阅读说明：本技术 一种氨氯地平半抗原、人工抗原、抗体及其制备方法和应用 (Amlodipine hapten, artificial antigen, antibody and preparation method and application thereof ) 是由 雷红涛 华启诚 李向梅 沈兴 李兆栋 韦晓群 王锦 林建浩 沈玉栋 孙远明 于 2020-09-25 设计创作，主要内容包括：本发明公开了一种氨氯地平半抗原、人工抗原、抗体及其制备方法和应用,本发明先制备得到了氨氯地平半抗原AN-SSA,应用该半抗原得到了氨氯地平人工完全抗原和氨氯地平多克隆抗体,该抗体对氨氯地平具有高灵敏度和高特异性的识别能力,半抑制浓度为0.31μg/kg,定量检测范围为0.04758～2.078μg/kg,检测限为0.085.μg/kg,对非洛地平、尼群地平的交叉反应率均低于1％。另外,本发明利用氨氯地平抗体建立了胶体金免疫层析技术,具有较宽的检测范围和较高的灵敏度,能够特异性识别氨氯地平的检测,可视检测线为20ng/mL,在保健食品中氨氯地平的免疫快速检测中具有较大应用前景。(The invention discloses AN amlodipine hapten, AN artificial antigen, AN antibody, a preparation method and AN application thereof, wherein the amlodipine hapten AN-SSA is prepared firstly, the hapten is used to obtain AN amlodipine artificial complete antigen and AN amlodipine polyclonal antibody, the antibody has high sensitivity and high specificity recognition capability on amlodipine, the half inhibition concentration is 0.31 mu g/kg, the quantitative detection range is 0.04758-2.078 mu g/kg, the detection limit is 0.085 mu g/kg, and the cross reaction rate on felodipine and nitrendipine is lower than 1%. In addition, the invention establishes a colloidal gold immunochromatographic assay technology by using the amlodipine antibody, has a wider detection range and higher sensitivity, can specifically identify the detection of amlodipine, has a visual detection line of 20ng/mL, and has a wider application prospect in the rapid immune detection of amlodipine in health foods.)

1. An amlodipine hapten which is characterized in that the structural formula is shown as a formula (I):

2. the method for preparing amlodipine hapten according to claim 1, wherein after amlodipine is dissolved in anhydrous dichloromethane, succinic anhydride and 4-dimethylaminopyridine are added for reaction, and a product is confirmed by a thin layer chromatography silica gel plate; and (3) after the reaction product is dried in a spinning mode, adding ethyl acetate and saturated saline solution for extraction, drying the ester layer by using anhydrous sodium sulfate, and finally concentrating to obtain the amlodipine hapten.

3. An amlodipine complete antigen, which is obtained by coupling a carrier protein to the carboxyl group of the amlodipine hapten according to claim 1.

4. Amlodipine complete antigen according to claim 3, wherein said coupling is by the active ester method.

5. Amlodipine complete antigen according to claim 3, wherein said carrier protein is bovine serum albumin, lactoferrin or chicken ovalbumin.

6. An amlodipine polyclonal antibody, which is characterized in that the amlodipine polyclonal antibody is obtained by emulsifying the amlodipine complete antigen of claim 3 and an immunological adjuvant, immunizing animals, separating serum and purifying.

7. Use of the amlodipine complete antigen as defined in claim 3 or the amlodipine polyclonal antibody as defined in claim 6 in the immunodetection of amlodipine in health food or in the preparation of amlodipine immunodetection products.

8. An enzyme linked immunosorbent assay kit for detecting amlodipine is characterized by comprising: the enzyme label plate coated with a coating antigen, an amlodipine standard solution, an amlodipine monoclonal antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a cleaning solution; the coating antigen is the amlodipine complete antigen of any one of claims 3 to 5; the enzyme conjugate is an amlodipine polyclonal antibody marked by horseradish peroxidase; the amlodipine besylate cloning antibody is the amlodipine besylate cloning antibody according to claim 6.

