A folic acid monoclonal antibody, its application, and a folic acid detection kit

Abstract

The present invention provides a folic acid monoclonal antibody, which has a high affinity with folic acid antigens, has strong recognition, and can be used to prepare products for detecting folic acid. The present invention provides a folic acid detection test strip; the test strip comprises a base plate and a sample pad, a fluorescent pad, a detection pad, and a sample suction pad stacked in sequence on the base plate. The present invention provides a folic acid detection kit, comprising the folic acid detection test strip and a shell, wherein the folic acid detection test strip is arranged inside the shell, and the kit has the advantages of achieving rapid detection, low cost, and high sensitivity. In addition, the kit described in the present invention also includes a folic acid sample processing liquid, which can achieve rapid release of folic acid and reduce the toxicity of the reagent, and the processing liquid contains components that protect folic acid, so that the folic acid in the sample after treatment is more stable and less susceptible to environmental factors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a folic acid sample processing solution and a folic acid monoclonal antibody, thereby obtaining a folic acid detection kit. Background Art

[0002] Folic acid is a small molecule present primarily in ionic form in many biochemical processes within the human body, playing a crucial physiological role. It supports cellular function, regulates gene activity, promotes red and white blood cell production, and promotes skin and intestinal renewal. Folic acid is also involved in the synthesis of several chemicals that regulate brain function. It is essential in a range of biochemical pathways involving one-carbon transfer, and plays a particularly crucial role in cell replication, where it is required for the synthesis of purines and pyrimidines. Growth and pregnancy increase the need for folic acid. Folic acid deficiency can lead to impaired cell replication and the accumulation of harmful metabolites. The former is primarily observed in rapidly replicating tissues (such as red blood cells), resulting in megaloblastic anemia, for example. Remethylation of homocysteine (Hcy) to methionine is closely linked to the metabolism of folic acid and other B vitamins. Inadequate folic acid intake can lead to elevated plasma Hcy levels, a risk factor for vascular disease. Furthermore, pre-pregnancy folic acid supplementation has been shown to prevent neural tube defects in fetuses.

[0003] The role of serum and red blood cell folate determination in diagnosing nutritional anemia and related diseases has been fully demonstrated. Both red blood cell folate and serum folate are indicators of the body's folate level. Red blood cell folate can reflect the body's folate storage status over the past three months. Low red blood cell folate indicates long-term folate deficiency. Red blood cell folate concentration = whole blood folate concentration / hematocrit Serum folate reflects the body's short-term folate level. Domestic and international researchers have conducted in-depth research on methods for detecting folate content in various products. Currently, there are a wide variety of methods for folate detection, including microbiological methods, colorimetry, high-performance liquid chromatography, thin-layer chromatography, ion capture, ultraviolet spectrophotometry, and chemiluminescence. Microbiological methods can only detect the total amount of folate compounds, which is limited by bacterial species and affected by antibiotics. Colorimetry has poor interference resistance and is therefore suitable for detecting pure folate. High-performance liquid chromatography (HPLC) instruments are expensive and somewhat complex to handle and operate. Thin-layer chromatography (TLC) alone has low sensitivity. Ultraviolet spectrophotometry, while advanced, currently has limited reagents and high costs, making it suitable only for detecting pure products. Chemiluminescence, the most widely used method in clinical practice, is however expensive, highly dependent on equipment and personnel, has poor interference resistance and reproducibility, and requires a long detection time.

[0004] In view of this, the present invention provides a monoclonal antibody with high affinity to folic acid and good specificity, and uses the monoclonal antibody to develop a fluorescent immunochromatographic detection kit. The method has low dependence on personnel and equipment, low cost and short detection time.

[0005] Folic acid testing requires sample pretreatment. Most folate in a sample is bound to endogenous proteins, with less than half being free. Most manufacturers use alkaline dissociation, which involves denaturing proteins with a strong base and simultaneously adding a specific reducing agent to break down disulfide bonds. The strong base is used to expose deeply buried disulfide bonds. This thorough treatment transforms proteins into a linear structure, fully releasing folate. Furthermore, since all proteins are destroyed, interfering protein factors in the serum, such as RF, heterophilic antibodies, and complement, are rendered ineffective, further ensuring the accuracy of the test results. However, the reducing agents used in the folate sample processing solutions of most commercially available products are toxic and volatile, posing potential hazards to operators. Whole blood samples require reagents to lyse red blood cells (RBCs). This lysis releases intracellular folate for detection, but most commercially available products require approximately 90 minutes for RBC lysis, making this a time-consuming step. Folic acid is easily destroyed in acidic solutions, is unstable to heat, and is easily destroyed by light. When the pH is lower than 4.5, it will be completely destroyed in 1 hour. Folic acid is stable to heat in alkaline or neutral solutions, but is easily decomposed under light (ultraviolet) conditions. Summary of the Invention

[0006] The present invention aims to overcome the deficiencies in the prior art by providing a novel monoclonal antibody for folic acid, and using the monoclonal antibody to prepare a new product for folic acid detection, while also providing a sample processing solution. The monoclonal antibody for folic acid provided by the present invention can recognize folic acid with high affinity, providing a higher sensitivity for the detection reagent; the sample processing solution can shorten the detection time and contains a protective component for folic acid, has low toxicity, and low volatility, providing a higher repeatability for the detection reagent; the folic acid detection kit has a short detection time, simple operation, and good repeatability.

[0007] To achieve the above objectives, the present invention provides a folate monoclonal antibody comprising the following heavy chain complementary determining regions: CDR1-VH, CDR2-VH, and CDR3-VH having amino acid sequences as shown in SEQ ID NO.1 to SEQ ID NO.3; and / or the following light chain complementary determining regions: CDR1-VL having an amino acid sequence as shown in SEQ ID NO.4, a CDR2-VL having an amino acid sequence of GAS, and a CDR3-VL having an amino acid sequence as shown in SEQ ID NO.5.

[0008] Preferably, the amino acid sequence of the heavy chain variable region of the folic acid monoclonal antibody is as shown in SEQ ID NO.6.

