Preparation method of folate-gamma-aminobutyric acid-bovine serum albumin conjugate and method and application of extracting folate binding protein

Abstract

The application discloses a preparation method of a folate-gamma-aminobutyric acid-bovine serum albumin conjugate and a method and application for extracting folate binding protein, and belongs to the technical field of in-vitro diagnostic reagents. The FA-GABA-BSA conjugate provided by the application can be used to extract folate binding protein from whey protein powder through an affinity chromatography method, so that the cost of the folate binding protein is greatly reduced; the folate binding protein extracted therefrom is good in specificity and high in purity, and can be used as a key raw material of a folate detection kit. The method for extracting folate binding protein provided by the application uses whey protein powder as a raw material, and extracts folate binding protein through an affinity chromatography column by using FA-GABA-BSA-4B agarose beads, so that the preparation process is simple, time consumption is short, and the method has good industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, and particularly relates to a method for preparing a folic acid-γ-aminobutyric acid-bovine serum albumin conjugate, as well as a method for extracting folic acid-binding protein and its application. Background Technology

[0002] Folic acid (FA) is a water-soluble B vitamin, also known as pteroylglutamic acid, with the molecular formula C. 19 H 19 N7O6, its coenzyme form, consists of some derivatives of tetrahydrofolate, formed by the combination of pteridine, para-aminobenzoic acid, glutamic acid, etc. Folic acid plays an important role in one-carbon unit metabolism. Folic acid deficiency increases the risk of many diseases, such as neural tube defects in newborns, megaloblastic anemia, epilepsy during pregnancy, cardiovascular disease, and cancer. Therefore, evaluating the folic acid content in the human body has become an important indicator in clinical testing.

[0003] Currently, the main methods for detecting folic acid include microbiological methods, radioimmunoassay, chromatographic analysis, and chemiluminescence. Among these, the microbiological method is not widely used due to its time-consuming and complex operation; radioimmunoassay can be used to detect and evaluate the nutritional status of folic acid, but from a quantitative perspective, it is difficult to obtain accurate folic acid content values; chromatographic analysis first uses chromatographic techniques to separate and extract folic acid, and then uses microbiological detection methods for quantitative detection. This method is not only complex and expensive (HPLC), but also requires large sample volumes, limiting its practical application. Chemiluminescence has good reproducibility, high sensitivity, and a wide linear range. It yields significantly better results for low-concentration folic acid samples than other methods and is convenient and rapid, making it the mainstream method.

[0004] Folate-binding proteins (FBPs) are basic proteins composed of 222 amino acid residues with a molecular weight between 30 and 35 kDa. FBPs are divided into two main categories: membrane-bound FBPs and soluble FBPs. Data shows that folic acid has a strong affinity for soluble FBPs. Using FBPs as the basis for immune responses, chemiluminescence immunoassay can be used to detect folic acid based on the principle of competitive binding receptor assays. However, current FBPs are expensive and difficult to obtain through gene expression; traditional methods for extracting FBPs from milk are cumbersome, time-consuming, and due to the complexity and diversity of FBP types and structures, it is impossible to obtain FBPs with high specificity and purity. Summary of the Invention

[0005] To address the above-mentioned technical problems, this invention provides a method for preparing a folic acid-γ-aminobutyric acid-bovine serum albumin conjugate (FA-GABA-BSA conjugate), as well as a method for extracting folic acid-binding protein and its applications. This folic acid-γ-aminobutyric acid-bovine serum albumin conjugate can be used to extract folic acid-binding protein from whey protein powder, reducing the production cost of folic acid-binding protein and yielding folic acid-binding protein with good specificity and high purity. The method for extracting folic acid-binding protein provided by this invention is simple, time-saving, and can be used for the industrial production of folic acid-binding protein, providing a key raw material for folic acid reagent kits.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing a folic acid-γ-aminobutyric acid-bovine serum albumin conjugate (FA-GABA-BSA conjugate), specifically comprising the following steps:

[0008] S1. Dissolve carbodiimide (EDC) in an aqueous solution of γ-aminobutyric acid (GABA), then add an aqueous solution of bovine serum albumin (BSA), mix well, and react at 22-28°C for at least 1 hour to obtain a γ-aminobutyric acid-bovine serum albumin complex (GABA-BSA complex); the mass ratio of γ-aminobutyric acid, bovine serum albumin, and carbodiimide is 1:0.6-0.8:0.4-0.7.

[0009] S2. Wash and dissolve the γ-aminobutyric acid-bovine serum albumin complex with borate buffer (BSB buffer) to obtain a borate buffer solution of the γ-aminobutyric acid-bovine serum albumin complex.

[0010] S3. Add N-hydroxysulfosuccinimide (Sulfo-NHS) and carbodiimide to the borate buffer solution of folic acid (FA), mix well, react at room temperature in the dark for at least 1 hour, centrifuge, and take the supernatant to obtain the activated folic acid solution; the molar ratio of folic acid, N-hydroxysulfosuccinimide and carbodiimide is 0.5-1.0:0.5-1.5:0.1-1.0;

[0011] S4. Add the activated folic acid solution to the borate buffer solution of the γ-aminobutyric acid-bovine serum albumin complex, react at room temperature in the dark for at least 1 hour, and then react at 2-8°C for at least 8 hours; after the reaction is completed, wash the reaction product with carbonate buffer (CBS buffer) to obtain the folic acid-γ-aminobutyric acid-bovine serum albumin conjugate.

[0012] In S1, the synthetic route for the γ-aminobutyric acid-bovine serum albumin complex is shown below:

[0013]

[0014] In S4, the synthetic route for the folic acid-γ-aminobutyric acid-bovine serum albumin conjugate is shown below:

[0015]

[0016] The folic acid-γ-aminobutyric acid-bovine serum albumin conjugate prepared by the above method can be used to extract folic acid-binding protein from whey protein powder via affinity chromatography, significantly reducing the production cost of folic acid-binding protein. Furthermore, the folic acid-binding protein extracted using this folic acid-γ-aminobutyric acid-bovine serum albumin conjugate exhibits high specificity and purity, making it a key raw material for folic acid detection kits.

[0017] In conjunction with the first aspect, the specific operation of S2 is as follows: The borate buffer solution is mixed with the γ-aminobutyric acid-bovine serum albumin complex solution and centrifuged. The supernatant is discarded, and the centrifuged product is dissolved in the borate buffer solution to obtain a borate buffer solution of the γ-aminobutyric acid-bovine serum albumin complex. The preferred concentration of the borate buffer solution is 0.01–0.1 M, and the pH is 6.0–9.0. The preferred centrifugation temperature is 2–8 °C, the rotation speed is 5000–10000 rpm, and the centrifugation time is 10–15 min.

[0018] In conjunction with the first aspect, the concentration of the borate buffer solution described in S3 is 0.01–0.1 M.

[0019] In conjunction with the first aspect, the centrifugation speed described in S3 is 5000-10000 rpm, and the centrifugation time is 10-15 min.

[0020] In conjunction with the first aspect, the volume ratio of the activated folic acid solution and the borate buffer solution of the γ-aminobutyric acid-bovine serum albumin complex in S4 is 1:1.5 to 2.5.

[0021] In conjunction with the first aspect, the concentration of the carbonate buffer solution described in S4 is 0.1–0.5 M, and the pH is 8.0–11.0.

[0022] Secondly, the present invention also provides a folic acid-γ-aminobutyric acid-bovine serum albumin conjugate, prepared according to the above preparation method.

[0023] Thirdly, the present invention also provides a method for extracting folic acid-binding protein from whey protein, wherein folic acid-γ-aminobutyric acid-bovine serum albumin-4B agarose beads are used to extract folic acid-binding protein from whey protein via an affinity chromatography column; wherein the folic acid-γ-aminobutyric acid-bovine serum albumin-4B agarose beads are 4B agarose beads crosslinked with the above-mentioned folic acid-γ-aminobutyric acid-bovine serum albumin conjugate.

