CN114409604B - Terazosin hydrochloride hapten, artificial antigen and antibody as well as preparation methods and applications thereof - Google Patents

Terazosin hydrochloride hapten, artificial antigen and antibody as well as preparation methods and applications thereof Download PDF

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CN114409604B
CN114409604B CN202111536235.9A CN202111536235A CN114409604B CN 114409604 B CN114409604 B CN 114409604B CN 202111536235 A CN202111536235 A CN 202111536235A CN 114409604 B CN114409604 B CN 114409604B
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terazosin hydrochloride
hapten
antibody
artificial antigen
hydrochloride
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雷红涛
潘康亮
关甜
王锦
李向梅
沈兴
徐振林
沈玉栋
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South China Agricultural University
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Abstract

The invention provides a terazosin hydrochloride hapten, an artificial antigen and an antibody, as well as preparation methods and applications thereof, the invention prepares two haptens, hapten T-2C and hapten T-SG, the hapten T-2C is used for obtaining artificial antigen T-2C, and further prepares a specific antibody for detecting the terazosin hydrochloride, the antibody has good sensitivity and specificity to the terazosin hydrochloride by using the artificial antigen T-SG-OVA as a coating antigen, the minimum detection limit is 0.38ng/mL, the half inhibition concentration is 5.72ng/mL, and an immunoassay method of the terazosin hydrochloride is established by using the antibody, thereby realizing rapid and accurate detection of the terazosin hydrochloride.

Description

Terazosin hydrochloride hapten, artificial antigen and antibody as well as preparation methods and applications thereof
Technical Field
The invention relates to the field of food detection, in particular to a terazosin hydrochloride hapten, an artificial antigen and an antibody, as well as a preparation method and application thereof.
Background
Teracontusion hydrochloride (terazosin hydrochloride), the chemical name 1- (4-amino-6, 7-dimethoxy-2-quinazolinyl) -4- [ (tetrahydrofuran-2-formyl) piperazine hydrochloride, which is the congener of prazosin, developed by Abbot corporation in the united states, was developed clinically in 1985 and is a postsynaptic α1-adrenergic receptor blocker. It can dilate blood vessel, reduce peripheral resistance of blood vessel, lower blood pressure, relax bladder neck and prostate capsule, and relieve symptoms of prostatic hyperplasia. Because of its good clinical performance, it was approved by the U.S. FDA for the treatment of hypertension in 1987, and later by the recommendations of the U.S. urology, the clinician treated it as the first drug for the treatment of benign prostatic hypertrophy in 1992. The terazosin hydrochloride is always used for treating mild and moderate hypertension and benign prostatic hypertrophy in China, and is characterized by no influence of food, good oral absorption, high bioavailability in human body, longer duration of drug effect and 2-3 times of that of prazosin in half-aging period, and good clinical curative effect.
The terazosin hydrochloride has quick response, definite curative effect and wide market application as a antihypertensive western medicine, but is easy to produce toxic and side effects when taken inappropriately, and needs to be taken under the guidance of a clinician.
For the detection of terazosin hydrochloride, there have been reported methods such as thin layer chromatography (thin layer chromatography, TLC), ultraviolet spectrophotometry, fluorescence, capillary electrophoresis, high performance liquid chromatography (high performance liquid chromatography, HPLC), and high performance liquid chromatography-tandem mass spectrometry (high performance liquid chromatography-tandem mass spectrometry, HPLC-MS/MS). However, the methods have the characteristics of high detection efficiency, high accuracy, strong anti-interference capability and the like; however, the apparatus and equipment required for detection are expensive, high in cost, complicated in sample pretreatment, and require professional operators, and do not meet the field detection requirements of a large number of samples.
Compared with the existing chromatographic-based method, the antigen-antibody specific molecule recognition-based immunodetection method has the advantages of being more advantageous in field detection, has the characteristics of rapidness, sensitivity, simplicity, convenience and the like, is low in cost, and has lower requirements on skills of operators. The key point of the development of the immunoassay method is to design a proper terazosin hydrochloride hapten to prepare an antibody with high sensitivity and strong specificity, but the related reports about the terazosin hydrochloride hapten, the artificial antigen and the antibody are not found in the center of the prior art.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the terazosin hydrochloride immunodetection method in the prior art, and provide a terazosin hydrochloride hapten, an artificial antigen and an antibody, as well as a preparation method and application thereof.