[0009] Preferably, in any of the above items, the amino acid sequence of the light chain variable region of the folic acid monoclonal antibody is as shown in SEQ ID NO.7.

[0010] The present invention also provides a coding sequence for the folate monoclonal antibody described in any one of the above items, wherein the coding sequence for the heavy chain variable region of the folate monoclonal antibody is nucleotides 1 to 363 of the nucleotide sequence shown in SEQ ID NO.10; and the coding sequence for the light chain variable region of the folate monoclonal antibody is nucleotides 1 to 336 of the nucleotide sequence shown in SEQ ID NO.11.

[0011] Preferably, in any of the above items, the gene segment encoding the heavy chain of the folate monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.10.

[0012] Preferably, in any of the above items, the gene segment encoding the light chain of the folate monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.11.

[0013] The present invention also provides use of any of the above-mentioned folic acid monoclonal antibodies in the preparation of a folic acid detection product.

[0014] The present invention also provides a folic acid test strip, which includes a base plate and a sample pad, a fluorescent pad, a detection pad and a sample suction pad stacked in sequence on the base plate, wherein the fluorescent pad contains any of the above-mentioned folic acid monoclonal antibodies, the folic acid monoclonal antibodies are marked with a marker, and the detection pad is provided with a detection line and a quality control line; the detection line is coated with a BSA-coupled folic acid antigen; and the quality control line is coated with a goat anti-mouse IgG polyclonal antibody.

[0015] Preferably, any of the above items is that the marker is a time-resolved fluorescent microsphere.

[0016] Preferably, any of the above items has a particle size of the time-resolved fluorescent microspheres of 100-300 nm; more preferably, 100, 150, 200, 250, 300 nm and ranges therebetween.

[0017] Preferably, in any of the above items, the mass ratio of the folic acid monoclonal antibody to the time-resolved fluorescent microspheres is (0.1-0.4):1; more preferably, it is 0.1:1, 0.2:1, 0.3:1, 0.4:1 and ranges therebetween.

[0018] Preferably, any of the above items is that the test line is coated with 0.5-2 mg / mL of the BSA-coupled folic acid antigen; more preferably, 0.5, 1.0, 1.5, 2.0 mg / mL and ranges therebetween.

[0019] Preferably, any of the above items is coated with 0.5-2 mg / mL of goat anti-mouse IgG polyclonal antibody; more preferably, 0.5, 1.0, 1.5, 2.0 mg / mL and ranges therebetween.

[0020] Preferably, any of the above items is that the diluent for the folic acid monoclonal antibody and the goat anti-mouse IgG polyclonal antibody is a 10-50 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose; further preferably, the concentration of trehalose in the PB buffer is 10, 20, 30, 40, 50 mM and ranges therebetween; further preferably, each 100 mL of the diluent contains 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 g and ranges therebetween.

[0021] The present invention also provides a folic acid detection kit, comprising any of the folic acid detection test strips described above, wherein the folic acid detection kit further comprises a shell, and the folic acid detection test strip is arranged inside the shell.

[0022] Preferably, any of the above items is that the housing comprises a detachably connected upper cover and a lower cover; the upper cover is provided with an observation window and a sample addition hole.

[0023] The present invention also provides a folic acid sample processing liquid, which is contained in the folic acid detection kit, or the folic acid sample processing liquid is used for sample processing in the folic acid detection kit or the folic acid detection test strip. Preferably, the folic acid sample processing liquid, the folic acid detection kit or the folic acid detection test strip of the present invention are applicable to at least one of whole blood, serum and plasma.

[0024] The folic acid sample processing solution contains three components: hemolysate, processing solution A, and processing solution B. The hemolysate can shorten the processing time for red blood cells in whole blood; processing solution B contains a folic acid-protecting component and a low-toxicity, low-volatility reducing agent, making the reagent safer and the folic acid in the treated sample more stable and less susceptible to environmental factors; processing solution A can convert bound folic acid into a detectable free state, while processing solution B neutralizes processing solution A while also stabilizing the free folic acid.

[0025] Preferably, any of the above items is that the hemolytic solution comprises one or more of sodium chloride, Triton X-100, SDS, sodium lauroyl sarcosinate, sodium deoxycholate, saponin, NH4Cl, and ascorbic acid.

[0026] Any of the above is preferably that the hemolyzed solution contains one or more of the following components in percentage by weight: 0.1%~1.0% sodium chloride, 0.1%~1% Triton X-100, 0.1%~0.5% SDS, 0.1%~2.0% sodium lauroyl sarcosinate, 0.5%~1% sodium deoxycholate, 0.1%~0.5% saponin, 50~100M NH4Cl, and 0.5%~5% ascorbic acid.

[0027] Preferably, any of the above items is characterized in that the hemolytic component comprises 0.01% Triton X-100, 0.3% sodium lauroyl sarcosinate, and 0.5% ascorbic acid.

[0028] Preferably, any of the above items is that the treatment liquid A contains one or more of NaOH, KOH, Na3PO4, K3PO4, Na2CO3, and NaHCO3.

[0029] Preferably, any of the above items is that the components of the treatment solution A include one or more of 0.1M~10M NaOH, 0.1M~10M KOH, 0.5M~5M K3PO4, 0.5M~5M Na3PO4, 0.5M~5M Na2HPO4, 0.5M~5M K2HPO4, 0.5M~5M Na2CO3, and 0.5M~5M NaHCO3.

[0030] Preferably, any of the above items is that the components of the treatment solution A include 0.4 M KOH and 0.3 M Na2CO3.

[0031] Preferably, any of the above items is that the treatment solution B contains one or more of DTT, TCEP, mercaptoethanol, cysteine, glutathione, ascorbic acid, vitamin E, coenzyme Q10, lipoic acid, EDTA, butylated hydroxytoluene, PMSF, glycerol, and sucrose.

[0032] Any of the above items is preferably that the components of the treatment solution B include one or more of 1~10mM DTT, 1~10mM TCEP, 5~50mM mercaptoethanol, 1~10mM cysteine, 0.5~5M glutathione, 0.5~5M ascorbic acid, 0.5~5M vitamin E, 0.01~0.1M coenzyme Q10, 0.01~0.1M lipoic acid, 0.1~0.2M EDTA, 0.01%~0.1% butylated hydroxytoluene, 0.1mM~0.1MPMSF, 5%~20% glycerol, and 0.1~0.5M sucrose.

