WO2022077824A1 - Haptène de picosulfate de sodium, antigène artificiel, anticorps, procédé de préparation de ceux-ci et leurs applications - Google Patents

Haptène de picosulfate de sodium, antigène artificiel, anticorps, procédé de préparation de ceux-ci et leurs applications Download PDF

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WO2022077824A1
WO2022077824A1 PCT/CN2021/077201 CN2021077201W WO2022077824A1 WO 2022077824 A1 WO2022077824 A1 WO 2022077824A1 CN 2021077201 W CN2021077201 W CN 2021077201W WO 2022077824 A1 WO2022077824 A1 WO 2022077824A1
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sodium picosulfate
sodium
antibody
picosulfate
artificial antigen
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PCT/CN2021/077201
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Chinese (zh)
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雷红涛
赖玮
李向梅
李兆栋
沈兴
徐振林
沈玉栋
杨金易
孙远明
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华南农业大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/28—Radicals substituted by singly-bound oxygen or sulphur atoms
    • C07D213/30—Oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/77—Ovalbumin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/795—Porphyrin- or corrin-ring-containing peptides
    • 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
    • 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
    • G01N33/577—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
    • 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
    • G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors

Definitions

  • the invention relates to the technical field of food and/or health care product safety detection, and more particularly, to a sodium picosulfate hapten, artificial antigen, antibody, and a preparation method and application thereof.
  • Sodium Picosulfate is a stimulant laxative, which can treat constipation by stimulating intestinal peristalsis and secretion, and inhibiting the absorption of water in the intestinal lumen. Due to its significant laxative effect, some unscrupulous merchants add sodium picosulfate to enzyme food or health food claiming to have weight loss function in order to achieve weight loss, bodybuilding and other effects. However, long-term consumption of excessive sodium picosulfate It may cause disturbance of the intestinal system or acute damage to the gastric mucosa.
  • the most common detection method for sodium picosulfate in food is mainly high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).
  • HPLC-MS/MS high performance liquid chromatography-tandem mass spectrometry
  • the Chinese Patent Publication No. CN110501438A "A Method for Detecting Sodium Picosulfate in Slimming Tea" discloses a method for detecting sodium picosulfate in slimming tea using triple quadrupole tandem mass spectrometry.
  • this method can accurately quantify sodium picosulfate in the sample, and the results are stable, it has disadvantages such as relatively complex pretreatment, long detection cycle, expensive equipment, and certain professional requirements for operators, making it difficult to achieve rapid on-site purpose of detection. Therefore, there is an urgent need to develop a rapid and simple method for the rapid detection of sodium picosulfate in weight loss health food.
  • immunodetection methods based on antigen-antibody specific molecular recognition have greater advantages in on-site detection, showing rapidity, sensitivity, simplicity, etc. Less demanding.
  • the key to the development of immunoassay methods is to design a suitable sodium picosulfate hapten and prepare an antibody with high sensitivity and specificity. related reports.
  • the primary purpose of the present invention is to overcome the above-mentioned defects and deficiencies existing in the prior art, and to provide two sodium picosulfate haptens.
  • the second object of the present invention is to provide two artificial antigens of sodium picosulfate.
  • the third object of the present invention is to provide a sodium picosulfate antibody.
  • the fourth object of the present invention is to provide an artificial antigen group for immunodetection of sodium picosulfate.
  • the fifth object of the present invention is to provide a sodium picosulfate immunoassay kit.
  • a kind of sodium picosulfate hapten is characterized in that, the structural formula of described sodium picosulfate hapten is as shown in formula (I) or formula (III):
  • the sodium picosulfate hapten of the formula (I) is named 4-((4-(carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenylsodium sulfate using systematic nomenclature;
  • the sodium picosulfate hapten of the formula (III) is named 4-((4-((5-carboxypentyl)oxy)phenyl)(pyridin-2-yl)methyl)benzene using systematic nomenclature Sodium sulfate.
  • the preparation method of the 4-((4-(carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfate (PC1) is as follows: Water dichloromethane is used as solvent, 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate is mixed with triethylamine, chlorosulfonic acid is added, and The reaction was carried out under stirring at room temperature, and 4-((4-(2-ethoxy-2-oxyethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfuric acid was obtained by separation and purification.
  • the molar ratio of ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate to triethylamine is 1:2-5.
  • the molar ratio of the ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate to triethylamine is 1:4.
