CN113933502B - Detection card and kit for quantitatively detecting folic acid by immunofluorescence chromatography - Google Patents
Detection card and kit for quantitatively detecting folic acid by immunofluorescence chromatography Download PDFInfo
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- CN113933502B CN113933502B CN202111214468.7A CN202111214468A CN113933502B CN 113933502 B CN113933502 B CN 113933502B CN 202111214468 A CN202111214468 A CN 202111214468A CN 113933502 B CN113933502 B CN 113933502B
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- 238000001514 detection method Methods 0.000 title claims abstract description 71
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 title claims abstract description 64
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 239000011724 folic acid Substances 0.000 title claims abstract description 32
- 229960000304 folic acid Drugs 0.000 title claims abstract description 32
- 235000019152 folic acid Nutrition 0.000 title claims abstract description 32
- 238000004587 chromatography analysis Methods 0.000 title claims abstract description 19
- 238000010166 immunofluorescence Methods 0.000 title claims abstract description 19
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 54
- 239000007788 liquid Substances 0.000 claims abstract description 40
- 229960002685 biotin Drugs 0.000 claims abstract description 27
- 235000020958 biotin Nutrition 0.000 claims abstract description 27
- 239000011616 biotin Substances 0.000 claims abstract description 27
- 239000008280 blood Substances 0.000 claims abstract description 24
- 210000004369 blood Anatomy 0.000 claims abstract description 23
- 239000000126 substance Substances 0.000 claims abstract description 22
- 229920001220 nitrocellulos Polymers 0.000 claims abstract description 20
- 102000036215 folic acid binding proteins Human genes 0.000 claims abstract description 19
- 108091011001 folic acid binding proteins Proteins 0.000 claims abstract description 19
- 239000000020 Nitrocellulose Substances 0.000 claims abstract description 16
- 239000012528 membrane Substances 0.000 claims abstract description 16
- 238000003908 quality control method Methods 0.000 claims abstract description 15
- 238000001914 filtration Methods 0.000 claims abstract description 14
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- 238000000034 method Methods 0.000 claims description 28
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- 238000002360 preparation method Methods 0.000 claims description 27
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 21
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- 230000005593 dissociations Effects 0.000 claims description 20
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 15
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 15
- 239000007983 Tris buffer Substances 0.000 claims description 15
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 15
- 239000005018 casein Substances 0.000 claims description 15
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 15
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- 238000001035 drying Methods 0.000 claims description 15
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- 230000008569 process Effects 0.000 claims description 14
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- 229930006000 Sucrose Natural products 0.000 claims description 12
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 12
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 238000003760 magnetic stirring Methods 0.000 claims description 9
- 239000011780 sodium chloride Substances 0.000 claims description 9
- 239000003398 denaturant Substances 0.000 claims description 8
- 239000007850 fluorescent dye Substances 0.000 claims description 8
- 238000005303 weighing Methods 0.000 claims description 8
- 239000003381 stabilizer Substances 0.000 claims description 7
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- 239000000872 buffer Substances 0.000 claims description 3
- HDFXRQJQZBPDLF-UHFFFAOYSA-L disodium hydrogen carbonate Chemical compound [Na+].[Na+].OC([O-])=O.OC([O-])=O HDFXRQJQZBPDLF-UHFFFAOYSA-L 0.000 claims description 3
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims description 3
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- 235000010378 sodium ascorbate Nutrition 0.000 claims description 3
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 3
- 229960005055 sodium ascorbate Drugs 0.000 claims description 3
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 3
- 230000001502 supplementing effect Effects 0.000 claims 2
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims 1
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- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 208000007502 anemia Diseases 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000003759 clinical diagnosis Methods 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
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- 238000002965 ELISA Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010016880 Folate deficiency Diseases 0.000 description 1
- 208000010188 Folic Acid Deficiency Diseases 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000037354 amino acid metabolism Effects 0.000 description 1
- 230000003432 anti-folate effect Effects 0.000 description 1
- 229940127074 antifolate Drugs 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
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- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- 239000004052 folic acid antagonist Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
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- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
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- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
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- 230000002194 synthesizing effect Effects 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a detection card and a kit for quantitatively detecting folic acid by an immunofluorescence chromatography, which belong to the technical fields of biology and immunodetection, wherein the detection card comprises an upper cover and a lower cover, a containing cavity is formed between the upper cover and the lower cover, a test strip is arranged in the containing cavity, the test strip comprises a PVC bottom plate, a sample pad, a blood filtering pad, a first combining pad, a second combining pad, a nitrocellulose membrane and absorbent paper, the sample pad, the blood filtering pad, the first combining pad, the second combining pad, the nitrocellulose membrane and the absorbent paper are sequentially overlapped in a staggered manner and then are adhered to the PVC bottom plate, the nitrocellulose membrane is sequentially coated with a detection T line and a quality control C line, and the first combining pad and the second combining pad are coated with solid phase liquid, 1mg/mL folic acid binding protein marked by fluorescent substances and folic acid-BSA marked by biotin substances.
