CN116574107A - Gelsemine colloidal gold detection test paper, preparation method and application - Google Patents

Gelsemine colloidal gold detection test paper, preparation method and application Download PDF

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Publication number
CN116574107A
CN116574107A CN202310533826.3A CN202310533826A CN116574107A CN 116574107 A CN116574107 A CN 116574107A CN 202310533826 A CN202310533826 A CN 202310533826A CN 116574107 A CN116574107 A CN 116574107A
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gelsemine
colloidal gold
monoclonal antibody
hapten
detection
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王磊
叶文才
王炳志
杨星星
黎尼平
麻楠
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SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD
Jinan University
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SHENZHEN BIOEASY BIOTECHNOLOGY CO LTD
Jinan University
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • 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
    • G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract

The invention discloses a gelsemine hapten, a colloidal gold labeled gelsemine monoclonal antibody and a preparation method and application of a gelsemine colloidal gold rapid detection test strip, and relates to the technical fields of biochemical engineering and biological detection. The structure of the gelsemine hapten is shown as I:

Description

Gelsemine colloidal gold detection test paper, preparation method and application
Technical Field
The invention belongs to the technical fields of biochemical engineering and biological detection, and particularly relates to a gelsemine hapten, a colloidal gold labeled gelsemine monoclonal antibody and a preparation method and application of a gelsemine colloidal gold rapid detection test strip.
Background
Gelsemium elegans (Gelsemium elegans) is a woody vine plant of the family loguaiaceae of the order gentianaea, also known as gelsemium elegans, large tea drugs, caulis hedyotis sinensis and the like, and is widely distributed in southern areas of China. The whole plant is extremely toxic, and people can die after eating a small amount of the whole plant by mistake. Because the flowers and roots of the gelsemium elegans plant are similar to the appearance of the commonly used Chinese herbal medicines such as honeysuckle, fiveleaf fissistigma herb and the like, the frequent occurrence of gelsemium elegans poisoning and death caused by miseating, misuse and the like. Gelsemium intoxication has become a frequent malignant poisoning event in southern areas of China. Therefore, development of a high-specificity, high-sensitivity, rapid and simple analysis method is needed, and technical support is provided for preventing miseating and misuse of gelsemium elegans and diagnosis and identification of poisoning thereof.
The alkaloid compound is a toxic component of gelsemine, wherein gelsemine is a characteristic component of gelsemine plants, and is distributed in roots, stems, leaves, flowers and fruits of the gelsemine plants and has high content. Currently, the method for detecting gelsemine includes spectrophotometry, high performance liquid chromatography, ultra-high performance liquid chromatography, liquid chromatography-tandem mass spectrometry and the like. The above methods are all instrumental analysis methods, and the sample preparation is complicated, the instrumental operation is complex, the analysis cost is expensive, the time consumption is long, and the requirements of rapid detection and accident scene detection of gelsemium poisoning prevention cannot be met. The immunoassay method based on antigen-antibody specificity recognition, such as a colloidal gold immunochromatography method, an enzyme-linked immunosorbent assay method and the like, has the characteristics of simplicity, rapidness, strong specificity, high sensitivity, good stability and the like, and can realize rapid detection of toxic substances. At present, no report on rapid detection test strips of gelsemium alkaloid exists.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide a gelsemine hapten, a colloidal gold labeled gelsemine monoclonal antibody and a gelsemine colloidal gold detection test strip.
The aim of the invention is achieved by the following technical scheme:
the structure of the gelsemine hapten is shown as a formula I:
the preparation method of the gelsemine hapten comprises the following steps:
3-mercaptopropionic acid, benzoin dimethyl ether and methylene dichloride are uniformly mixed in a test tube. The gelsemine is weighed into a quartz crucible, and the solution is added into the crucible. Under the magnetic stirring, the ultraviolet lamp is used for irradiation reaction for 30 to 60 minutes. Evaporating the solvent after the reaction is completed, and purifying by silica gel column chromatography to obtain the gelsemine hapten.
The gelsemine monoclonal antibody marked by the colloidal gold is obtained by coupling the colloidal gold and the gelsemine monoclonal antibody;
the gelsemine monoclonal antibody is obtained by animal immunization through gelsemine antigen;
the gelsemine antigen is obtained by coupling gelsemine hapten and carrier protein, wherein the carrier protein comprises one or more of bovine serum albumin, human serum albumin and chicken egg albumin.
The assembly of the gelsemine colloidal gold detection test paper comprises a test paper strip, micropores and a straw, wherein the micropores comprise a colloidal gold labeled gelsemine monoclonal antibody, and the colloidal gold is obtained by coupling the gelsemine monoclonal antibody with the colloidal gold;