  • the molar ratio of ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate, triethylamine and chlorosulfonic acid is 1 ⁇ 1.5:2 ⁇ 5:1 ⁇ 2.
  • the mol ratio of described 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate, triethylamine and chlorosulfonic acid is 1: 4:1.5.
  • the molar ratio of the 4-((4-(2-ethoxy-2-oxyethoxy)phenyl)(pyridin-2-yl)methyl)phenylsulfuric acid to methanol is 1 ⁇ 2:1 to 3.
  • the mol ratio of described 4-((4-(2-ethoxy-2-oxyethoxy) phenyl) (pyridin-2-yl) methyl) phenylsulfuric acid and methanol is 1: 1.
  • the preparation of sodium 4-((4-((5-carboxypentyl)oxy)phenyl)(pyridin-2-yl)methyl)phenyl sulfate (PC3)
  • the method is to use dichloromethane as a solvent, mix ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate and triethylamine, add chlorosulfonic acid The acid was reacted under stirring at room temperature for 3 to 5 h, and ethyl 6-(4-(pyridin-2-yl(4-(sulfooxy)phenyl)methyl)phenoxy)hexanoate was obtained by separation and purification.
  • the molar ratio of ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate to triethylamine is 1:2 ⁇ 5 .
  • the molar ratio of ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate to triethylamine is 1:4.
  • the molar ratio of ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate, triethylamine and chlorosulfonic acid is 1 ⁇ 1.5:2 ⁇ 5:1 ⁇ 2.
  • the molar ratio of ethyl 6-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)hexanoate, triethylamine and chlorosulfonic acid is 1 :4:1.5.
  • the molar ratio of the 4-(((4-((6-ethoxy-6-oxyhexyl)oxy)phenyl)(pyridin-2-yl)methyl)phenylsulfuric acid to methanol It is 1 ⁇ 2:1 ⁇ 3.
  • a kind of sodium picosulfate hapten is characterized in that, the structural formula of described sodium picosulfate hapten is as shown in formula (II) or formula (IV):
  • the sodium picosulfate hapten of formula (II) is named 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid by systematic nomenclature.
  • the sodium picosulfate hapten of formula (IV) is named 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid using systematic nomenclature .
  • the preparation method of the 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid is as follows: 2- (4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy) ethyl acetate, dissolved in methanol, and then added 1 mol/L sodium hydroxide in a volume ratio of 1:1 The aqueous solution was stirred for reaction at room temperature, and after the reaction was completed, the pH was adjusted to 6-7 with 1 mol/L hydrochloric acid to obtain 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy ) acetic acid.
  • the molar ratio of ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate to methanol is 1-2:1-3.
  • the molar ratio of ethyl 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetate to sodium hydroxide is 1-1.5:1-2.
  • the preparation method of the 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid is as follows : Fully dissolve 4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenol in DMF, add cesium carbonate and ethyl bromoacetate, and react at 50 ⁇ 60°C for 3 ⁇ 5h After the reaction, the solvent DMF was removed, extracted with water and ethyl acetate, the organic phases were combined, dried with anhydrous sodium sulfate, and the ethyl acetate was removed by rotary evaporation to obtain the intermediate product 2-(4-((4-(benzyloxy) )phenyl)(pyridin-2-yl)methyl)phenoxy)ethyl acetate.
  • the molar ratio of 4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenol, cesium carbonate and ethyl bromoacetate is 1 ⁇ 2:1 ⁇ 1.5 : 1 to 2.
  • the molar ratio of 4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenol, cesium carbonate and ethyl bromoacetate is 1:1.2:1.
  • the molar ratio of ethyl 2-(4-((4-(benzyloxy)phenyl)(pyridin-2-yl)methyl)phenoxy)acetate to sodium hydroxide is 1 ⁇ 2:1-2.
  • the invention also provides a sodium picosulfate artificial antigen PC1-carrier protein or PC3-carrier protein, which is obtained by coupling the carrier protein with the sodium picosulfate hapten PC1 or PC3;
  • the preparation method of the artificial antigen PC1-carrier protein of sodium picosulfate specifically comprises the following steps:
  • the mass ratio of PC1, NHS and EDC in step (1) is 1:1.1-2:1-2.1.
  • the mass ratio of PC1, NHS and EDC in step (1) is 1:1.4:1.6.
  • the mass-volume ratio of the carrier protein to the PBS buffer in step (2) is 10 mg: 1 mL.