9. An amlodipine colloidal gold rapid detection card, which is characterized by comprising a bottom plate, and a sample pad, a combination pad, a cellulose membrane and a water absorption pad which are sequentially arranged on the bottom plate, wherein the combination pad is internally adsorbed with a colloidal gold-labeled amlodipine polyclonal antibody according to claim 6, the cellulose membrane is printed with an invisible detection line and an invisible quality control line, the invisible detection line is printed by a coating antigen solution, and the invisible quality control line is printed by a goat anti-rabbit antibody; the coating antigen is the amlodipine complete antigen of any one of claims 3 to 5.

10. Use of the enzyme linked immunosorbent assay kit of claim 8 or the colloidal gold rapid detection card of claim 9 in the rapid immunodetection of amlodipine in health food.

Technical Field

The invention relates to the technical field of food safety detection, in particular to an amlodipine hapten, an artificial antigen and an antibody as well as a preparation method and application thereof.

Background

Amlodipine is currently the most common antihypertensive drug, and is a third-generation dihydropyridine calcium channel blocker. Amlodipine acts by relaxing the smooth muscle of the arterial wall, reducing the total peripheral resistance and thereby lowering blood pressure. Illegal merchants add amlodipine and other medicaments into health-care food in order to increase sales and gain violence so as to achieve good effect of reducing blood pressure to attract consumers, and the method violates food of ChinaSafety lawIt also seriously impairs the interests and physical and mental health of the consumers. 2019The food safety office, the public security department and the market supervision bureau of the state department jointly publish 8 major cases of food health care food treatment fraud and false propaganda, wherein the cases relate to illegal addition of amlodipine medicines in health care food. In view of the above, it is urgent to enhance the detection of illegal drug addition in health food.

Research shows that amlodipine tends to cause a series of side effects such as leg or ankle swelling, physical fatigue, stomachache, nausea, dizziness and the like when being taken excessively. Because the diet is unscientific, the number of people suffering from hypertension in China is rapidly increased, and therefore, the OTC health food market for hypertension patients in China is various. The fish and the dragon are mixed, and western medicines are added into pure food most commonly. The addition of medicines in the health food is prohibited. Amlodipine is an illicit drug in health foods. Therefore, establishing a reliable and rapid detection method for amlodipine has important significance for monitoring illegal addition in health-care food.

Currently, the main detection methods of amlodipine include spectrophotometry, High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), and the like. For example, chinese patent CN111579539A discloses that amlodipine reacts with sodium nitrite under acidic condition to generate diazonium salt, then reacts with N, N-dimethyl-1-naphthylamine to generate strongly fluorescent derivative, and the derivative is directly measured by fluorescence method; chinese patent CN108051534A discloses high performance liquid chromatography-tandem mass spectrometry detection of amlodipine in the health care product; although the methods have reliable results and high accuracy, instruments and equipment are expensive, the pretreatment of samples is complex and tedious, the detection cost is high, professional personnel are required to operate, and the requirements of field detection on low cost, rapidness, accuracy and large-batch detection are not met. Therefore, there is an urgent need to establish a high-sensitivity immunoassay method capable of detecting amlodipine. The key point of establishing the method is to design a proper amlodipine hapten and obtain an antibody with high sensitivity and strong specificity, but related reports about the amlodipine hapten, the artificial antigen and the antibody are not found in the prior art.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide an amlodipine hapten.

The second object of the present invention is to provide an artificial complete antigen of amlodipine.

The third purpose of the invention is to provide an amlodipine polyclonal antibody.

The fourth purpose of the invention is to provide the application of the amlodipine artificial antigen and the polyclonal antibody.

The above object of the present invention is achieved by the following technical solutions:

an amlodipine hapten, the structural formula of which is shown as the formula (I):

the amlodipine hapten is named by adopting a systematic nomenclature method:

(4- ((2- ((4- (2-chlorophenyl) -3- (ethoxycarbonyl) -5- (methoxycarbonyl) -6-methyl-1, 4-dihydropyridin-2-yl) methoxy) ethyl) amino) -4-oxobutanoic acid 4- ((2- ((4- (2-chlorophenyl) -3- (ethoxycarbonyl) -5- (methoxycarbonyl) -6-methyl-1, 4-dihydryl-2-yl) methoxy) ethyl) amino) -4-oxobutanoic acid.