Description
Technical Field
The invention relates to the technical fields of biology and immunodetection, in particular to a detection card and a kit for quantitatively detecting folic acid by using an immunofluorescence chromatography.
Background
Folic acid belongs to hapten, is a small molecular substance, can not induce immune response alone, and has no immunogenicity. As coenzyme of one-carbon single enzyme system in vivo biochemical reaction, the main physiological functions in human body are: 1) Participating in the synthesis of purine and thymine, and further synthesizing DNA and RNA; participating in amino acid metabolism; 2) Involved in synthesis of hemoglobin and methyl compounds such as epinephrine, choline, creatine, and the like; 3) Providing energy for the organism, improving immunity and helping to subside and anxiety; 4) Promoting the nuclear maturation and growth of erythrocytes is important for the formation of erythrocytes.
Currently, the folic acid determination method includes: enzyme-linked immunosorbent assay, radioimmunoassay, liquid chromatography-mass spectrometry, chemiluminescence method, etc. The method has the advantages of sensitive and accurate instrument, strong specificity and good separation degree, can simultaneously measure various medicines, but requires expensive instruments, complex pretreatment of samples, is tedious and time-consuming, has higher detection cost, can not be operated on site, and requires professional personnel to operate, and has long detection time.
Disclosure of Invention
Aiming at the defects, the invention provides the detection card and the kit for quantitatively detecting folic acid by using the immunofluorescence chromatography, which are used for batch and rapid detection of folic acid, and have the advantages of high detection sensitivity, good specificity, rapid and accurate detection, convenient use, simple operation, portability and convenient storage and transportation.
In order to solve the technical problems, the invention adopts the following technical scheme: the detection card comprises an upper cover and a lower cover, wherein the upper cover and the lower cover are mutually buckled and connected, a containing cavity is formed between the upper cover and the lower cover, a test strip is arranged in the containing cavity, the test strip comprises a PVC bottom plate, a sample pad, a blood filtering pad, a first combining pad, a second combining pad, a nitrocellulose membrane and absorbent paper, the sample pad, the blood filtering pad, the first combining pad, the second combining pad, the nitrocellulose membrane and the absorbent paper are sequentially overlapped in a staggered manner and then are adhered to the PVC bottom plate, the nitrocellulose membrane is sequentially coated with a detection T line and a quality control C line, the first combining pad and the second combining pad are coated with solid phase liquid, 1mg/mL folic acid binding protein marked by fluorescent substances and folic acid-BSA marked by biotin substances, and the first combining pad and the second combining pad are placed in an air blast drying box and dried for 4 hours at 37 ℃.
As a further improvement of the technical scheme, the fluorescent substance labeling 1mg/mL folic acid binding protein process comprises the following steps:
(1) Preparation of antibodies: the folic acid binding protein was removed and allowed to equilibrate at room temperature for 20min.
(2) Preparation of fluorescent dye: the fluorochrome was dissolved to 1mol/L with PBS for use.
(3) And (3) marking: mu.L of fluorescent dye and 25. Mu.L of 4mg/mL folic acid binding protein are added to each 50. Mu.L of system, and the residual volume is treated with NaHCO with pH value of 8.0 and 1mol/L 3 Complement, shake the reaction for 3 hours in the shaking table of room temperature after mixing.
(4) And (3) dialyzing: and (3) labeling the antibody, dialyzing in a buffer solution for 36 hours, and replacing the dialyzate every 4 hours to obtain the labeled fluorescent antibody.
As a further improvement of the above technical scheme, the biotin substance-labeled folic acid-BSA process comprises the following steps:
(1) Preparation of protein: the folic acid-BSA was taken out and allowed to equilibrate at room temperature for 20min.
(2) Preparation of biotin: taking out biotin, and standing at room temperature for 20min for balancing.
(3) And (3) marking: add 5.0mg/mL folic acid-BSA 15. Mu.L and biotin 2. Mu.L to 50. Mu.L system, the remaining volume was replaced with NaHCO at pH 8.0, 1mol/L 3 Complement, shake the reaction for 3 hours in the shaking table of room temperature, mix fully.
(4) And (3) dialyzing: and (3) labeling the antibody, dialyzing in a buffer solution for 36 hours, and replacing the dialysate every 4 hours to obtain the labeled biotin antigen.
As a further improvement of the technical scheme, 3% of BSA by mass, 1.5% of sucrose by mass, 0.1% of trehalose by mass and 0.5% of KCl by mass are added in the dialysis steps of a folic acid binding protein labeling process with a fluorescent substance of 1mg/mL and a folic acid-BSA labeling process with a biotin substance.