  • the mass ratio of PC1 in step (1) to the carrier protein in step (2) is 1-2:1-4.
  • the mass ratio of PC1 in step (1) to the carrier protein in step (2) is 1:3.
  • the preparation method of the sodium picosulfate artificial antigen PC3-carrier protein is the same as the preparation method of PC1-carrier protein, that is, PC1 is replaced by PC3 for the preparation of PC3-carrier protein.
  • the invention also provides a sodium picosulfate artificial antigen PC2-carrier protein or PC4-carrier protein, which is obtained by coupling the carrier protein with the sodium picosulfate hapten PC2 or PC4;
  • the preparation method of the artificial antigen PC2-carrier protein of sodium picosulfate specifically comprises the following steps:
  • the mass ratio of PC2 in step (1) to the carrier protein in step (2) is 1-2:1-2.
  • the mass ratio of PC2 in step (1) to the carrier protein in step (2) is 1:1.6.
  • the sodium picosulfate artificial antigen is characterized in that the carrier protein is bovine serum albumin (Bovine serum albumin, BSA), keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), lactoferrin (Lactoferrin, LF) or chicken egg albumin (ovalbumin, OVA) any one or more.
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • lactoferrin lactoferrin
  • OVA chicken egg albumin
  • the preparation method of the sodium picosulfate artificial antigen PC4-carrier protein is the same as the preparation method of PC2-carrier protein, that is, PC2 is replaced by PC4 for the preparation of PC4-carrier protein .
  • the present invention also provides a sodium picosulfate antibody, which is prepared by using the artificial antigen of sodium picosulfate described in PC1-carrier protein, PC2-carrier protein, PC3-carrier protein or PC4-carrier protein;
  • the sodium picosulfate antibody is any one or more of monoclonal antibodies and polyclonal antibodies.
  • the preparation method of the sodium picosulfate polyclonal antibody specifically comprises the following steps:
  • PC1-LF immunogen PC1-conjugated lactoferrin
  • immune adjuvant incomplete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations
  • emulsified uniformly and immunized animals New Zealand white rabbits weighing 2.5 to 3 kg were immunized by subcutaneous injection on the back, subcutaneous in various parts, leg muscle and ear vein. The second immunization was performed after 4 weeks, and the booster immunization was performed every 3 weeks thereafter.
  • blood was collected from the marginal ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, boost the immunization with the ear vein.
  • the present invention also provides any one or more of the sodium picosulfate artificial antigens and/or sodium picosulfate antibodies in food and/or health care products containing sodium picosulfate for rapid immune detection, and/or Application in the preparation of sodium picosulfate immunoassay products.
  • the present invention also provides an artificial antigen group for immunodetection of sodium picosulfate, the artificial antigen group contains PC1-carrier protein or PC3-carrier protein as immunogen and PC2-carrier protein or PC4 as coating original - Carrier protein.
  • the immunogen in the artificial antigen group is the artificial antigen of the sodium picosulfate hapten PC1 coupled lactoferrin (PC1-LF); the coating was originally the sodium picosulfate hapten PC2 coupled chicken egg Artificial antigen for albumin (PC2-OVA).
  • PC1-LF sodium picosulfate hapten PC1 coupled lactoferrin
  • PC2-OVA sodium picosulfate hapten PC2 coupled chicken egg Artificial antigen for albumin
  • the invention also provides the application of the artificial antigen group in the rapid immune detection of sodium picosulfate in food and/or health care products, or in the preparation of sodium picosulfate immune detection products.
  • the present invention also provides a sodium picosulfate immunoassay kit, which is prepared from the above artificial antigen group.
  • the sodium picosulfate immunoassay kit is an enzyme-linked immunosorbent assay kit and/or a colloidal gold rapid detection kit.
  • the sodium picosulfate immunoassay kit is an enzyme-linked immunosorbent assay kit containing:
  • ELISA plate coated with artificial antigen sodium picosulfate standard solution, sodium picosulfate antibody, enzyme conjugate concentrate, enzyme conjugate dilution, substrate color developing solution, stop solution, washing solution;
  • the artificial antigen is the above-mentioned coated original PC2-carrier protein;
  • the enzyme conjugate is the sodium picosulfate antibody labeled with horseradish peroxidase.
  • the sodium picosulfate antibody is a sodium picosulfate polyclonal antibody prepared by using the immunogen PC1-LF.