The invention aims to provide a terazosin hydrochloride hapten.
The invention also aims to provide the application of the terazosin hydrochloride hapten in preparing the terazosin hydrochloride artificial antigen.
The invention also aims to provide the terazosin hydrochloride artificial antigen: artificial antigen T-2C and/or artificial antigen T-SG.
The invention also aims to provide application of the terazosin hydrochloride artificial antigen in preparation of terazosin hydrochloride artificial antibodies.
The invention also aims to provide the terazosin hydrochloride antibody.
The invention also aims at providing an immunoassay method for detecting the terazosin hydrochloride.
The invention also aims to provide a kit for detecting the terazosin hydrochloride.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a terazosin hydrochloride hapten which is hapten T-2C or hapten T-SG, wherein the structural formula of hapten T-2C is shown as a formula (I),
the hapten T-2C is named as follows by adopting a system naming method: (6, 7-dimethoxy-2- (4- (tetrahydrofuran-2-carbonyl) piperazin-1-yl) quinazolin-4-yl) glycine, (6, 7-dimethoxy-2- (4- (tetrahydrofuran-2-carbonyl) piperazin-1-yl) quinazolin-4-yl) glycine;
the structural formula of the hapten T-SG is shown as a formula (II),
the hapten T-SG is named as follows by adopting a system naming method: 4- ((6, 7-dimethoxy-2- (4- (tetrahydrofuran-2-carbonyl) piperazin-1-yl) quinazolin-4-yl) amino) -4-oxobutanoic acid; namely 4- ((6, 7-dimethoxy-2- (4- (tetrahydrofuran-2-carbonyl) piperazin-1-yl) quinazolin-4-yl) a mino) -4-oxoglutarate acid.
The preparation method of hapten T-2C comprises the following steps:
the method comprises the steps of (1) fully reacting terazosin hydrochloride, ethyl bromoacetate, cesium carbonate and sodium iodide with a solvent N, N-Dimethylformamide (DMF); separating and purifying, dissolving the separated and purified reactant in methanol, fully hydrolyzing, and regulating the pH value to 6-7.
Preferably, terazosin hydrochloride, ethyl bromoacetate, cesium carbonate and sodium iodide are stirred with the solvent N, N-Dimethylformamide (DMF) overnight at 70 ℃; separating and purifying the reactant, dissolving the separated and purified reactant in methanol, then stirring the methanol and a sodium hydroxide aqueous solution for 3 to 5 hours at room temperature, and adjusting the pH value to 6 to 7 after the reaction is finished to obtain hapten T-2C.
Preferably, the molar ratio of the terazosin hydrochloride to the ethyl bromoacetate is 1:1-3.
Further preferably, the molar ratio of the terazosin hydrochloride to the ethyl bromoacetate is 1:1.2.
Preferably, the molar ratio of the terazosin hydrochloride to the cesium carbonate is 1-1.5:4-6.
Further preferably, the molar ratio of terazosin hydrochloride to cesium carbonate is 1.5:5.
Preferably, the molar ratio of the terazosin hydrochloride to the sodium iodide is 1-2:0.1-1.
Further preferably, the molar ratio of the terazosin hydrochloride to the sodium iodide is 1:0.2.
The structural formula of the terazosin hydrochloride is as follows:
the preparation method of the hapten T-SG comprises the following steps:
the method comprises the steps of fully reacting terazosin hydrochloride, succinic anhydride, triethylamine and 4-Dimethylaminopyridine (DMAP) with a solvent N, N-Dimethylformamide (DMF), and separating and purifying to obtain hapten T-SG.
Preferably, the molar ratio of the terazosin hydrochloride to the succinic anhydride is 1:1-3.
Further preferably, the molar ratio of the terazosin hydrochloride to the succinic anhydride is 1:1.3.
Preferably, the mol ratio of the terazosin hydrochloride to the triethylamine is 1-1.5:2-4.
Further preferably, the molar ratio of the terazosin hydrochloride to the triethylamine is 1.5:3.