[0024] Folic acid has a strong affinity for folic acid-binding proteins, but as a small molecule, folic acid cannot be directly linked to affinity chromatography packing materials. Therefore, this invention uses bovine serum albumin (BSA) as a carrier and γ-aminobutyric acid (GABA) as the linker between folic acid and BSA. An affinity chromatography column containing a folic acid-γ-aminobutyric acid-BSA conjugate is used as a ligand to extract folic acid-binding proteins from whey protein powder. This preparation process is simple and time-efficient, overcoming the shortcomings of existing traditional processes for extracting folic acid-binding proteins from milk. This method can obtain high-purity, high-affinity folic acid-binding proteins with high sensitivity and specificity, and can be used to prepare folic acid detection kits.

[0025] In conjunction with the third aspect, the preparation method of the folic acid-γ-aminobutyric acid-bovine serum albumin-4B agarose beads includes the following operations: hydrogen bromide activated agarose gel beads (CNBr-Sepharose 4B) are swollen with hydrochloric acid solution, added to the carbonate buffer solution of the folic acid-γ-aminobutyric acid-bovine serum albumin conjugate, kept at 2-8℃ for more than 8 hours, washed with carbonate buffer, blocked with glycine buffer, and then washed to obtain folic acid-γ-aminobutyric acid-bovine serum albumin-4B agarose beads.

[0026] After hydrogen bromide-activated agarose gel beads swelled in hydrochloric acid solution, they first underwent a cross-linking reaction with the folic acid-γ-aminobutyric acid-bovine serum albumin conjugate. Then, they were blocked with glycine buffer. The amino group in the glycine molecule blocked the excess carboxyl groups on the 4B agarose beads that were not bound to the amino groups of the folic acid-γ-aminobutyric acid-bovine serum albumin conjugate, forming a glycine protecting group. In subsequent reactions, this glycine protecting group protects the blocked carboxyl molecules from attack by other amino molecules.

[0027] Preferably, the concentration of the hydrochloric acid solution is 0.0005 to 0.01 M, and more preferably a 0.001 M hydrochloric acid solution is used.

[0028] Preferably, the swelling time of the hydrogen bromide-activated agarose gel with hydrochloric acid solution shall not exceed 15 minutes, and more preferably 10 to 15 minutes.

[0029] Preferably, the concentration of the carbonate buffer solution is 0.1–0.5 M, and the pH is 8.0–11.0. A 0.1 M carbonate buffer solution with a pH of 9.6 is preferred.

[0030] Preferably, the concentration of the glycine buffer is 0.1-1.0M, and more preferably 0.5M glycine buffer is used.

[0031] Preferably, the washing process involves alternating rinsing with carbonate buffer, urea solution, and glycine buffer in sequence to remove unbound and weakly bound folic acid conjugate proteins, followed by washing with phosphate buffer.

[0032] More preferably, the carbonate buffer solution used for cleaning has a concentration of 0.01–0.5 M, a pH of 8.0–11.0, and contains 5–10% wt sodium hydroxide; the urea solution used for cleaning has a concentration of 0.1–0.5 M and contains 3–15% wt sodium chloride; the glycine buffer solution used for cleaning has a concentration of 0.01–0.1 M, a pH of 2.0–4.0, and contains 1–20% wt hydrochloric acid; and the phosphate buffer solution used for cleaning has a concentration of 0.01–0.1 M and a pH of 6.5–7.5.

[0033] In conjunction with the third aspect, the procedures for extracting folic acid-binding proteins include:

[0034] Dissolve whey protein in carbonate buffer and store at 2–8°C for at least 8 hours. Centrifuge, collect the supernatant, filter, and mix with folic acid-γ-aminobutyric acid-bovine serum albumin-4B agarose beads for at least 1 hour. After filtering off excess whey protein solution, pack it into a chromatography column, wash with carbonate buffer and equilibrate.

[0035] Using TBS buffer (pH 7.5–8.5, 0.5–1.0 M) as the eluent, elute at a flow rate of 1.0–3.0 mL / min. Once the absorbance of the eluent at 280 nm stabilizes, elute with glycine buffer. Collect the eluent when the absorbance at 280 nm begins to show a significant upward trend. Stop collecting before the absorbance at 280 nm drops back to the baseline level.

[0036] Preferably, the concentration of the carbonate buffer solution is 0.1–0.5 M, and the pH is 8.0–11.0.

[0037] Preferably, the concentration of the dissolved whey protein is 40–50 mg / mL.

[0038] Preferably, the centrifugation speed is 5000-10000 rpm and the centrifugation time is 10-15 min.

[0039] Preferably, the TBS buffer contains 1.0–2.0 M sodium chloride and 0.01–0.05 M imidazole.

[0040] Preferably, the concentration of the glycine buffer solution is 0.05-0.2M and the pH is 2.0-4.0, and more preferably, a glycine buffer solution with a concentration of 0.05M and a pH of 2.6 is used.

[0041] In conjunction with the third aspect, the preparation method further includes neutralizing and concentrating the extracted folic acid-binding protein.

[0042] In conjunction with the third aspect, the preparation method further includes purifying the extracted folic acid-binding protein.

[0043] Preferably, the obtained folic acid-binding protein is purified using a gel.

[0044] Optionally, purification can be performed as follows: the obtained folic acid-binding protein solution is neutralized and concentrated, then purified using a Superdex 200 gel filtration column, with PBS buffer as the eluent. Preferably, the concentration of the PBS buffer is 0.01–0.1 M, and the pH is 6.5–7.5. More preferably, the concentration of the PBS buffer is 0.01 M, and the pH is 7.4.

[0045] Fourthly, this invention also provides the application of the above method in the preparation of a folic acid detection kit: folic acid-binding protein is extracted from whey protein according to the above method, and the obtained folic acid-binding protein is used as a raw material for preparing the folic acid detection kit. The folic acid-binding protein obtained by the above method has high affinity and good specificity. The folic acid detection kit prepared using it as a raw material has high sensitivity, strong specificity, and good stability. It can accurately quantify the folic acid content in serum or plasma on a chemiluminescence platform, and its results are highly correlated with those of commercially available reagents.

[0046] In conjunction with the fourth aspect, the reagents in the kit also include pretreatment reagent 1, pretreatment reagent 2, magnetic bead working solution, biotinylated folic acid-binding protein working solution, enzyme-labeled folic acid conjugate working solution, luminescent reagent, washing solution, and calibrators; pretreatment reagent 1 is a sodium 2-mercaptoethanesulfonate solution with a concentration of 2.0–10.0 mg / mL, and pretreatment reagent 2 is a sodium hydroxide solution with a concentration of 10–100 mg / mL; the magnetic bead working solution consists of biotinylated antibody-coated magnetic beads and 0.01–0.1 M PBS buffer at pH 6.5–7.5, with a magnetic bead concentration of 0.1–1.5 mg / mL; the folic acid-binding protein working solution consists of biotinylated folic acid-binding protein and 0.05–0.2 M PBS buffer at pH 7.0–8.5. The enzyme-labeled folic acid conjugate working solution consists of a Tris buffer solution containing a biotin-labeled folate-binding protein concentration of 0.1–1.5 μg / mL; the working solution consists of a FA-GABA-BSA conjugate labeled with alkaline phosphatase and a 0.05–0.2 M Tris buffer at pH 7.0–8.5, with the FA-GABA-BSA conjugate concentration of 0.1–1.5 μg / mL; the luminescent reagent consists of AMPPD and Dioxetane Buffer, with the AMPPD concentration of 0.1–1.5 mg / mL; the washing buffer is a 0.01–0.1 M PBST buffer at pH 6.5–7.5; and the calibrators are folic acid antigens diluted in PBS buffer containing 0.1% ProClin 300 and 5% BSA at concentrations of 5.0 ± 0.5 ng / mL and 15.0 ± 2.0 ng / mL.

[0047] The preferred concentrations of the sodium 2-mercaptoethanesulfonate solution are 5.0 mg / mL; the preferred concentrations of the sodium hydroxide solution are 25 mg / mL; the preferred pH of the PBS buffer is 7.4 and 0.01 M; the preferred concentration of the magnetic beads is 0.75 mg / mL; the preferred pH of the Tris buffer is 7.4 and 0.05 M; the preferred concentration of the biotin-labeled folic acid-binding protein is 0.25 μg / mL; the preferred concentration of the FA-GABA-BSA conjugate labeled with alkaline phosphatase is 0.6 μg / mL; the preferred concentration of AMPPD is 0.1 mg / mL; and the preferred pH of the PBST buffer is 7.4 and 0.01 M.

