CN113501781B - Hapten, artificial antigen and antibody for detecting chlorpheniramine maleate, and preparation methods and applications thereof - Google Patents
Hapten, artificial antigen and antibody for detecting chlorpheniramine maleate, and preparation methods and applications thereof Download PDFInfo
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Abstract
The invention discloses hapten, artificial antigen and antibody for detecting chlorpheniramine maleate, and a preparation method and application thereof. The present invention provides two haptens: hapten 1 and hapten 2, wherein the structural formula of hapten 1 is shown as formula (I); the structural formula of hapten 2 is shown as formula (II). The antibody for detecting chlorpheniramine maleate is prepared by using hapten 2, and the artificial antigen 1 for coating is prepared by using hapten 1, so that the antibody has high sensitivity and high specific recognition capability to chlorpheniramine maleate, the minimum detection limit is 0.06ng/mL, the detection limit required by market supervision is met, and the core raw material is provided for establishing an immune detection method for specifically detecting chlorpheniramine maleate, thereby having good application prospect.
Description
Technical Field
The invention belongs to the technical field of food safety detection, and particularly relates to hapten, artificial antigen and antibody for detecting chlorpheniramine maleate, and a preparation method and application thereof.
Background
Chlorpheniramine maleate (Chlorphenamine maleate), also known as chlorphenamine, is chemically known as N, N-dimethyl-7- (4-chlorophenyl) -2-pyridinylpropylamine maleate, is a histamine Hl receptor antagonist, can resist telangiectasia caused by anaphylaxis, has obvious central inhibition effect, can increase the effects of anesthetics, analgesics, hypnotics and local anesthetics, is clinically used for skin allergy, and is also commonly used for allergic rhinitis, drug and food allergy and the like. However, excessive use of chlorpheniramine maleate may result in palpitations, skin ecchymosis, and bleeding tendencies. In the detection bulletin published by the market supervision bureau, some illegal merchants are driven by benefits, and illegal addition of chlorpheniramine maleate to herbal tea brings potential harm to the body of consumers.
At present, no standard detection method for chlorpheniramine maleate is established in China. In the related literature at home and abroad, the detection technology of chlorpheniramine maleate mainly comprises a spectrum method, a non-aqueous acid-base titration method, a mass spectrometry method, a chromatography method, a combination technology thereof and the like (Li Jianding, wang Pei. The detection method of chlorpheniramine maleate [ J ]. The scientific field, 2015 (17): 176+205.). Although the accuracy of the method is high, the instrument and equipment are expensive, the sample pretreatment is complex and complicated, the detection cost is high, the operation of specialized personnel is needed, and the method does not meet the requirement of on-site instant detection, and Chinese patent No. 101865857A discloses a detection method for detecting chlorpheniramine maleate in non-biological materials by utilizing precipitation, and the method is applicable to on-site rapid detection, but is only used for detecting samples with slightly high chlorpheniramine maleate content in the samples; chinese patent No. 102565042A discloses a method for rapidly screening chlorpheniramine maleate-doped Chinese patent medicine and health food and a rapid semi-quantitative determination method, which can realize rapid screening, but can only realize the concentration of chlorpheniramine maleate in semi-quantitative detection samples, and is difficult to meet the food supervision requirement of on-site rapid detection. Therefore, there is an urgent need to develop a rapid, accurate, efficient and stable method for detecting chlorpheniramine maleate in foods.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of chlorpheniramine maleate detection methods in the prior art, and provides hapten, artificial antigen and antibody for detecting chlorpheniramine maleate, and preparation methods and applications thereof.
The invention aims to provide two hapten, hapten 1 and hapten 2 for detecting chlorpheniramine maleate.
The invention also aims to provide application of the hapten 1 and the hapten 2 in preparation of artificial antigens for detecting chlorpheniramine maleate.
The invention also aims to provide the artificial antigen 1 and the artificial antigen 2 for detecting chlorpheniramine maleate.
The invention also aims to provide application of the hapten 1, the artificial antigen 1, the hapten 2 or the artificial antigen 2 in preparation of an artificial antibody for detecting chlorpheniramine maleate.
The invention also aims to provide an antibody for detecting chlorpheniramine maleate.
It is also an object of the present invention to provide an artificial antigen set for the immunological detection of chlorpheniramine maleate.
The invention also aims to provide a kit for detecting chlorpheniramine maleate.
The invention also aims at providing an immunoassay method for detecting chlorpheniramine maleate.
The above object of the present invention is achieved by the following technical means:
the invention provides a hapten 1 for detecting chlorpheniramine maleate, wherein the structural formula of the hapten 1 is shown as a formula (I):
the hapten 1 adopts a systematic nomenclature as follows: 4- ((4-chlorophenyl) (pyridin-2-yl) methoxy) -4-oxobutanoic acid.
The preparation method of the hapten 1 comprises the following steps:
dissolving (4-chlorophenyl) (pyridine-2-yl) methanol, triethylamine and 4-dimethylaminopyridine in anhydrous pyridine solvent, adding succinic anhydride, stirring for reaction, and separating and purifying to obtain hapten 1.
Preferably, the temperature of the stirring reaction is 80-90 ℃ and the time is 3-5 h.
The application of the hapten 1 in preparing an artificial antigen for detecting chlorpheniramine maleate is also within the protection scope of the invention.
The invention also provides hapten 2 for detecting chlorpheniramine maleate, which has a structural formula shown in formula (II):
the hapten 2 adopts a systematic nomenclature as follows: 4- ((4-chlorophenyl) (pyridin-2-yl) methoxy) methyl) benzoic acid.
The preparation method of the hapten 2 comprises the following steps:
s1, dissolving (4-chlorophenyl) (pyridine-2-yl) methanol and sodium hydride in anhydrous acetonitrile serving as a solvent, stirring for reaction, adding 4-bromomethyl benzoate, continuing stirring for reaction, and separating and purifying;
s2, dissolving the product separated and purified in the step S1 in methanol serving as a solvent, adding sodium hydroxide/alkaline aqueous solution, stirring for reaction, and regulating acid after the reaction is finished to obtain hapten 2.
