the appearance of immature megaloblasts and a decrease in the life span of erythrocytes. Pernicious anemia is a megaloblastic anemia caused by folate deficiency due to the lack of intrinsic factor.

[0005] At present, the methods for quantitative determination of folic acid include microbiological method, high performance liquid chromatography (HPLC), enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay (CLIA) and radioimmunoassay (RIA). Folic acid exists in the body in combination with proteins, so when testing biological samples such as serum or plasma, the samples need to be pre-treated to release folic acid from the binding protein before testing. Existing reagents generally have two or three components when pre-treating samples. The reducing agent and the denaturing agent cannot coexist, resulting in more reagent components and a more complicated process. Existing single-component reducing agents are prone to incomplete dissociation, resulting in inaccurate test results and poor repeatability. In addition, existing dissociation agents are mostly strong acids or strong bases, which are unstable in storage and easily affected by air. Summary of the invention

[0006] The purpose of the present invention is to provide an efficient, simple and stable release agent for detecting folic acid content in human serum or heparin plasma, using a mixed solution of sodium salicylate, guanidine isothiocyanate and tri-(2-formylethyl)phosphine hydrochloride, which can effectively denature and dissociate proteins within the pH range of 5.0 to 7.0, and the mixed solution can be stably stored, and mild, efficient and sufficient dissociation can be achieved, thereby improving the sensitivity of folic acid detection. The release agent is used in the corresponding immunoassay method to solve the technical problems of the existing product process being complicated or incomplete release, poor sensitivity and repeatability, unstable treatment solution and unsatisfactory correlation of sample comparison.

[0007] Based on the above purpose, the technical solution adopted by the present invention is:

[0008] A release agent for detecting folic acid content in human serum or heparin plasma. The pH value of the treatment liquid R1 is 5.0-7.0, and the treatment liquid R1 comprises: 2-(N-morpholine)ethanesulfonic acid (MES) with a concentration of 5.0-15.0 g / L; tri-(2-formylethyl)phosphine hydrochloride with a concentration of 13.3-15.3 g / L; guanidine isothiocyanate with a concentration of 45.0-55.0 g / L; and sodium salicylate with a concentration of 4.0-6.0 g / L.

[0009] Preferably, the treatment solution R1 has a pH value of 6.0 and includes: 2-(N-morpholine)ethanesulfonic acid (MES) at a concentration of 10.0 g / L; tri-(2-formylethyl)phosphine hydrochloride at a concentration of 14.3 g / L; guanidine isothiocyanate at a concentration of 50.0 g / L; sodium salicylate at a concentration of 5.0 g / L; and the rest is purified water;

[0010] Preparation of treatment liquid R1: Weigh 800 g of purified water, add 10.0 g of 2-(N-morpholine)ethanesulfonic acid (MES), 14.3 g of tri-(2-formylethyl)phosphine hydrochloride, 50.0 g of guanidine isothiocyanate, and 5.0 g of sodium salicylate in sequence, mix well, adjust the pH to within the range of 6.0±0.05, and weigh to 1 L with purified water.

[0011] A magnetic microparticle chemiluminescent detection reagent for determining human folic acid content, using the above-mentioned release agent, includes a treatment solution R1, a folic acid reagent R2 (referred to as reagent R2), a magnetic separation reagent, a calibration product, a quality control product, and a chemiluminescent substrate solution. In the following examples, the folic acid reagent R2, magnetic separation reagent, calibration product, quality control product, and chemiluminescent substrate used are all reagents produced by Leadman.

[0012] A method for detecting human folic acid content using a magnetic particle chemiluminescent detection reagent comprises the following steps:

[0013] (1) Add the sample to be tested, calibrator or quality control material and the processing solution R1 component into the reaction tube, mix and incubate at 37°C for 15 minutes;

[0014] (2) adding reagent R2 to the sample after incubation in step (1), and incubating at 37°C for 5 min;

[0015] (3) adding the magnetic separation reagent to the sample after incubation in step (2), and continuing incubation at 37° C. for 15 min;

[0016] (4) washing to remove unbound antibodies and impurities;

[0017] (5) adding chemiluminescent substrate solution, and measuring the relative luminescence intensity (RLU) after ALP catalyzes the substrate to emit light;

[0018] Within a certain range, the relative luminescence intensity RLU is inversely proportional to the folic acid antigen concentration. The folic acid content of the sample to be tested can be read from the standard curve by interpolation.