[0048] For example, the method for detecting folic acid using the above-mentioned folic acid detection kit can be carried out as follows: 20–100 μL of the test sample, 20–100 μL of pretreatment reagent 1, and 20–100 μL of pretreatment reagent 2 are co-incubated for 10–15 min to release the bound endogenous folic acid; then, 20–100 μL of biotinylated folic acid binding protein working solution and 20–100 μL of magnetic bead working solution are added to the pretreated sample and incubated for 10–15 min to form a folic acid binding protein-folic acid complex, allowing the entire complex to bind to the solid-phase magnetic beads; 20–100 μL of enzyme-labeled folic acid conjugate working solution is added and incubated for 10–15 min to competitively bind the biotinylated folic acid binding protein blank sites. Then, the magnetic particles of the reaction solution are adsorbed onto the electrode using a magnet, unbound substances are washed away, and a luminescent substrate is added to cause the folic acid complex to generate a light signal, which is detected by a photomultiplier tube, and the detection result is automatically output from the instrument.

[0049] The beneficial effects of this invention are as follows:

[0050] The method for preparing the folic acid-γ-aminobutyric acid-bovine serum albumin conjugate provided by this invention can produce a folic acid-γ-aminobutyric acid-bovine serum albumin conjugate that can be used to extract folic acid-binding protein from whey protein powder. This conjugate can be used to extract folic acid-binding protein from whey protein powder through a simple and easy affinity column chromatography. This method not only significantly reduces the production cost of folic acid-binding protein and simplifies the process, but also provides folic acid-binding protein with high specificity and purity. It can be used to prepare folic acid detection kits based on biotin-biotin antibody reaction systems. The resulting folic acid detection kits have good performance and can accurately quantify the folic acid content in serum or plasma on a chemiluminescence platform, with a high correlation to the detection results of commercially available reagents. Attached Figure Description

[0051] Figure 1 Example 4 of the present invention illustrates the change in absorbance of the eluent at 280 nm during the extraction of FBP using an affinity chromatography column.

[0052] Figure 2 This is the SDS-PAGE electrophoresis pattern of the FBP purified in Example 4 of this invention;

[0053] Figure 3 The correlation between the folic acid detection kit in Example 10 of this invention and the clinical samples of the benchmark reagent (n=95);

[0054] Figure 4 The results of sample homology detection in Example 10 of the present invention (n=33). Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0056] Unless otherwise specified, the reagents used in the following examples are all commercially available or obtained using methods known in the art.

[0057] Example 1

[0058] This invention provides a folic acid-γ-aminobutyric acid-bovine serum albumin conjugate and its preparation method.

[0059] 1. Preparation of γ-aminobutyric acid-bovine serum albumin complex

[0060] 1) Preparation

[0061] Weigh 500 mg of γ-aminobutyric acid (Aladdin) and dissolve it in 10 mL of ultrapure water to obtain a 50 mg / mL GABA solution. Weigh 300 mg of bovine serum albumin (Sigma) and dissolve it in 3.0 mL of ultrapure water to obtain a 100 mg / mL BSA solution for later use.

[0062] 2) Reaction

[0063] 200 mg of carbodiimide (Thermo Fisher Scientific) was weighed and dissolved thoroughly in 7.5 mL of GABA solution. Then, 2.5 mL of BSA solution was added and the mixture was shaken and stirred. The mixture was placed on a rotary reactor and reacted at 22–28 °C for 2 h to obtain the γ-aminobutyric acid-bovine serum albumin complex (GABA-BSA). The mass ratio of GABA:BSA:EDC in this reaction was 1:0.67:0.53.

[0064] 3) Ultrafiltration

[0065] Add 5.0 mL of 0.05 M borate buffer (BSB, pH = 8.0) and 3.0 mL of GABA-BSA complex to the inner tube of a 15 mL ultrafiltration tube, and cap the tube. Centrifuge at 5000 rpm for 10 min at 2–8 °C. Add another 5.0 mL of BSB buffer (pH = 8.0) and centrifuge again. After centrifugation, remove the inner tube, invert it into a clean centrifuge tube, and centrifuge at 5000 rpm for 10 min at 2–8 °C to transfer the desalted GABA-BSA complex from the ultrafiltration inner tube to a collection tube. Store at 2–8 °C for later use. Measure the GABA-BSA complex using a BCA protein quantification kit.

[0066] 2. Preparation of folic acid-γ-aminobutyric acid-bovine serum albumin (FA-GABA-BSA) conjugate

[0067] 1) Folic acid activation

[0068] Accurately weigh 15 mg of folic acid and dissolve it in 4.5 mL of 0.05 M BSB buffer. Dissolve the folic acid thoroughly in the dark, and then add 0.5 mL of BSB buffer. Add 5 mg of N-hydroxysulfosuccinimide (Sulfo-NHS) and 3.5 mg of EDC (FA:Sulfo-NHS:EDC molar ratio = 1.0:0.68:0.53). Sonicate to mix thoroughly, then place the mixture in a rotary kinematic reactor in the dark. React at room temperature for 3 hours, then centrifuge at 10,000 rpm for 10 minutes. Collect the supernatant and discard the dicyclohexylurea reaction byproduct.

[0069] 2) Preparation of FA-GABA-BSA

[0070] 1.0 mL of activated FA was added to 2.0 mL of GABA-BSA complex solution within 1 min. After ultrasonic mixing, the mixture was placed in a rotary reactor in the dark and reacted at room temperature for 3 h, followed by reaction at 2–8 °C for 25 h. The reaction solution was collected and subjected to two ultrafiltrations with 0.2 M carbonate buffer (CBS, pH = 9.6) to remove unreacted folic acid esters and byproduct impurities. The buffer was then replaced with pH 9.6, 0.2 M CBS buffer to obtain FA-GABA-BSA. The protein concentration was determined to be 10.9 mg / mL using the BCA method.

[0071] 3) Calculation of FA-GABA-BSA concentration and coupling ratio

[0072] Because folic acid has significant absorption peaks at both 280 nm and 360 nm, while protein has strong absorption at 280 nm, a standard curve for folic acid (FA) was established by detecting its OD value at 360 nm to calculate the FA concentration; according to the formula: Calculate the coupling ratio of FA to GABA-BSA in the FA-GABA-BSA conjugate, where M FA / M 载体 : The coupling ratio of FA to GABA-BSA vector; C FA FA concentration; MW FA FA molecular weight; C 载体 GABA-BSA carrier concentration; MW 载体 The molecular weight of the GABA-BSA carrier is shown in Table 1. The final coupling ratio of FA to GABA-BSA was 10.02.

[0073] Table 1. FA-GABA-BSA concentrations and coupling ratios (unit: mg / mL)

[0074] name <![CDATA[OD 360 ]]> Dilution factor FA concentration protein concentration Molecular weight Mw Coupling ratio FA 0.689 20 0.725 / 441.4 / FA-GABA-BSA 0.576 20 0.598 10.9 66446 10.02

[0075] Example 2

[0076] This invention provides a folic acid-γ-aminobutyric acid-bovine serum albumin conjugate and its preparation method.

[0077] 1. Preparation of γ-aminobutyric acid-bovine serum albumin complex

[0078] 1) Preparation

[0079] Weigh 450 mg of γ-aminobutyric acid (Aladdin) and dissolve it in 10 mL of ultrapure water to obtain a 45 mg / mL GABA solution. Weigh 270 mg of bovine serum albumin (Sigma) and dissolve it in 3.0 mL of ultrapure water to obtain a 90 mg / mL BSA solution.

[0080] 2) Reaction

[0081] 135 mg of carbodiimide (Thermo Fisher Scientific) was weighed and dissolved thoroughly in 7.5 mL of GABA solution. Then, 2.25 mL of BSA solution was added and the mixture was shaken and stirred. The mixture was placed on a rotary reactor and reacted at 22–28 °C for 3 h to obtain the γ-aminobutyric acid-bovine serum albumin complex (GABA-BSA). The mass ratio of GABA:BSA:EDC in this reaction was 1:0.6:0.4.