Preferably, the solution of anhydrous acetonitrile in the step S1 is stirred at normal temperature for 30min, methyl 4-bromomethylbenzoate is added, stirred for 1h, and then heated to 75 ℃ for 2h.
Preferably, the concentration of the aqueous sodium hydroxide solution in the step S2 is 1mol/L.
Preferably, the stirring reaction in the step S2 is performed at room temperature for 3-5 hours.
Preferably, in the step S2, the pH is adjusted to 6-7 by using 1mol/L hydrochloric acid.
The application of the hapten 2 in preparing an artificial antigen for detecting chlorpheniramine maleate is also within the protection scope of the invention.
The invention also provides two artificial antigens for detecting chlorpheniramine maleate, namely an artificial antigen 1 and an artificial antigen 2, wherein the structural formula of the artificial antigen 1 is shown in a formula (III):
the structural formula of the artificial antigen 2 is shown as a formula (IV):
the preparation method of the artificial antigen 1 is characterized in that the hapten 1 (PEO) is coupled with carrier protein by a carbodiimide method.
The preparation method of the artificial antigen 2 is characterized in that the hapten 2 (PB 1) is coupled with carrier protein by an active ester method to obtain the artificial antigen.
Preferably, the carrier protein is any one or more of bovine serum albumin (Bovine serum albumin, BSA), keyhole limpet hemocyanin (Keyhole limpet hemocyanin, KLH), lactoferrin (LF) or chicken Ovalbumin (OVA).
As a specific embodiment of the above method, the method for preparing artificial antigen 1 comprises the following steps:
s1, dissolving carrier protein in PBS buffer solution (0.01 moL/L, pH=7.4) to obtain carrier protein solution;
s2, dissolving hapten 1 (PEO) in N, N-Dimethylformamide (DMF) to obtain hapten PEO solution;
s3, mixing the hapten PEO solution in the step S2 with the carrier protein solution in the step S1, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) dry powder, and stirring for 12 hours at room temperature in a dark place;
s4, dialyzing the reaction solution obtained in the step S3 by using PBS buffer solution to obtain the artificial antigen 1.
Preferably, the dialysis in step S4 is dialysis for 2 days, and the fluid is changed 4 times per day.
Preferably, the molar mass ratio of hapten 1 (PEO) to EDC in step (1) is 1:1.5-3.
More preferably, the molar mass ratio of hapten 1 (PEO) to EDC in step (1) is 1:2.
Preferably, the ratio of the carrier protein to the phosphate buffer in step (2) is 10 mg/1 mL.
Preferably, the molar mass ratio of hapten 1 (PEO) in step (1) to carrier protein in step (2) is 1-2:1-4.
More preferably, the molar mass ratio of hapten 1 (PEO) in step (1) to carrier protein in step (2) is 1:2.
As a specific embodiment of the above method, the method for preparing artificial antigen 2 comprises the following steps:
(1) Dissolving hapten 2 (PB 1), NHS and EDC in DMF, and stirring for 2-4 hours at room temperature in a dark place to obtain hapten PB1 activating solution;
(2) The carrier protein was added to PBS buffer (0.01 moL/L, ph=7.4);
(3) Slowly and dropwise adding hapten PB1 activating solution into the carrier protein solution in the step (2), and reacting for 12 hours at 4 ℃;
(4) And (3) dialyzing the reaction solution obtained in the step (3) by using PBS buffer solution to obtain the artificial antigen 2.
Preferably, the molar mass ratio of hapten 2, NHS and EDC in step (1) is 1:1.1-2:1-2.1.
More preferably, the molar mass ratio of hapten 2, NHS to EDC in step (1) is 1:1.4:1.6.
Preferably, the ratio of the carrier protein to the phosphate buffer in step (2) is 10 mg/1 mL.
Preferably, the molar mass ratio of hapten 2 in step (1) to carrier protein in step (2) is 1-2:1-4.
More preferably, the molar mass ratio of hapten 2 (PB 1) in step (1) to carrier protein in step (2) is 1:2.
An artificial antigen group for detecting chlorpheniramine maleate, which takes carrier protein as artificial antigen 1 (PEO-KLH) or artificial antigen 2 (PB 1-KLH) of keyhole limpet hemocyanin or artificial antigen 1 (PEO-LF) of carrier protein as lactoferrin as immunogen and takes carrier protein as artificial antigen 1 (PEO-BSA or PEO-LF) of bovine serum albumin or lactoferrin as coating antigen.
Preferably, the artificial antigen group uses an artificial antigen 2 (PB 1-KLH) of which the carrier protein is keyhole limpet hemocyanin as an immunogen and uses an artificial antigen 1 (PEO-BSA) of which the carrier protein is bovine serum albumin as a coating antigen.
The application of the artificial antigen group in detecting chlorpheniramine maleate is also within the protection scope of the invention.
Preferably, the use of said artificial antigen group for detecting chlorpheniramine maleate in food products.
A specific antibody for detecting chlorpheniramine maleate, which is prepared by immunizing an animal with artificial antigen 1 or artificial antigen 2.
Preferably, the antibodies are prepared from an artificial antigen 2 immunized animal.
Preferably, the specific antibody is one or two of monoclonal antibody and polyclonal antibody.
The application of the antibody in detecting chlorpheniramine maleate is also within the protection scope of the invention.
Preferably, the antibody is used for detecting chlorpheniramine maleate in food.
A kit for detecting chlorpheniramine maleate comprises an artificial antigen 1 (PEO-KLH) or an artificial antigen 2 (PB 1-KLH) taking carrier protein as key hole hemocyanin or an artificial antigen 1 (PEO-LF) taking carrier protein as lactoferrin as an immunogen, an artificial antigen group taking carrier protein as bovine serum albumin or lactoferrin (PEO-BSA or PEO-LF) as a coating antigen and a specific antibody prepared by immunizing animals with the artificial antigen 1 (PEO-KLH or PEO-LF) or the artificial antigen 2 (PB 1-KLH).