[0019] The methodological identification data when using the above folic acid detection reagent to determine the folic acid content in the human body can reach the following indicators:

[0020] Sensitivity: minimum detection amount is 0.10ng / mL;

[0021] Repeatability: The precision of the quality control product QC1 is 1.97% (n=10, the number of repetitions), and the precision of the quality control product QC2 is 1.85% (n=10, the number of repetitions). The precision is far lower than the industry requirement, indicating that the detection reagent using the diluent of the present invention has good repeatability during the experiment;

[0022] Correlation: The reagent in the embodiment was used to simultaneously detect 42 samples of the imported chemiluminescent immunoassay kit, and the folic acid concentration determined by the reagent of the present invention was used as the ordinate, and the result of the imported chemiluminescent immunoassay kit was used as the abscissa for regression analysis. The correlation equation was y=1.0168x-0.0574, and the correlation coefficient R 2 =0.9773. The statistical results show that the correlation is good.

[0023] As can be seen from the above, the advantages of the present invention are:

[0024] 1. The sample treatment liquid in the existing folic acid kit contains strong acid or strong base, which is easy to oxidize when exposed to air, unstable during storage, and easy to fail for a long time. The treatment liquid R1 in the present invention is a mixed solution of tri-(2-formylethyl)phosphine hydrochloride, sodium salicylate, and guanidine isothiocyanate in the 2-(N-morpholine)ethanesulfonic acid (MES) system, wherein tri-(2-formylethyl)phosphine hydrochloride can reduce the disulfide bonds in proteins more quickly at room temperature, has strong specificity and small pH range limitations, and has its own structural stability and resistance to air oxidation, so that no precautions are required during processing and storage. Guanidine isothiocyanate in the treatment liquid R1 of the present invention is a mild and effective denaturant that can effectively denature proteins within a specific concentration range; sodium salicylate optimizes the dissociation effect, and the sensitivity and repeatability of the reagent, as well as the correlation of sample comparison, are better improved, and the effect is ideal; the entire reaction system does not use strong acid and strong base for denaturation, which has little effect on the reaction system; the treatment liquid uses a single-component mode to simplify the process.

[0025] 2. After the folic acid releaser R1 provided by the present invention denatures the folic acid binding protein, the magnetic particle chemiluminescence detection is realized. The folic acid releaser and the corresponding detection reagent and detection method can effectively and completely release the folic acid in the sample. The detection result has high sensitivity, can detect low-concentration folic acid, has good repeatability, and the performance can reach the level of international products. In addition, the sample pretreatment requirements are low, and a large number of samples can be quickly and high-throughput detected (batch is 180 tests / hour), which is convenient for clinical application.

[0026] 3. The release agent provided by the present invention is used in a magnetic microparticle chemiluminescence platform and can be used in conjunction with a fully automatic chemiluminescence instrument. The operation steps are greatly simplified, the detection speed and detection flux are increased, the detection efficiency is improved, and errors caused by human operation can be avoided. The magnetic microspheres are used as solid phase carriers, which are easy to clean and can effectively remove non-specific binding. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The graph is a concentration-luminescence value curve of a standard substance when the release agent of Comparative Example 1 is used to detect folic acid;

[0028] Figure 2 The graph is a concentration-luminescence value curve of a standard substance when the release agent of Comparative Example 2 is used to detect folic acid;

[0029] Figure 3 The graph is a concentration-luminescence value curve of a standard substance when the release agent of Comparative Example 3 is used to detect folic acid;

[0030] Figure 4 The graph is a concentration-luminescence value curve of a standard substance when the release agent of Comparative Example 4 is used to detect folic acid;

[0031] Figure 5 The graph is a concentration-luminescence value curve of a standard substance when the release agent of Example 2 of the present invention is used to detect folic acid;

[0032] Figure 6 When the release agent in Comparative Example 1 detects folic acid, a scatter plot of the correlation between folic acid concentration and the comparative reagent;

[0033] Figure 7 When the release agent in Comparative Example 2 detects folic acid, a scatter plot of the correlation between folic acid concentration and the comparative reagent;

[0034] Figure 8 When the release agent in Comparative Example 3 detects folic acid, a scatter plot of the correlation between folic acid concentration and the comparative reagent;

[0035] Fig. 9 When the release agent in Comparative Example 4 detects folic acid, a scatter plot of the correlation between folic acid concentration and the comparative reagent;

[0036] Fig.10This is a scatter plot of the correlation between the folic acid concentration detected by the detection reagent obtained in Example 4 of the present invention and the comparison reagent. DETAILED DESCRIPTION

[0037] The present invention is further explained below in conjunction with the embodiments and drawings, but this is not intended to limit the scope of protection of the present application.