[0033] Preferably, in any of the above items, the buffer solution of the treatment solution B is one or more of 20-500 mM Tris-HCl, 0.01M-0.2M PBS, 20 mM-200 mM HEPES, 20mM-200mM MES, and 20mM-200mM MOPS.

[0034] Preferably, any of the above items is that the components of treatment solution B include 300 mM Tris-HCl, 0.1 M EDTA, 10 mM cysteine, 5% glycerol, and 0.05 M coenzyme Q10.

[0035] Any of the above is preferably that the specific method of using the folic acid sample processing solution is: if the folic acid sample is a whole blood sample, 100 μL of the collected whole blood is added to 2 mL of hemolyzed blood, and the solution is allowed to stand at room temperature in the dark for 30 minutes to obtain a hemolyzed solution; 100 μL of the hemolyzed solution is added to 30 μL of treatment solution A, mixed evenly, and then 30 μL of treatment solution B is added, mixed evenly, and then the solution is allowed to stand at room temperature for 5 minutes before detection; if the folic acid sample is serum or plasma, 100 μL of serum or plasma is added to 30 μL of treatment solution A, mixed evenly, and then 30 μL of treatment solution B is added, mixed evenly, and then the solution is allowed to stand at room temperature for 5 minutes before detection.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The folic acid monoclonal antibody provided by the present invention has high affinity with folic acid antigen and can be used to prepare products for detecting folic acid.

[0038] The folic acid test strips and kit provided by the present invention have the advantages of simple operation, rapid reaction, high sensitivity, suitability for on-site rapid detection, and economy and practicality. They can perform rapid screening and quickly, accurately and safely indicate whether the person being tested is deficient in folic acid. They can also serve as an auxiliary basis for the diagnosis of megaloblastic anemia, enabling patients to receive early diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the electrophoresis diagram of the folic acid monoclonal antibody in the preferred embodiment 1 of the present invention.

[0040] Figure 2 This is the upper cover of the folic acid detection kit in preferred embodiment 3 of the present invention.

[0041] Figure 3 This is the lower cover of the folic acid detection kit in preferred embodiment 3 of the present invention.

[0042] Figure 4 This is a structural diagram of a folic acid test strip in preferred embodiment 3 of the present invention.

[0043] Markings in the accompanying drawings: 1-observation window; 2-sample loading hole; 3-test card strip area; 4-bottom plate; 5-test pad; 6-sample suction pad; 7-fluorescence pad; 8-sample pad; 9-quality control line; 10-test line. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] It should be noted that the "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of an antibody. The variable domain of the heavy chain can be referred to as "VH". The variable domain of the light chain can be referred to as "VL". These domains are usually the most variable parts of the antibody and contain the antigen binding site. The light or heavy chain variable region is composed of a framework region interrupted by three hypervariable regions called "complementarity determining regions" or "CDRs". The framework region of an antibody, that is, the combined framework region of the constituent light and heavy chains, plays a role in positioning and aligning the CDRs, which are primarily responsible for binding to the antigen.

[0046] "Framework" or "FR" regions refer to the regions of an antibody variable domain excluding those defined as CDRs. Each antibody variable domain framework can be further subdivided into contiguous regions (FR1, FR2, FR3, and FR4) separated by CDRs.

[0047] Typically, the variable regions VL / VH of the heavy and light chains can be obtained by connecting the following numbered CDRs and FRs in the following combinations: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0048] In the present invention, CDR1-VH, CDR2-VH and CDR3-VH refer to the three hypervariable regions of the heavy chain variable region, respectively. Correspondingly, CDR1-VL, CDR2-VL and CDR3-VL refer to the three hypervariable regions of the light chain variable region, respectively.

[0049] In a first aspect, the present invention provides a folic acid monoclonal antibody;

[0050] The amino acid sequence of the heavy chain complementary determining region of the folic acid monoclonal antibody is: CDR1-VH, CDR2-VH, and CDR3-VH as shown in SEQ ID NO.1 to SEQ ID NO.3;

[0051] The amino acid sequence of the complementary determining regions of the light chain of the folic acid monoclonal antibody is as follows: CDR1-VL with an amino acid sequence as shown in SEQ ID NO.4, CDR2-VL with an amino acid sequence as GAS, and CDR3-VL with an amino acid sequence as shown in SEQ ID NO.5;

[0052] The amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.5 are shown in Table 1.

[0053] Table 1

[0054]

[0055] In a preferred embodiment of the present invention, the folic acid monoclonal antibody is preferably of murine origin.

[0056] Preferably, the folic acid monoclonal antibody comprises a heavy chain variable region VH having an amino acid sequence as shown in SEQ ID NO.6.

[0057] Preferably, the amino acid sequence of the light chain variable region VL is as shown in SEQ ID NO.7.

[0058] The amino acid sequences represented by SEQ ID NO.6 and SEQ ID NO.7 are shown in Table 2.

[0059] Table 2

[0060]

[0061] Preferably, the light chain constant region of the folic acid monoclonal antibody is as shown in SEQ ID NO.8.

[0062] Preferably, the heavy chain constant region of the folic acid monoclonal antibody is as shown in SEQ ID NO.9.

[0063] Preferably, the amino acid sequence of the heavy chain of the folic acid monoclonal antibody consists of the amino acid sequences shown in SEQ ID NO.6 and SEQ ID NO.9 in sequence;

[0064] Preferably, the amino acid sequence of the light chain of the folic acid monoclonal antibody consists of the amino acid sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8 in sequence.

[0065] The amino acid sequences represented by SEQ ID NO.8 and SEQ ID NO.9 are shown in Table 3.

[0066] Table 3

[0067]

[0068] The folic acid monoclonal antibody provided by the present invention has high affinity and specificity to folic acid antigen and can be used to prepare products for detecting folic acid.

[0069] In a second aspect, the present invention provides the use of the folic acid monoclonal antibody in the preparation of a folic acid detection product.