[0082] 3) Ultrafiltration

[0083] Add 5.0 mL of 0.01 M borate buffer (BSB, pH = 6.0) and 3.0 mL of GABA-BSA complex to the inner tube of a 15 mL ultrafiltration tube, and cap the tube. Centrifuge at 10,000 rpm for 10 min at 2–8 °C. Add another 5.0 mL of BSB buffer (pH = 6.0) and centrifuge again. After centrifugation, remove the inner tube, invert it into a clean centrifuge tube, and centrifuge at 10,000 rpm for 10 min at 2–8 °C to transfer the desalted GABA-BSA complex from the ultrafiltration inner tube to a collection tube. Store at 2–8 °C for later use. Measure the GABA-BSA complex using a BCA protein quantification kit.

[0084] 2. Preparation of folic acid-γ-aminobutyric acid-bovine serum albumin (FA-GABA-BSA) conjugate

[0085] 1) Folic acid activation

[0086] Accurately weigh 15 mg of folic acid and dissolve it in 4.5 mL of 0.01 M BSB buffer. Dissolve the folic acid thoroughly in the dark, and then add 0.5 mL of BSB buffer. Add 7.4 mg of N-hydroxysulfosuccinimide (Sulfo-NHS) and 1.3 mg of EDC (FA:Sulfo-NHS:EDC molar ratio = 0.5:0.5:0.1). Mix thoroughly by sonication, and place the mixture in a rotary kinematic reactor in the dark. React at room temperature for 5 h, then centrifuge at 5000 rpm for 15 min. Collect the supernatant and discard the dicyclohexylurea reaction byproduct.

[0087] 2) Preparation of FA-GABA-BSA

[0088] 1.0 mL of activated FA was added to 2.0 mL of GABA-BSA complex solution within 1 min. After ultrasonic mixing, the mixture was placed in a rotary reactor in the dark and reacted at room temperature for 5 h, followed by reaction at 2–8 °C for 28 h. The reaction solution was collected and subjected to two ultrafiltrations with 0.1 M carbonate buffer (CBS, pH = 8.0) to remove unreacted folic acid activated esters and byproduct impurities. The buffer was then replaced with pH 8.0, 0.1 M CBS buffer to obtain FA-GABA-BSA.

[0089] Example 3

[0090] This invention provides a folic acid-γ-aminobutyric acid-bovine serum albumin conjugate and its preparation method.

[0091] 1. Preparation of γ-aminobutyric acid-bovine serum albumin complex

[0092] 1) Preparation

[0093] Weigh 550 mg of γ-aminobutyric acid (Aladdin) and dissolve it in 10 mL of ultrapure water to obtain a 55 mg / mL GABA solution. Weigh 330 mg of bovine serum albumin (Sigma) and dissolve it in 3.0 mL of ultrapure water to obtain a 110 mg / mL BSA solution for later use.

[0094] 2) Reaction

[0095] 288.75 mg of carbodiimide (Thermo Fisher Scientific) was weighed and dissolved thoroughly in 7.5 mL of GABA solution. Then, 3.0 mL of BSA solution was added and the mixture was shaken and stirred. The mixture was placed on a rotary reactor and reacted at 22–28 °C for 1 h to obtain the γ-aminobutyric acid-bovine serum albumin complex (GABA-BSA). The mass ratio of GABA:BSA:EDC in this reaction was 1:0.8:0.7.

[0096] 3) Ultrafiltration

[0097] Add 5.0 mL of 0.1 M borate buffer (BSB, pH = 9.0) and 3.0 mL of GABA-BSA complex to the inner tube of a 15 mL ultrafiltration tube, and cap the tube. Centrifuge at 5000 rpm for 15 min at 2–8 °C. Add another 5.0 mL of BSB buffer (pH = 9.0) and centrifuge again. After centrifugation, remove the inner tube, invert it into a clean centrifuge tube, and centrifuge at 5000 rpm for 15 min at 2–8 °C to transfer the desalted GABA-BSA complex from the ultrafiltration inner tube to a collection tube. Store at 2–8 °C for later use. Measure the GABA-BSA complex using a BCA protein quantification kit.

[0098] 2. Preparation of folic acid-γ-aminobutyric acid-bovine serum albumin (FA-GABA-BSA) conjugate

[0099] 1) Folic acid activation

[0100] Accurately weigh 15 mg of folic acid and dissolve it in 4.5 mL of 0.1 M BSB buffer. Dissolve the folic acid thoroughly in the dark, and then add 0.5 mL of BSB buffer. Add 11.1 mg of N-hydroxysulfosuccinimide (Sulfo-NHS) and 6.5 mg of EDC (FA:Sulfo-NHS:EDC molar ratio = 1.0:1.5:1.0). After sonicating and mixing, place the mixture in a rotary kinematic reactor in the dark and react at room temperature for 1 h. Centrifuge at 10000 rpm for 10 min, collect the supernatant, and discard the dicyclohexylurea reaction byproduct.

[0101] 2) Preparation of FA-GABA-BSA

[0102] 1.0 mL of activated FA was added to 2.0 mL of GABA-BSA complex solution within 1 min. After ultrasonic mixing, the mixture was placed in a rotary reactor in the dark and reacted at room temperature for 1 h, followed by reaction at 2–8 °C for 22 h. The reaction solution was collected and subjected to two ultrafiltrations with 0.5 M carbonate buffer (CBS, pH = 11.0) to remove unreacted folic acid activated esters and byproduct impurities. The buffer was then replaced with pH 11.0, 0.5 M CBS buffer to obtain FA-GABA-BSA.

[0103] Example 4

[0104] This embodiment provides a method for extracting folic acid-binding protein from whey protein powder, the specific steps of which are as follows:

[0105] 1. Preparation of FA-GABA-BSA affinity chromatography column

[0106] 1) Activation: Weigh 4.0g of CNBr-Sepharose 4B (GE Healthcare) and soak it in 0.001M HCl in a sintered glass funnel for 10 minutes. Repeatedly aspirate with 50 column volumes (CV, approximately 500mL) of 0.001M HCl to allow it to swell (1g of activated 4B dry powder beads will have a volume of approximately 3.5mL after soaking). Keep the activation time within 15 minutes.

[0107] 2) Cross-linking: 12 mL of activated agarose beads were added to 12 mL of pH 9.6, 0.1 M CBS buffer containing 40 mg of FA-GABA-BSA conjugate, and reacted in a rotary reactor at 2–8 °C for 25 h. The cross-linked FA-GABA-BSA-4B agarose beads were washed with 0.1 M CBS in a sintered glass funnel, and the filtrate was collected. The protein concentration in the filtrate was tested by the BCA method, and the content of FA-GABA-BSA bound to the agarose beads was calculated to be 32.41 mg, with a cross-linking rate of 81.03%.

[0108] 3) Blocking: Add 5 column volumes (approximately 60 mL) of 0.5 M glycine buffer to the agarose beads for blocking, and rotate the reaction at room temperature for 30 min.

[0109] 4) Washing: After sealing, repeatedly wash the FA-GABA-BSA-4B agarose beads with 10 column volumes (approximately 100 mL) of pH 10.0, 0.05 M MCBS buffer (containing 7.5% wt sodium hydroxide), 0.1 M urea (9% wt sodium chloride), and pH 2.6, 0.05 M glycine buffer (containing 10% wt hydrochloric acid) using a sintered glass funnel. Finally, wash the FA-GABA-BSA-4B agarose beads with pH 7.4, 0.05 M PBS buffer and store at 2–8°C protected from light.

[0110] 2. Extraction of folic acid-binding protein

[0111] Folic acid-binding protein was extracted from whey protein powder using an FA-GABA-BSA-4B affinity chromatography column. The specific procedure is as follows:

[0112] 1) Sample preparation: Accurately weigh 15.0 g of whey protein powder (NUTREND) into a 500 mL beaker, add 300 mL of pH 9.6, 0.1 M CBS equilibration buffer to dissolve it, so that the protein concentration is 50 mg / mL, and stir at 2–8 °C for 25 h. After centrifugation at 10,000 rpm for 15 min, collect the supernatant and filter it through a 0.45 μm membrane for later use.