The buffer solution is 50mM PBS buffer solution, the pH value of the buffer solution is 7.0, and the preparation method of the buffer solution comprises the following steps: weighing Na 2 HPO 4 ·12H 2 O 8.7g、NaH 2 PO 4 ·12H 2 0.9g of O, 26.4g of NaCl, 30g of BSA, 15g of sucrose, 3g of trehalose and 3g of KCl are dissolved in distilled water and are evenly mixed under magnetic stirring, and the volume is fixed to 3000 mL.
As a further improvement of the technical scheme, the quality control C line is coated with biotin-BSA, the concentration is 0.6mg/mL, the liquid dividing amount is 0.5 mu L/cm, and the film dividing speed is 80mm/s; the detection T line is coated with avidin, the concentration is 2.5mg/mL, the liquid dividing amount is 1.5 mu L/cm, the film dividing speed is 80mm/s, the coated nitrocellulose film is placed in a blast drying oven, the temperature is 37 ℃ for 30min for standby, sealing liquid is sprayed on the detection T line after drying, the liquid dividing amount is 2.5 mu L/cm, the film dividing speed is 80mm/s, and the nitrocellulose film sprayed with the sealing liquid is placed in the blast drying oven, and the temperature is 37 ℃ for 30min for standby.
As a further improvement of the above technical solution, the preparation method of the sealing liquid comprises: 1.2g of Tris, 2.0g of BSA, 0.5g of casein, 0.1g of PVP, 9 2g of S and 1g of trehalose are weighed and dissolved in 800mL of distilled water, concentrated HCl is dropwise added to the solution under magnetic stirring until the pH value is 8.0, the volume is fixed to 100mL, and then 0.1mL of Tween is added and uniformly mixed for later use, wherein the buffer solution is 100mM of Tris and the pH value is 8.0.
As a further improvement of the technical scheme, the sample pad, the first bonding pad and the second bonding pad are respectively immersed into corresponding treatment liquid, the shaking table is vibrated for 30min, and the sample pad, the first bonding pad and the second bonding pad are placed in a forced air drying oven to be dried for 4 hours and stored for standby.
As a further improvement of the above technical solution, the preparation method of the treatment liquid of the sample pad comprises: weighing 0.3g of Tris, 0.9g of NaCl, 2g of casein and 20000 1g of PEG, dissolving in 80mL of distilled water, dropwise adding concentrated HCl to a pH value of 8.0 under magnetic stirring, fixing the volume to 100mL, adding 0.1mL of Tween, and uniformly mixing for later use, wherein the buffer solution is 25mM of Tris, and the pH value is 8.0;
the preparation method of the treatment fluid of the first bonding pad and the second bonding pad comprises the following steps: weighing Na 2 HPO 4 ·12H 2 O 0.29g、NaH 2 PO 4 ·12H 2 0.03g of O, 0.88g of NaCl, 2.0g of PVP, 2.0g of casein, 5g of BSA, 0.1g of trehalose and 0.5g of sucrose are dissolved in 100mL of distilled water, 0.1mL of Tween is added and mixed for standby, 10 mM of PBS is added, and the pH value of the buffer solution is 7.0.
As a further improvement of the technical scheme, the preparation method of the solid phase liquid comprises the following steps: 2.0g of casein, 2.5g of BSA, 0.8g of trehalose and 0.5g of sucrose are weighed and dissolved in 100mL of distilled water, and 0.1mL of Tween is added for uniform mixing for standby.
A kit for quantitative detection of folic acid by immunofluorescence chromatography, the kit comprising a sample dissociation solution and the detection card, the sample dissociation solution comprising a stabilizer, a denaturant and a neutralizer, the stabilizer comprising 25mM MES, 1% tcep, 0.5% mercaptoethanol, 0.2% pc300 and 2.5% sodium ascorbate, the denaturant comprising 0.1mM sodium carbonate-sodium bicarbonate buffer, 5% sodium hydroxide, 0.1% methanol and 0.2% pc300, the neutralizer comprising 50mM Tris buffer, 0.9% NaCl and 0.5% casein, the percentages being mass percentages.
The invention adopts the technical proposal and has the following beneficial effects:
1. the detection card and the kit for quantitatively detecting folic acid by using the immunofluorescence chromatography are convenient to operate, convenient to observe, high in sensitivity, accurate in result, short in detection and sample dissociation time and suitable for on-site rapid detection.
2. According to the invention, an amplification system is introduced based on fluorescent dye by utilizing the principle of a competition method, each avidin molecule has four biotin binding sites, and the amplification system has a multistage amplification effect, so that the detection sensitivity is improved; the binding between avidin and biotin has extremely high affinity, the reaction is highly specific, and the binding characteristics of the avidin and biotin are not affected by the high dilution of the reaction reagent, so that the nonspecific action of the reaction reagent can be reduced to the greatest extent in practical application; the affinity constant between avidin and biotin is extremely high, and the dissociation constant of a complex formed by combination is very small, so that the complex is irreversibly reactive; and the combination of the acid, the alkali, the denaturant and the like is not affected, so that the stability is high.