Preferably, the kit comprises an artificial antigen group taking carrier protein as keyhole limpet hemocyanin (PB 1-KLH) as an immunogen, and taking bovine serum albumin (PEO-BSA) as an artificial antigen 1 of the carrier protein as a coating antigen and a specific antibody prepared by immunizing animals with the artificial antigen 2 (PB 1-KLH).
Further preferably, the kit comprises: the kit comprises an ELISA plate coated with an artificial antigen, chlorpheniramine maleate standard solution, chlorpheniramine maleate antibody, enzyme conjugate concentrated solution, enzyme conjugate diluent, substrate color development solution, stop solution and washing solution; the coating artificial antigen is PEO-carrier protein; the enzyme conjugate is horse radish peroxidase marked chlorpheniramine maleate antibody.
The chlorpheniramine maleate colloidal gold immunochromatography test strip comprises a lining board 1, and a sample pad 2, a gold-labeled conjugate pad 3, a cellulose membrane 4 and a water-absorbing pad 7 which are sequentially arranged on the lining board 1, wherein colloidal gold-labeled artificial antigen 1 (PEO-KLH or PEO-LF) or specific antibodies prepared by immunizing animals with the artificial antigen 2 (PB 1-KLH) are adsorbed in the gold-labeled conjugate pad 3, a invisible detection line 5 and an invisible quality control line 6 are printed on the cellulose membrane 4, the invisible detection line 5 is printed by adopting a coating antigen solution, and the invisible quality control line 6 is printed by adopting goat anti-rabbit antibodies; the coating antigen is artificial antigen 1 (PEO-BSA or PEO-LF) taking carrier protein as bovine serum albumin or lactoferrin.
Preferably, the gold-labeled conjugate pad 3 is internally adsorbed with a specific antibody prepared by immunizing an animal with the colloidal gold-labeled artificial antigen 2 (PB 1-KLH); the coating antigen is artificial antigen 1 (PEO-BSA) taking carrier protein as bovine serum albumin.
An immunoassay method for detecting chlorpheniramine maleate takes carrier protein as artificial antigen 1 (PEO-BSA or PEO-LF) of bovine serum albumin or lactoferrin as a coating antigen, takes carrier protein as artificial antigen 1 (PEO-KLH) or artificial antigen 2 (PB 1-KLH) of keyhole limpet hemocyanin or takes carrier protein as artificial antigen 1 (PEO-LF) of lactoferrin as an antibody for detection, and uses antibodies prepared by immunization of animals.
Preferably, the specific antibody prepared by immunizing an animal with the artificial antigen 1 (PEO-BSA) with the carrier protein as bovine serum albumin as a coating antigen and the artificial antigen 2 (PB 1-KLH) with the carrier protein as keyhole limpet hemocyanin as an immunogen is used as a detection antibody for detection.
Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensor, immune colloidal gold, and the like.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the chlorpheniramine maleate hapten 1 and hapten 2 are prepared, the hapten 1 and hapten 2 are used for coupling carrier proteins to obtain artificial antigens, the hapten 2 is used for preparing an antibody for detecting chlorpheniramine maleate, the hapten 1 is used for preparing an artificial antigen 1 for coating, the antibody has high sensitivity and high specificity recognition capability on chlorpheniramine maleate, the half inhibition concentration is 1.16ng/mL, the minimum detection limit is 0.06ng/mL, the analogue has no cross reaction or the cross reaction rate is less than 0.01%, the interference of the analogue can be effectively eliminated, and a core material is provided for establishing an enzyme-linked immunosorbent assay method of chlorpheniramine maleate. The invention establishes an immune rapid detection method of chlorpheniramine maleate in herbal tea, has the advantages of strong specificity, high sensitivity, short detection time, low cost, low requirements on skills of operators and the like, and is suitable for rapid detection of a large number of samples on site. The key of the method is to design proper chlorpheniramine maleate hapten and artificial antigen and obtain the antibody with high sensitivity and strong specificity. In addition, the invention develops the application of the chlorpheniramine maleate immunoassay kit in the immune rapid detection of chlorpheniramine maleate in food by utilizing the chlorpheniramine maleate antibody. The ELISA kit and the colloidal gold immunochromatography test strip developed by the invention can specifically identify chlorpheniramine maleate and have high detection sensitivity on chlorpheniramine maleate.
Drawings
FIG. 1 shows the synthetic route for chlorpheniramine maleate hapten 1 (PEO).
FIG. 2 shows the synthetic route for chlorpheniramine maleate hapten 2 (PB 1).
FIG. 3 shows the synthetic route of chlorpheniramine maleate artificial antigen PB 1-KLH.
FIG. 4 is a synthetic route for chlorpheniramine maleate artificial antigen PEO-BSA.
FIG. 5 is an ultraviolet spectrum of KLH, PB1 and PB1-KLH of example 4.
FIG. 6 is a UV spectrum of BSA, PEO and PEO-BSA from example 4.
FIG. 7 is a standard inhibition curve of chlorpheniramine maleate antibody against chlorpheniramine maleate.
FIG. 8 is a schematic side view of a chlorpheniramine maleate colloidal gold immunochromatographic strip, wherein 1: a lining plate; 2: a sample pad; 3: a gold label conjugate pad; 4: a cellulose film; 5: invisible detection lines; 6: invisible quality control line; 7: a water absorbing pad.
FIG. 9 is a graph showing the judgment of the detection result of chlorpheniramine maleate colloidal gold immunochromatography test strip.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 Synthesis and identification of chlorpheniramine maleate hapten
Based on the characteristics of chlorpheniramine maleate structure, 2 artificial haptens are designed.
1. Synthesis and identification of hapten PEO (synthetic route see FIG. 1)
Taking (4-chlorophenyl) (pyridine-2-yl) methanol (1 mol), triethylamine (3 mol), 4-dimethylaminopyridine (3 mol), taking anhydrous pyridine as a solvent, reacting with succinic anhydride (1.5 mol) for 3-5 h under stirring at 80-90 ℃, separating and purifying to obtain hapten PEO.