Preferably, the molar ratio of the terazosin hydrochloride to the DMAP is 1-2:0.3-1.5.
Further preferably, the molar ratio of the terazosin hydrochloride to the DMAP is 1:0.5.
The application of the hapten T-2C and/or hapten T-SG in preparing the terazosin hydrochloride artificial antigen is also within the protection scope of the invention.
An artificial antigen of terazosin hydrochloride is obtained by coupling hapten T-2C or hapten T-SG with carrier protein, the structural formula of the artificial antigen T-2C obtained by coupling hapten T-2C with carrier protein is shown as formula (III), wherein P is carrier protein,
the structural formula of the artificial antigen T-SG obtained by coupling the hapten T-SG with carrier protein is shown as a formula (IV), wherein P is the carrier protein,
preferably, the carrier protein (P) 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).
According to the preparation method of the artificial antigen T-2C or the artificial antigen T-SG, the hapten T-2C or the hapten T-SG is utilized to couple the carrier protein through an active ester method.
As a specific embodiment of the method, the preparation method of the artificial antigen T-2C comprises the following steps:
(1) Hapten T-2C and N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) are dissolved in N, N-Dimethylformamide (DMF), and are stirred for 2-4 hours at room temperature under the condition of avoiding light, so as to obtain hapten T-2C activating solution;
(2) Adding a carrier protein to the PBS buffer;
(3) Slowly and dropwise adding the hapten T-2C activating solution in the step (1) 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 T-2C.
Preferably, the mass ratio of hapten T-2C, NHS to EDC in step (1) is 1:0.5-1.5:1-3.
More preferably, the mass ratio of hapten T-2C, NHS to EDC in step (1) is 1:0.8:1.9.
Preferably, the mass volume ratio of the carrier protein to the PBS buffer in the step (2) is 7-8 mg:1-2 mL.
More preferably, the ratio of the carrier protein to PBS buffer in step (2) is 8 mg/1 mL.
Preferably, the mass ratio of hapten T-2C in step (1) to carrier protein in step (2) is 1-2:1-4.
More preferably, the mass ratio of hapten T-2C in step (1) to carrier protein in step (2) is 2:3.
The preparation method of the artificial antigen T-SG is the same as that of the artificial antigen T-2C.
The application of the terazosin hydrochloride artificial antigen in the preparation of the terazosin hydrochloride antibody is also within the protection scope of the invention.
An artificial antigen combination of terazosin hydrochloride comprises an immunogen and a coating antigen, wherein the immunogen is obtained by coupling a carrier protein with a hapten T-2C, namely an artificial antigen T-2C; the coating antigen is the terazosin hydrochloride artificial antigen.
Preferably, the coating antigen is derived from the hapten T-SG coupled carrier protein, i.e., the artificial antigen T-SG.
Further preferably, the immunogen is derived from the hapten T-2C coupled carrier protein Bovine Serum Albumin (BSA), i.e., artificial antigen T-2C-BSA; the coating antigen is obtained from the T-SG coupling carrier protein chicken Ovalbumin (OVA), namely artificial antigen T-SG-OVA.
The application of the artificial antigen combination in the preparation of the terazosin hydrochloride antibody and/or the detection of the terazosin hydrochloride is also within the protection scope of the invention.
The terazosin hydrochloride antibody is prepared by using an artificial antigen T-2C immune animal obtained by coupling hapten T-2C with carrier protein.
Preferably, the terazosin hydrochloride antibody is prepared by using an artificial antigen T-2C-BSA immune animal obtained by coupling a hapten T-2C with a carrier protein Bovine Serum Albumin (BSA).
Preferably, the terazosin hydrochloride antibody is a monoclonal antibody or a polyclonal antibody.
A preparation method of a terazosin hydrochloride polyclonal antibody is characterized in that an artificial antigen T-2C immune experimental animal obtained by coupling hapten T-2C with carrier protein is utilized.
Preferably, the artificial antigen T-2C-BSA immune experimental animal obtained by utilizing the hapten T-2C coupled carrier protein Bovine Serum Albumin (BSA) is prepared.