[0038] Example 1

[0039] In this embodiment, a releasing agent used for detecting the folic acid content in human serum or heparin plasma is the treatment liquid R1.

[0040] The treatment liquid R1 includes: 2-(N-morpholine)ethanesulfonic acid (MES), the concentration is 5.0-15.0 g / L; tri-(2-formylethyl)phosphine hydrochloride, the concentration is 13.3-15.3 g / L; guanidine isothiocyanate, the concentration is 45.0-55.0 g / L; sodium salicylate, the concentration is 4.0-6.0 g / L; the rest is purified water. The pH value of the treatment liquid R1 is 5.0-7.0.

[0041] Example 2

[0042] In this embodiment, a releasing agent used for detecting the folic acid content in human serum or heparin plasma is a treatment solution R1 (also called reagent R1).

[0043] The pH value of the treatment liquid R1 is 6.0, and it includes: 2-(N-morpholine)ethanesulfonic acid (MES) with a concentration of 10.0 g / L; tri-(2-formylethyl)phosphine hydrochloride with a concentration of 14.3 g / L; guanidine isothiocyanate with a concentration of 50.0 g / L; sodium salicylate with a concentration of 5.0 g / L; and the rest is purified water. Preparation of treatment liquid R1: weigh 800 g of purified water, add 10.0 g of 2-(N-morpholine)ethanesulfonic acid (MES), 14.3 g of tri-(2-formylethyl)phosphine hydrochloride, 50.0 g of guanidine isothiocyanate, and 5.0 g of sodium salicylate in sequence, mix well, adjust the pH to within the range of 6.0±0.05, and weigh to 1 L with purified water.

[0044] Comparative Example 1

[0045] The treatment solution R1 of this comparative example includes a reducing agent, a denaturing agent and an enhancing agent, wherein the composition of the reducing agent is different from that of Example 2. The reducing agent in this comparative example is a DTT (dithiothreitol) buffer solution of 2-(N-morpholino)ethanesulfonic acid (MES) at a concentration of 1.0 g / L.

[0046] Comparative Example 2

[0047] The treatment solution R1 of this comparative example only includes the reducing agent and the denaturing agent in Example 2, and the enhancing agent sodium salicylate is not added.

[0048] Comparative Example 3

[0049] The treatment solution R1 of this comparative example includes a reducing agent, a denaturing agent and an enhancing agent, wherein the concentration of the denaturing agent is different from that of Example 2. In this comparative example, the concentration of the denaturing agent is 25.0 g / L guanidine thiocyanate.

[0050] Comparative Example 4

[0051] The treatment solution R1 of this comparative example includes a reducing agent, a denaturing agent and an enhancing agent, wherein the concentration of the denaturing agent is different from that of Example 2. In this comparative example, the concentration of the denaturing agent is 100.0 g / L guanidine thiocyanate.

[0052] Example 3

[0053] This embodiment provides a method for preparing a magnetic microparticle chemiluminescent detection reagent for determining human folic acid content. In this embodiment, the release agent composition adopts the formula in Example 2, and the detection reagent includes a treatment solution R1, a reagent R2, a magnetic separation reagent, a calibration product, a quality control product, and a chemiluminescent substrate solution. The preparation of the detection reagent includes the following steps:

[0054] Preparation of reagent R2: 1) The buffer of reagent R2 uses commercial AP Conjugate Stabilizer with a pH of 8.0; 2) prepare alkaline phosphatase-labeled anti-folate antibody; 3) dilute the alkaline phosphatase-labeled anti-folate antibody with the buffer of reagent R2.

[0055] Preparation of magnetic separation reagent: 1) The magnetic particle buffer is commercially available AP Conjugate Stabilizer, pH 7.0; 2) Prepare folic acid-BSA (bovine serum albumin) coated magnetic particle concentrate; 3) Dilute the magnetic particle concentrate with the magnetic particle buffer of the magnetic separation reagent to obtain the magnetic separation reagent.

[0056] Preparation of calibrators / quality control products: After dissolving the folic acid antigen, prepare it into different concentrations using the calibrator / quality control buffer to obtain calibrators and quality control products QC1 and QC2 of different concentrations.

[0057] Prepare chemiluminescent substrate solution: 1) prepare 0.2 M Tris-HCl buffer with a pH value of 9.3, and add 0.3 mg / mL dioxane; 2) dissolve the chemiluminescent substrate catalyzed by alkaline phosphatase in the buffer.

[0058] Among them, anti-folate antibody coupled with alkaline phosphatase is a commonly used coupling process for chemiluminescent platforms.