[0113] 2) Sample loading: Due to the large sample volume, sample incubation was performed by stirring. FA-GABA-BSA-4B agarose beads were added to 253 mL of the filtrate obtained in step 1), and stirred at room temperature for 2 hours to ensure sufficient contact between the packing material and the whey protein solution. The mixture was then filtered through a glass frit funnel, and the eluent was collected in a clean container and labeled for later use to prevent FBP loss due to operational errors.

[0114] 3) Column packing: Select a Φ1.0cm×15cm chromatography column (Kimble Company), fill it with filtered FA-GABA-BSA-4B agarose gel to a height of 9.8mL (i.e., 1CV = 9.8mL), add pH 9.6, 0.1M CBS equilibration buffer at a flow rate of 1.0mL / min to wash away unbound whey protein and equilibrate the chromatography column, and store it in the dark.

[0115] 4) Washing: Use 3 column volumes (approximately 30 mL) of pH 8.0, 1.0 M TBS buffer (containing 1.5 M sodium chloride and 0.03 M imidazole) to wash away non-specific proteins at a flow rate of 1.0 mL / min. Collect 2.0 mL of the eluted fraction from each tube and detect the absorbance of the eluent fraction at 280 nm. This fraction is not usable.

[0116] 5) Elution: After the baseline stabilizes, elute the FBP with approximately 60 mL of pH 2.6, 0.05 M glycine buffer, collecting 2.0 mL of the eluted fraction per tube, and then transfer it to A. 280 Test results. Figure 1 As shown.

[0117] 6) Preservation: Add 4 column volumes (approximately 40 mL) of pH 7.4, 0.01 M PBS buffer to preserve the affinity chromatography column.

[0118] 7) Neutralization: Neutralize 34 mL (tubes 21-37, 17 tubes total) of the elution fraction containing FBP with 3.0 M Tris buffer.

[0119] 8) Calculation: Using the corresponding buffer solution as a blank control, A 280 Detection, reading OD 280 =1.648, FBP protein concentration: 1.03 mg / mL, the total amount of FBP obtained is: 1.03 mg / mL × 34 mL = 35.06 mg.

[0120] 9) Concentration: Filter the FBP elution fraction using a 10KD MWCO membrane, concentrate the FBP to obtain an FBP protein concentration of 5.07 mg / mL, filter through a 0.45 μm membrane, and store at 2–8 °C for later use.

[0121] 3. Purification of folic acid-binding protein

[0122] Five mL (approximately 25 mg) of crude folic acid-binding protein obtained by affinity chromatography was further purified using a Superdex 200 gel filtration column (GE Healthcare). Elution was performed by adding 0.01 M PBS buffer (pH 7.4) at a flow rate of 1.0 mL / min, and the eluent was collected. The final FBP yield was 22.8 mg, with a recovery rate of 91.0%.

[0123] 4. Identification of FBP molecular weight and purity

[0124] SDS-PAGE analysis showed that the molecular weight of folic acid-binding protein was approximately 32 kDa. The results are as follows: Figure 2 As shown in the figure. HPLC analysis shows that its purity can reach over 98%.

[0125] Example 5

[0126] This embodiment provides a method for extracting folic acid-binding protein from whey protein powder, the specific steps of which are as follows:

[0127] 1. Preparation of FA-GABA-BSA affinity chromatography column

[0128] 1) Activation: Weigh 4.0g of CNBr-Sepharose 4B (GE Healthcare) and soak it in 0.0005M HCl in a sintered glass funnel for 15 minutes. Repeatedly aspirate with 50 column volumes (CV, approximately 500mL) of 0.0005M HCl to allow it to swell (1g of activated 4B dry powder beads will have a volume of approximately 3.5mL after soaking). Keep the activation time within 15 minutes.

[0129] 2) Crosslinking: Add 12 mL of activated agarose beads to 12 mL of pH 8.0, 0.1 M CBS buffer containing 40 mg FA-GABA-BSA conjugate, and react in a rotary reactor at 2–8 °C for 22 h. The crosslinked FA-GABA-BSA-4B agarose beads are then washed with 0.1 M CBS in a sintered glass funnel.

[0130] 3) Blocking: Add 5 column volumes (approximately 60 mL) of 0.1 M glycine buffer to the agarose beads for blocking, and rotate the reaction at room temperature for 60 min.

[0131] 4) Washing: After sealing, repeatedly wash the FA-GABA-BSA-4B agarose beads with 10 column volumes (approximately 100 mL) of pH 8.0, 0.01 M MCBS buffer (containing 5% wt sodium hydroxide), 0.1 M urea (3% wt sodium chloride), and pH 2.0, 0.01 M glycine buffer (containing 20% ​​wt hydrochloric acid) using a sintered glass funnel. Finally, wash the FA-GABA-BSA-4B agarose beads with pH 6.5, 0.1 M PBS buffer and store at 2–8°C protected from light.

[0132] 2. Extraction of folic acid-binding protein

[0133] Folic acid-binding protein was extracted from whey protein powder using an FA-GABA-BSA-4B affinity chromatography column. The specific procedure is as follows:

[0134] 1) Sample preparation: Accurately weigh 12.0 g of whey protein powder (NUTREND) and place it in a 500 mL beaker. Add 300 mL of pH 8.0, 0.1 M CBS equilibration buffer to dissolve the protein to a concentration of 40 mg / mL. Incubate at 2–8 °C and stir for 22 h. Centrifuge at 10,000 rpm for 10 min, collect the supernatant, and filter through a 0.45 μm membrane for later use.

[0135] 2) Sample loading: Due to the large sample volume, incubation was performed by stirring. FA-GABA-BSA-4B agarose beads were added to 253 mL of the filtrate obtained in step 1), and stirred at room temperature for 1 hour to ensure sufficient contact between the packing material and the whey protein solution. The mixture was then filtered through a glass frit funnel, and the eluent was collected in a clean container and labeled for later use to prevent FBP loss due to operational errors.

[0136] 3) Column packing: Select a Φ1.0cm×15cm chromatography column (Kimble Company), fill it with filtered FA-GABA-BSA-4B agarose gel to a height of 9.8mL (i.e., 1CV = 9.8mL), add pH 8.0, 0.1M CBS equilibration buffer at a flow rate of 1.0mL / min to wash away unbound whey protein and equilibrate the chromatography column, and store it in the dark.

[0137] 4) Washing: Use 3 column volumes (approximately 30 mL) of pH 7.5, 0.5 M TBS buffer (containing 1.0 M sodium chloride and 0.01 M imidazole) to wash away non-specific proteins at a flow rate of 3.0 mL / min. Collect 2.0 mL of the eluted fraction from each tube and detect the absorbance of the eluted fraction at 280 nm. This fraction is not usable.

[0138] 5) Elution: After the baseline stabilizes, elute the FBP with approximately 60 mL of pH 2.0, 0.2 M glycine buffer, collecting 2.0 mL of the eluted fraction per tube, and then transfer it to A. 280 Testing.

[0139] 6) Preservation: Add 4 column volumes (approximately 40 mL) of pH 6.5, 0.1 M PBS buffer to preserve the affinity chromatography column.

[0140] 7) Neutralization: Neutralize the elution fraction containing FBP with 2.5M Tris buffer.

[0141] 8) Concentration: Filter the FBP eluent using a 10KD MWCO membrane, concentrate the FBP, filter it again using a 0.45μm membrane, and store it at 2-8℃ for later use.

[0142] 3. Purification of folic acid-binding protein

[0143] Take 5 mL (approximately 25 mg) of crude folic acid-binding protein obtained by affinity chromatography and further purify it using a Superdex 200 gel filtration column (GE Healthcare). Elute with 0.01 M PBS buffer (pH 7.5) at a flow rate of 1.0 mL / min and collect the eluent.