3. The invention provides a sample dissociation solution, which has short dissociation time, only needs 5min, is suitable for three samples of serum, plasma and whole blood, has wide application range, is safe and nontoxic, has stable performance, and is characterized in that most of the reagent boxes on the current market are prepared by separately preparing two serum and whole blood.
4. The test strip for quantitatively detecting folic acid by using the immunofluorescence chromatography improves the superposition method and the width of a sample pad, a blood filtering pad and a combining pad, and has the advantages of good result repeatability, few interference factors, strong specificity, high sensitivity, simple operation and convenient detection.
5. The kit for quantitatively detecting folic acid by using the immunofluorescence chromatography can accurately and sensitively detect different blood samples, and can detect serum, whole blood and plasma, and the pretreatment process of the sample is simple, consumes less time, can rapidly produce results, and has high efficiency and low cost.
6. The invention provides a T-line detection sealing liquid, which improves the binding capacity of avidin and a nitrocellulose membrane, improves the stability of a detection sample due to the existence of the sealing liquid, solves the storage problem of a kit and is beneficial to the later process control.
7. The invention provides a fluorescent marking process, which solves the problem of unstable marked fluorescent substances and is beneficial to the control of the later process. The content of the to-be-detected object can be accurately measured through the fluorescent immunoassay reader, high sensitivity is achieved, the detection range is wide, the method is simple, convenient and rapid, the content of the phylloic acid in the human serum, the plasma and the whole blood sample can be rapidly detected within 15-18min, the whole blood sample is filtered through the blood filtering pad, the fluorescent immunoassay technology is utilized, a small amount of blood sample and sample liquid are mixed and added, and the result can be rapidly shown by inserting the fluorescent immunoassay reader. The method is reliable in theory, practical and feasible, can be completed in a secondary biosafety laboratory, and has the characteristics of high sensitivity, wide detection range, rapidness, simplicity and the like.
8. The kit for quantitatively detecting folic acid by using the immunofluorescence chromatography is used for determining the content of folic acid in pregnant women, and the kit for quantitatively detecting the content of folic acid in serum, plasma or whole blood by using the immunofluorescence chromatography has an auxiliary effect on clinical diagnosis and treatment of diseases such as anemia.
The invention is further described below with reference to the drawings and the detailed description.
Drawings
FIG. 1 is a schematic diagram of the internal structure of a detection card for quantitatively detecting folic acid by immunofluorescence chromatography;
FIG. 2 is a schematic diagram of the structure of a test strip of a detection card for quantitatively detecting folic acid by using an immunofluorescence chromatography method;
FIG. 3 is a schematic diagram of the structure of the upper cover of a detection card for quantitatively detecting folic acid by immunofluorescence chromatography;
FIG. 4 is a graph comparing the folic acid measurement values of the experimental part of the present invention, wherein the x-axis is the serum folic acid measurement value of the chemiluminescence method, and the y-axis is the serum folic acid measurement value of the method.
In the drawing the view of the figure,
1-upper cover, 2-lower cover, 3-PVC bottom plate, 4-sample pad, 5-blood filter pad, 6-first combination pad, 7-second combination pad, 8-nitrocellulose membrane, 9-detection T line, 10-quality control C line, 11-absorbent paper, 12-sample adding hole, 13-detection window.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples, in which specific conditions are not noted, according to conventional conditions or manufacturer's recommended conditions. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The described embodiments are intended to be illustrative of only some, but not all embodiments of the invention, and all other embodiments that may be made by one of ordinary skill in the art without inventive faculty are intended to be within the scope of the invention.
Example 1:
as shown in fig. 1-3, a detection card for quantitatively detecting folic acid by using an immunofluorescence chromatography method comprises an upper cover 1 and a lower cover 2, wherein the upper cover 1 and the lower cover 2 are mutually connected in a buckling way, a containing cavity is formed between the upper cover 1 and the lower cover 2, a test paper strip is arranged in the containing cavity, the test paper strip comprises a PVC bottom plate 3, a sample pad 4, a blood filtering pad 5, a first bonding pad 6, a second bonding pad 7, a nitrocellulose membrane 8 and a water absorbing paper 11, the sample pad 4, the blood filtering pad 5, the first bonding pad 6, the second bonding pad 7, the nitrocellulose membrane 8 and the water absorbing paper 11 are sequentially overlapped with each other in a staggered way, then are adhered to the PVC bottom plate 3, the size of the staggered way is 1mm, one end of the sample pad 4 is adhered to the PVC bottom plate 3, the other end of the sample pad 4 is overlapped to one end of the blood filtering pad 5, one end of the blood filtering pad 5 is adhered to one end of the PVC bottom plate 3, the other end of the first bonding pad 6 is overlapped to one end of the first bonding pad 6, one end of the first bonding pad 6 is adhered to one end of the PVC bottom plate 3, the other end of the first bonding pad 6 is adhered to one end of the second bonding pad 7 is overlapped to one end of the PVC bottom plate 3, the other end of the nitrocellulose membrane 8 is adhered to the other end of the second bonding pad 7 is overlapped with the other end 11, and the nitrocellulose membrane is overlapped with one end 8 is overlapped with one end of the other end 11; the nitrocellulose membrane 8 is sequentially coated with a detection T line 9 and a quality control C line 10, the width of the test strip is 3-4mm, and the test strip is sealed and preserved at normal temperature by adding a drying agent.