[0059] The preparation method of the magnetic particle reagent concentrate comprises the following steps:

[0060] (1) Prepare coupling buffer 1: 0.1 M sodium borate buffer, pH 9.5, stored at 2-8°C;

[0061] (2) Prepare coupling buffer 2: 3 M ammonium sulfate buffer, pH 9.5, stored at 2-8°C;

[0062] (3) Prepare coupling blocking solution: 0.2 M phosphate buffer, pH 7.5, containing 1% bovine serum albumin, 0.1% Tween 20, and 10% sucrose;

[0063] (4) Preparation of washing and storage buffer: 0.2 M phosphate buffer, pH 7.5, containing 1% bovine serum albumin and 10% sucrose (mass percentage);

[0064] (5) resuspending the tosyl-modified magnetic beads by vortexing;

[0065] (6) Transfer 100uL of magnetic beads to a 2mL centrifuge tube and add 1mL of coupling buffer 1;

[0066] (7) Place the centrifuge tube on a magnetic separator until the magnetic beads migrate to the side close to the magnet and the solution becomes clear in about 5 minutes;

[0067] (8) Carefully remove the supernatant to avoid mixing the magnetic beads;

[0068] (9) Move the centrifuge tube away from the magnet and resuspend the magnetic beads in 1 mL of coupling buffer 1 by vortexing for 5 minutes;

[0069] (10) Repeat steps (7) to (9) three times;

[0070] (11) Use coupling buffer 1 to weigh the magnetic beads to 100 uL, add 322 uL of coupling buffer 2 and 400 ug of folic acid-BSA, and then use coupling buffer 1 to weigh to 1 mL;

[0071] (12) After thorough shaking and mixing, incubate in a 37°C incubator overnight;

[0072] (13) In a fume hood, add 5 uL of ethanolamine to the coupling reaction system after overnight incubation in step (12), vortex and mix on a vortex mixer for 30 minutes to terminate the reaction;

[0073] (14) Place the centrifuge tube on a magnetic separator until the microspheres migrate to the side close to the magnet and the solution becomes clear in about 5 minutes;

[0074] (15) Carefully remove the supernatant to avoid mixing the magnetic beads;

[0075] (16) Move the centrifuge tube away from the magnet and resuspend the magnetic beads in 1 mL of coupling buffer 1 by vortexing for 5 minutes;

[0076] (17) Repeat steps (14) to (16) three times;

[0077] (18) Add 1 mL of coupling blocking solution, shake on a vortex mixer for 5 minutes to resuspend the magnetic beads, and then incubate in a 37 °C incubator overnight;

[0078] (19) Place the centrifuge tube on a magnetic separator until the microspheres migrate to the side close to the magnet and the solution becomes clear in about 5 minutes;

[0079] (20) Carefully remove the supernatant to avoid mixing the magnetic beads;

[0080] (21) Remove the centrifuge tube from the magnet and resuspend the magnetic beads in 1 mL of washing and storage buffer by vortexing for 5 minutes;

[0081] (22) Repeat steps (19)-(21) three times, weigh the coupled magnetic beads to 1 mL with washing and storage buffer, and store for later use;

[0082] The magnetic particle concentrate prepared above is 10 mg / mL, and the technical data of the reaction system is 1 mL. The large sample is coupled according to this (the corresponding parameters and the amount of coupling buffer added increase proportionally with the reaction system).

[0083] Example 4

[0084] The folic acid content in the human body was detected using the magnetic microparticle chemiluminescent detection reagent for folic acid content prepared in Example 3, and the detection method was as follows:

[0085] (1) First, 20 μl of the calibration series of Example 3 (with concentrations of 0, 1.0, 2.0, 5.0, 10.0, and 20.0 ng / mL, respectively) and 30 μl of the treatment solution R1 of Example 2 were added to a reaction tube, mixed and incubated at 37° C. for 15 min;

[0086] (2) The above reagent series and 30 μl of reagent R2 in Example 3 were added to the reaction tubes in sequence, mixed and incubated at 37° C. for 5 min;

[0087] (3) The reagent series obtained in step (2) were combined with 25 μl of the magnetic separation reagent of Example 3 and incubated at 37° C. for 15 min;

[0088] (4) washing three times with cleaning solution to remove unbound antibodies and impurities;

[0089] (5) 150 μl of the luminescent substrate solution of Example 3 was added, and after ALP catalyzed the substrate to emit light, the relative luminescence intensity (RLU) was measured using a Leadman chemiluminescence detector to obtain a series of numerical values ​​of folic acid concentration-luminescence value.