  • the coating is originally PC2-OVA.
  • the sodium picosulfate immunoassay kit is a colloidal gold rapid detection kit
  • the colloidal gold rapid detection kit contains:
  • the above-mentioned sodium picosulfate antibody labeled with colloidal gold is adsorbed in the binding pad, and invisible detection lines are printed on the cellulose membrane and invisible quality control line, the invisible detection line is printed with artificial antigen solution, and the invisible quality control line is printed with goat anti-rabbit antibody; the artificial antigen is that the artificial antigen is the above-mentioned coated original PC2-carrier protein .
  • the sodium picosulfate antibody is a sodium picosulfate polyclonal antibody prepared by using the immunogen PC1-LF.
  • the coating is originally PC2-OVA.
  • the present invention also provides the application of the sodium picosulfate immunological detection kit in the rapid immunological detection of sodium picosulfate in food and/or health care products.
  • the enzyme-linked immunosorbent assay kit and/or the colloidal gold rapid detection card in the sodium picosulfate immunoassay kit is provided for the rapid immunoassay of sodium picosulfate in food and/or health products applications in .
  • the present invention has the following beneficial effects:
  • the invention prepares the sodium picosulfate haptens PC1, PC2, PC3 and PC4, and uses the haptens PC1, PC2, PC3 and PC4 to couple with the carrier protein to obtain artificial antigens, wherein the hapten PC1 or PC3 is coupled with the carrier protein to obtain immunity
  • the original; PC2 or PC4 is coupled to the carrier protein to obtain the coating original, and the immunogen is used to prepare the sodium picosulfate antibody, which has high sensitivity and high specificity for the recognition of sodium picosulfate, and the half inhibitory concentration is 5ng/mL.
  • the minimum detection limit is 0.10ng/mL
  • the cross-reaction rates to structural analogs are all less than 10%, indicating that the sodium picosulfate antibody has extremely high specificity for sodium picosulfate and can effectively exclude its analogs
  • the interference of sodium picosulfate provides a core reagent for the establishment of an enzyme-linked immunosorbent assay for sodium picosulfate.
  • the present invention utilizes the sodium picosulfate antibody to develop the application of the sodium picosulfate immunodetection kit in the rapid immunological detection of sodium picosulfate in food and/or health care products.
  • the enzyme-linked immune kit and the colloidal gold rapid detection kit developed by the invention can specifically identify the sodium picosulfate, and have high detection sensitivity for the sodium picosulfate.
  • Fig. 1 is the synthetic route of sodium picosulfate immunogen PC1-LF.
  • Figure 2 shows the UV spectra of LF, PC1 and PC1-LF.
  • Figure 3 shows the UV spectra of LF, PC3 and PC3-LF.
  • Fig. 4 is the synthetic route of the original PC2-OVA coated with sodium picosulfate.
  • Figure 5 shows the UV spectra of OVA, PC2 and PC2-OVA.
  • Figure 6 shows the UV spectra of OVA, PC4 and PC4-OVA
  • Figure 7 is a standard inhibition curve of sodium picosulfate antibody to sodium picosulfate.
  • Figure 8 is a schematic side view of a sodium picosulfate colloidal gold immunochromatographic test strip, wherein 1: PVC bottom plate; 2: Sample pad; 3: Binding pad; 4: NC membrane; 5: Detection line (T point); 6 : quality control line (point C); 7: absorbent pad.
  • FIG. 9 is a diagram showing the determination of the detection result of the sodium picosulfate colloidal gold immunochromatographic test strip.
  • the present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • the synthetic method of sodium picosulfate artificial antigen comprises the following steps:
  • LF
  • PBS buffer Na 2 HPO 4 ⁇ 12H 2 O 2.90g, NaCl 8.50g, KCl 0.20g, KH 2 PO 4 0.20g, add distilled water to make up to 1000mL.
  • the best combination of two artificial antigens of sodium picosulfate is artificial antigen 4-((4-(carboxymethoxy)phenyl)(pyridin-2-yl)methyl)phenylsodium sulfate-LF (PC1 -LF) (see Fig. 1 for the synthetic route) and 2-(4-((4-hydroxyphenyl)(pyridin-2-yl)methyl)phenoxy)acetic acid-OVA(PC2-OVA) (see the synthetic route Figure 4) (see Example 4 for details).
  • LF, PC1, and PC1-LF were identified by ultraviolet (200-350 nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of the sodium picosulfate immunogen PC1-LF was similar to that of the carrier.