Hapten PEO mass spectrometry results: MS: c (C) 16 H 14 ClNO 4 ：319.06，ESI - [M-H] - :：318.0。
Hapten PEO nuclear magnetic results: 1 H NMR(500MHz,Chloroform-d)δ8.66(dd,J＝4.4,1.7Hz,1H),7.65(td,J＝7.2,1.7Hz,1H),7.57–7.48(m,2H),7.40–7.30(m,4H),7.05(s,1H),2.79–2.68(m,2H),2.68–2.57(m,2H).
the structural formula of hapten PEO is shown as formula (I):
hapten PEO was named by systematic nomenclature: 4- ((4-chlorophenyl) (pyridin-2-yl) methoxy) -4-oxobutanoic acid.
2. Synthesis and identification of hapten PB1 (synthetic route see FIG. 2)
(1) Taking (4-chlorophenyl) (pyridine-2-yl) methanol (1 mol), sodium hydride (1.3 mol), taking anhydrous acetonitrile as a solvent, stirring at normal temperature for reaction for 30min, adding 4-bromomethyl benzoate (1.5 mol), stirring for reaction for 1h, heating to 75 ℃ for continuous reaction for 2h, stopping the reaction, and separating and purifying.
(2) Dissolving the separated and purified product in 5mL of methanol, adding 1mol/L sodium hydroxide aqueous solution, stirring at room temperature for reaction for 3-5 h, and adjusting the pH to 6-7 by using 1mol/L hydrochloric acid after the reaction is finished to obtain the hapten PB1.
Hapten PB1 mass spectrometry results: MS: c (C) 20 H 16 ClNO 3 ：353.08，ESI - [M-H] - ：：352.1。
Hapten PB1 nuclear magnetic resonance results: 1 H NMR(500MHz，Chloroform-d)δ9.63(s，1H)，8.65(dd，J＝4.4，1.7Hz，1H)，7.99-7.93(m，2H)，7.63(td，J＝7.3，1.7Hz，1H)，7.51(ddd，J＝7.1，4.4，1.3Hz，1H)，7.45(dd，J＝7.4，1.4Hz，1H)，7.42-7.30(m，6H)，5.98(s，1H)，4.71(t，J＝1.0Hz，2H).
the structural formula of the hapten PB1 is shown as a formula (II):
hapten PB1 was named by systematic nomenclature: 4- ((4-chlorophenyl) (pyridin-2-yl) methoxy) methyl) benzoic acid.
EXAMPLE 2 Synthesis of chlorpheniramine maleate artificial antigen 1
1. Synthesis of chlorpheniramine maleate artificial antigen 1
The synthesis method of chlorpheniramine maleate artificial antigen 1 comprises the following steps:
hapten PEO was coupled to the carrier protein by the carbodiimide method. 4moL of carrier protein was dissolved in 500. Mu.L of PBS buffer (0.01 moL/L, pH=7.4) to obtain a carrier protein solution; 1moL of hapten PEO is weighed and dissolved in 300 mu L of DMF to obtain PEO solution; mixing PEO solution with carrier protein solution, adding 1.5mol EDC dry powder into the mixed solution, and stirring for 12h at room temperature in dark place; and then dialyzing with PBS buffer solution for 2 days and 4 times per day, and after the dialysis is finished, obtaining chlorpheniramine maleate artificial antigen PEO-carrier protein, subpackaging in a centrifuge tube, and preserving at-20 ℃ for later use.
(2) Coupling hapten PB1 with carrier protein by an active ester method, weighing 1moL of hapten PB1,1.1moL of NHS and 1moL of EDC, dissolving in 50-200 mu L of DMF, and stirring at room temperature for 2-4 hours in a dark place to obtain PB1 activating solution; 4moL of carrier protein is dissolved in 1mL of PBS buffer solution (0.01 moL/L, pH=7.4), PB1 activating solution is slowly and dropwise added at room temperature, and the reaction is carried out for 12 hours at 4 ℃; dialyzing with PBS buffer solution for 2 days and 4 times per day, and packaging into centrifuge tube, and storing at-20deg.C.
Wherein, the formula of PBS buffer solution: na (Na) 2 HPO 4 ·12H 2 O 2.90g，NaCl 8.50g，KCl 0.20g，KH 2 PO 4 0.20g, distilled water was added to a volume of 1000mL.
EXAMPLE 3 Synthesis of chlorpheniramine maleate artificial antigen
1. Synthesis of chlorpheniramine maleate artificial antigen
The synthesis method of chlorpheniramine maleate artificial antigen comprises the following steps:
(1) Hapten PEO was coupled to the carrier protein by the carbodiimide method. 0.5moL of carrier protein was dissolved in 500. Mu.L of PBS buffer (0.01 moL/L, pH=7.4) to obtain a carrier protein solution; 1moL of hapten PEO is weighed and dissolved in 300 mu L of DMF to obtain PEO solution; mixing PEO solution with carrier protein solution, adding 3mol EDC dry powder into the mixed solution, and stirring for 12h at room temperature in dark place; and then dialyzing with PBS buffer solution for 2 days and 4 times per day, and after the dialysis is finished, obtaining chlorpheniramine maleate artificial antigen PEO-carrier protein, subpackaging in a centrifuge tube, and preserving at-20 ℃ for later use.
(2) Coupling hapten PB1 with carrier protein by an active ester method, weighing 1moL of hapten PB1,2moL of NHS and 2.1moL of EDC, dissolving in 50-200 mu L of DMF, and stirring at room temperature for 2-4 hours in a dark place to obtain PB1 activating solution; 0.5moL of carrier protein is dissolved in 1mL of PBS buffer solution (0.01 moL/L, pH=7.4), PB1 activating solution is slowly added dropwise at room temperature, and the reaction is carried out for 12 hours at 4 ℃; dialyzing with PBS buffer solution for 2 days and 4 times per day, and packaging into centrifuge tube, and storing at-20deg.C.
Wherein, the formula of PBS buffer solution: na (Na) 2 HPO 4 ·12H 2 O 2.90g，NaCl 8.50g，KCl 0.20g，KH 2 PO 4 0.20g, distilled water was added to a volume of 1000mL.