[0070] In a third aspect, the present invention provides a folic acid test strip, comprising a base plate and a sample pad, a fluorescent pad, a detection pad, and a sample suction pad stacked sequentially on the base plate;

[0071] The fluorescent pad contains any one of the above-mentioned folic acid monoclonal antibodies, which is labeled with a marker, and can identify the location or concentration of the marker through detection.

[0072] The folic acid test strip provided by the present invention has the advantages of simple operation, rapid reaction, high sensitivity, strong specificity, suitability for on-site rapid detection, and economy and practicality. It can be used for rapid screening, quickly, accurately and safely indicating whether the person being tested is folic acid deficient, and serve as an auxiliary basis for the diagnosis of megaloblastic anemia, enabling patients to receive early diagnosis and early treatment.

[0073] In a preferred embodiment of the present invention, the marker is a time-resolved fluorescent microsphere.

[0074] Preferably, the particle size of the time-resolved fluorescent microspheres can be, for example, but not limited to, 100 nm, 150 nm, 200 nm, 250 nm or 300 nm;

[0075] Preferably, the mass ratio of the folic acid monoclonal antibody to the time-resolved fluorescent microspheres is: (0.1-0.4):1.

[0076] The present invention makes the sensitivity of the test strip higher by adjusting the particle size and dosage of the marker.

[0077] In a preferred embodiment of the present invention, a detection line (T line) and a quality control line (C line) are provided on the detection pad;

[0078] The test line was coated with 0.5-2 mg / mL of the BSA-conjugated folate antigen purchased from Sigma-Aldrich. The folate antigen used in the present invention is not limited to its preparation method; any folate capable of forming an antigen-antibody specific binding with the folate monoclonal antibody provided herein can be used as a folate antigen. The method of conjugating the folate antigen with BSA is conventional in the art and will not be described in detail here.

[0079] Preferably, the quality control line is coated with 0.5-2 mg / mL of goat anti-mouse IgG polyclonal antibody.

[0080] Preferably, the diluent for the BSA-coupled folic acid antigen and goat anti-mouse IgG polyclonal antibody is a 10-50 mM PB buffer solution containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose.

[0081] The test strip provided by this invention utilizes the principle of antigen-antibody reaction. After labeling a monoclonal antibody against folate with time-resolved fluorescent microspheres, the label is solidified onto a detection pad, preferably a nitrocellulose membrane (NC membrane). The pad (e.g., NC membrane) is coated with a BSA-conjugated folate antigen. Based on the principle of a competitive reaction, detection can be performed using a matching instrument within the test time. If folate is present in the sample, a weaker light signal is generated in the instrument; if it is absent, a stronger light signal is generated. The intensity of the light signal is used to predict the amount of analyte loaded.

[0082] By using the test strip provided by the present invention for testing, the test results can be obtained within 10-20 minutes of the entire process, which is fast and efficient, and helps medical personnel to obtain the test results in a timely manner, make comprehensive judgments based on the results, and take timely measures.

[0083] In a fourth aspect, the present invention provides a folic acid sample processing solution comprising a hemolysate, a processing solution A, and a processing solution B. The processing solution accelerates the release of folic acid from a sample while ensuring the stability of the folic acid. The hemolysate promotes the rapid release of folic acid from cells in the sample, the processing solution A converts bound folic acid into a detectable free state, and the processing solution B neutralizes the processing solution A while also stabilizing the free folic acid.

[0084] In a preferred embodiment of the present invention, the hemolyzed solution comprises the following components in percentage by weight: one or more of 0.1% to 1.0% sodium chloride, 0.1% to 1% Triton X-100, 0.1% to 0.5% SDS, 0.1% to 2.0% sodium lauroyl sarcosinate, 0.5% to 1% sodium deoxycholate, 0.1% to 0.5% saponin, 50 to 100 M NH4Cl, and 0.5% to 5% ascorbic acid;

[0085] Preferably, the hemolytic component comprises 0.01% Triton X-100, 0.3% sodium lauroyl sarcosinate, and 0.5% ascorbic acid.

[0086] In a preferred embodiment of the present invention, the treatment solution A comprises one or more of 0.1M~10M NaOH, 0.1M~10M KOH, 0.5M~5M K3PO4, 0.5M~5M Na3PO4, 0.5M~5M Na2HPO4, 0.5M~5M K2HPO4, 0.5M~5MNa2CO3, and 0.5M~5M NaHCO3.

[0087] Preferably, the components of the treatment solution A include 0.4 M KOH and 0.3 M Na2CO3.

[0088] In a preferred embodiment of the present invention, the effective components of the treatment solution B include one or more of 1~10mM DTT, 1~10mM TCEP, 5~50mM mercaptoethanol, 1~10mM cysteine, 0.5~5M glutathione, 0.5~5M ascorbic acid, 0.5~5M vitamin E, 0.01~0.1M coenzyme Q10, 0.01~0.1M lipoic acid, 0.1~0.2M EDTA, 0.01%~0.1% butylated hydroxytoluene, 0.1mM~0.1M PMSF, 5%~20% glycerol, and 0.1~0.5M sucrose; the buffer used in the treatment solution B is 20~500mM Tris-HCl, 0.01M~0.2M PBS, 20mM~200mM HEPES, 20mM~200mM One or more of MES, 20mM~200mM MOPs.

[0089] Preferably, the components of treatment solution B include 300 mM Tris-HCl, 0.1 M EDTA, 10 mM cysteine, 5% glycerol and 0.05 M coenzyme Q10.

[0090] The specific procedure is as follows: If the sample to be tested is whole blood, add 100 μL of collected whole blood to 2 mL of hemolyzed solution and incubate in the dark at room temperature for 30 minutes to obtain a hemolyzed solution. Then, add 30 μL of treatment solution A to 100 μL of the hemolyzed solution, mix thoroughly, and then add 30 μL of treatment solution B. After mixing, incubate at room temperature for 5 minutes before testing. Alternatively, if the sample to be tested is serum or plasma, add 100 μL of collected serum or plasma to 30 μL of treatment solution A, mix thoroughly, and then add 30 μL of treatment solution B. After mixing, incubate at room temperature for 5 minutes before testing.