The invention also provides a preparation method of the amlodipine hapten, after amlodipine is dissolved in anhydrous dichloromethane, succinic anhydride and 4-dimethylaminopyridine are added for reaction, and a thin-layer chromatography silica gel plate is used for confirming a product; and (3) after the reaction product is dried in a spinning mode, adding ethyl acetate and saturated saline solution for extraction, drying the ester layer by using anhydrous sodium sulfate, and finally concentrating to obtain the amlodipine hapten.

Preferably, the mass-to-volume ratio of amlodipine to anhydrous dichloromethane is 30-50 mg:1 mL.

More preferably, the mass-to-volume ratio of amlodipine to anhydrous dichloromethane is 45mg:1 mL.

Preferably, the mass ratio of the amlodipine, the succinic anhydride and the 4-dimethylaminopyridine is 4-5: 1.

More preferably, the mass ratio of amlodipine, succinic anhydride and 4-dimethylaminopyridine is 4.5:5: 1.

Preferably, the developing solvent for thin layer chromatography is methanol: ethyl acetate (methanol: ethyl acetate ═ 1: 5).

The invention also provides an amlodipine complete antigen which is obtained by coupling carrier protein on the carboxyl of the amlodipine hapten.

Preferably, the coupling is by an active ester method.

Preferably, the carrier protein is Bovine Serum Albumin (BSA), Lactoferrin (LF), or chicken Ovalbumin (OVA). Wherein the structural formula of the complete antigen coupled with BSA or LF is as follows:

as shown.

The invention also provides an amlodipine polyclonal antibody, which is obtained by emulsifying any amlodipine complete antigen and an immunologic adjuvant, immunizing animals, separating serum and purifying.

Preferably, the animal is immunized by using the artificial antigen obtained by coupling the amlodipine hapten and the lactoferrin as an immunogen.

As a preferable embodiment, the method for preparing the amlodipine polyclonal antibody specifically comprises the following steps:

(1) 2 adult New Zealand white rabbits (female and male) are selected for back immunization, each complete antigen is 500 mu L of hapten-lactoferrin conjugate (AN-SSA-LF) (1mg/mL), and the hapten-lactoferrin conjugate (AN-SSA-LF) is evenly emulsified with Freund complete adjuvant according to the volume ratio of 1:1 during primary immunization to immunize the New Zealand white rabbits. Emulsifying AN-SSA-LF with Freund incomplete adjuvant at a volume ratio of 1:1, and performing boosting immunization every three weeks for 4 times;

(2) after the third immunization, blood is taken from the marginal ear vein of the rabbit at an interval of 7 days, and the supernatant is obtained by centrifugation and is stored at minus 20 ℃ for later use.

The invention also provides the application of the amlodipine complete antigen or the amlodipine polyclonal antibody in the immune rapid detection of amlodipine in health food or in the preparation of amlodipine immune detection products.

The invention also provides an enzyme linked immunosorbent assay kit for detecting amlodipine, which comprises: the enzyme label plate coated with a coating antigen, an amlodipine standard solution, an amlodipine monoclonal antibody, an enzyme conjugate concentrated solution, an enzyme conjugate diluent, a substrate developing solution, a stop solution and a cleaning solution; the coating antigen is any one of the amlodipine complete antigen; the enzyme conjugate is an amlodipine polyclonal antibody marked by horseradish peroxidase; the amlodipine polyclonal antibody is any one of the amlodipine polyclonal antibodies.

The invention also provides an amlodipine colloidal gold rapid detection card, which comprises a bottom plate, and a sample pad, a combination pad, a cellulose membrane and a water absorption pad which are sequentially arranged on the bottom plate, wherein any amlodipine polyclonal antibody marked by colloidal gold is adsorbed in the combination pad, the cellulose membrane is printed with an invisible detection line and an invisible quality control line, the invisible detection line is printed by coating antigen solution, and the invisible quality control line is printed by goat anti-rabbit antibody; the coating antigen is any one of the amlodipine complete antigens.