EXAMPLE 4 Synthesis and identification of chlorpheniramine maleate artificial antigen
1. Synthesis of chlorpheniramine maleate artificial antigen
The synthesis method of chlorpheniramine maleate artificial antigen comprises the following steps:
(1) Hapten PEO was coupled to the carrier protein by the carbodiimide method. 2moL of carrier protein was dissolved in 500. Mu.L of PBS buffer (0.01 moL/L, pH=7.4) to obtain a carrier protein solution; 1moL of hapten PEO is weighed and dissolved in 300 mu L of DMF to obtain PEO solution; mixing PEO solution with carrier protein solution, adding 2mol EDC dry powder into the mixed solution, and stirring for 12h at room temperature in dark place; and then dialyzing with PBS buffer solution for 2 days and 4 times per day, and after the dialysis is finished, obtaining chlorpheniramine maleate artificial antigen PEO-carrier protein, subpackaging in a centrifuge tube, and preserving at-20 ℃ for later use.
(2) Coupling hapten PB1 with carrier protein by an active ester method, weighing 1moL of hapten PB1,1.4moL of NHS and 1.6moL of EDC, dissolving in 50-200 mu L of DMF, and stirring at room temperature for 2-4 hours in a dark place to obtain PB1 activating solution; 2moL of carrier protein is dissolved in 1mL of PBS buffer (0.01 moL/L, pH=7.4), PB1 activating solution is slowly added dropwise at room temperature, and the reaction is carried out for 12h at 4 ℃; dialyzing with PBS buffer solution for 2 days and 4 times per day, and packaging into centrifuge tube, and storing at-20deg.C.
Wherein, the formula of PBS buffer solution: na (Na) 2 HPO 4 ·12H 2 O 2.90g，NaCl 8.50g，KCl 0.20g，KH 2 PO 4 0.20g, distilled water was added to a volume of 1000mL.
Of these, the best combination of two chlorpheniramine maleate artificial antigens was 4- ((4-chlorophenyl) (pyridin-2-yl) methoxy) methyl) benzoic acid-KLH (PB 1-KLH) (see FIG. 3 for the synthetic route) and 4- ((4-chlorophenyl) (pyridin-2-yl) methoxy) -4-oxobutanoic acid-BSA (PEO-BSA) (see FIG. 4 for the synthetic route) (see example 6 for details).
Specifically, fig. 3 shows that hapten 4- ((4-chlorophenyl) (pyridin-2-yl) methoxy) methyl) benzoic acid is coupled with Keyhole Limpet Hemocyanin (KLH) by an active ester method, and the artificial antigen 4- ((4-chlorophenyl) (pyridin-2-yl) methoxy) methyl) benzoic acid-KLH is synthesized.
FIG. 4 is a chart showing the coupling of hapten 4- ((4-chlorophenyl) (pyridin-2-yl) methoxy) -4-oxobutanoic acid to Bovine Serum Albumin (BSA) by the carbodiimide method to synthesize 4- ((4-chlorophenyl) (pyridin-2-yl) methoxy) -4-oxobutanoic acid-BSA.
2. Identification of chlorpheniramine maleate artificial antigen
(1) The obtained PB1-KLH was subjected to ultraviolet scanning, and the results are shown in FIG. 5.
Specifically, the KLH, PB1 and PB1-KLH are respectively subjected to ultraviolet (200-400 nm) scanning identification, and the absorption curve of the chlorpheniramine maleate immunogen PB1-KLH is found to be obviously different from that of the carrier protein KLH by comparing the highest absorption values of all substances before and after coupling, PB1 has an obvious characteristic absorption value at 280nm, and after coupling reaction, the absorption value of PB1-KLH is increased at 280nm and is obviously higher than that of KLH at 280 nm. Since the small molecule components such as unreacted drugs are removed by dialysis after the coupling, the change of absorbance of the coupled product is contributed by the protein-bound drug molecules, thus indicating that the reaction product is a complex of carrier protein and PB1, and the coupling is successful.
(2) The above-synthesized PEO-BSA was subjected to ultraviolet scanning, and the results are shown in FIG. 6.
Specifically, BSA, PEO, PEO-BSA was identified by ultraviolet (200-400 nm) scanning, and by comparing the highest absorbance values of the substances before and after coupling, it was found that the absorption curve of chlorpheniramine maleate immunogen PEO-BSA was significantly different from that of carrier protein BSA, PEO had a characteristic peak at 230nm and 260nm, and after coupling reaction, the absorbance values of PEO-BSA were not significantly changed at 240nm and 260nm, but the absorbance values were significantly increased at 240nm and also significantly increased at 280nm as seen by comparing the curves of PEO. Since the small molecule components such as unreacted drugs are removed by dialysis after the coupling, the change of absorbance of the coupled product is contributed by the protein-bound drug molecules, thus indicating that the reaction product is a complex of carrier protein and PEO, and the coupling is successful.
EXAMPLE 5 preparation of chlorpheniramine maleate antibody
1. Polyclonal antibody preparation
The artificial antigen PB1-KLH prepared in example 4 was uniformly emulsified with an immunological adjuvant (complete Freund's adjuvant for the first immunization and incomplete Freund's adjuvant for the subsequent booster immunization) in a volume ratio of 1:1 to immunize New Zealand white rabbits. The New Zealand white rabbits have a weight of 2.5-3 kg, are subjected to subcutaneous multipoint injection at the neck and back, are subjected to secondary immunization after 4 weeks, and are subjected to booster immunization every 3 weeks. The ear margin vein was bled 1 week after the third boost and serum titers were determined using an indirect competition ELISA. When the potency no longer increases, the ear margin vein is used to boost the immunity. After one week heart blood was collected and serum was obtained from the collected blood in the following manner: the mixture is subjected to warm bath for 0.5 to 1 hour at 37 ℃, then is kept stand overnight at 4 ℃, and the separated serum is sucked by a rubber head dropper, and is centrifuged at 3000 to 5000rpm for 10 minutes at 4 ℃ to obtain the supernatant. The antiserum is purified by adopting an octanoic acid-ammonium sulfate precipitation method to obtain a polyclonal antibody, and the polyclonal antibody is frozen at the temperature of minus 20 ℃ for standby.