[0091] In a fifth aspect, the present invention provides a folic acid detection kit, comprising the folic acid sample processing solution, a folic acid detection test strip and a shell, wherein the folic acid detection test strip is arranged inside the shell.

[0092] The folic acid detection kit provided by the present invention contains a folic acid detection test strip and a folic acid sample processing solution, and thus has all the beneficial effects of the folic acid detection test strip and the folic acid sample processing solution.

[0093] In a preferred embodiment of the present invention, the housing includes an upper cover and a lower cover that are detachably connected;

[0094] The upper cover is provided with an observation window and a sample addition hole.

[0095] The shapes of the observation window and the sample addition hole are not specifically limited in the present invention. The observation window can be, for example, square and located above the test line and the quality control line of the test strip for observing the test results. The sample addition hole can be, for example, a circular hole with a diameter of 0.5-1 cm and located above the sample pad.

[0096] Add test sample to the kit:

[0097] It should be noted that the diluent for the folic acid monoclonal antibody and goat anti-mouse IgG polyclonal antibody of the present invention is a 10-50 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose; unless otherwise specified in the following examples, preferably, the diluent for the folic acid monoclonal antibody and goat anti-mouse IgG polyclonal antibody is a 20 mM PB buffer containing trehalose, and each 100 mL of the diluent contains 0.5 g of trehalose.

[0098] The method for preparing a folic acid monoclonal antibody of the present invention is a conventional method for preparing a monoclonal antibody in the prior art, which is briefly described as follows:

[0099] (1) Animal immunization:

[0100] A: Folic acid purchased from Sigma-Aldrich was used as the folic acid antigen. It was mixed with equal volumes of Freund's adjuvant to an appropriate volume and emulsified completely. Mice were immunized by intraperitoneal injection. Each mouse was injected with 50 μg of the immunogen in a volume of 100 μL, once a week.

[0101] B: After immunization 4 times, the titer of antibodies in the serum was tested by indirect ELISA method with folic acid coating, and mice with an OD value greater than 1.0 detected by 16,000-fold serum dilution were screened.

[0102] (2) Preparation of monoclonal antibodies:

[0103] A: Splenocytes from screened mice were fused with myeloma cells, and the fused cells were plated and cultured using the limiting dilution method.

[0104] B: Screen the monoclonal cell wells and culture and expand them. The monoclonal cell well with the highest OD value detected in the cell culture supernatant is used as the target hybridoma cell, culture and expand it to obtain the hybridoma cell line and freeze it;

[0105] C: Isolation of antibody variable region genes from hybridoma cells using RT-PCR: After homogenization of the hybridoma cells, add cell lysis buffer for RNA extraction. Precipitate RNA from the aqueous phase with isopropanol. Wash the precipitated RNA after centrifugation to remove impurities, resuspend it, and perform reverse transcription to obtain cDNA.

[0106] D: PCR was performed using mouse-specific primers known in the art, using hybridoma cell cDNA as a template, to amplify the heavy and light chain variable region genes of the antibody. A 50 μL system contained 5 μL of cDNA, HotStarTaq Plus enzyme, dNTPs, and 0.5 μM of specific primers. PCR amplification was performed under the following conditions: initial denaturation at 94°C for 5 minutes; 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 50 seconds; and 72°C for 7 minutes. The resulting PCR products were identified by 1% agarose gel electrophoresis, and the target fragments were recovered and sent for sequencing. Based on the antibody gene sequences obtained by sequencing, the amino acid sequences of the heavy chain variable region of the folate monoclonal antibody as shown in SEQ ID NO.6 and the light chain variable region of the folate monoclonal antibody as shown in SEQ ID NO.7 were further obtained. The amino acid sequence of the light chain constant region of the folate monoclonal antibody is shown in SEQ ID NO.8, and the amino acid sequence of the heavy chain constant region of the folate monoclonal antibody is shown in SEQ ID NO.9.

[0107] E: Construction of monoclonal antibody expression vector:

[0108] The antibody gene sequence obtained by sequencing in step D was used to construct a monoclonal antibody expression vector using conventional methods in the art. Homologous recombination primers were used to add homologous recombination arms to both ends of the antibody heavy chain variable region gene and the light chain variable region gene, respectively. The expression plasmid containing the mouse antibody heavy and light chain IgG1 constant regions was linearized using a dual enzyme to generate homologous recombination arms. The variable region gene fragments added with the homologous recombination arms and the linearized plasmid were ligated by homologous recombination to form a complete expression vector, pCDNA3.4. The recombinant product was transformed into TOP10 competent Escherichia coli and the plasmid was amplified to obtain the pCDNA3.4-folate monoclonal antibody heavy chain plasmid and the pCDNA3.4-folate monoclonal antibody light chain plasmid.

[0109] The gene fragment encoding the heavy chain of the folic acid monoclonal antibody inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.10, wherein nucleotides 1 to 363 are the coding sequence for the variable region of the heavy chain of the folic acid monoclonal antibody, and nucleotides 364 to 1335 are the coding sequence for the constant region of the heavy chain of the folic acid monoclonal antibody.

[0110] The gene fragment encoding the light chain of the folic acid monoclonal antibody inserted into the pCDNA3.4 vector is the nucleotide sequence shown in SEQ ID NO.11, wherein nucleotides 1 to 336 are the coding sequence for the light chain variable region of the folic acid monoclonal antibody, and nucleotides 337 to 657 are the coding sequence for the light chain constant region of the folic acid monoclonal antibody.