The application of the terazosin hydrochloride antibody in the detection of the terazosin hydrochloride and/or the preparation of the test kit for detecting the terazosin hydrochloride is also within the protection scope of the invention.
An immunoassay method for detecting terazosin hydrochloride takes the terazosin hydrochloride artificial antigen as an antigen and takes an antibody prepared from an artificial antigen T-2C immunized animal obtained by coupling hapten T-2C with carrier protein as a detection antibody for detection; the immunoassay method is a method of non-diagnostic therapeutic interest.
Preferably, the artificial antigen T-SG obtained by coupling hapten T-SG with carrier protein is used as antigen.
Further preferably, the detection is performed by using an antibody prepared by immunizing an animal with an artificial antigen T-SG-OVA of chicken Ovalbumin (OVA) as an antigen and an artificial antigen T-2C-BSA of Bovine Serum Albumin (BSA) as a carrier protein as an immunogen as a detection antibody.
Such immunoassay methods include, but are not limited to, enzyme immunoassay, immunochromatography, immunosensor, immune colloidal gold, and the like.
A kit for detecting terazosin hydrochloride comprises the terazosin hydrochloride artificial antigen and the terazosin hydrochloride antibody.
Preferably, the kit comprises an artificial antigen T-SG obtained by coupling the hapten T-SG with a carrier protein and an antibody prepared by immunizing an animal with the artificial antigen T-2C of the hapten T-2C coupled with the carrier protein.
Further preferably, the kit comprises an artificial antigen T-SG-OVA obtained by coupling carrier protein chicken Ovalbumin (OVA) with hapten T-SG and an antibody prepared by immunizing animals with artificial antigen T-2C-BSA of Bovine Serum Albumin (BSA) with hapten T-2C.
Preferably, the kit further comprises one or more of an ELISA plate, a terazosin hydrochloride standard, an enzyme conjugate, a chromogenic solution, a stop solution or a washing solution.
Preferably, the kit comprises an ELISA plate coated with the terazosin hydrochloride artificial antigen, a terazosin hydrochloride standard, an enzyme conjugate, a chromogenic solution, a stop solution and a concentrated washing solution.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides two terazosin hydrochloride hapten, hapten T-2C and hapten T-SG, wherein artificial antigen T-2C prepared by coupling hapten T-2C with Bovine Serum Albumin (BSA) is used as immunogen, thus a terazosin hydrochloride antibody is prepared, and artificial antigen T-SG-OVA prepared by coupling hapten T-SG with chicken Ovalbumin (OVA) is used as coating antigen; the obtained antibody has high titer, strong specificity and high affinity, the minimum detection limit LOD of terazosin hydrochloride is 0.38ng/mL, and the half inhibition concentration IC is high 50 5.72ng/mL, the quantitative detection range is 1.04-31.44 ng/mL, the detection sensitivity is high, and the linear range is wide; the antibody of the invention has the characteristics of simplicity, convenience, rapidness, strong specificity, wide linear range and high sensitivity; the terazosin hydrochloride artificial antigen and the antibody can be used for rapidly and accurately detecting the terazosin hydrochloride.
Drawings
FIG. 1 is a synthetic scheme for hapten T-2C of example 1 of the present invention.
FIG. 2 is a synthetic scheme for hapten T-SG of example 1 of the present invention.
FIG. 3 is an ultraviolet scan of hapten T-2C, BSA, T-2C-BSA of example 2 of the present application.
FIG. 4 is an ultraviolet scan of hapten T-SG, OVA, T-SG-OVA of example 2 of the present application.
FIG. 5 is a standard curve of an indirect competition ELISA using the terazosin hydrochloride antibody of example 4 of the present application.
Detailed Description
The invention is further illustrated in the following drawings and 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 terazosin hydrochloride hapten
1. Synthesis and identification of terazosin hydrochloride hapten T-2C
1.1 Synthesis of terazosin hydrochloride hapten T-2C
Terazosin hydrochloride (1 mol), ethyl bromoacetate (1.2 mol), cesium carbonate (4 mol) and sodium iodide (0.2 mol) were stirred with solvent N, N-dimethylformamide (DMF, 5 mL) at 70 ℃ overnight; separating and purifying the reactant, dissolving the separated and purified reactant in 5mL of methanol, then adding 1mol/L sodium hydroxide aqueous solution, stirring for 3-5 h at room temperature, and adjusting the pH to 6-7 by using hydrochloric acid with the concentration of 1mol/L after the reaction is finished, thus obtaining the hapten T-2C. The synthetic route for hapten T-2C is shown in FIG. 1.