2. Monoclonal antibody preparation
Female Bal b/c mice were immunized with the artificial antigen PB1-KLH prepared in example 4. The mice were immunized by subcutaneous multipoint injection in the abdomen after the artificial antigen PB1-KLH was uniformly emulsified with an equal volume of immunoadjuvant (Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the booster immunization), and after 1 week of each booster immunization, the tail blood was taken to determine the antiserum titer. When the titer is stable and unchanged, selecting the mice with the best immune effect to boost one-time immunity, and taking spleen cells for fusion after 3 days to prepare the monoclonal antibody.
EXAMPLE 6 sensitivity and specificity determination of chlorpheniramine maleate antibodies
1. Chlorpheniramine maleate immunogen and coating antigen combination selection
And detecting the titer and inhibition rate of each combination by adopting an indirect competition ELISA method, and screening the optimal combination according to the result.
TABLE 1 potency and inhibition data for chlorpheniramine maleate 4-group immunogen and coating antigen combinations
The potency and inhibition rate test results of chlorpheniramine maleate 4-group immunogen and coating antigen combinations are shown in Table 1. After screening different combinations of immunogens and coating precursors, the optimal combinations are: PB1-KLH was used as immunogen and PEO-BSA was used as coating antigen. Under the combination, the antibody can specifically recognize target analyte chlorpheniramine maleate, and has better antibody sensitivity.
2. Chlorpheniramine maleate antibody sensitivity assay
Determination of chlorpheniramine maleate antibody sensitivity by establishing a chlorpheniramine maleate antibody (ELISA) standard curve and determining the median inhibitory concentration (IC 50 ) To represent.
The standard curve establishment specific steps comprise:
(1) The chlorpheniramine maleate polyclonal antibody prepared in example 3 was diluted 1:8000 with PBST, while blank wells (replaced with PBST) were provided;
(2) Diluting chlorpheniramine maleate artificial antigen PEO-BSA to 125ng/mL with coating liquid, coating 96-well ELISA plates, adding 100 mu L of each well, incubating for 12h in a constant-temperature water bath at 37 ℃, discarding the coating liquid, washing 2 times with PBST (0.01M PBS,0.06%Tween-20 (v/v)), and drying by beating;
(3) Adding 120 μl of sealing solution (1% fish gelatin protein solution) into each hole, sealing at 37deg.C for 3 hr, discarding sealing solution, clapping the plate, and oven drying at 37deg.C;
(4) Chlorpheniramine maleate was diluted to 200000, 10000, 500, 25,1.25,0.0625,0.003125ng/mL with PBST;
(5) 50 μl of chlorpheniramine maleate diluent was added to each row at concentrations of 200000, 10000, 500, 25,1.25,0.0625,0.003125ng/mL (three groups in parallel), 50 μl/well of PBST diluent was added to each well at a concentration of 0ng/mL, and then 50 μl of chlorpheniramine maleate antibody diluent described in step (1) was added to each well. After incubation at 37℃for 40min, the wells were discarded, washed 5 times with PBST (0.01M PBS,0.06%Tween-20 (v/v)) and patted dry;
(6) Adding goat anti-rabbit secondary anti-IgG-HRP (5000-fold dilution), incubating at 37deg.C for 30min, discarding the liquid in the wells, washing with PBST (0.01M PBS,0.06%Tween-20 (v/v)) for 5 times, and drying;
(7) Adding 100 mu L of color development liquid into each hole, and incubating at 37 ℃ for 10min for color development;
(8) mu.L of stop solution (10% H) was added to each well 2 SO 4 ) Terminating the reaction, and reading the OD value at 450nm by using an enzyme-labeled instrument;
wherein the formula of PBST is as follows: na (Na) 2 HPO 4 ·12H 2 O 14.50g，NaCl 42.50g，KCl 1.00g，KH 2 PO 4 1.00g, tween-20.0 mL, and distilled water was added to a volume of 5000mL.
1% of fish gelatin protein solution: 0.01g of fish gelatin protein powder is dissolved in 1mL of PBST, and the specific dosage is calculated according to actual dosage.
Taking an OD value as an ordinate and a corresponding standard concentration logarithmic value as an abscissa, performing curve fitting on the function by applying four parameters of origin software: y= (a-D)/[ 1+ (X/C) B]+D, wherein A and D represent the minimum and maximum absorbance (OD) of the drug concentration, respectively, C is the midpoint concentration, OD when the standard concentration is equal to C is (A+D)/2, at the inflection point of the curve, half the inhibitory concentration is IC 50 B represents the steepness of the curve, called the slope factor: in standard curve IC 10 The corresponding drug concentration is the detection limit, and IC is used 20 ～IC 80 Is the detection range.
ELISA standard curve is established by taking chlorpheniramine maleate as a standard substance, the minimum detection limit is 0.06ng/mL, and the half inhibition concentration is 1.16ng/mL. As can be seen from fig. 7, the standard curve established by using chlorpheniramine maleate as the standard has a typical S-shaped curve, and the detection sensitivity is good.
3. Chlorpheniramine maleate antibody specificity assay
The specificity of chlorpheniramine maleate antibody is determined by cross-reaction experiments of chlorpheniramine maleate and analogues thereof, and the specificity of the antibody is expressed by the cross-reaction rate (CR), and the smaller the cross-reaction, the better the specificity.
Chlorpheniramine maleate and analogues thereof are used as competitive antigens, serial dilutions are respectively carried out, an indirect competition ELISA method is adopted for measurement, and the steps refer to an experimental method for sensitivity verification, thus obtaining the IC of each analogue 50 Values. The cross-reactivity (CR) of chlorpheniramine maleate and various analogues was calculated using the following formula:
the results of the cross-reaction experiments of chlorpheniramine maleate and its analogues are shown in Table 2.
TABLE 2 Cross-reaction test results of chlorpheniramine maleate and its analogues
Note that: NR indicates no reaction.