[0090] (6) Fitting is performed based on the folic acid concentration-luminescence value series values ​​to obtain a folic acid concentration-luminescence value standard curve based on the standard substance, such as Figure 2 shown.

[0091] (7) Calculate the concentration of the sample to be tested based on the fitted folic acid concentration-luminescence value standard curve.

[0092] Within a certain range, RLU is inversely proportional to the concentration of folate antigen, and the folate content of the sample to be tested can be read from the standard curve by interpolation.

[0093] According to the above method, the release agents in Comparative Examples 1 to 4 are used to obtain the corresponding concentration-luminescence value curves, such as Figure 1 — Figure 4 The release agent in Example 2 obtains its corresponding concentration-luminescence value curve, as shown in Figure 5 The comparison results of the standard curve are as follows:

[0094] Table 1 Standard curve (Comparative Example 1)

[0095] Calibrator (ng / mL) 0 1.0 2.0 5.0 10.0 20.0 RLU(100000) 28.811 25.323 18.762 8.798 4.846 3.539

[0096] Table 2 Standard curve (Comparative Example 2)

[0097] Calibrator (ng / mL) 0 1.0 2.0 5.0 10.0 20.0 RLU(100000) 30.133 25.764 18.612 9.051 4.683 4.265

[0098] Table 3 Standard curve (Comparative Example 3)

[0099] Calibrator (ng / mL) 0 1.0 2.0 5.0 10.0 20.0 RLU(100000) 30.094 23.840 15.708 7.279 3.698 2.564

[0100] Table 4 Standard curve (Comparative Example 4)

[0101] Calibrator (ng / mL) 0 1.0 2.0 5.0 10.0 20.0 RLU(100000) 35.392 29.110 18.503 9.400 4.818 3.323

[0102] Table 5 Standard curve (Example 2)

[0103] Calibrator (ng / mL) 0 1.0 2.0 5.0 10.0 20.0 RLU(100000) 34.675 24.462 16.233 8.089 4.330 2.873

[0104] 20 μl of the sample to be tested / quality control product and 30 μl of reagent R1 of Example 2 were added to the reaction tube, mixed and incubated at 37° C. for 15 min; the above steps (2) to (5) were repeated to determine the relative luminescence intensity RLU of the sample to be tested;

[0105] Then, the folic acid concentration value corresponding to the RLU of the sample to be tested is calculated according to the folic acid concentration-luminescence value standard curve.

[0106] Using the above detection method, the methodological identification data can reach the following indicators:

[0107] Sensitivity: minimum detection amount is 0.10ng / mL;

[0108] Repeatability: The precision of the quality control product QC1 is 1.97% (n=10), and the precision of QC2 is 1.85% (n=10), which is far lower than the national requirement, indicating that the detection reagent of the present invention has good repeatability during the experiment;

[0109] Correlation: 42 samples were tested simultaneously using the detection reagents obtained by the detection reagents in this embodiment and the release agents in the comparative example, and the imported chemiluminescent immunoassay kit, and correlation analysis was performed respectively: the folic acid concentration measured by the reagents of the present invention was used as the ordinate, and the result measured by the imported chemiluminescent immunoassay kit was used as the abscissa for regression analysis. Fig.10 As shown, the correlation equation is y=1.0168x-0.0574, and the correlation coefficient R2=0.9773. The statistical analysis results show that the correlation is good. The correlation analysis results obtained using the release agents of Comparative Examples 1 to 4 are as follows: Figure 6 — Fig. 9 As shown, the correlation is poor.

[0110] Table 6 Sensitivity (Comparative Example 1)

[0111]

[0112] It can be seen from Table 6 that the mean luminescence value of point A is X=2859156, SD=150880, X-2SD=2557396; the mean luminescence value of point B is 2432275. The fitting equation of point AB is y=294151x+2883980, R2=0.9996.

[0113] According to the linear fitting equation of concentration-luminescence value at points AB, the luminescence value of M-2SD was substituted into the above equation to obtain the corresponding concentration value, which is the minimum detection limit. That is, the minimum detection limit of the detection reagent is 0.71 ng / mL.

[0114] Table 7 Sensitivity (Comparative Example 2)

[0115]

[0116] It can be seen from Table 7 that the mean luminous value of point A is X=2976662, SD=138914, X-2SD=2698834; the mean luminous value of point B is 2576372. The fitting equation of point AB is y=409201x+3016270, R 2 =0.9950.