1.2 identification of terazosin hydrochloride hapten T-2C
Nuclear magnetic identification of hapten T-2C: 1 H NMR(600MHz,Methanol-d4)δ8.44(s,2H), 7.28(s,1H),6.79(s,1H),3.99(s,2H),3.83(s,3H),3.80-3.78(m,4H),3.37(s,2H),3.22(p,J＝1.7Hz,8H),3.14(s,2H),1.80(s,2H).
the mass spectrum of hapten T-2C resulted in the following: MS: c (C) 21 H 27 N 5 O 6 ：445.20，ESI-[M-H] + ：446.2。
The structural formula of hapten T-2C is shown as formula (I):
hapten T-2C was named by systematic nomenclature: (6, 7-dimethoxy-2- (4- (tetrahydrofuran-2-carbonyl) piperazin-1-yl) quinazolin-4-yl) glycine, i.e., (6, 7-dimethoxy-2- (4- (tetrahydrofuran-2-carbonyl) piperazin-1-yl) quinazolin-4-yl) glycine.
2. Synthesis and identification of terazosin hydrochloride hapten T-SG
2.1 Synthesis of terazosin hydrochloride hapten T-SG
Terazosin hydrochloride (1 mol), succinic anhydride (1.3 mol), triethylamine (1.5 mol) and 4-Dimethylaminopyridine (DMAP) (0.5 mol) are reacted with solvent N, N-dimethylformamide (DMF, 5 ml), and hapten T-SG is obtained after separation and purification. The synthetic route pattern of hapten T-SG is shown in FIG. 2.
2.2 identification of terazosin hydrochloride hapten T-SG
Nuclear magnetic identification result of hapten T-SG: 1 H NMR(600MHz,Methanol-d4)δ10.44(s, 1H),7.45(s,2H),3.95(s,3H),3.86(s,3H),3.80-3.78(m,4H),3.37(s,2H),3.22(p,8H),3.14(s,2H),2.55(d,2H)1.80(s,2H).
the mass spectrum of hapten T-SG results were: MS: c (C) 23 H 29 N 5 O 7 ：487.21，ESI-[M-H] + ：488.2。
The structural formula of hapten T-SG is shown as formula (II):
hapten T-SG is named by systematic nomenclature: 4- ((6, 7-dimethoxy-2- (4- (tetrahydrofuran-2-carbonyl) piperazin-1-yl) quinazolin-4-yl) amino) -4-oxobutanoic acid; i.e.
4-((6,7-dimethoxy-2-(4-(tetrahydrofuran-2-carbonyl)piperazin-1-yl)quinazolin-4-yl)a mino)-4-oxobutanoic acid。
EXAMPLE 2 Synthesis and identification of terazosin hydrochloride artificial antigen
1. Synthesis of terazosin hydrochloride artificial antigen
The hapten T-2C and hapten T-SG prepared in example 1 were coupled to Bovine Serum Albumin (BSA) and chicken Ovalbumin (OVA) by an active ester method.
1moL of hapten T-2C prepared in example 1 is weighed, dissolved in 50-200 mu L N and N-Dimethylformamide (DMF) with 1.4moL of N-hydroxysuccinimide (NHS) and 1.6moL of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and stirred at room temperature for 2-4 hours in a dark place to obtain hapten T-2C activating solution; 10mg BSA was added to 1mL PBS buffer (0.01 moL/L, pH=7.4); slowly and dropwise adding hapten T-2C activating solution into BSA buffer solution, and reacting at 4 ℃ for 12 hours; dialyzing with PBS buffer solution for 3 days and 3 times per day, and after the dialysis is finished, obtaining the terazosin hydrochloride artificial antigen T-2C, subpackaging in a centrifuge tube, and preserving at-20deg.C for use.
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.
The artificial antigen T-SG is prepared as artificial antigen T-2C, except that the carrier protein is chicken Ovalbumin (OVA).