the preparation of the colloidal gold comprises the following steps:
heating chloroauric acid to boiling, adding trisodium citrate solution, continuously boiling for 15min when the color of the solution is changed into bright red, and naturally cooling to room temperature to obtain a colloidal gold solution;
the preparation of the colloidal gold labeled monoclonal antibody comprises the following steps:
regulating the colloidal gold solution to be weak alkaline, adding a monoclonal antibody of gelsemine, then adding Bovine Serum Albumin (BSA) for continuous reaction to obtain a colloidal gold-labeled monoclonal antibody precipitate, centrifuging and re-suspending to obtain the colloidal gold-labeled monoclonal antibody;
the components of the test strip comprise a bottom plate, a sample pad, a nitrocellulose membrane and an absorption pad, wherein the bottom plate, the sample pad, the nitrocellulose membrane and the absorption pad are sequentially connected;
the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a gelsemine coupling antigen which can be combined with a gelsemine monoclonal antibody, and the quality control line is coated with a goat anti-mouse IgG secondary antibody which can be combined with the gelsemine monoclonal antibody; the coating streak is prepared by a spot film instrument, and the coating quantity of the gelsemine coupling antigen is 1.5 mug/cm 2 The coating concentration of the goat anti-mouse IgG secondary antibody is 1.5 mug/cm 2 ；
The test strip is assembled by sequentially overlapping a sample pad, a nitrocellulose membrane and an absorption pad on a bottom plate, and respectively pressing the sample pad and the absorption pad above the nitrocellulose membrane by 3mm;
the detection principle of the gelsemine colloidal gold detection test strip is that the competition method immunochromatography technology is adopted, so that the gelsemine in the sample to be detected and the gelsemine antigen coated on the detection line are combined with the gelsemine monoclonal antibody marked by the colloidal gold in a competition mode. If the sample to be detected contains gelsemine, the gelsemine monoclonal antibody marked by colloidal gold is combined in the micropore incubation process, so that the combination of the gelsemine monoclonal antibody marked by colloidal gold and the gelsemine antigen coated on the detection line is inhibited, and the detection result can be obtained through the color comparison of the detection line and the quality control line.
Compared with the prior art, the invention has the following advantages and effects:
(1) The gelsemine hapten and the colloidal gold labeled monoclonal antibody can be applied to detection in immunochromatography, and provide a basis for establishing a rapid, simple, specific and sensitive detection method of the gelsemine.
(2) The gelsemine colloidal gold detection test strip disclosed by the invention is used for detecting the residual quantity of the gelsemine in a sample by comparing the colors of the detection line and the quality control line in the test strip by applying the principle of colloidal gold immunochromatography, can be used for rapidly and accurately detecting whether the sample contains the gelsemine or not, can meet the requirements of rapid detection on the gelsemine poisoning site, and has the characteristics of simplicity and convenience in operation, rapid detection, strong specificity and low cost, and has a wide application prospect.
Drawings
FIG. 1 is a flow chart of the preparation of gelsemine hapten;
fig. 2 is a mass spectrum of gelsemine (a) and gelsemine hapten (B);
FIG. 3 is a schematic diagram of a gelsemine colloidal gold test strip;
FIG. 4 is a schematic diagram of test strip result determination.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 preparation of gelsemine hapten
3-mercaptopropionic acid (250 mg,2.355 mmol), benzoin dimethyl ether (35 mg,0.137 mmol) and methylene chloride (9 mL) were mixed well in a test tube. Gelsteine (65 mg,0.202 mmol) was weighed into a 50mL quartz crucible and the above solution was added to the crucible. Under the magnetic stirring, the ultraviolet lamp is used for irradiation reaction for 30 to 60 minutes. After the reaction was completed, the solvent was evaporated to dryness and purified by silica gel column chromatography to give a colorless oil. The obtained product was subjected to high resolution mass spectrometry, the results are shown in FIG. 2, and the exact mass numbers thereof are shown in the figure(m/z 429.4[M+H] + ，C 23 H 28 N 2 O 4 S + ) The mass number of the gelsemine is increased by 106Da, which indicates that the synthesis of the target substance is successful and accords with the expected result.
Example 2 preparation of immunogens
10mg of gelsemine hapten is weighed and 1mL of anhydrous N, N-dimethylformamide is added for dissolution. Then, 6mg of N-hydroxysuccinimide and 7mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride were added and reacted overnight at room temperature to give hapten solution A. The solution B was obtained by weighing 35mg of bovine serum albumin, adding 3.5mL of 0.1M boric acid buffer solution for dissolution, and dropwise adding 1mL of N, N-dimethylformamide. Dripping the solution A into the solution B, reacting overnight at room temperature, dialyzing for 2-3 d by using normal saline to obtain the gelsemine-bovine serum albumin conjugate, namely the immunogen, centrifuging and split charging, and preserving at-20 ℃ for later use.