[0117] According to the linear fitting equation of concentration-luminescence value at points AB, the luminescence value of M-2SD was substituted into the above equation to obtain the corresponding concentration value, which is the minimum detection limit. That is, the minimum detection limit of the detection reagent is 0.69 ng / mL.

[0118] Table 8 Sensitivity (Comparative Example 3)

[0119]

[0120] It can be seen from Table 8 that the mean luminous value of point A is X=2954675, SD=175193, X-2SD=2604288; the mean luminous value of point B is 2383993. The fitting equation of point AB is y=203706x+3012450, R 2 =0.9993.

[0121] According to the concentration-luminescence value linear fitting equation of point AB, the luminescence value of M-2SD was substituted into the above equation to obtain the corresponding concentration value, which is the minimum detection limit, that is, the minimum detection limit of the detection reagent is 0.61 ng / mL.

[0122] Table 9 Sensitivity (Comparative Example 4)

[0123]

[0124] It can be seen from Table 9 that the mean luminous value of point A is X=3536411, SD=185181, X-2SD=3166049; the mean luminous value of point B is 2911023. The fitting equation of point AB is y=266768x+3542750, R 2 =0.9965.

[0125] According to the concentration-luminescence value linear fitting equation of point AB, the luminescence value of M-2SD was substituted into the above equation to obtain the corresponding concentration value, which is the minimum detection limit, that is, the minimum detection limit of the detection reagent is 0.59 ng / mL.

[0126] Table 10 Sensitivity (Example 2)

[0127]

[0128] It can be seen from Table 10 that the mean luminous value of point A is X=3466167, SD=45297, X-2SD=3375573; the mean luminous value of point B is 2489803. The fitting equation of point AB is y=196619x+3471010, R 2 =0.9997.

[0129] According to the concentration-luminescence value linear fitting equation of point AB, the luminescence value of M-2SD was substituted into the above equation to obtain the corresponding concentration value, which is the minimum detection limit, that is, the minimum detection limit of the detection reagent is 0.09 ng / mL.

[0130] Conclusion: The addition of tri-(2-formylethyl)phosphine hydrochloride in the treatment solution R1 has better sensitivity to the reagent than other reducing agents, and the sensitivity is better improved when the denaturant concentration is 50.0 g / L than guanidine thiocyanate at other concentrations. At the same time, the addition of sodium salicylate has a significant effect on improving the sensitivity compared with no addition of sodium salicylate.

[0131] Table 11 Repeatability (Comparative Example 1):

[0132]

[0133] As can be seen from Table 11, the CV of 10 replicates of the quality control product QC1 with a target concentration range of 5.0±20% (ng / mL) was 6.57%, and the CV of 10 replicates of the quality control product QC2 with a target concentration range of 10.0±20% (ng / mL) was 6.27%.

[0134] Table 12 Repeatability (Comparative Example 2):

[0135]

[0136] As can be seen from Table 12, the CV of 10 replicates of the quality control product QC1 with a target concentration range of 5.0±20% (ng / mL) was 6.99%, and the CV of 10 replicates of the quality control product QC2 with a target concentration range of 10.0±20% (ng / mL) was 7.01%.

[0137] Table 13 Repeatability (Comparative Example 3):

[0138]

[0139] As can be seen from Table 13, the CV of 10 replicates of the quality control product QC1 with a target concentration range of 5.0±20% (ng / mL) was 6.05%, and the CV of 10 replicates of the quality control product QC2 with a target concentration range of 10.0±20% (ng / mL) was 5.90%.

[0140] Table 14 Repeatability (Comparative Example 4):

[0141]

[0142] As can be seen from Table 14, the CV of 10 replicates of the quality control product QC1 with a target concentration range of 5.0±20% (ng / mL) was 5.94%, and the CV of 10 replicates of the quality control product QC2 with a target concentration range of 10.0±20% (ng / mL) was 5.65%.

[0143] Table 15 Repeatability (Example 2):

[0144]

[0145] As can be seen from Table 15, the CV of 10 replicates of the quality control product QC1 with a target concentration range of 5.0±20% (ng / mL) was 1.97%, and the CV of 10 replicates of the quality control product QC2 with a target concentration range of 10.0±20% (ng / mL) was 1.85%.

[0146] Conclusion: The addition of tri-(2-formylethyl)phosphine hydrochloride to the treatment solution R1 has better reagent repeatability than other reducing agents, and the denaturant concentration of 50.0 g / L has better repeatability than other concentrations of guanidine thiocyanate. At the same time, the addition of sodium salicylate also significantly improves the repeatability compared with no addition of sodium salicylate.