2. Identification of terazosin hydrochloride artificial antigen
Ultraviolet scanning was performed on BSA, hapten T-2C and synthesized T-2C-BSA as described above. The ultraviolet scan results are shown in fig. 3.
The BSA, the T-2C and the T-2C-BSA are respectively subjected to ultraviolet (200-350 nm) scanning identification, and the absorption curve of the T-2C-BSA is obviously different from that of the carrier protein BSA by comparing the highest absorbance values of all substances before and after coupling, the T-2C has a characteristic peak at 240nm and 260nm respectively, and after the BSA is coupled, the absorption peak of the T-2C-BSA is obviously higher than that of the BSA at 240nm and 280nm, and the absorption curve of the T-2C is obviously shifted relative to that of hapten. Since the small molecule components such as unreacted drugs are removed by dialysis after the coupling, the drug characteristic peak of the coupled product is contributed by the protein-bound drug molecules, so that the reaction product is a complex of carrier protein BSA and T-2C, and the successful coupling of the T-2C-BSA is shown.
Ultraviolet scanning is carried out on OVA, hapten T-SG and synthesized T-SG-OVA. The ultraviolet scan results are shown in fig. 4.
The OVA, hapten T-SG and T-SG-OVA are respectively subjected to ultraviolet (200-350 nm) scanning identification, and the absorption curve of the T-SG-OVA is found to be obviously different from that of carrier protein OVA by comparing the highest light absorption values of all substances before and after coupling, the hapten T-SG has a characteristic peak at 245nm, and after coupling of the OVA, the absorption peak of the T-SG-OVA is obviously higher than that of the OVA at 240nm and 260nm, and the absorption curve of the T-SG-OVA is obviously shifted relative to that of the hapten T-SG. The small molecular components such as unreacted medicines and the like are completely removed by dialysis in the dialysis process of the coupling reaction, so that the characteristic peak of the medicines appearing in the coupling product is contributed by the medicine molecules combined by proteins, and the reaction product is a complex of carrier protein OVA and hapten T-SG, and the success of the coupling of the T-SG-OVA is indicated.
Example 3 preparation of antibodies
1. Preparation of polyclonal antibodies
T-2C-BSA prepared in example 2 was used as an immunogen and an immunological adjuvant (incomplete Freund's adjuvant for the first immunization and Freund's incomplete adjuvant for the subsequent booster immunization) were uniformly emulsified in a volume ratio of 1:1, and the body weight of the immunized New Zealand white rabbit was 2.5-3 kg. The immunization was performed a second time after 4 weeks, followed by a 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 at 4 ℃ for overnight, and the separated serum is sucked by a suction pipe, and is centrifuged at 3000 to 5000rpm for 10 minutes at 4 ℃ to obtain the supernatant. The antiserum is purified to polyclonal antibody by ammonium sulfate precipitation method, and frozen at-20deg.C for use.
2. The monoclonal antibody is prepared by the following specific steps:
female Bal b/C mice were immunized with T-2C-BSA prepared in example 2. The method comprises the steps of uniformly emulsifying artificial antigen T-2C-BSA and an equal volume of immunological adjuvant (complete Freund's adjuvant is used for primary immunization and incomplete Freund's adjuvant is used for boosting), immunizing mice by adopting an abdomen subcutaneous multipoint injection method, and measuring the titer of antisera by tail blood after 1 week of boosting. When the titer is stable and unchanged, selecting the mice with the best immune effect to strengthen one-time immunity, and taking spleen cells for fusion after 3 days to prepare the monoclonal antibody.
Example 4 optimization of the combination of the terazosin hydrochloride immunogen and the coating antigen
The invention also prepares artificial antigen T-2C-LF, artificial antigen T-SG-LF and artificial antigen T-2C-OVA which take lactoferrin (OVA) as carrier proteins according to the preparation method of T-2C-BSA in example 2, and the artificial antigen T-2C-LF, artificial antigen T-SG-LF and artificial antigen T-2C-OVA which take Ovalbumin (OVA) as carrier proteins are coupled successfully.