Preferably, the coating antigen is an artificial antigen obtained by coupling amlodipine hapten and bovine serum albumin.

The invention also provides the application of the enzyme linked immunosorbent assay kit or the colloidal gold rapid detection card in the immune rapid detection of amlodipine in health food.

Compared with the prior art, the invention has the following beneficial effects:

the amlodipine hapten AN-SSA is prepared, the amlodipine artificial complete antigen and the amlodipine polyclonal antibody are obtained by applying the hapten, the antibody has high sensitivity and high specificity recognition capability on amlodipine, the semi-inhibitory concentration is 0.31 mug/kg, the quantitative detection range is 0.04758-2.078 mug/kg, the detection limit is 0.085 mug/kg, the cross reaction rate on felodipine and nitrendipine is lower than 1%, and a core reagent is provided for establishing AN immunochromatography detection method of amlodipine. In addition, the invention establishes a colloidal gold immunochromatographic technique by using the amlodipine antibody, has a wider detection range and higher sensitivity, can specifically identify the detection of amlodipine, and has a visual detection line of 20 ng/mL.

Drawings

FIG. 1 is a flow chart of preparation of amlodipine hapten, artificial antigen, antibody and ELISA.

FIG. 2 is a schematic diagram of the synthesis process of amlodipine hapten AN-SSA according to the present invention.

Fig. 3 is an ultraviolet scanning identification chart of amlodipine complete antigen of the present invention.

Fig. 4 is an indirect competition ELISA standard curve of amlodipine antibody of the present invention.

Fig. 5 is a schematic structural diagram of the test strip for rapidly detecting amlodipine colloidal gold.

Fig. 6 is a schematic diagram showing the results of the test strip for rapidly detecting amlodipine colloidal gold.

Wherein: a is a negative sample detection result, B is a weak positive sample detection result, C is a positive sample detection result, and D and E are test strip invalid results.

FIG. 7 shows the analysis results of the labeled samples of the health food in the amlodipine detection test strip in the embodiment of the present invention. (negative sample N: extract of health food containing no drug).

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

The following examples include the synthesis of amlodipine haptens, amlodipine artificial antigens and the preparation of amlodipine antibodies, the flow chart of which is shown in fig. 1.

Example 1 Synthesis and characterization of amlodipine hapten AN-SSA

1. Test method

450mg of amlodipine is dissolved in 10mL of anhydrous dichloromethane, then 500mg of succinic anhydride and 100mg of 4-dimethylaminopyridine are added to the above solution, heated and stirred at 60 ℃ for 20 hours, the reaction process is monitored by TLC (thin layer chromatography silica gel plate) and finally the product is confirmed, the developing solvent thereof is methanol: ethyl acetate 1: 5. After the reaction product is spin-dried, a proper amount of ethyl acetate and saturated saline solution are added for extraction for three times, 100mg of anhydrous sodium sulfate is added into AN ethyl acetate layer for drying, and finally, amlodipine hapten AN-SSA is obtained after concentration, wherein the synthesis process is shown in figure 2.

2. Identification

Performing nuclear magnetic identification by using an amlodipine standard, purifying a target product, dissolving the purified target product in deuterated methanol, and performing nuclear magnetic hydrogen spectrum identification and mass spectrum identification. The nuclear magnetic resonance hydrogen spectrum result chart of the target product is as follows:

¹H NMR(600 MHz,Chloroform-d)δ7.20(d,J＝7.4 Hz,1H),4.78–4.58(m, 2H),4.34–4.25(m,0H),4.10(d,J＝6.8 Hz,0H),4.02(s,2H),3.55(d,J＝49.2 Hz, 5H),2.66(s,2H),2.46(s,2H),2.32(s,2H),2.03(s,0H),1.77–1.66(m,0H),1.59(s, 0H),1.46–1.12(m,12H),0.90(d,J＝48.8 Hz,2H),0.06(s,19H).