[0144] Example 6

[0145] This embodiment provides a method for extracting folic acid-binding protein from whey protein powder, the specific steps of which are as follows:

[0146] 1. Preparation of FA-GABA-BSA affinity chromatography column

[0147] 1) Activation: Weigh 4.0g of CNBr-Sepharose 4B (GE Healthcare) and soak it in 0.01M HCl in a sintered glass funnel for 10 minutes. Repeatedly aspirate with 50 column volumes (CV, approximately 500mL) of 0.01M HCl to allow it to swell (1g of activated 4B dry powder beads will have a volume of approximately 3.5mL after soaking). Keep the activation time within 10 minutes.

[0148] 2) Crosslinking: Add 12 mL of activated agarose beads to 12 mL of pH 11.0, 0.5 M CBS buffer containing 40 mg FA-GABA-BSA conjugate, and react in a rotary reactor at 2–8 °C for 28 h. The crosslinked FA-GABA-BSA-4B agarose beads are then washed with 0.5 M CBS in a sintered glass funnel.

[0149] 3) Blocking: Add 5 column volumes (approximately 60 mL) of 1.0 M glycine buffer to the agarose beads for blocking, and rotate the reaction at room temperature for 30 min.

[0150] 4) Washing: After sealing, repeatedly wash the FA-GABA-BSA-4B agarose beads with 10 column volumes (approximately 100 mL) of pH 11.0, 0.5 M MCBS buffer (containing 10% wt sodium hydroxide), 0.5 M urea (15% wt sodium chloride), and pH 4.0, 0.1 M glycine buffer (containing 1% wt hydrochloric acid) using a sintered glass funnel. Finally, wash the FA-GABA-BSA-4B agarose beads with pH 7.5, 0.01 M PBS buffer and store at 2–8°C protected from light.

[0151] 2. Extraction of folic acid-binding protein

[0152] Folic acid-binding protein was extracted from whey protein powder using an FA-GABA-BSA-4B affinity chromatography column. The specific procedure is as follows:

[0153] 1) Sample preparation: Accurately weigh 15.0 g of whey protein powder (NUTREND) into a 500 mL beaker, add 300 mL of pH 11.0, 0.5 M CBS equilibration buffer to dissolve it, so that the protein concentration is 50 mg / mL, and stir at 2–8 °C for 28 h. After centrifugation at 5000 rpm for 15 min, collect the supernatant and filter it through a 0.45 μm membrane for later use.

[0154] 2) Sample loading: Due to the large sample volume, incubation was performed by stirring. FA-GABA-BSA-4B agarose beads were added to 253 mL of the filtrate obtained in step 1), and stirred at room temperature for 5 hours to ensure sufficient contact between the packing material and the whey protein solution. The mixture was then filtered through a glass frit funnel, and the eluent was collected in a clean container and labeled for later use to prevent FBP loss due to operational errors.

[0155] 3) Column packing: Select a Φ1.0cm×15cm chromatography column (Kimble Company), fill it with filtered FA-GABA-BSA-4B agarose gel to a height of 9.8mL (i.e., 1CV = 9.8mL), add pH11.0, 0.5M CBS equilibration buffer at a flow rate of 1.0mL / min to wash away unbound whey protein and equilibrate the chromatography column, and store it in the dark.

[0156] 4) Washing: Use 3 column volumes (approximately 30 mL) of pH 8.5, 1.0 M TBS buffer (containing 2.0 M sodium chloride and 0.05 M imidazole) to wash away non-specific proteins at a flow rate of 1.0 mL / min. Collect 2.0 mL of the eluted fraction from each tube and detect the absorbance of the eluted fraction at 280 nm. This fraction is not usable.

[0157] 5) Elution: After the baseline stabilizes, elute the FBP with approximately 60 mL of pH 4.0, 0.05 M glycine buffer, collecting 2.0 mL of the eluted fraction per tube, and then transfer it to A. 280 Testing.

[0158] 6) Preservation: Add 4 column volumes (approximately 40 mL) of pH 7.5, 0.01 M PBS buffer to preserve the affinity chromatography column.

[0159] 7) Neutralization: Neutralize the elution fraction containing FBP with 3.0M Tris buffer.

[0160] 8) Concentration: Filter the FBP eluent using a 10KD MWCO membrane, concentrate the FBP, filter it again using a 0.45μm membrane, and store it at 2-8℃ for later use.

[0161] 3. Purification of folic acid-binding protein

[0162] Take 5 mL (approximately 25 mg) of crude folic acid-binding protein obtained by affinity chromatography and further purify it using a Superdex 200 gel filtration column (GE Healthcare). Elute with 0.1 M PBS buffer (pH 6.5) at a flow rate of 1.0 mL / min and collect the eluent.

[0163] Example 7

[0164] This embodiment provides a folic acid detection kit, the reagents of which include:

[0165] The following reagents were prepared: pretreatment reagent 1, pretreatment reagent 2, magnetic bead working solution, biotinylated folic acid-binding protein working solution, enzyme-labeled folic acid conjugate working solution, luminescent reagent, washing solution, and negative / positive calibrators. Pretreatment reagent 1 was a sodium 2-mercaptoethanesulfonate (MESNA) solution with a concentration of 5.0 mg / mL; pretreatment reagent 2 was a NaOH solution with a concentration of 25 mg / mL; the magnetic bead working solution consisted of biotinylated antibody-coated magnetic beads and 0.01 M PBS buffer at pH 7.4, with a magnetic bead concentration of 0.75 mg / mL; the biotinylated folic acid-binding protein working solution consisted of biotinylated folic acid-binding protein (prepared in Example 4) and 0.05 M Tris buffer at pH 7.4, with a biotinylated folic acid-binding protein concentration of 0.25 μg / mL; the enzyme-labeled folic acid conjugate working solution consisted of FA-GABA-BSA conjugate labeled with alkaline phosphatase and 0.05 M Tris buffer at pH 7.4. The solution consists of Tris buffer and an enzyme-labeled folic acid conjugate working solution with a FA-GABA-BSA conjugate labeled with alkaline phosphatase at a concentration of 0.6 μg / mL. The luminescent solution consists of AMPPD and Dioxetane Buffer, with an AMPPD concentration of 0.1 mg / mL. The washing buffer is 0.01 MPa BST buffer at pH 7.4. The calibrators are folic acid antigens diluted in PBS buffer containing 0.1% ProClin 300 and 5% BSA at concentrations of 5.0 ± 0.5 ng / mL and 15.0 ± 2.0 ng / mL.

[0166] Example 8

[0167] This embodiment provides a folic acid detection kit, the reagents of which include:

[0168] The following reagents were included: pretreatment reagent 1, pretreatment reagent 2, magnetic bead working solution, biotinylated folic acid-binding protein working solution, enzyme-labeled folic acid conjugate working solution, luminescent reagent, washing solution, and negative / positive calibrators. Pretreatment reagent 1 consisted of a solution of sodium 2-mercaptoethanesulfonate (MESNA) at a concentration of 2.0 mg / mL; pretreatment reagent 2 was a NaOH solution at a concentration of 10 mg / mL; the magnetic bead working solution consisted of biotinylated antibody-coated magnetic beads and 0.01 M PBS buffer at pH 7.5, with a magnetic bead concentration of 0.1 mg / mL; the biotinylated folic acid-binding protein working solution consisted of biotinylated folic acid-binding protein (prepared in Example 5) and 0.05 M Tris buffer at pH 7.0, with a biotinylated folic acid-binding protein concentration of 0.1 μg / mL; the enzyme-labeled folic acid conjugate working solution consisted of a FA-GABA-BSA conjugate labeled with alkaline phosphatase and pH 7.0. The working solution consisted of 0.05% Tris buffer at pH 7.0, with the concentration of the FA-GABA-BSA conjugate labeled with alkaline phosphatase at 0.1 μg / mL. The luminescent solution consisted of AMPPD and Dioxetane Buffer, with the AMPPD concentration at 0.1 mg / mL. The washing buffer was 0.01 M PBST buffer at pH 7.5. The calibrators were 5.0 ± 0.5 ng / mL and 15.0 ± 2.0 ng / mL folate antigen diluted in PBS buffer containing 0.1% ProClin 300 and 5% BSA.