[0147] Table 16 Clinical sample comparison correlation (Comparative Example 1):

[0148]

[0149]

[0150] As can be seen from Table 16, the treatment solution R1 of this comparative example includes a reducing agent, a denaturing agent and an enhancing agent, wherein the composition of the reducing agent is different from that of Example 2. The reducing agent in this comparative example is a DTT buffer solution of 2-(N-morpholine)ethanesulfonic acid (MES). 42 samples were simultaneously tested with the imported chemiluminescent immunoassay kit, and the folic acid concentration determined by the reagent of the present invention was used as the ordinate, and the result of the imported chemiluminescent immunoassay kit was used as the abscissa for regression analysis. The correlation equation is y=1.267x-0.0921, and the correlation coefficient R2=0.8223. The statistical processing results show that the correlation is slightly poor.

[0151] Table 17 Clinical sample comparison correlation (Comparative Example 2):

[0152]

[0153]

[0154] As can be seen from Table 17, the comparative example treatment liquid R1 includes a reducing agent and a denaturing agent. Unlike Example 2, no enhancer component is added. 42 samples are simultaneously tested using the imported chemiluminescent immunoassay kit, and the folic acid concentration determined by the reagent of the present invention is used as the ordinate, and the result of the imported chemiluminescent immunoassay kit is used as the abscissa for regression analysis. The correlation equation is y=1.2949x+0.7675, and the correlation coefficient R2=0.7645. The results of statistical processing show that the correlation is slightly poor.

[0155] Table 18 Clinical sample comparison correlation (Comparative Example 3):

[0156]

[0157]

[0158] As can be seen from Table 18, the treatment liquid R1 of this comparative example includes a reducing agent, a denaturing agent and an enhancing agent, wherein the concentration of the denaturing agent is different from that in Example 2. In this comparative example, the denaturing agent concentration is 25.0 g / L guanidine thiocyanate. The 42 samples were simultaneously tested with the imported chemiluminescent immunoassay kit, and the folic acid concentration determined by the reagent of the present invention was used as the ordinate, and the result of the imported chemiluminescent immunoassay kit was used as the abscissa for regression analysis. The correlation equation is y=1.1735x+0.1135, and the correlation coefficient R2=0.8024. The results of statistical processing show that the correlation is slightly poor.

[0159] Table 19 Clinical sample comparison correlation (Comparative Example 4):

[0160]

[0161]

[0162] As can be seen from Table 19, the treatment liquid R1 of this comparative example includes a reducing agent, a denaturing agent and an enhancing agent, wherein the concentration of the denaturing agent is different from that in Example 2. In this comparative example, the denaturing agent concentration is 100.0 g / L guanidine thiocyanate. The 42 samples were simultaneously tested with the imported chemiluminescent immunoassay kit, and the folic acid concentration determined by the reagent of the present invention was used as the ordinate, and the result of the imported chemiluminescent immunoassay kit was used as the abscissa for regression analysis. The correlation equation is y=1.0194x+0.0143, and the correlation coefficient R2=0.8187. The results of statistical processing show that the correlation is slightly poor.

[0163] Table 20 Clinical sample comparison correlation (Example 2):

[0164]

[0165]

[0166] As can be seen from Table 20, the reducing agent component (tri-(2-formylethyl)phosphine hydrochloride) and 50.0 g / L denaturing agent component (guanidine isothiocyanate) in the embodiment were added, and the enhancing agent component (sodium salicylate) was added, and 42 samples of the imported chemiluminescent immunoassay kit were tested simultaneously. The folic acid concentration determined by the reagent of the present invention was used as the ordinate, and the result of the imported chemiluminescent immunoassay kit was used as the abscissa for regression analysis. The correlation equation was y=1.0168x-0.0574, and the correlation coefficient R2=0.9773. The statistical processing results showed that the correlation was good.

[0167] Conclusion: The correlation was significantly improved by adding tri-(2-formylethyl)phosphine hydrochloride and 50.0 g / L guanidine isothiocyanate and sodium salicylate to the treatment solution R1.

[0168] Example 5

[0169] The composition of the release agent in this embodiment is: 2-(N-morpholine)ethanesulfonic acid (MES), with a concentration of 10.0 g / L; tri-(2-formylethyl)phosphine hydrochloride, with a concentration of 15.3 g / L; guanidine isothiocyanate, with a concentration of 55.0 g / L; sodium salicylate, with a concentration of 6.0 g / L, and a pH of 6.0±0.05.