As can be seen from Table 2, the cross-reactivity of chlorpheniramine maleate antibody to chlorpheniramine maleate was 100%, IC 50 The value is 1.16 ng-The mL, but the structural analogues have no cross reaction or the cross reaction rate is lower than 0.01%, which indicates that the chlorpheniramine maleate antibody has extremely high specificity to chlorpheniramine maleate, can effectively eliminate the interference of the analogues, and can be specially used for detecting the chlorpheniramine maleate.
The above results illustrate: the chlorpheniramine maleate antibody prepared by the invention has strong detection specificity to chlorpheniramine maleate.
EXAMPLE 7 development of chlorpheniramine maleate ELISA kit
1. Enzyme conjugates
The chlorpheniramine maleate antibody prepared in example 3 was labeled with horseradish peroxidase.
2. Preparation of ELISA plate
PEO-BSA was diluted to 1. Mu.g/mL with coating buffer, 100. Mu.L of each well was added, incubated overnight at 37℃in the absence of light, the wells were decanted, washed 2 times with wash solution for 30s each time, patted dry, then 120. Mu.L of blocking solution was added to each well, incubated at 25℃in the absence of light for 2h, the wells were decanted, patted dry, dried and stored in vacuum with aluminum film.
3. Construction of enzyme linked immunosorbent assay kit for detecting chlorpheniramine maleate
An enzyme-linked immunosorbent assay kit for detecting chlorpheniramine maleate is composed to contain the following components:
(1) Coating an ELISA plate treated by original PEO-BSA;
(2) 6 bottles of chlorpheniramine maleate standard solution with the concentration of 0 mug/L, 1.25 mug/L, 25 mug/L, 500 mug/L, 10000 mug/L and 200000 mug/L respectively.
(3) Enzyme conjugate: horse radish peroxidase labeled chlorpheniramine maleate antibody.
(4) The substrate color development liquid consists of liquid A and liquid B, wherein the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(5) The stop solution is 10% sulfuric acid;
(6) The washing liquid is pH 7.4 and contains 0.5-1.0% Tween-20, 0.01-0.03% sodium azide preservative and 0.1-0.3 mol/L phosphate buffer solution, wherein the percentages are weight volume percentages;
4. actual sample detection
And numbering corresponding micropores of the sample and the standard substance in sequence, making 2 holes of each sample and each standard substance in parallel, and recording the positions of the standard holes and the sample holes. The enzyme conjugate concentrate was diluted with enzyme conjugate diluent in the required amount at a 1:11 volume ratio (i.e., 1 part enzyme conjugate concentrate was added to 11 parts enzyme conjugate diluent, ready to use). Adding 50 mu L of standard substance/sample into corresponding microwells, adding 50 mu L of enzyme conjugate working solution into the microwells, gently shaking and mixing, and placing the microwells in a light-shielding environment at 25 ℃ for reaction for 35min after covering with a cover plate film. And spin-drying the liquid in the holes, adding 300 mu L/hole of washing working solution, washing for 4-5 times, and pouring out the washing solution in the holes at intervals of 10s each time, and drying by a piece of absorbent paper (the bubbles which are not removed after drying can be pricked by an unused gun head). Adding 50 mu L/hole of substrate solution A, adding 50 mu L/hole of substrate solution B, gently shaking and mixing, and placing the mixture in a light-shielding environment at 25 ℃ for reaction for 10min after covering with a cover plate film. Adding 50 mu L of stop solution per hole, gently shaking and mixing, setting an enzyme label at 450nm, and measuring the OD value of each hole.
5. Analysis of detection results
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 multiplied by 100 to give the percent absorbance value of the standard or sample. And drawing a standard curve graph by taking the percentage absorbance of the standard substance as an ordinate and taking the logarithm of the concentration (mug/L) of the chlorpheniramine maleate standard substance as an abscissa. Substituting the percentage absorbance of the sample into a standard curve, reading the concentration corresponding to the sample from the standard curve, and multiplying the concentration by the dilution multiple corresponding to the standard curve to obtain the actual concentration of chlorpheniramine maleate in the sample.
As can be seen from FIG. 7, the half Inhibitory Concentration (IC) of chlorpheniramine maleate antibody 50 ) 1.16ng/mL, and the lowest detection limit is 0.06ng/mL; the chlorpheniramine maleate antibody prepared by the method can meet the detection requirement, has high-sensitivity identification capability on chlorpheniramine maleate, and has high detection sensitivity on chlorpheniramine maleate.
6. Additive recovery experiments
The commercial bulk herbal tea is selected as a standard sample, 4 types of cold tea, cold throat tea, pharyngitis tea and heat-clearing and detoxifying tea are adopted, 3 concentrations of 0.05, 0.10 and 0.20mg/L are respectively used for standard adding, an unlabeled sample is arranged, and the sample is verified to contain no chlorpheniramine maleate. The sample extraction method comprises the following steps: 10g (accurate to 0.001 g) of the sample was taken in a 100mL dry beaker, 40g of ultra-pure water was accurately added, shaking was performed, and then the sample was allowed to stand for 10 minutes, and the supernatant was taken for use.
The recovery rate is calculated according to the following formula: recovery = (labeled sample chlorpheniramine maleate detected concentration-unlabeled sample chlorpheniramine maleate detected concentration)/labeled concentration x 100%.
The results of the addition recovery experiment are shown in table 3.
TABLE 3 sample addition recovery test results
The results show that the addition recovery rates of 4 samples of cold tea, cold throat tea, pharyngitis tea and heat-clearing and detoxicating tea are all between 80% and 120%, the variation coefficients are all less than 10%, and the kit has high precision and good stability, and can meet the detection requirement of chlorpheniramine maleate.
EXAMPLE 8 development of chlorpheniramine maleate colloidal gold immunochromatography test strip
1. Preparation of gold-labeled antibodies and gold-labeled conjugate pads
The colloid Jin Xuanfu liquid with the average diameter of 40nm is prepared by adopting a method of reducing chloroauric acid by trisodium citrate.