[0169] Example 9

[0170] This embodiment provides a folic acid detection kit, the reagents of which include:

[0171] The following reagents were included: pretreatment reagent 1, pretreatment reagent 2, magnetic bead working solution, biotinylated folic acid-binding protein working solution, enzyme-labeled folic acid conjugate working solution, luminescent reagent, washing solution, and negative / positive calibrators. Pretreatment reagent 1 was a sodium 2-mercaptoethanesulfonate (MESNA) solution with a concentration of 10.0 mg / mL; pretreatment reagent 2 was a NaOH solution with a concentration of 100 mg / mL; the magnetic bead working solution consisted of biotinylated antibody-coated magnetic beads and 0.1 M PBS buffer at pH 6.5, with a magnetic bead concentration of 1.5 mg / mL; the biotinylated folic acid-binding protein working solution consisted of biotinylated folic acid-binding protein (prepared in Example 6) and 0.2 M Tris buffer at pH 8.5, with a biotinylated folic acid-binding protein concentration of 1.5 μg / mL; the enzyme-labeled folic acid conjugate working solution consisted of FA-GABA-BSA conjugate labeled with alkaline phosphatase and pH 8.5. The working solution consisted of 0.2% Tris buffer at pH 8.5, with the concentration of the FA-GABA-BSA conjugate labeled with alkaline phosphatase at 1.5 μg / mL. The luminescent solution consisted of AMPPD and Dioxetane Buffer, with the AMPPD concentration at 1.5 mg / mL. The washing buffer was 0.1% MPBST buffer at pH 6.5. The calibrators were 5.0 ± 0.5 ng / mL and 15.0 ± 2.0 ng / mL folic acid antigen diluted in PBS buffer containing 0.1% ProClin 300 and 5% BSA.

[0172] Example 10

[0173] This embodiment provides a method for detecting folic acid using the kit from Example 7. The specific operation is as follows:

[0174] 40 μL of the test sample, 25 μL of pretreatment reagent 1, and 25 μL of pretreatment reagent 2 were co-incubated for 10 min to release the bound endogenous folic acid. Then, 50 μL of biotinylated folic acid-binding protein working solution and 50 μL of magnetic bead working solution were added to the pretreated sample and incubated for 10 min to form a folic acid-binding protein-folate complex, which was then bound to the solid-phase magnetic beads. Next, 50 μL of enzyme-labeled folic acid conjugate working solution was added and incubated for 10 min to competitively bind the biotinylated folic acid-binding protein blank sites. The magnetic particles of the reaction solution were then adsorbed onto the electrode using a magnet. Unbound substances were washed away, and a luminescent substrate was added to induce light emission from the folic acid complex. This light signal was detected by a photomultiplier tube, and the results were automatically output from the instrument.

[0175] The analytical sensitivity, accuracy, linear range, specificity, stability, homology, and correlation of the folic acid detection kit of Example 7, tested using the above method, are as follows:

[0176] 1) Analytical sensitivity

[0177] Using a zero-concentration calibrator as the test sample, the folic acid detection kit of Example 7 was used for testing. The measurements were repeated 20 times, and the relative luminescence intensity (RLU) of the 20 measurements was obtained. The mean (M) and standard deviation (SD) were calculated, yielding M-2SD. A linear equation was obtained by performing a two-point regression fitting based on the concentration (0.5 ng / mL) of the zero-concentration calibrator and the relative luminescence intensity (RLU) of adjacent concentration calibrators. Substituting the relative luminescence intensity corresponding to M-2SD into the fitted linear equation, the corresponding concentration value was calculated to be 0.28 ng / mL. Therefore, the sensitivity of the folic acid detection kit of Example 7 is 0.28 ng / mL, which is superior to the sensitivity of conventional folic acid detection kits, whose analytical sensitivity can reach 0.6 ng / mL.

[0178] 2) Accuracy

[0179] Within the measurement range specified by the kit, an appropriate buffer system was selected to prepare two concentration points for the international standard (No.: 95 / 528): 4.0 ng / mL and 12.0 ng / mL. Each point was measured in triplicate, and the average value (M) was taken. The ratio of the measured concentration to the labeled concentration was calculated. The measurement results are detailed in Table 2. Table 2 shows that the accuracy of the folic acid detection kit prepared using the above-mentioned FBP is 101%–105%, within the range of 90%–110%, indicating that the folic acid detection kit of Example 7 has high accuracy.

[0180] Table 2. Accuracy test results (unit: ng / mL)

[0181]

[0182] 3) Linear detection

[0183] Using the folic acid test kit of Example 7, high-value samples X8 with concentrations exceeding the upper limit of linearity and eight samples diluted with zero-value calibrator X0 were measured. The samples were randomly arranged, and the measurements were repeated three times. The average value was calculated, and the theoretical concentration value of each sample was calculated based on the dilution ratio. A linear regression was performed on the average value of the measurements, and the slope, intercept, and correlation coefficient r were calculated. The results are shown in Table 3. r = 0.9996, indicating a high linear correlation within the detection range.

[0184] Table 3. Linearity detection results (unit: ng / mL)

[0185]

[0186] 4) Specific detection

[0187] The folic acid assay kit of Example 7 was used to test folic acid structural analogs: aminopterin, methotrexate, and folinic acid. The assays were repeated three times, and the cross-reactivity rate was calculated. The results in Table 4 show that the folic acid assay kit does not easily cross-react with folic acid structural analogs, further demonstrating that the folic acid binding protein purified in this invention has high specificity.

[0188] Table 4 Cross-reactivity rate detection (unit: ng / mL)

[0189]

[0190]

[0191] 5) Correlation detection

[0192] The folic acid detection kit of Example 7 was compared and validated with Roche's folic acid electrochemiluminescence detection kit, and correlation analysis was performed. The results are as follows: Figure 3 As shown, the correlation coefficient r ≥ 0.9843 indicates that the test results from these two test kits have a good correlation.

[0193] 6) Homology detection

[0194] The folic acid detection kit from Example 7 was used to test homologous serum and plasma samples, and correlation analysis was performed. The results are as follows: Figure 4 As shown, the correlation coefficient r ≥ 0.9857 between the two indicates that the results of the kit in detecting homologous serum and plasma samples are consistent.

[0195] 7) Stability testing

[0196] The biotinylated folic acid-binding protein solution in the folic acid detection kit of Example 7 was treated as follows: a: three freeze-thaw cycles at -20℃; b: seven freeze-thaw cycles at -20℃; c: accelerated heat treatment at 37℃ for seven days; d: accelerated heat treatment at 37℃ for 14 days; e: storage at 2-8℃ for seven days; f: storage at 2-8℃ for one month; g: storage at -20℃ for three months. Then, low-value samples (concentration 5.0 ng / mL) and high-value samples (concentration 15.0 ng / mL) were tested using the biotinylated folic acid-binding protein solution treated under the above different conditions, with each test performed three times. The bias and coefficient of variation were calculated, and the results are shown in Table 5.

[0197] Table 5. Stability test results (unit: ng / mL)

[0198]

[0199]

[0200] As shown in Table 5, the deviation (B5%) and coefficient of variation (CV) of the biotinylated folic acid binding protein (FBP-Bio) solution treated under the above different conditions were both less than 5% when detecting low / high value samples. This indicates that the FBP-Bio solution in the folic acid detection kit of Example 7 has good stability and the kit prepared using folic acid binding protein has good precision.

[0201] Furthermore, using the folic acid test kit of Example 7, the deviations in the test results for low-value samples and high-value samples after 14 days of accelerated heat treatment at 37°C were -3.95% and -0.50%, respectively. These deviations are superior to those of conventional folic acid test kits after 14 days of accelerated heat treatment at 37°C (specifically, the detection deviation for low-value samples in conventional folic acid test kits is 5.81%, and the detection deviation for high-value samples is 7.24%). This indicates that the biotinylated folic acid-binding protein solution in the folic acid test kit of Example 7 has high thermal stability at 37°C, and the folic acid test kit of Example 7 has a long shelf life.