[0111] F: Expression and purification of monoclonal antibodies:

[0112] The monoclonal antibody heavy and light chain expression plasmids obtained in step E were added to Opti-Mem transfection medium at a ratio of 1:1. After thorough mixing, PEI transfection reagent (4 times the mass of DNA) was added. After mixing, the mixture was placed in the dark at room temperature for 10 minutes, and then added to 293T cells. After incubation for 6 hours, the transfection system was removed, and FreeStyleTM293 expression medium was added. After culturing for 5 days, the cell culture supernatant was collected and the expressed cell culture supernatant was purified by affinity purification (Protein A) to obtain the monoclonal antibody. The specific steps are as follows:

[0113] (1) Centrifuge the expressed antibody supernatant at 2500 × g for 10 min at room temperature to remove the precipitate;

[0114] (2) The affinity purification column containing Protein A was thoroughly washed with 10 volumes of binding buffer;

[0115] (3) The expression supernatant was passed through the purification column at a flow rate of 5 mL / min;

[0116] (4) Wash the purification column thoroughly with 20 times the volume of the purification column binding buffer;

[0117] (5) Elute the purification column with 0.1 M pH = 3.0-3.5 citric acid buffer until the elution peak drops to equilibrium, and adjust the pH to 7.0 with 1 M pH = 9.0 Tris-HCl buffer;

[0118] (6) The purified monoclonal antibody was concentrated using a centrifugal column, PBS was used as the buffer for antibody storage, and the concentration of the concentrated antibody was measured using an ultra-micro UV spectrophotometer.

[0119] In a preferred embodiment of the present invention, a pCDNA3.4-folate monoclonal antibody heavy chain plasmid and a pCDNA3.4-folate monoclonal antibody light chain plasmid were co-transfected at a 1:1 ratio, and expression and purification were performed according to the method in Step F to obtain a folate monoclonal antibody. The obtained folate monoclonal antibody was verified by SDS-PAGE and ELISA, respectively, using the methods described in Example 1. The folate monoclonal antibody exhibited two characteristic bands of approximately 25 kD and 50 kD, representing the light and heavy chains of IgG, respectively. The indirect titer determination by ELISA demonstrated that the folate monoclonal antibody specifically recognized folate.

[0120] The methods used in the present invention for antibody preparation, antibody gene sequencing, construction and identification of monoclonal antibody expression vectors, etc., are all conventional methods in molecular biology. The primers and other related sequences involved therein can be obtained through existing technologies, such as relevant information recorded in gene databases or existing literature, or can be obtained from the target gene fragment of the heavy chain or light chain of the folate monoclonal antibody, such as the nucleotide sequence shown in SEQ ID NO. 10 or SEQ ID NO. 11, and are not described in detail here.

[0121] Example 1

[0122] The variable region sequences of the folic acid monoclonal antibody are shown in Table 2.

[0123] A 12% SDS-PAGE gel was prepared according to conventional methods, and 5 μg of the above antibody was loaded and electrophoresed using a protein molecular weight standard as a reference. The results showed that the folic acid monoclonal antibody showed two characteristic bands of about 25 kD and 50 kD, which were the light chain and heavy chain of IgG, respectively ( Figure 1 After scanning and analysis, the antibody content of the strips was above 90%. Figure 1 In the examples below, M is a marker and I is a monoclonal antibody against folic acid. The antibodies used in the following examples are the same as those in Example 1.

[0124] Example 2

[0125] Folic acid monoclonal antibody affinity verification:

[0126] A microplate was coated with BSA-conjugated folate antigen at a concentration of 100 ng / well. 100 μL of diluted folate monoclonal antibody was added to each well at concentrations of 100, 20, 4, and 0.8 ng / mL. The plates were incubated at 37°C for 1 hour, then washed three times. 100 μL of a 1:5000 dilution of goat anti-mouse IgG-HRP (Beijing Solebold Technology Co., Ltd., Cat. No. SE131, original concentration 1 mg / mL) was added to each well. The plates were incubated at 37°C for 30 minutes, then washed three times. 100 μL of TMB was added to each well for color development. The plates were incubated at 37°C for 15 minutes, then stopped with stop solution and read on a microplate reader. The results are shown in the table below, where OD1, OD2, and OD3 represent triplicate readings of the same sample.

[0127] Table 4

[0128]

[0129] As shown in the table above, when the concentration of the folic acid monoclonal antibody provided by the present invention is less than 4 ng / mL, the detection OD exceeds 0.500, indicating strong recognition and affinity with the folic acid antigen.

[0130] Example 3

[0131] A folic acid detection kit, such as Figures 2 to 4 As shown, 1 is an observation window, 2 is a sample loading hole, 3 is a test card strip area, 4 is a bottom plate, 5 is a test pad, 6 is a sample suction pad, 7 is a fluorescent pad, 8 is a sample pad, 9 is a quality control line, and 10 is a test line. The folic acid test kit includes a folic acid test strip and a housing, with the folic acid test strip disposed within the housing. The folic acid test strip includes a bottom plate 4 and a sample pad 8, a fluorescent pad 7, a test pad 5, and a sample suction pad 6 stacked sequentially on the bottom plate; the test pad 5 is provided with a test line 10 and a quality control line 9; the housing includes a detachably connected upper cover and lower cover; the upper cover is provided with an observation window 1 and a sample loading hole 2, and the lower cover is provided with a test card strip area 3.

[0132] Example 4

[0133] Example 4 is similar to the folic acid detection kit provided in Example 3, except that the folic acid detection kit provided in Example 4 also includes a folic acid sample processing solution, which includes a hemolysate, a processing solution A, and a processing solution B. The processing solutions accelerate the release of folic acid from the sample while ensuring the stability of the folic acid. The hemolysate promotes the rapid release of folic acid from sample cells, the processing solution A converts bound folic acid into a detectable free state, and the processing solution B neutralizes the processing solution A while also stabilizing the free folic acid.

[0134] The hemolysate comprises one or more of the following components in percentage by weight: 0.1% to 1.0% sodium chloride, 0.1% to 1% Triton X-100, 0.1% to 0.5% SDS, 0.1% to 2.0% sodium lauroyl sarcosinate, 0.5% to 1% sodium deoxycholate, 0.1% to 0.5% saponin, 50 to 100 M NH4Cl, and 0.5% to 5% ascorbic acid;

[0135] Preferably, the mass percentage of Triton X-100 in the hemolyzed solution is 0.01%;

[0136] Preferably, the mass percentage of sodium lauroyl sarcosinate in the hemolysate is 0.3%;

[0137] Preferably, the mass percentage of ascorbic acid in the hemolysate is 0.5%.