The first binding pad 6 and the second binding pad 7 are coated with solid phase liquid, folic acid binding protein marked by fluorescent substances and folic acid-BSA marked by biotin substances, and the materials are placed in a blast drying box and dried for 4 hours at 37 ℃.
The upper cover 1 is provided with a sample adding hole 12 and a detection window 13, the position of the sample adding hole 12 corresponds to the position of the blood filtering pad 5, so that a sample can be dripped onto the blood filtering pad 5, the position of the detection window 13 corresponds to the positions of the detection T line 9 and the quality control C line 10, and the upper cover 1 and the lower cover 2 are both made of plastic materials.
The process for labeling folic acid binding protein by fluorescent substances comprises the following steps:
(1) Preparation of antibodies: the folic acid binding protein was removed and allowed to equilibrate at room temperature for 20min.
(2) Preparation of fluorescent dye: the fluorochrome was dissolved to 1mol/L with PBS for use.
(3) And (3) marking: mu.L of fluorescent dye and 25. Mu.L of 4mg/mL folic acid binding protein are added to each 50. Mu.L of system, and the residual volume is treated with NaHCO with pH value of 8.0 and 1mol/L 3 Complement, shake the reaction for 3 hours in the shaking table of room temperature after mixing.
(4) And (3) dialyzing: and (3) labeling the antibody, dialyzing in a buffer solution for 36 hours, and replacing the dialyzate every 4 hours to obtain the labeled fluorescent antibody.
The biotin substance-labeled folic acid-BSA process comprises the following steps:
(1) Preparation of protein: the folic acid-BSA was taken out and allowed to equilibrate at room temperature for 20min.
(2) Preparation of biotin: taking out biotin, and standing at room temperature for 20min for balancing.
(3) And (3) marking: add 5.0mg/mL folic acid-BSA 15. Mu.L and biotin 2. Mu.L to 50. Mu.L system, the remaining volume was replaced with NaHCO at pH 8.0, 1mol/L 3 Complement, shake the reaction for 3 hours in the shaking table of room temperature, mix fully.
(4) And (3) dialyzing: and (3) labeling the antibody, dialyzing in a buffer solution for 36 hours, and replacing the dialysate every 4 hours to obtain the labeled biotin antigen.
3% of BSA by mass, 1.5% of sucrose by mass, 0.1% of trehalose by mass and 0.5% of KCl by mass are added in the dialysis steps of the fluorescent substance-labeled folic acid binding protein process and the biotin substance-labeled folic acid-BSA process.
The buffer solution is 50mM PBS buffer solution, the pH value of the buffer solution is 7.0, and the preparation method of the buffer solution comprises the following steps: weighing Na 2 HPO 4 ·12H 2 O 8.7g、NaH 2 PO 4 ·12H 2 0.9g of O, 26.4g of NaCl, 30g of BSA, 15g of sucrose, 3g of trehalose and 3g of KCl are dissolved in distilled water and are evenly mixed under magnetic stirring, and the volume is fixed to 3000 mL.
The quality control C line 10 is coated with biotin-BSA, the concentration is 0.6mg/mL, the liquid dividing amount is 0.5 mu L/cm, and the film dividing speed is 80mm/s; the detection T line 9 is coated with avidin, the concentration is 2.5mg/mL, the liquid dividing amount is 1.5 mu L/cm, the film dividing speed is 80mm/s, and the detection T line is placed in a blast drying oven and dried at 37 ℃ for 30min for standby.
And (3) after the coating avidin of the detection T line 9 is dried, spraying sealing liquid on the detection T line 9, wherein the liquid dividing amount is 2.5 mu L/cm, the film dividing speed is 80mm/s, and placing the film in a blast drying oven for drying at 37 ℃ for 30min for standby.
The preparation method of the sealing liquid comprises the following steps: 1.2g of Tris, 2.0g of BSA, 0.5g of casein, 0.1g of PVP, 9 2g of S and 1g of trehalose are weighed and dissolved in 800mL of distilled water, concentrated HCl is dropwise added to the solution under magnetic stirring until the pH value is 8.0, the volume is fixed to 100mL, and then 0.1mL of Tween is added and uniformly mixed for later use, wherein the buffer solution is 100mM of Tris and the pH value is 8.0.