Example 3 preparation of coating Material
10mg of gelsemine hapten is weighed and 1mL of anhydrous N, N-dimethylformamide is added for dissolution. Then adding 6uL of diisopropylethylamine, adding 5uL of isobutyl chloroformate under ice bath condition, and reacting for 30min at 0 ℃ to obtain hapten solution A. 70mg of ovalbumin is weighed, 7mL of 0.1M boric acid buffer solution is added for dissolution, and 1mL of N, N-dimethylformamide is added dropwise to obtain solution B. And (3) dropwise adding the solution A into the solution B, reacting overnight at room temperature, dialyzing with PBS buffer solution for 2-3 d to obtain a coating raw material, centrifuging and split charging, and preserving at-20 ℃ for later use.
Example 4 preparation of gelsemine monoclonal antibody
The gelsemine immunogen is mixed with Freund's complete adjuvant in equal volume, fully emulsified, immunized for 6 weeks old BaLb/c mice, spleen cells of the immunized mice are fused with SP 2/0-Ag14 myeloma cells, hybridoma cell strains capable of stably secreting specific monoclonal antibodies of the gelsemine are obtained through screening, cloning, subcloning and other procedures, and ascites is prepared. Standing ascites for 1h at room temperature, standing at 4deg.C overnight, centrifuging to collect middle clear layer, purifying with saturated ammonium sulfate to obtain gelsemine monoclonal antibody solution, and preserving at-20deg.C.
Example 5 preparation of gelsemine monoclonal antibody-colloidal gold-labeled substance
100mL of a 0.01% chloroauric acid solution was heated to boiling and 2.0mL of a 1% trisodium citrate solution was added with rapid stirring. When the chloroauric acid aqueous solution turns bright red, the heating reaction is continued for 15min, and after natural cooling, the chloroauric acid aqueous solution is preserved in a dark place at 4 ℃ for standby. With magnetic stirring, using 0.1moL/L K 2 CO 3 The pH of the colloidal gold solution was adjusted to 7.5, 80. Mu.g of gelsemine monoclonal antibody was added to each ml of colloidal gold solution, and after mixing, the mixture was reacted at room temperature for 10 minutes, and 10% BSA aqueous solution was added. Vortex shaking, mixing, standing for 10min, centrifuging, washing, re-suspending, and preserving at 4deg.C for use.
Example 6 preparation of microwell reagent
100 mu L of gelsemine monoclonal antibody-colloidal gold marker is added into a microwell plate, and the microwell plate is placed in a freeze dryer for prefreezing for 3 hours and vacuum drying for 20 hours, thus obtaining the microwell reagent.
Example 7 preparation of gelsemine colloidal gold test strip
1. The gelsemine coating raw material is coated on the detection line (T line) of the nitrocellulose membrane by a spot film tester, the goat anti-mouse IgG secondary antibody is coated on the quality control line (C line) of the nitrocellulose membrane, and the mixture is dried in an oven at 37 ℃ for 5 hours.
2. The sample pad, nitrocellulose membrane, and absorbent pad were sequentially laminated with a PVC plate as a backing (fig. 3). Cutting the compounded plastic sheet into strips with the length of 5mm multiplied by 85mm, and placing the strips into a container with a drying agent for sealing and preserving for later use.
Example 8 detection of gelsemine in samples
1. Sample pretreatment: adding 100mL of ultrapure water into 1g of dried fresh gelsemium elegans branches and leaves, refluxing for 1h, recovering the decoction to room temperature, mixing uniformly, and standing for 1min, wherein the supernatant is a liquid to be detected of a gelsemium elegans sample A; adding 1000mL of ultrapure water into 1g of dried fresh gelsemium elegans branches and leaves, refluxing for 1h, recovering the decoction to room temperature, mixing uniformly, and standing for 1min, wherein the supernatant is a liquid to be detected of a gelsemium elegans sample B; the Wanglaoji herbal tea is the liquid C to be detected.
2. And (3) detection: 200 mu L of the liquid to be detected is taken to be placed in the red reagent micropore, pumped for 5 to 10 times, evenly mixed and incubated for 3 minutes at room temperature. The test strips were inserted into the microwells and incubated at room temperature for 6min. The test strip was removed from the microwell, the sample pad at the lower end of the test strip was gently scraped off, and the result was interpreted (FIG. 4), and after 5min, the result was not valid.
3. Interpretation of the results:
4. specificity experiments
The test strip is used for detecting diphenhydramine, chlorpheniramine maleate and other drug standard substance solutions with the mass concentration of 50 mg/kg. The detection results are negative, which shows that the test strip has no cross to the above medicines and has better specificity to gelsemine.
5. Shelf life experiment
Three batches of conventionally produced test strips were used for shelf life experiments and stored at room temperature. A certain number of test strips are randomly selected for each month to detect quality control samples containing 0mg/kg, 0.5mg/kg, 1mg/kg and 2mg/kg of gelsemine, and each group is repeated for 3 times and continuously measured for 18 months. The test result of the test strip in the first 13 months is not abnormal, and in the test of the 14 th to 18 th months, the test strip has false positive results, so that the shelf life is determined to be 12 months.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims (10)