[0202] Example 11

[0203] This embodiment provides a method for detecting folic acid using the kit from Example 8. The specific operation is as follows:

[0204] 20 μL of the test sample, 20 μL of pretreatment reagent 1, and 20 μL of pretreatment reagent 2 were co-incubated for 10 min to release the bound endogenous folic acid. Then, 20 μL of biotinylated folic acid-binding protein working solution and 20 μL of magnetic bead working solution were added to the pretreated sample and incubated for 10 min to form a folic acid-binding protein-folate complex, which was then bound to the solid-phase magnetic beads. Next, 20 μL of enzyme-labeled folic acid conjugate working solution was added and incubated for 10 min to competitively bind the biotinylated folic acid-binding protein blank sites. The magnetic particles of the reaction solution were then adsorbed onto the electrode using a magnet. Unbound substances were washed away, and a luminescent substrate was added to induce light emission from the folic acid complex. This light signal was detected by a photomultiplier tube, and the results were automatically output from the instrument.

[0205] Example 12

[0206] This embodiment provides a method for detecting folic acid using the kit from Example 9. The specific operation is as follows:

[0207] 100 μL of the test sample, 100 μL of pretreatment reagent 1, and 100 μL of pretreatment reagent 2 were co-incubated for 15 min to release the bound endogenous folic acid. Then, 100 μL of biotinylated folic acid-binding protein working solution and 100 μL of magnetic bead working solution were added to the pretreated sample and incubated for 15 min to form a folic acid-binding protein-folate complex, which was then bound to the solid-phase magnetic beads. Next, 100 μL of enzyme-labeled folic acid conjugate working solution was added and incubated for 15 min to competitively bind the biotinylated folic acid-binding protein blank sites. The magnetic particles of the reaction solution were then adsorbed onto the electrode using a magnet. Unbound substances were washed away, and a luminescent substrate was added to induce light emission from the folic acid complex. This light signal was detected by a photomultiplier tube, and the results were automatically output from the instrument.

[0208] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a folic acid-γ-aminobutyric acid-bovine serum albumin conjugate, characterized in that, Specifically, the following steps are included: S1. Dissolve carbodiimide in an aqueous solution of γ-aminobutyric acid, then add an aqueous solution of bovine serum albumin, mix well, and react at 22-28°C for at least 1 hour to obtain a γ-aminobutyric acid-bovine serum albumin complex; the mass ratio of γ-aminobutyric acid, bovine serum albumin and carbodiimide is 1:0.6-0.8:0.4-0.

7. S2. Wash and dissolve the γ-aminobutyric acid-bovine serum albumin complex with borate buffer to obtain a borate buffer solution of the γ-aminobutyric acid-bovine serum albumin complex. S3. Add N-hydroxysulfosuccinimide and carbodiimide to the folic acid borate buffer solution, mix well, react at room temperature in the dark for at least 1 hour, centrifuge, and take the supernatant to obtain the activated folic acid solution; the molar ratio of folic acid, N-hydroxysulfosuccinimide and carbodiimide is 0.5-1.0:0.5-1.5:0.1-1.0; S4. Add the activated folic acid solution to the borate buffer solution of the γ-aminobutyric acid-bovine serum albumin complex, react at room temperature in the dark for at least 1 hour, and then react at 2-8°C for at least 8 hours; after the reaction is completed, wash the reaction product with carbonate buffer to obtain the folic acid-γ-aminobutyric acid-bovine serum albumin conjugate.

2. The method for preparing the folic acid-γ-aminobutyric acid-bovine serum albumin conjugate according to claim 1, characterized in that, The specific operation of S2 is as follows: after mixing the borate buffer with the γ-aminobutyric acid-bovine serum albumin complex solution, centrifuge, discard the supernatant, and dissolve the centrifuged product with borate buffer to obtain a borate buffer solution of the γ-aminobutyric acid-bovine serum albumin complex. and / or The concentration of the borate buffer solution described in S3 is 0.01–0.1 M; and / or The centrifugation temperature described in S3 is 2–8℃, the rotation speed is 5000–10000 rpm, and the centrifugation time is 10–15 min; and / or The volume ratio of the activated folic acid solution and the borate buffer solution of the γ-aminobutyric acid-bovine serum albumin complex in S4 is 1:1.5 to 2.5; and / or The carbonate buffer solution described in S4 has a concentration of 0.1–0.5 M and a pH of 8.0–11.

0.

3. A folic acid-γ-aminobutyric acid-bovine serum albumin conjugate, characterized in that, Prepared according to the preparation method described in claim 1 or 2.

4. A method for extracting folic acid-binding protein from whey protein, characterized in that, Folic acid-binding protein was extracted from whey protein using folic acid-γ-aminobutyric acid-bovine serum albumin-4B agarose beads via affinity chromatography; the folic acid-γ-aminobutyric acid-bovine serum albumin-4B agarose beads were 4B agarose beads cross-linked with the folic acid-γ-aminobutyric acid-bovine serum albumin conjugate described in claim 3.

5. The method for extracting folic acid-binding protein from whey protein according to claim 4, characterized in that, The preparation method of the folic acid-γ-aminobutyric acid-bovine serum albumin-4B agarose beads includes the following steps: hydrogen bromide activated agarose gel beads are swollen with hydrochloric acid solution, added to a carbonate buffer solution of folic acid-γ-aminobutyric acid-bovine serum albumin conjugate, kept at 2-8℃ for more than 8 hours, washed with carbonate buffer, blocked with glycine buffer, and then washed to obtain folic acid-γ-aminobutyric acid-bovine serum albumin-4B agarose beads.

6. The method for extracting folic acid-binding protein from whey protein according to claim 5, characterized in that, The concentration of the hydrochloric acid solution is 0.0005–0.01 M; and / or The swelling time of the hydrogen bromide-activated agarose gel with hydrochloric acid solution should not exceed 15 minutes. and / or The carbonate buffer solution has a concentration of 0.1–0.5 M and a pH of 8.0–11.0; and / or The concentration of the glycine buffer solution is 0.1–1.0 M; and / or The cleaning process involves alternating rinsing with carbonate buffer, urea solution, and glycine buffer in sequence, followed by rinsing with phosphate buffer.

7. The method for extracting folic acid-binding protein from whey protein according to claim 4, characterized in that, The procedures for extracting folic acid-binding protein include: Dissolve whey protein in carbonate buffer and store at 2–8°C for at least 8 hours. Centrifuge, collect the supernatant, filter, and mix with folic acid-γ-aminobutyric acid-bovine serum albumin-4B agarose beads for at least 1 hour. After filtering off excess whey protein solution, pack it into a chromatography column, wash with carbonate buffer and equilibrate. Using TBS buffer (pH 7.5–8.5, 0.5–1.0 M) as the eluent, elute at a flow rate of 1.0–3.0 mL / min. Once the absorbance of the eluent at 280 nm stabilizes, elute with glycine buffer. Collect the eluent when the absorbance at 280 nm begins to show a significant upward trend. Stop collecting before the absorbance at 280 nm drops back to the baseline level.

8. The method for extracting folic acid-binding protein from whey protein according to claim 7, characterized in that, The carbonate buffer solution has a concentration of 0.1–0.5 M and a pH of 8.0–11.0; and / or The concentration of the dissolved whey protein is 40–50 mg / mL; and / or The centrifugation speed is 5000–10000 rpm, and the centrifugation time is 10–15 min; and / or The TBS buffer contains 1.0–2.0 M sodium chloride and 0.01–0.05 M imidazole; and / or The glycine buffer solution has a concentration of 0.05–0.2 M and a pH of 2.0–4.0; and / or The preparation method also includes neutralizing and concentrating the extracted folic acid-binding protein.

9. The method for extracting folic acid-binding protein from whey protein according to claim 4, characterized in that, The preparation method also includes purifying the extracted folic acid-binding protein.

10. The application of the method for extracting folic acid-binding protein from whey protein according to any one of claims 4 to 9 in the preparation of a folic acid detection kit, characterized in that, Folic acid-binding protein was extracted from whey protein using the method described above, and the resulting folic acid-binding protein was used as a raw material for preparing the folic acid detection kit.

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