  • the protein LF is obviously different.
  • PC1 has a characteristic peak at 240nm and 300nm. After the coupling reaction, the absorption peak of PC1-LF is significantly higher than that of LF at 240nm and 300nm. displacement.
  • LF, PC3, and PC3-LF were identified by ultraviolet (200-350 nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of the sodium picosulfate immunogen PC3-LF was similar to that of the carrier.
  • the protein LF is obviously different.
  • PC3 has a characteristic peak at 240nm and 260nm. After the coupling reaction, the absorption peak of PC3-LF is significantly higher than that of LF at 240nm and 260nm. displacement.
  • OVA, PC2, and PC2-OVA were identified by ultraviolet (200-350 nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of the original PC2-OVA coated with sodium picosulfate was similar to that of the original PC2-OVA.
  • the carrier protein OVA is obviously different.
  • PC2 has a characteristic peak at 350nm, and the carrier protein OVA only has a characteristic peak at 280nm. After the coupling reaction, PC2-OVA has an obvious absorption peak at 350nm, and the curve of PC2 can be seen. significant displacement occurred.
  • OVA, PC4, and PC4-OVA were identified by UV (200-350 nm) scanning respectively, and by comparing the highest absorbance values of each substance before and after coupling, it was found that the absorption curve of the original PC4-OVA coated with sodium picosulfate was the same as that of the original PC4-OVA.
  • the carrier protein OVA is obviously different.
  • PC4 has a characteristic peak at 210nm, and carrier protein OVA has characteristic peaks at 240nm and 280nm.
  • PC4-OVA has obvious absorption peaks at 220nm, 240nm and 260nm, and Comparing the curves for PC4 shows a significant shift.
  • the prepared immunogen PC1-LF and immune adjuvant (incomplete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations) were emulsified uniformly in a volume ratio of 1:1, and New Zealand white rabbit.
  • the New Zealand white rabbit weighs 2.5-3 kg, and is injected subcutaneously at multiple points on the neck and back.
  • the second immunization is performed after 4 weeks, and the booster immunization is performed every 3 weeks thereafter.
  • One week after the third booster immunization blood was collected from the marginal ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises, boost the immunization with the ear vein.
  • the test results of the titer inhibition rate of the antiserum obtained by immunizing New Zealand white rabbits are shown in Table 1.
  • the antiserum produced by the rabbits immunized with different sodium picosulfate artificial antigens as immunogens has a certain titer.
  • the antiserum obtained has different degrees of inhibitory effect on the target analyte sodium picosulfate.
  • the antiserum titer of 1:32000 and the inhibition rate of 75.0% shown by the combination of the immunogen and coating original structure number 1 is the best combination. Under this combination, the sodium picosulfate antibody can not only specifically recognize the target analysis Sodium picosulfate, and the antibody sensitivity is good.
  • Both the antiserum titers and inhibition rates were higher than those of the immunogen and coat structure combinations numbered 2, 3, and 4, although the inhibition rates shown by the immunogen and coat structure combinations of numbers 5 and 6 were higher than those of the immunogen combination number 1.
  • the inhibition rate shown by the combination of the original and the original coating structure is similar, but the antiserum titer of these two combinations is much lower than the antiserum titer of the combination of No. 1, so the combination of the immunogen of No. 1 and the original coating structure is the best. best combination.
  • Inhibition rate (OD value of titer-OD value of inhibition)/OD value of inhibition*100%
  • the determination of the sensitivity of sodium picosulfate antibody is expressed by establishing a standard curve of sodium picosulfate antibody (ELISA) and calculating the half-inhibitory concentration as IC50 .
  • the specific steps of standard curve establishment include:
  • Example 3 The sodium picosulfate antibody prepared in Example 3 was diluted to 1:8000 with PBST, and a blank control well (replaced with PBST) was set at the same time;
  • PBST The formula of PBST is: Na 2 HPO 4 ⁇ 12H 2 O 14.50 g, NaCl 42.50 g, KCl 1.00 g, KH 2 PO 4 1.00 g, Tween-20 3.0 mL, and distilled water was added to make up to 5000 mL.
  • 1% isinglass solution preparation such as dissolving 0.01g of isinglass powder in 1mL of PBST, the specific calculation is based on the actual amount.