[0138] The treatment solution A comprises one or more of 0.1M~10M NaOH, 0.1M~10M KOH, 0.5M~5M K3PO4, 0.5M~5M Na3PO4, 0.5M~5M Na2HPO4, 0.5M~5M K2HPO4, 0.5M~5M Na2CO3, and 0.5M~5M NaHCO3;

[0139] Preferably, in the treatment solution A, the concentration of KOH is preferably 0.4 M;

[0140] Preferably, the concentration of Na2CO3 in the treatment solution A is preferably 0.3 M.

[0141] The effective components of the treatment solution B include one or more of 1-10 mM DTT, 1-10 mM TCEP, 5-50 mM mercaptoethanol, 1-10 mM cysteine, 0.5-5 M glutathione, 0.5-5 M ascorbic acid, 0.5-5 M vitamin E, 0.01-0.1 M coenzyme Q10, 0.01-0.1 M lipoic acid, 0.1-0.2 M EDTA, 0.01%-0.1% butylated hydroxytoluene, 0.1 mM-0.1 M PMSF, 5%-20% glycerol, and 0.1-0.5 M sucrose; the buffer used in the treatment solution B is 20-500 mM Tris-HCl, 0.01 M-0.2 M PBS, 20 mM-200 mM HEPES, 20 mM-200 mM MES, 20 mM-200 mM One or more of MOPS;

[0142] Preferably, the components of treatment solution B include 300 mM Tris-HCl, 0.1 M EDTA, 10 mM cysteine, 5% glycerol and 0.05 M coenzyme Q10.

[0143] The specific operation process is as follows:

[0144] The sample to be tested is whole blood. Take 100 μL of collected whole blood and add it to 2 mL of hemolyzed blood. Let it stand at room temperature in the dark for 30 minutes to obtain a hemolyzed solution diluted to 21 times. Take 100 μL of hemolyzed solution and add 30 μL of treatment solution A, mix well, and then add 30 μL of treatment solution B. After mixing, let it stand at room temperature for 5 minutes before testing.

[0145] If the sample to be tested is serum or plasma, take 100 μL of serum or plasma, add 30 μL of treatment solution A, mix well, then add 30 μL of treatment solution B, let it stand at room temperature for 5 minutes before testing.

[0146] Example 5

[0147] Example 5 provides a folic acid detection kit prepared by time-resolved fluorescent microsphere-labeled antibodies.

[0148] The folic acid monoclonal antibody of the present invention is used to prepare a folic acid detection kit. The kit includes a detection card and a test strip. The detection card is divided into an upper cover and a lower base. The test strip is embedded with a folic acid monoclonal antibody labeled with time-resolved fluorescent microspheres on a fluorescent pad. The detection line is coated with BSA-coupled folic acid antigen. The folic acid content in the sample is quantitatively detected by a competitive method.

[0149] 1. Kit preparation process:

[0150] The folic acid monoclonal antibody prepared by the method of the present invention is surface-labeled with time-resolved fluorescent microspheres. The specific implementation method is as follows:

[0151] Time-resolved fluorescent microsphere antibody labeling: 1 mL of 1% carboxyl time-resolved fluorescent microspheres was added to 9 mL of MES buffer, followed by 25 μL of 10 mg / mL EDC solution and 25 μL of 10 mg / mL NHS solution. The mixture was shaken at room temperature for 30 minutes, and the precipitate was collected by centrifugation. After adding HEPES rehydration solution and ultrasonically dispersing the mixture, 1 mL of 1 mg / mL folic acid monoclonal antibody was added. The mixture was shaken at room temperature for 120 minutes, and the precipitate was collected by centrifugation. 1 mL of blocking buffer was added, and the mixture was shaken at room temperature for 120 minutes. The microsphere precipitate was collected by centrifugation and reconstituted with rehydration solution.

[0152] Preparation of the fluorescent pad: Dilute the labeled time-resolved fluorescent microspheres with the microsphere reconstitution solution and spray the fluorescent pad using a gold sprayer at a rate of 3 μL / cm with a spray spacing of 6 mm. After spraying, dry the pad at 37°C in a low humidity (<30%) oven for 2 h.

[0153] NC membrane-coated CT lines: T lines use BSA-coupled folic acid antigens at a concentration of 1.5 mg / mL, and C lines use goat anti-mouse IgG antibodies at a concentration of 1 mg / mL. 1 μL / cm lines are drawn. After completion, the lines are placed in a 37°C low humidity (<30%) oven for 24 hours.

[0154] Sample pad treatment: The sample pad treatment solution consists of buffer salt, sustained-release agent, cosolvent, blocking agent, etc. The specific formula is 20mM Tris buffer, and each 100mL Tris buffer contains 1g BSA, 0.5g Tween 20, and 2g sucrose. 2 Treat with 1 mL of sample treatment solution. After evenly treating, place in a 37°C, low humidity (<30%) oven dry for 2 hours.

[0155] Test strip assembly: Attach the NC film, sample pad, fluorescent pad, and sample pad to the base plate (PVC board) in that order, with the sample pad and fluorescent pad each pressing 1-2 mm against the NC film, and the sample pad pressing 1-2 mm against the fluorescent pad. After assembly, cut the test strip into widths of 4 ± 0.4 mm and install the card holder. Place the card holder and desiccant in an aluminum foil bag and seal. Label and box the finished test card.

[0156] Test sample: Folic acid: purchased from Sigma-Aldrich.

[0157] The MES buffer, EDC solution, NHS solution, HEPES reconstitution solution, blocking solution, microsphere reconstitution solution, etc. are all conventional reagents in the art and can be prepared and obtained by methods described in the prior art or purchased through commercial channels.

[0158] 2 Kit detection process

[0159] Place the test card on a clean, flat surface, draw 80-100 μL of treated serum, plasma or hemolysis solution and drop it into the sample well of the test card.