The colloidal gold is firstly treated with 0.2mol of K 2 CO 3 The pH of the solution was adjusted to 8.5, then the amount of antibody labeling was determined by classical NaCl titration, and finally 20. Mu.g of chlorpheniramine maleate antibody prepared in example 3 and 1mL of colloidal gold solution were selected for labeling to obtain gold-labeled antibody, which was stored at 4 ℃. Spraying 4% BSA solution on glass wool at an amount of 8 μL/cm with XYZ-3000 three-dimensional film spraying instrument, drying at 42deg.C for 50min, and adding gold-labeled antibodySpraying 6 mu L/cm on glass wool, drying at 42 deg.C for 50min, and vacuum drying for preservation.
2. Conjugated antigen sheep anti-rabbit coated cellulose membrane
Coating antigen with the concentration of 1mg/mL is sprayed on the lower side of the cellulose membrane in the amount of 1.2 mu L/cm by using an XYZ-3000 three-dimensional film spraying instrument to serve as a detection line. Sheep anti-rabbit IgG with a concentration of 120. Mu.g/L was sprayed on the upper side of the cellulose membrane in an amount of 1.2. Mu.L/cm using an XYZ-3000 three-dimensional film spraying apparatus as a control line, with the two lines being 8mm apart.
3. Assembling of quick test paper strip
As shown in fig. 8, the cellulose film 4 was stuck to the middle portion of the backing plate 1, and the water absorbing pad 7 was stuck to the upper side of the cellulose film 4 and overlapped with the cellulose film 4 by 1mm. The gold-labeled conjugate pad 3 was adhered under the cellulose membrane 4 to overlap by 1mm. The sample pad 2 is adhered under the gold-labeled conjugate pad 3 to overlap by 2mm. The assembled test paper board was cut into test strips 3.05mm wide with a chopper.
4. Preparation of detection sample solution
1) Preparation of liquid samples 1 to 4
5. Quick detection test strip detection and judgment
After the sample solution to be tested is added into the test paper strip or the test paper card test end, the sample solution to be tested drives the sample to be tested and the gold-labeled antibody in the gold-labeled conjugate pad 3 to diffuse towards the cellulose membrane 4 together through the siphon action, and finally permeates into the end of the water absorption pad 7. In the diffusion process, if an object to be detected exists in a sample, the object to be detected is combined with the gold-labeled antibody, so that an antigen binding point on the gold-labeled antibody is occupied, the combination of the gold-labeled antibody and a invisible detection line 5 (a conjugate of hapten and carrier protein) on a cellulose membrane 4 is prevented, and the invisible detection line 5 is not developed or developed very weakly, namely, positive or weak positive of the detection sample is indicated; if the sample to be detected is not contained in the sample, the gold-labeled antibody shows a clear red line when encountering the invisible detection line 5 in the upward moving process, and the detection sample is negative. Similarly, the gold-labeled antibody is also combined with the invisible quality control line 6 (goat anti-rabbit IgG) on the cellulose membrane 4, so that the invisible quality control line 6 is red. The presence or absence of the color of the invisible quality control line 6 indicates the validity or invalidity of the test strip, and the determination result is shown in fig. 9.
6. Determination of detection Limit
Adding a series of standard chlorpheniramine maleate medicines with concentration into the liquid samples 1-4, pretreating the samples, detecting the samples by using the colloidal gold test strip, and determining the visual detection limit by naked eye qualitative judgment. The specific results are shown in Table 4.
TABLE 4 detection results of chlorpheniramine maleate test strip in sample
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. Two hapten for detecting chlorpheniramine maleate, which is characterized by comprising hapten 1 and hapten 2, wherein the structural formula of hapten 1 is shown as formula (I):
the structural formula of the hapten 2 is shown as a formula (II):
2. use of the hapten of claim 1 for the preparation of an artificial antigen for the detection of chlorpheniramine maleate.
3. Two artificial antigens for detecting chlorpheniramine maleate are characterized by comprising an artificial antigen 1 and an artificial antigen 2, wherein the structural formula of the artificial antigen 1 is shown as a formula (III):
the structural formula of the artificial antigen 2 is shown as a formula (IV):
the carrier protein is bovine serum albumin or keyhole limpet hemocyanin.
4. The method for preparing the artificial antigen according to claim 3, wherein the artificial antigen 1 is obtained by coupling a carrier protein from the hapten 1 by a carbodiimide method; the artificial antigen 2 is obtained by coupling the hapten 2 with carrier protein through an active ester method.
5. An artificial antigen group for detecting chlorpheniramine maleate, characterized in that the artificial antigen 1 or the artificial antigen 2 taking the carrier protein as keyhole limpet hemocyanin as the immunogen in claim 3; an artificial antigen 1 using the carrier protein of claim 3 as bovine serum albumin as a coating antigen.
6. The chlorpheniramine maleate colloidal gold immunochromatography test strip is characterized by comprising a lining plate (1) and a sample pad (2), a gold-labeled conjugate pad (3), a cellulose membrane (4) and a water absorption pad (7) which are sequentially arranged on the lining plate (1), wherein specific antibodies prepared by immunizing animals with an artificial antigen 1 or an artificial antigen 2, which are marked by colloidal gold and take carrier proteins as key holes in claim 3, are adsorbed in the gold-labeled conjugate pad (3), a invisible detection line (5) and an invisible quality control line (6) are printed on the cellulose membrane (4), the invisible detection line (5) is printed by adopting a coating antigen solution, and the invisible quality control line (6) is printed by adopting goat anti-rabbit antibodies; the coating antigen is an artificial antigen 1 taking the carrier protein as bovine serum albumin in claim 3.
7. An immunoassay method for detecting chlorpheniramine maleate for non-diagnostic purposes is characterized in that an antibody prepared by immunizing an animal with an artificial antigen 1 with carrier protein as bovine serum albumin in claim 3 as a coating antigen and an antibody prepared by immunizing an animal with the artificial antigen 1 or the artificial antigen 2 with carrier protein as keyhole limpet hemocyanin in claim 3 as an immunogen is used as a detection antibody for detection.
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