1. The gelsemine hapten is characterized in that the structure of the gelsemine hapten is shown as a formula I:
2. the gelsemine hapten of claim 1, wherein the preparation of the gelsemine hapten comprises the following steps:
uniformly mixing 3-mercaptopropionic acid, benzoin dimethyl ether and methylene dichloride in a test tube, weighing gelsemine in a quartz crucible, and adding the solution into the crucible; under the condition of magnetic stirring, using an ultraviolet lamp to irradiate and react for 30-60 min, evaporating the solvent after the reaction is finished, and purifying by silica gel column chromatography to obtain the gelsemine hapten.
3. The gelsemine monoclonal antibody is characterized in that the gelsemine monoclonal antibody is obtained by coupling a gelsemine monoclonal antibody with colloidal gold, the gelsemine monoclonal antibody is obtained by animal immunization through gelsemine total antigen, the gelsemine total antigen is obtained by coupling a gelsemine hapten and carrier protein according to claim 1, and the carrier protein is selected from one or more of bovine serum albumin, human serum albumin or chicken egg serum albumin.
4. Use of a gelsemine hapten according to claim 1 or 2 or a gelsemine hapten according to claim 3 for the detection of gelsemine.
5. Use of a gelsemine hapten according to claim 1 or 2 or a gelsemine hapten according to claim 3 for the preparation of a gelsemine-specific antibody.
6. Use of a colloidal gold-labeled gelsemine monoclonal antibody or a gelsemine monoclonal antibody according to claim 3 for the detection of gelsemine.
7. The use of a colloidal gold-labeled gelsemine monoclonal antibody or gelsemine monoclonal antibody according to claim 3 in the preparation of a gelsemine immunochromatographic test strip.
8. Use of a gelsemine monoclonal antibody or gelsemine monoclonal antibody labeled by colloidal gold according to claim 3 in the preparation of a gelsemine enzyme-linked immunoassay kit.
9. The gelsemine colloidal gold detection test strip is characterized in that components of the colloidal gold detection test strip comprise a bottom plate, a sample pad, a nitrocellulose membrane, an absorption pad, micropores and a straw, wherein the micropores contain the colloidal gold-labeled gelsemine monoclonal antibody according to claim 3.
10. The gelsemine colloidal gold detection test strip according to claim 9, wherein a detection line and a quality control line are arranged on the nitrocellulose membrane, the detection line is coated with a gelsemine coupling antigen, and the quality control line is coated with a goat anti-mouse IgG secondary antibody.
CN202310533826.3A 2023-05-12 2023-05-12 Gelsemine colloidal gold detection test paper, preparation method and application Pending CN116574107A (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102199156A (en) * 2010-03-26 2011-09-28 上海张江中药现代制剂技术工程研究中心 Preparation of gelsemine from gelsemium extract by using chromatography
CN110256305A (en) * 2019-07-24 2019-09-20 中国药科大学 A kind of naphthalenesulfonamide compound, preparation method and application
CN111057064A (en) * 2020-03-18 2020-04-24 中国农业科学院蜜蜂研究所 14-hydroxyl gelsemine hapten and artificial antigen as well as preparation method and application thereof
CN113025581A (en) * 2021-05-28 2021-06-25 中国农业科学院蜜蜂研究所 Hybridoma cell strain secreting gelsemine monoclonal antibody and application thereof
CN114903956A (en) * 2022-07-19 2022-08-16 广东省计量科学研究院（华南国家计量测试中心） Chinese herbal medicine composition for preventing and treating pig diseases and preparation method and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102199156A (en) * 2010-03-26 2011-09-28 上海张江中药现代制剂技术工程研究中心 Preparation of gelsemine from gelsemium extract by using chromatography
CN110256305A (en) * 2019-07-24 2019-09-20 中国药科大学 A kind of naphthalenesulfonamide compound, preparation method and application
CN111057064A (en) * 2020-03-18 2020-04-24 中国农业科学院蜜蜂研究所 14-hydroxyl gelsemine hapten and artificial antigen as well as preparation method and application thereof
CN113025581A (en) * 2021-05-28 2021-06-25 中国农业科学院蜜蜂研究所 Hybridoma cell strain secreting gelsemine monoclonal antibody and application thereof
CN114903956A (en) * 2022-07-19 2022-08-16 广东省计量科学研究院（华南国家计量测试中心） Chinese herbal medicine composition for preventing and treating pig diseases and preparation method and application thereof

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