[0160] Import the standard curve (lgX-Y four-parameter fitting) from the test strip. After adding the sample for 10-15 minutes, scan the detection area using a fluorescence immunoassay analyzer to obtain a fluorescent signal. The corresponding folate concentration in the processed serum, plasma, or hemolyzed solution is displayed. The standard curve and the test results for serum sample S1, plasma sample S2, and whole blood sample S3 (21-fold diluted hemolyzed solution, 50% hematocrit) are shown in the table below. The calculated concentration for serum sample S1 is 4.073 ng / mL, and the calculated concentration for plasma sample S2 is 3.748 ng / mL. The red blood cell folate concentration in whole blood sample S3 is calculated as follows: folate concentration in hemolyzed solution × dilution factor ÷ hematocrit = 5.995 ng / mL × 21 ÷ 50% = 251.79 ng / mL.

[0161] This example also determined the sensitivity of the test kit by testing serum samples with concentrations ranging from 0 to 1 ng / mL. When the folate concentration in the serum sample reached 0.78 ng / mL, the detection signal T / C ratio was 11.51, which was significantly different from the T / C ratio of 12.88 for the 0 ng / mL sample. This indicates that the detection limit can reach approximately 0.78 ng / mL. The lower limit of the normal range of folate in serum is approximately 2 ng / mL. Therefore, the folate detection product provided by the present invention can meet the requirements of serum testing.

[0162] Table 5

[0163]

[0164] Table 6

[0165]

[0166] Example 6

[0167] Example 6 provides verification of the treatment effect of hemolysis.

[0168] Add 100 μL of whole blood to 2 mL of hemolyzed solution (0.01% Triton X-100, 0.3% sodium lauroyl sarcosinate, 0.5% ascorbic acid). Mix thoroughly, incubate at room temperature in the dark for 0, 10, 20, 30, 40, 50, and 60 minutes. Read the absorbance at 540 nm on a spectrophotometer. Complete red blood cell disruption is indicated when the absorbance does not increase over time. The results are shown in the table below.

[0169] Table 7

[0170]

[0171] From the data in the above table, we can see that when the standing time reaches 30 minutes, the absorbance no longer increases with time, proving that the red blood cells have been completely broken.

[0172] Example 7

[0173] Example 7 provides a verification of the effect of treatment solution A.

[0174] 100 μL of each of three serum samples (S1, S2, and S3) with high folate levels was added to 30 μL of treatment solution A (0.4 M KOH, 0.3 M Na₂CO₃) and mixed thoroughly. Then, 30 μL of treatment solution B (300 mM Tris-HCl, 0.1 M EDTA, 10 mM cysteine, 5% glycerol, 0.05 M Coenzyme Q₁₀) was added and mixed thoroughly. The samples were allowed to stand at room temperature for 5 minutes, 30 minutes, 1 hour, and 3 hours before being added to the test card for testing. The results are shown in the table below.

[0175] Table 8

[0176]

[0177] From the data in the above table, we can see that the folic acid content of the treated samples remains unchanged as the standing time increases (5min~3h).

[0178] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A folic acid monoclonal antibody, characterized in that: It includes the heavy chain complementary determining regions whose amino acid sequences are shown below in sequence: CDR1-VH, CDR2-VH, and CDR3-VH as shown in SEQ ID NO.1 to SEQ ID NO.3; and the light chain complementary determining regions whose amino acid sequences are shown below in sequence: CDR1-VL as shown in SEQ ID NO.4, CDR2-VL with an amino acid sequence of GAS, and CDR3-VL as shown in SEQ ID NO.

5.

2. The folic acid monoclonal antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the folic acid monoclonal antibody is shown in SEQ ID NO.6; and / or the amino acid sequence of the light chain variable region of the folic acid monoclonal antibody is shown in SEQ ID NO.

7.

3. The nucleic acid encoding the folic acid monoclonal antibody according to claim 1 or 2, characterized in that: The nucleotide sequence of the nucleic acid encoding the heavy chain variable region of the folate monoclonal antibody is shown in nucleotides 1 to 363 of SEQ ID NO.10; the nucleotide sequence of the nucleic acid encoding the light chain variable region of the folate monoclonal antibody is shown in nucleotides 1 to 336 of SEQ ID NO.

11.

4. The nucleic acid encoding the folic acid monoclonal antibody according to claim 3, characterized in that The nucleotide sequence of the nucleic acid encoding the heavy chain of the folic acid monoclonal antibody is shown in SEQ ID NO.10; the nucleotide sequence of the nucleic acid encoding the light chain of the folic acid monoclonal antibody is shown in SEQ ID NO.

11.

5. Use of the folic acid monoclonal antibody according to claim 1 or 2 in the preparation of a folic acid detection product, characterized in that: The folic acid detection product is a test strip or a detection kit.

6. A folic acid test strip, comprising a base plate and a sample pad, a fluorescent pad, a detection pad, and a sample suction pad stacked on the base plate in sequence, characterized in that: The fluorescent pad contains the folic acid monoclonal antibody according to any one of claims 1 or 2, and the folic acid monoclonal antibody is marked with a marker. The detection pad is provided with a detection line and a quality control line; the detection line is coated with BSA-coupled folic acid antigen; the quality control line is coated with goat anti-mouse IgG polyclonal antibody.

7. The test strip according to claim 6, wherein The marker is time-resolved fluorescent microspheres.

8. The test strip according to claim 6, wherein The diluent for the folic acid monoclonal antibody and the goat anti-mouse IgG polyclonal antibody is a 10-50 mM PB buffer solution containing trehalose, and each 100 mL of the diluent contains 0.1-1.0 g of trehalose.

9. A folic acid detection kit, characterized in that: The folic acid test strip according to any one of claims 6 to 8 is included, and the folic acid test kit further comprises a shell, wherein the folic acid test strip is arranged inside the shell.

10. The folic acid detection kit according to claim 9, wherein The folic acid detection kit also includes a folic acid sample processing solution, which contains three components: hemolysate, processing solution A, and processing solution B; the hemolysate contains 0.01% Triton X-100, 0.3% sodium lauroyl sarcosinate, and 0.5% ascorbic acid; the processing solution A contains 0.4M KOH and 0.3M Na2CO3; the processing solution B contains 300mM Tris-HCl, 0.1M EDTA, 10mM cysteine, 5% glycerol, and 0.05M coenzyme Q10.

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