The sample pad 4, the first bonding pad 6 and the second bonding pad 7 are respectively immersed in the corresponding treatment liquid, the shaking table oscillates for 30min, and the sample pad, the first bonding pad 6 and the second bonding pad 7 are placed in a blast drying oven to be dried for 4 hours and stored for standby.
The preparation method of the treatment liquid of the sample pad 4 comprises the following steps: 0.3g of Tris, 0.9g of NaCl, 2g of casein and 20000 1g of PEG are weighed and dissolved in 80mL of distilled water, concentrated HCl is added dropwise under magnetic stirring until the pH value is 8.0, the volume is fixed to 100mL, then 0.1mL of Tween is added and mixed evenly for later use, and the buffer solution is 25mM of Tris and the pH value is 8.0.
The preparation method of the treatment liquid for the first bonding pad 6 and the second bonding pad 7 comprises the following steps: weighing Na 2 HPO 4 ·12H 2 O 0.29g、NaH 2 PO 4 ·12H 2 0.03g of O, 0.88g of NaCl, 2.0g of PVP, 2.0g of casein, 5g of BSA, 0.1g of trehalose and 0.5g of sucrose are dissolved in 100mL of distilled water, 0.1mL of Tween is added and mixed for standby, 10 mM of PBS is added, and the pH value of the buffer solution is 7.0.
The preparation method of the solid phase liquid comprises the following steps: 2.0g of casein, 2.5g of BSA, 0.8g of trehalose and 0.5g of sucrose are weighed and dissolved in 100mL of distilled water, and 0.1mL of Tween is added for uniform mixing for standby.
Example 2:
a kit for quantitative detection of folic acid by immunofluorescence chromatography, the kit comprising a sample dissociation liquid and a detection card as described in example 1, the sample dissociation liquid comprising a stabilizer, a denaturant and a neutralizing agent, the stabilizer comprising 25mM MES, 1% tcep, 0.5% mercaptoethanol, 0.2% pc300 and 2.5% sodium ascorbate, the denaturant comprising 0.1mM sodium carbonate-sodium bicarbonate buffer, 5% sodium hydroxide, 0.1% methanol and 0.2% pc300, the neutralizing agent comprising 50mM Tris buffer, 0.9% NaCl and 0.5% casein, the percentages being mass percentages.
The detection method of the kit comprises the following steps:
the sample to be tested is serum, whole blood or plasma, and the sample is dissociated by a sample dissociation liquid, wherein the sample dissociation process comprises the following steps: mixing 25 mu L of stabilizer with 15 mu L of denaturant, adding 40 mu L of sample to be detected, standing at 37 ℃ for 5min, adding 50 mu L of neutralizer, and fully mixing to obtain sample mixed solution; adding 100 mu L of sample mixed solution into a sample adding hole 12, reacting for 16min at room temperature, combining folic acid in the sample mixed solution with fluorescent substance-marked folic acid binding protein antibodies coated on a first binding pad 6 and a second binding pad 7, combining the residual fluorescent substance-marked folic acid binding protein with biotin substance-marked folic acid-BSA, combining the fluorescent substance-marked folic acid binding protein with biotin-marked folic acid-BSA conjugate with avidin coated on a detection T line 9 under the chromatographic action, and detecting the relative content of antifolate by a fluorescence immunoassay analyzer, wherein the more folic acid in the sample, the lower the fluorescence signal.
Experiment
The detection card is used for detecting quality control products in enterprises, folic acid standard products with different concentrations are added into a sample adding area, the concentrations are respectively 0.5ng/mL, 1ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL and 20ng/mL, 3 parallels are carried out on each concentration, after 15-18min of reaction, a fluorescence analyzer reads signals of a quality control C line 10 and a detection T line 9, concentration values of the folic acid are obtained, the process is repeated for 3 times, and the detection results are shown in the following table:
from the table above, it can be seen that the linear range of the kit for quantitative detection of folic acid by immunofluorescence chromatography of the invention is 0.5ng/mL to 40ng/mL, and the kit is repeated three times, three times in parallel, and the repeatability is controlled within 6%.
Dissociation was performed using the sample dissociation solution of example 2, and the serum of the rogowski value was compared with the sample dissociation solution of the present invention and the plate folic acid values, and the results are shown in the following table:
the results of the detection of the folic acid sample dissociation liquid and the plate of the embodiment 2 of the invention are adopted as a reference, the linear equation of the detection results is y= 0.9946 x+ 0.1365, and the correlation coefficient R 2 Taken together, 0.9848 shows that the sample dissociation solution of example 2 works well, with good agreement with the positive control, as shown in fig. 4.