[0170] The folic acid content was tested by magnetic particle chemiluminescence method. After testing, the sensitivity of the detection reagent of this embodiment was: the minimum detection amount was 0.08 ng / mL; repeatability: the precision of the quality control product QC1 with a target concentration range of 5.0±20% (ng / mL) was 1.82%, and the precision of the quality control product QC2 with a target concentration range of 10.0±20% (ng / mL) was 1.80%.

[0171] The treatment solution of the present invention is used, and the principle of combining chemiluminescence detection technology with magnetic microparticle immunoseparation technology is utilized to quantitatively detect the folic acid content in human serum or heparin plasma samples. The dissociation is effectively driven by the synergistic effect of specific reducing agents, denaturants, and enhancers, so that the dissociation is more thorough, thereby enabling the denaturant and reducing agent to achieve more accurate folic acid detection in the same system, and the treatment solution can be stably stored, ensuring the sensitivity and repeatability of the detection, and the sample pretreatment requirements are low, and large batches of samples can be detected quickly and at high throughput, which is convenient for clinical application. The present invention provides a detection method for dissociating bound folic acid in human serum or heparin plasma into free folic acid, which provides a simpler process, more sufficient release, better repeatability, and more stable reagents.

[0172] The preparation of the human folic acid releaser and the magnetic microparticle chemiluminescence detection reagent for content determination provided by the present invention can be used in conjunction with a fully automatic chemiluminescence analyzer, which greatly simplifies the operation steps, increases the detection speed and detection throughput, improves the detection efficiency, and avoids errors caused by human operation.

[0173] Those skilled in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples; under the concept of the present invention, the technical features in the above embodiments or different embodiments can also be combined, and there are many other changes in different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

[0174] Any matters not described in the present invention are applicable to the prior art.

Claims

1. A releasing agent for detecting folic acid content in human body, characterized in that: Used for quantitatively detecting the folic acid content in human serum or plasma samples, the release agent is the treatment solution R1; the pH value of the treatment solution R1 is 5.0-7.0, and includes: 2-(N-morpholine)ethanesulfonic acid (MES) with a concentration of 5.0-15.0 g / L; tri-(2-formylethyl)phosphine hydrochloride with a concentration of 13.3-15.3 g / L; guanidine isothiocyanate with a concentration of 45.0-55.0 g / L; sodium salicylate with a concentration of 4.0-6.0 g / L.

2. The release agent according to claim 1, characterized in that The treatment solution R1 has a pH value of 6.0 and includes: 2-(N-morpholine)ethanesulfonic acid (MES) at a concentration of 10.0 g / L; tri-(2-formylethyl)phosphine hydrochloride at a concentration of 14.3 g / L; guanidine isothiocyanate at a concentration of 50.0 g / L; sodium salicylate at a concentration of 5.0 g / L; and the rest is purified water; Preparation of treatment liquid R1: Weigh 800 g of purified water, add 10.0 g of 2-(N-morpholine)ethanesulfonic acid (MES), 14.3 g of tri-(2-formylethyl)phosphine hydrochloride, 50.0 g of guanidine isothiocyanate, and 5.0 g of sodium salicylate in sequence, mix well, adjust the pH to within the range of 6.0±0.05, and weigh to 1 L with purified water.

3. A magnetic particle chemiluminescent detection reagent for determining the folic acid content in human body, characterized in that: Using the release agent described in claim 1 or 2, comprising a treatment solution R1 and a folic acid reagent R2, a magnetic separation reagent, a calibration product, a quality control product, and a chemiluminescent substrate solution; The sensitivity of the detection reagent is: the minimum detection amount is 0.10 ng / mL; repeatability: the precision of the quality control product QC1 with a target concentration range of 5.0±20% (ng / mL) is 1.97%, and the precision of the quality control product QC2 with a target concentration range of 10.0±20% (ng / mL) is 1.85%.

4. A method for detecting the magnetic microparticle chemiluminescence detection reagent for determining the human folic acid content according to claim 3, characterized in that: The steps include: (1) Add the sample to be tested, calibrator or quality control material and the processing solution R1 component into the reaction tube, mix and incubate at 37°C for 15 minutes; (2) adding reagent R2 to the sample after incubation in step (1), and incubating at 37°C for 5 min; (3) adding the magnetic separation reagent to the sample after incubation in step (2), and continuing incubation at 37° C. for 15 min; (4) washing to remove unbound antibodies and impurities; (5) adding chemiluminescent substrate solution, and measuring the relative luminescence intensity (RLU) after ALP catalyzes the substrate to emit light; Within a certain range, the relative luminescence intensity RLU is inversely proportional to the concentration of folate antigen, and the folate content of the sample to be tested is read from the standard curve by interpolation.

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