The prepared T-2C-BSA is used as immunogen, terazosin hydrochloride antibodies prepared by immunizing New Zealand white rabbits according to the method of example 3 are screened as coating sources to prepare T-2C-LF and T-SG-LF and the T-SG-OVA prepared in example 2 are used as coating sources, and the titer and inhibition ratio of antisera obtained by immunizing the New Zealand white rabbits are detected by ELISA.
The specific operation steps are as follows:
(1) The terazosin hydrochloride artificial antigen T-2C-OVA, T-2C-LF, T-SG-LF and T-SG-OVA are respectively diluted to the concentration of 250ng/mL by coating liquid (0.05M carbonate buffer solution, pH 9.6), 96-hole ELISA plates are coated, 100 mu L of the plates are added into each hole, incubated overnight in a 37 ℃ constant-temperature water bath, the coating liquid is discarded, and PBST (0.01M PBS,0.06%Tween-20 (v/v)) is used for washing for 2 times;
(2) Adding 120 μl of sealing solution (1 wt% fish gelatin protein) into each hole, sealing at 37deg.C for 3 hr, discarding sealing solution, clapping the plate, and oven drying at 37deg.C;
(3) The terazosin hydrochloride polyclonal antibodies were diluted 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000 with PBST, while blank wells (replaced with PBST) were set; diluting 1mg/mL of terazosin hydrochloride drug 1000 times with PBST to 1 mug/mL;
the titers are as follows: adding 50 mu L of PBST into each hole, diluting the obtained terazosin hydrochloride antibody by multiple ratio, sequentially adding 50 mu L of the obtained terazosin hydrochloride antibody into the holes, and replacing the last hole with 50 mu L of PBST without adding the antibody;
inhibition column: adding 50 mu L of medicine into each hole, diluting the obtained terazosin hydrochloride antibody by a multiple ratio, sequentially adding 50 mu L of the terazosin hydrochloride antibody into the holes, and replacing the last hole with 50 mu L of PBST without adding the antibody; incubating at 37deg.C for 40min, washing for 5 times, and clapping the plate;
(4) Adding goat anti-rabbit secondary antibody Ig-HRP (5000-fold dilution), incubating for 30min at 37 ℃, washing for 5 times, and clapping;
(5) Adding a color development solution, and incubating at 37 ℃ for color development for 10min;
(6) Adding in10％H 2 SO 4 The reaction was terminated and OD values were read at 450 nm;
the potency is OD 450 The dilution of the antiserum was about 1.0.
Inhibition ratio = (OD value of potency-OD value of inhibition)/OD value of inhibition × 100%
The titers and inhibition rates of the antisera of the 4-group immunogen and coating antigen combinations are shown in table 1.
Table 1 4 titers and inhibition rates of antisera of group immunogen and coating antigen combinations
As can be seen from Table 1, different terazosin hydrochloride artificial antigens have certain titers as antisera generated by immunized New Zealand white rabbits, and the obtained antisera has different degrees of inhibition effects on target analytes of terazosin hydrochloride. Wherein, the antiserum potency 1:128000 and inhibition ratio 90.85% shown by the immunogen and coating antigen structure combination of the number 4 are the optimal combination; under the combination, the terazosin hydrochloride antibody not only can specifically recognize the target analyte terazosin hydrochloride, but also has good antibody sensitivity; the antigen and coating antigen combinations of numbers 4 are the best combinations, since the antiserum titers and inhibition rates are higher than the antigen and coating antigen combinations of numbers 1, 2 and 3. Namely T-2C-BSA as immunogen and T-SG-OVA as coating antigen.