Stability experiment investigation:
the kit provided by the invention is subjected to destructive test at 37 ℃ for 6 months in a detection period, and the stability of the kit is detected on the 1 st day, the 3 rd day, the 7 th day, the 10 th day, the 15 th day, the 30 th day, the 45 th day, the 60 th day, the 90 th day, the 120 th day, the 150 th day and the 180 th day respectively, and the standard is as follows:
1. physical state of sample dissociation liquid
Appearance: colorless transparent clear liquid, no particles, floccules, precipitation and no volatility.
2. Performance index
(1) Negative quality control product compliance rate: 10 parts of negative quality control materials are used for detection, the detection results are shown in table 1, all negative results are obtained, and the accuracy of the kit meets the requirements.
(2) Positive quality control product compliance rate: and detecting by using 10 positive (including strong, medium and weak positive) quality control products, wherein the detection results are shown in table 2, all positive results are obtained, and the accuracy of the kit meets the requirements.
Minimum detection limit: the detection is performed with the lowest detection limit reference S, and the result should be positive.
Repeatability: the coefficient of variation of the results from day 1 to day 180 should be no more than 15% for negative reference, positive reference and minimum detection limit.
The stability of the kit is examined as follows:
sample dissociation liquid physical state stability results:
accelerated disruption test results of the kit at 37 ℃):
through experiments, the composition can be stabilized for at least 15 days at 37 ℃, and according to the principle of stability experiments, the Arrhenius formula: d (Ink)/dT=Ea/RT 2 Ea, and is stored for 10 months at normal temperature, which is equivalent to breaking for 15 days at 37 ℃, so that the clinical requirements of hospital consultation and health quarantine departments can be met, and the method can also be used for disease diagnosis research of colleges and universities and scientific research institutions.
The coincidence rate of the kit and the detection kit by the Roche electrochemiluminescence method is as follows:
100 serum of patients with definite folic acid deficiency and 300 serum of healthy examination are collected from clinic, and the kit and the detection kit (the kit on the market) of the Roche electrochemiluminescence method are used for simultaneous detection, and the specificity and the detection rate are shown in the following table:
clinical comparison with the marketed kit
Total compliance of two kits for 400 samples
As shown in the table, the specificity of the kit is not greatly different from that of the kit on the market, the detection rate is about 98%, the total coincidence rate is basically consistent, the total coincidence rate is up to 99%, the self-made sample dissociation solution dissociation sample effect of the kit is obvious, the specificity of the detection result is good, the coincidence rate is high, and the kit can play an auxiliary role in clinical diagnosis and treatment of anemia.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the invention, but is merely illustrative of the embodiments, and all the details not described herein are common knowledge of a person skilled in the art, so that it is possible for a person skilled in the art to modify the technical solutions described in the foregoing embodiments or to make equivalent substitutions for some technical features thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (2)
1. A kit for quantitative detection of folic acid by immunofluorescence chromatography is characterized in that: the sample dissociation solution comprises a stabilizer, a denaturant and a neutralizer, wherein the stabilizer comprises 25mM MES, 1% TCEP, 0.5% mercaptoethanol, 0.2% PC300 and 2.5% sodium ascorbate, the denaturant comprises 0.1mM sodium carbonate-sodium bicarbonate buffer, 5% sodium hydroxide, 0.1% methanol and 0.2% PC300, and the neutralizer comprises 50mM Tris buffer, 0.9% NaCl and 0.5% casein, and the percentages are mass percentages;
the detection card comprises an upper cover and a lower cover, wherein the upper cover and the lower cover are mutually connected in a buckling manner, a containing cavity is formed between the upper cover and the lower cover, a test strip is arranged in the containing cavity, the test strip comprises a PVC bottom plate, a sample pad, a blood filtering pad, a first bonding pad, a second bonding pad, a nitrocellulose membrane and absorbent paper, the sample pad, the blood filtering pad, the first bonding pad, the second bonding pad, the nitrocellulose membrane and the absorbent paper are sequentially overlapped in a staggered manner and then are adhered to the PVC bottom plate, the nitrocellulose membrane is sequentially coated with a detection T line and a quality control C line, the first bonding pad and the second bonding pad are coated with solid phase liquid, 1mg/mL folic acid binding protein marked by fluorescent substances and folic acid-BSA marked by biotin substances, the first bonding pad and the second bonding pad are placed in an air blast drying box, and the air blast drying is carried out at 37 ℃ for 4h;