  • the half-inhibitory concentration is IC 50
  • B represents the steepness of the curve, which is called the slope factor: IC 10 is the detection limit, and IC 20 to IC 80 is the detection range.
  • the standard curve of ELISA was established with sodium picosulfate as the standard, the lowest detection limit was 0.10ng/mL, and the half inhibitory concentration was 5ng/mL. Combining with Fig. 7, it can be seen that the standard curve established with sodium picosulfate as the standard has a typical S-shaped curve, and the detection sensitivity is good.
  • the specificity of the sodium picosulfate antibody was determined by the cross-reaction experiment between sodium picosulfate and its analogs, and the specificity of the antibody was expressed by the cross-reactivity ratio (CR). The smaller the cross-reaction, the better the specificity.
  • the sodium picosulfate antibody prepared in Example 3 was labeled with horseradish peroxidase.
  • An ELISA kit for the detection of sodium picosulfate was constructed, including the following components:
  • Enzyme conjugate horseradish peroxidase-labeled sodium picosulfate antibody.
  • the substrate color developing solution is composed of A solution and B solution, A solution is carbamide peroxide, and B solution is tetramethylbenzidine;
  • the stop solution is 10% sulfuric acid
  • the washing solution is pH 7.4, containing 0.5% ⁇ 1.0% Tween-20, 0.01 ⁇ 0.03 ⁇ sodium azide preservative, 0.1 ⁇ 0.3mol/L phosphate buffer, and the percentage is weight volume percentage;
  • the percent absorbance of the standard or sample is equal to the average of the absorbance values of the standard or sample (double well) divided by the average of the absorbance values of the first standard (0 standard), and then multiplied by 100% to obtain the standard
  • the percent absorbance value of the product or sample Take the percentage absorbance of the standard product as the ordinate and the logarithm of the standard concentration of sodium picosulfate ( ⁇ g/L) as the abscissa to draw the standard curve. Substitute the percent absorbance of the sample into the standard curve, read the concentration corresponding to the sample from the standard curve, and multiply the corresponding dilution factor to obtain the actual concentration of sodium picosulfate in the sample.
  • the half inhibitory concentration (IC 50 ) of the sodium picosulfate antibody is 5 ng/mL, and the minimum detection limit is 0.10 ng/mL; it shows that the sodium picosulfate antibody prepared by the present invention can meet the detection requirements, and is suitable for the detection of sodium picosulfate.
  • Sodium picosulfate has a high-sensitivity identification ability, and the detection sensitivity of sodium picosulfate is high.
  • Enzyme foods including jelly, candy, preserves and beverages
  • three concentrations of 2, 10, and 20 ⁇ g/kg were selected as spiked samples, spiked with three concentrations of 2, 10, and 20 ⁇ g/kg, respectively.
  • Unspiked samples were set and verified to be free of sodium picosulfate.
  • the samples were pretreated according to the sample extraction method in "Determination of Sodium Picosulfate in Food-BJS 201911".
  • recovery rate (detected concentration of sodium picosulfate in spiked sample-detected concentration of sodium picosulfate in unspiked sample)/spiked concentration ⁇ 100%.
  • Embodiment 7 The rapid detection method of sodium picosulfate colloidal gold
  • a colloidal gold suspension with an average diameter of 40 nm was prepared by reducing chloroauric acid with trisodium citrate. Under reflux conditions, heat 100 mL of 0.01% chloroauric acid solution to boiling, keep stirring, and quickly add 1.1 mL of 1% trisodium citrate. When the color of the reaction solution became grape red, heating and stirring were continued for 5 min. After cooling to room temperature, 0.05% sodium azide was added and stored at 4°C.
  • the pH was adjusted to about 8.2 with 0.2 mol of K 2 CO 3 solution, and 30 ⁇ g of the antibody was labeled with 1 mL of colloidal gold solution by the classical NaCl titration method. Then, label according to the optimal labeling amount. After labeling for 1 hour, add 10% BSA under stirring (to make the final BSA concentration 1%), incubate for 1 hour, centrifuge at 10000 rpm at 4°C for 25 minutes, and remove the supernatant. Add 5% BSA solution with the same volume of colloidal gold solution to resuspend, centrifuge at 10000rpm for 25min at 4°C, repeat twice.