the mass spectrometry results were as follows: MS: C₂₄H_29CLN₂O₈:508.95，ESI^-[M-H]:506.9。

Both results indicate that the derivatization site was correct and successful. Amlodipine hapten (AN-SSA) is successfully synthesized, and the structural formula is shown as follows:

the amlodipine hapten adopts a systematic nomenclature as follows:

(4- ((2- ((4- (2-chlorophenyl) -3- (ethoxycarbonyl) -5- (methoxycarbonyl) -6-methyl-1, 4-dihydropyridin-2-yl) methoxy) ethyl) amino) -4-oxobutanoic acid 4- ((2- ((4- (2-chlorophenyl) -3- (ethoxycarbonyl) -5- (methoxycarbonyl) -6-methyl-1, 4-dihydryl-2-yl) methoxy) ethyl) amino) -4-oxobutanoic acid.

Example 2 Synthesis and identification of amlodipine complete antigen

1. Coupling of amlodipine complete antigen and carrier protein

(1) Sequentially weighing 8mg of amlodipine hapten, 3mg of NHS and 4mg of EDC, dissolving in 200 mu L of DMF, stirring for 4h at room temperature, and fully activating;

(2) 10mg of Bovine Serum Albumin (BSA) was weighed into 2mL of PBS buffer (0.1M, pH 7.4);

(3) slowly dropwise adding the solution obtained in the step (1) into the solution obtained in the step (2), and stirring for 7h:

dialyzing with 0.1M (PH 7.4) buffer solution for two days, changing the solution 4 times every day, and obtaining amlodipine complete antigen (AN-SSA-BSA) after the dialysis is finished, wherein the PBS buffer solution has the formula: na2HPO4 & 12H2O 2.90.90 g, NaCl 8.50g, KCl 0.20g, KH2PO40.20g, and distilled water to reach volume of 1000 mL.

Similarly, Lactoferrin (LF) and chicken Ovalbumin (OVA) are adopted to replace BSA to serve as carrier protein, so that target products AN-SSA-LF and AN-SSA-OVA are obtained, and the process is the same as that for preparing AN-SSA-BSA complete antigen.

2. Identification

Taking an amlodipine complete antigen, carrying out ultraviolet scanning full wavelength, and carrying out ultraviolet scanning identification (200-400 nm) scanning identification on the BSA, LF and OVA complete antigens respectively as shown in figure 3, comparing the maximum absorption wavelengths of the substances before and after coupling, wherein the absorption curve of the amlodipine complete antigen is obviously different from the curve of the carrier protein, the maximum absorption wavelength is shifted, and a characteristic absorption peak of the amlodipine complete antigen appears at 375nm, so that the ultraviolet absorption curve of the amlodipine complete antigen is the accumulated absorption characteristic of the amlodipine complete antigen and indicates that the amlodipine complete antigen is successfully coupled.

Example 3 preparation of amlodipine polyclonal antibody

(1) 2 (female and male) adult healthy New Zealand white rabbits are selected for immunization, the dosage of each rabbit is 500 mu L of complete antigen AN-SSA-LF (1mg/mL), and the AN-SSA-LF is emulsified with Freund complete adjuvant according to the volume ratio of 1:1 during primary immunization to immunize the New Zealand white rabbits. Emulsifying AN-SSA-LF with a Freund incomplete adjuvant according to the volume ratio of 1:1, and performing boosting immunization once every three weeks for four times;

(2) after the third immunization, blood was collected from the ear vein of the rabbit at an interval of 7 days, and the supernatant was centrifuged and stored at-20 ℃ for further use.