the process for labeling 1mg/mL folic acid binding protein by using a fluorescent substance comprises the following steps:
(1) Preparation of antibodies: taking out folic acid binding protein, and standing at room temperature for 20min for balancing;
(2) Preparation of fluorescent dye: dissolving the fluorescent dye to 1mol/L with PBS for standby;
(3) And (3) marking: mu.L of fluorescent dye and 25. Mu.L of 4mg/mL folic acid binding protein are added to each 50. Mu.L of system, and the residual volume is treated with NaHCO with pH value of 8.0 and 1mol/L 3 Supplementing, mixing, and vibrating and reacting for 3 hours at room temperature by a shaking table;
(4) And (3) dialyzing: labeling the antibody, dialyzing in a buffer solution for 36 hours, and replacing the dialyzate every 4 hours to obtain a labeled fluorescent antibody;
the biotin substance-labeled folic acid-BSA process comprises the following steps:
(1) Preparation of protein: taking out folic acid-BSA, and balancing at room temperature for 20min;
(2) Preparation of biotin: taking out biotin, and standing at room temperature for balancing for 20min;
(3) And (3) marking: add 5.0mg/mL folic acid-BSA 15. Mu.L and biotin 2. Mu.L to 50. Mu.L system, the remaining volume was replaced with NaHCO at pH 8.0, 1mol/L 3 Supplementing, vibrating and reacting for 3 hours at room temperature by a shaking table, and fully and uniformly mixing;
(4) And (3) dialyzing: the antibody is marked, the dialysis is carried out in buffer solution for 36 hours, and the dialysis solution is replaced every 4 hours, so that the marked biotin antigen is obtained;
the quality control C line is coated with biotin-BSA, the concentration is 0.6mg/mL, the liquid dividing amount is 0.5 mu L/cm, and the film dividing speed is 80mm/s; the detection T line is coated with avidin, the concentration is 2.5mg/mL, the liquid dividing amount is 1.5 mu L/cm, the film dividing speed is 80mm/s, the coated nitrocellulose film is placed in a blast drying oven, the temperature is 37 ℃ for 30min for standby, sealing liquid is sprayed on the detection T line after drying, the liquid dividing amount is 2.5 mu L/cm, the film dividing speed is 80mm/s, and the nitrocellulose film sprayed with the sealing liquid is placed in the blast drying oven, and the temperature is 37 ℃ for 30min for standby;
the preparation method of the sealing liquid comprises the following steps: 1.2g of Tris, 2.0g of BSA, 0.5g of casein, 0.1g of PVP, 9 2g of S and 1g of trehalose are weighed and dissolved in 800mL of distilled water, concentrated HCl is dropwise added to the solution under magnetic stirring until the pH value is 8.0, the volume is fixed to 100mL, and then 0.1mL of Tween is added and uniformly mixed for later use, wherein the buffer solution is 100mM of Tris and the pH value is 8.0;
the sample pad, the first bonding pad and the second bonding pad are respectively immersed in corresponding treatment liquid, the shaking table oscillates for 30min, and the sample pad, the first bonding pad and the second bonding pad are placed in a blast drying oven to be dried for 4 hours and stored for standby;
the preparation method of the treatment fluid of the sample pad comprises the following steps: weighing 0.3g of Tris, 0.9g of NaCl, 2g of casein and 20000 1g of PEG, dissolving in 80mL of distilled water, dropwise adding concentrated HCl to a pH value of 8.0 under magnetic stirring, fixing the volume to 100mL, adding 0.1mL of Tween, and uniformly mixing for later use, wherein the buffer solution is 25mM of Tris, and the pH value is 8.0;
the preparation method of the treatment fluid of the first bonding pad and the second bonding pad comprises the following steps: weighing Na 2 HPO 4 ·12H 2 O 0.29g、NaH 2 PO 4 ·12H 2 Dissolving 0.03g of O, 0.88g of NaCl, 2.0g of PVP, 2.0g of casein, 5g of BSA, 0.1g of trehalose and 0.5g of sucrose in 100mL of distilled water, adding 0.1mL of Tween, uniformly mixing for later use, 10 mM of PBS, and enabling the pH value of a buffer solution to be 7.0;
the preparation method of the solid phase liquid comprises the following steps: 2.0g of casein, 2.5g of BSA, 0.8g of trehalose and 0.5g of sucrose are weighed and dissolved in 100mL of distilled water, and 0.1mL of Tween is added for uniform mixing for standby.
2. The kit for quantitative detection of folic acid by immunofluorescence chromatography according to claim 1, characterized in that: adding 3% of BSA (BSA) by mass, 1.5% of sucrose by mass, 0.1% of trehalose by mass and 0.5% of KCl by mass into dialysis steps of a fluorescent substance labeled 1mg/mL folic acid binding protein process and a biotin substance labeled folic acid-BSA process;
the buffer solution is 50mM PBS buffer solution, the pH value of the buffer solution is 7.0, and the preparation method of the buffer solution comprises the following steps: weighing Na 2 HPO 4 ·12H 2 O 8.7g、NaH 2 PO 4 ·12H 2 0.9g of O, 26.4g of NaCl, 30g of BSA, 15g of sucrose, 3g of trehalose and 3g of KCl are dissolved in distilled water and are evenly mixed under magnetic stirring, and the volume is fixed to 3000 mL.
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| CN117214445B (en) * | 2023-11-09 | 2024-01-26 | 山东子峰生物技术有限公司 | Quantum dot fluorescence detection reagent for joint inspection of folic acid, vitamin B12 and ferritin and application thereof |
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