  • TB solution containing 3% BSA, 3% sucrose, 0.01 mol/L sodium borate and 0.05% sodium azide
  • TB solution containing 3% BSA, 3% sucrose, 0.01 mol/L sodium borate and 0.05% sodium azide
  • XYZ-3000 three-dimensional film sprayer to spray 4% BSA solution on glass wool at a volume of 8 ⁇ L/cm
  • a drying oven to dry at 42°C for 50 min
  • spray the gold-labeled antibody at a volume of 6 ⁇ L/cm on the glass wool.
  • dried in a drying oven at 42 °C for 50 min and stored under vacuum.
  • the coated antigen with a concentration of 1 mg/mL was sprayed on the lower side of the cellulose membrane at a concentration of 1.2 ⁇ L/cm using the XYZ-3000 three-dimensional spray film instrument as the detection line.
  • Goat anti-rabbit IgG with a concentration of 120 ⁇ g/L was sprayed on the upper side of the cellulose membrane at a concentration of 1.2 ⁇ L/cm using an XYZ-3000 three-dimensional film sprayer, as a control line, with an interval of 8 mm between the two lines.
  • the cellulose film 4 was pasted on the middle part of the backing plate 1 , and the water-absorbing pad 7 was pasted on the upper side of the cellulose film 4 and overlapped with the cellulose film 4 by 1 mm.
  • the gold-labeled conjugate pad 3 is pasted under the cellulose film 4 and overlapped by 1 mm.
  • the sample pad 2 is pasted under the gold-labeled conjugate pad 3 and overlapped by 2mm.
  • the assembled test board was cut into 3.05mm wide test strips with a cutter.
  • Figure 9 shows the results of the rapid detection of the test strips.
  • the specific detection and judgment method is as follows: when the sample solution to be tested is added to the test strip or the test end of the test paper card, the solution to be tested drives the object to be tested and the gold-labeled antibody in the gold-labeled conjugate pad 3 to the cellulose by siphoning The membrane 4 diffuses and eventually penetrates the end of the absorbent pad 7 .
  • the analyte binds to the gold-labeled antibody, which in turn occupies the antigen binding point on the gold-labeled antibody, preventing the gold-labeled antibody from interacting with the invisible detection line 5 on the cellulose membrane 4 ( The combination of the hapten and the carrier protein) makes the invisible detection line 5 show no color or very weak color, which means that the test sample is positive or weakly positive; if there is no sample to be tested in the sample, the gold-labeled antibody is in the process of moving up. , when the invisible detection line 5 shows a clear red line, it means that the test sample is negative.
  • the gold-labeled antibody also binds to the invisible control line 6 (goat anti-rabbit IgG) on the cellulose membrane 4, making the invisible control line 6 appear red.
  • the presence or absence of the color of the invisible control line 6 respectively indicates the validity or invalidity of the test strip.
  • the detection limits of sodium picosulfate colloidal gold immunochromatographic test strips for different samples are shown in Table 4.
  • a series of concentrations of standard drugs were added to the blank solid and liquid samples. After sample pretreatment, the samples were detected with the above-mentioned colloidal gold test strips, and the visual detection limit was determined by qualitative judgment with the naked eye.
  • Solid sample detection limit ( ⁇ g/g) Liquid sample detection limit ( ⁇ g/g) Sodium Picosulfate 0.05 0.2

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Abstract

L'invention concerne un haptène de picosulfate de sodium, un antigène artificiel, un anticorps, un procédé de préparation de ceux-ci et leurs applications. Selon l'invention, des haptènes de picosulfate de sodium PC1, PC2, PC3 et PC4 sont préparés en premier lieu, une protéine porteuse conjuguée à l'haptène est appliquée pour produire l'antigène artificiel et un animal immunisé acquiert un anticorps dirigé contre le picosulfate de sodium. De préférence, un conjugué PC1-lactoferrine sert d'immunogène, l'anticorps acquis par immunisation présente une capacité de reconnaissance hautement sensible et hautement spécifique concernant le picosulfate de sodium, la CI50 étant de 5 ng/ml et les taux de réaction croisée concernant des analogues structuraux étant inférieurs à 10 %. L'invention concerne également un kit de réactifs de dosage immunoenzymatique de picosulfate de sodium, un kit de réactifs de dosage immunochromatographique à l'or colloïdal et des applications de ceux-ci dans des produits alimentaires et/ou des produits de soins de santé.
PCT/CN2021/077201 2020-10-14 2021-02-22 Haptène de picosulfate de sodium, antigène artificiel, anticorps, procédé de préparation de ceux-ci et leurs applications WO2022077824A1 (fr)

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