Example 4 ELISA detection of polyclonal antibodies to amlodipine

1. ELISA detection

(1) Serum was diluted 1:1000 with PBST while blank control wells were set (no serum added, PBST substituted);

(2) diluting amlodipine artificial antigen AN-SSA-OVA to the concentration of 1 mug/mL by using a coating solution, coating a 96-hole enzyme label plate, adding 100 mug L of the coating solution into each hole, incubating for 12h at 37 ℃, discarding the coating solution, and washing for 2 times by using PBST;

(3) adding 120 μ L of sealing solution (1% fish skin collagen) into each well, sealing at 37 deg.C for 3 hr, discarding the sealing solution, beating, drying at 37 deg.C for 1 hr;

(4) serum was diluted 1000-fold with PBST and amlodipine drug was diluted to 100, 33.33, 11.11, 3.703, 1.23, 0.411, 0.137, 0.045, 0.0152, 0.005, 0.00166 ng/mL;

(5) adding 50 μ L of amlodipine drug diluent (three groups are parallel) in each row, adding 50 μ L of serum diluent/hole, incubating at 37 deg.C for 40min, and washing for 5 times;

(6) adding goat anti-rabbit secondary antibody IgG-HRP (diluted by 5000 times), incubating for 30min at 37 ℃, washing for 5 times, and clapping and drying;

(7) adding color development liquid to incubate for 10min at 37 ℃;

(8) 50 μ L of 10% H was added₂SO₄The reaction was stopped and the OD read at 450 nm;

(9) the drug amlodipine is replaced by felodipine and nitrendipine, the test is carried out according to the same dilution times, and the cross reaction rate of the antibody to other analogues with the structure of the amlodipine is determined.

2. As a result, an indirect competition ELISA standard curve of amlodipine antibody at the semi-Inhibitory Concentration (IC) of amlodipine is shown in FIG. 4₅₀) 0.31ng/mL, linear range of quantitative determination (IC)₂₀～IC₈₀) 0.047-2.07 ng/mL, the lowest detection limit is 0.085ng/mL, and the cross-reaction rates of the felodipine and nitrendipine are both lower than 1%.

Embodiment 5 an amlodipine colloidal gold rapid detection test strip

1. Preparation of gold-labeled antibody and gold-labeled conjugate pad

(1) Preparation of colloidal gold labeled amlodipine polyclonal antibody

Colloidal gold suspension with average diameter of 40nm was prepared by reducing chloroauric acid with trisodium citrate. Under the condition of heating reflux, 200mL of film-coated ultrapure water is heated to boiling and stirred continuously, 8mL of 1% chloroauric acid solution is poured into the reactor rapidly, and 9.25mL of 1% trisodium citrate is added rapidly after the chloroauric acid solution is boiled again. Heating and stirring were continued for 10min when the reaction solution became reddish-red in color. After cooling to room temperature, 0.05% sodium azide was added and stored at 4 ℃.

The colloidal gold is labeled with 0.2mol of K before being labeled with the antibody₂CO₃The solution was adjusted to pH 9.6 and 50. mu.g of antibody-labeled 1mL of colloidal gold solution was determined by classical NaCl titration. Then labeling according to the optimal labeling amount, after labeling for 1 hour, adding 30 μ L10% BSA under stirring, standing at room temperature for 1 hour, sealing, centrifuging at 4 ℃ 10000rpm for 15min, and discarding the supernatant liquid. Adding 5% BSA solution with the same volume of colloidal gold solution for resuspension, centrifuging at 4 deg.C and 10000rpm for 15min, and repeating twice. Finally, the suspension was resuspended in 1/5 volumes of a colloidal gold solution of TB (containing 0.5% BSA, 1% sucrose, 0.5% pvp, 0.01mol/L sodium borate and 0.05% sodium azide), dispersed by sonication, and stored in a freezer at 4 ℃.

(1) Preparation of gold-labeled conjugate pad

Spraying gold-labeled antibody onto glass fiber cotton in an amount of 0.8 μ L/cm by using XYZ-3000 three-dimensional film spraying instrument, drying at 45 deg.C for 120min, and vacuum drying for storage.

2. Coupled antigen goat anti-rabbit coated cellulose membrane

AN amount of 1.0. mu.L/cm of coating antigen (AN-SSA-BSA) at a concentration of 0.1mg/mL was sprayed onto the lower side of the cellulose membrane as a detection line using AN XYZ-3000 three-dimensional spray coater. Goat anti-rabbit IgG with concentration of 0.01mg/mL was sprayed on the upper side of the cellulose membrane in an amount of 0.8. mu.L/cm by using an XYZ-3000 three-dimensional film spraying instrument as a quality control line, and the distance between the two lines was 8 mm.

3. Assembly of quick test paper strip

The cellulose membrane is stuck on the middle part of the lining plate, and the water absorption pad is stuck on the upper side of the cellulose and is overlapped with the cellulose membrane by 2 mm. Gold-labeled conjugate pads were stuck under the cellulose membrane with an overlap of 2 mm. The sample pad was stuck under the gold-labeled conjugate pad with an overlap of 2 mm. The assembled test paper board is cut into test paper strips with the width of 4mm by a cutting machine, and the structural schematic diagram of the assembled test paper strip for rapidly detecting amlodipine and colloidal gold is shown in fig. 5.

After the sample solution to be tested is dripped to the test end of the test strip or 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 diffuse together to the cellulose membrane 4 through the siphon action and finally permeate into the end 7 of the water absorption pad. In the diffusion process, if a substance to be detected exists in the sample, the gold-labeled antibody is firstly combined with the substance to be detected, so that an antigen binding site on the gold-labeled antibody is occupied, the combination of the gold-labeled antibody and the invisible detection line 5 (a combination of a hapten and a carrier protein) on the cellulose membrane 4 is prevented, and the invisible detection line 5 is not colored or is weakly colored, namely, the detection sample is positive or weakly positive; if the sample to be detected does not exist in the sample, a clear red line is displayed when the gold-labeled antibody meets the invisible detection line 5 in the upward moving process, and the detection sample is negative. Similarly, the excess gold-labeled antibody also binds to the invisible quality control line 6 (goat anti-rabbit IgG) on the cellulose membrane 4, so that the invisible control line 6 is red. The presence or absence of the color of the invisible control line 6 indicates the effectiveness or ineffectiveness of the test strip, respectively, as shown in fig. 6.

Example 6 detection of sample by amlodipine colloidal gold fast test strip

1. Detection of health food samples

(1) Preparation of test sample solution

And (4) pretreatment of the health food sample solution. Weighing 5g of capsule sample, removing capsule coat, grinding, crushing, transferring into a 50 mL centrifuge tube, adding 20mL of 80% methanol water, vortex and shaking for 1min, ultrasonically extracting for 3min, centrifuging at 4000rpm for 5min, taking 1mL of supernatant, diluting by 5 times with working buffer solution (PBS), and determining the content of amlodipine in the solution.

(2) Detection and result determination

Inserting the sample end of the colloidal gold test strip into a pretreated sample to be detected, and driving the object to be detected and the gold-labeled antibody in the gold-labeled conjugate pad to diffuse together to the NC membrane through the siphoning action of the solution to be detected, and finally permeating into the water absorption pad. In the diffusion process, if a sample contains a large amount of drugs to be detected, the gold-labeled antibody is firstly combined with the large amount of the drugs to be detected, and the gold-labeled antibody cannot be combined with the coating antigen of the T line, so that the T line is not developed, and the result is positive. When the sample contains a small amount of the drug to be detected, the gold-labeled antibody is firstly combined with the drug to be detected, a small amount of the antibody is combined with the coating antigen, the T line is red, and the result is weak positive. When the sample does not contain the drug to be detected, the T line for combining the gold-labeled antibody and the coating antigen is red, and the result is negative. And judging the detection result in 5-10 minutes of the whole detection.

Adding a series of standard drugs with concentration into blank health product capsule samples, pretreating the samples, detecting the sample solution with the colloidal gold test strip, and determining cut-off value by naked eye qualitative judgment, as shown in FIG. 7. The visual detection line of the amlodipine detection test strip is 20ng/mL, and the experimental result shows that the method has the advantages of high detection speed, intuition, convenient operation and the like.

2. Stability test

The test strip is put into an aluminum platinum bag for vacuum exhaust packaging, and is stored at 37 ℃, 22 ℃ and 4 ℃, all indexes are stable after 3 months, namely, the sensitivity is basically unchanged, the detection color development depth is uniform, and the reaction results are consistent.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.