EXAMPLE 5 establishment of an Indirect competitive ELISA detection method for terazosin hydrochloride
1. Experimental method
An indirect competition ELISA method for detecting terazosin hydrochloride, which comprises the following steps:
(1) The artificial antigen T-SG-OVA prepared in example 2 is used as a coating source, the coating liquid is diluted to 62.5ng/mL, a 96-well ELISA plate is coated, 100 mu L of the coating liquid is added to each well, and the mixture is incubated overnight at 37 ℃ for 12 hours;
(2) Removing the coating liquid, washing twice, and beating to dryness;
(3) 120 mu L of blocking solution (namely 1wt% of fish skin collagen) is added into each hole, and the mixture is blocked for 3 hours at 37 ℃;
(4) Discarding the sealing liquid, beating the plate, drying at 37 ℃ for 30min, taking out, and bagging for standby by self-sealing;
(5) With PBST 1: 4000-fold dilution of the polyclonal antibody prepared in example 3, and the terazosin hydrochloride drug was diluted to 1000ng/mL, 250ng/mL, 62.5ng/mL, 15.63ng/mL, 3.9ng/mL, 0.98ng/mL, 0.244ng/mL, 0.06ng/mL, 0.015ng/mL;
(6) Adding 50 mu L of terazosin hydrochloride drug diluent to be detected (three groups are parallel) into each row, adding 50 mu L of polyclonal antibody diluent prepared in the example 3 per hole, incubating for 40min at 37 ℃, washing for five times, and beating to dryness;
(7) Adding goat anti-rabbit secondary antibody-HRP (5000-fold dilution) 100 μl/well, incubating at 37deg.C for 30min, washing five times, and drying;
(8) Adding a developing solution, and developing for 10min at 100 mu L per hole;
(9) 50. Mu.L of 10% H was added 2 SO 4 The reaction was stopped and the OD was read at 450 nm.
2. Experimental results
The standard curve of the indirect competition ELISA for the detection of terazosin hydrochloride is shown in FIG. 5, and the half Inhibitory Concentration (IC) of the antibody for the detection of terazosin hydrochloride can be seen from FIG. 5 50 ) 5.72ng/mL, the quantitative detection range is 1.04-31.44 ng/mL, and the lowest limit of detection (LOD) is 0.38ng/mL; the antibody for detecting the terazosin hydrochloride prepared by the invention can meet the detection requirement.
Example 6 evaluation of specificity of antibodies for detection of terazosin hydrochloride
1. Experimental method
The specificity of the antibody for detecting the terazosin hydrochloride is determined by a cross reaction experiment of the terazosin hydrochloride antibody and the terazosin hydrochloride drug and analogues thereof, the specificity of the antibody is expressed by a cross reaction rate (CR), and the smaller the cross reaction rate is, the stronger the specificity is. The terazosin hydrochloride and analogues thereof such as Phenbutamine (PHE), phenbutamine (CPT), benzomorphine (PHM) and buprofeprinopine (AFP) are respectively preparedThe dilution was performed by the indirect competition ELISA method, and the IC of each analogue was obtained in the same manner as in example 5 50 The value, the terazosin hydrochloride cross-reaction rate (CR) was calculated according to the following formula
CR(％)＝IC 50 (terazosin hydrochloride)/IC 50 (analog) ×100%
2. Experimental results
The results of the cross-reaction of the terazosin hydrochloride polyclonal antibody prepared in example 3 with the terazosin hydrochloride drug and analogues thereof are shown in table 2,
TABLE 2 Cross-reaction results of terazosin hydrochloride polyclonal antibodies with terazosin hydrochloride and analogues thereof
Note that: NR represents no reaction
As is clear from Table 2, the cross reaction rate of the polyclonal antibody for detecting terazosin hydrochloride to terazosin hydrochloride was 100%, and IC 50 5.72ng/mL, has no cross to the analogue of terazosin hydrochloride, i.e. prazosin, doxazosin and tolazoline; the method has the advantages that the antibody for detecting the terazosin hydrochloride has high recognition capability and strong specificity on the terazosin hydrochloride, can effectively eliminate the interference of the terazosin hydrochloride analogs of prazosin, doxazosin and tolazoline on the detection of the terazosin hydrochloride, and can be specially used for detecting the terazosin hydrochloride.
EXAMPLE 7 development of kit for detecting terazosin hydrochloride
1. Composition of the kit
The kit for detecting the terazosin hydrochloride comprises the following parts:
(1) Preparing an ELISA plate coated with a coating source: the terazosin hydrochloride artificial antigen T-SG-OVA prepared in example 2 is used as a coating raw material, the coating raw material is diluted to 31.25 mug/L by using a coating buffer solution, 100 mug/L is added into each hole, the mixture is incubated overnight at 37 ℃ in a dark place, liquid in the hole is poured out, the mixture is washed for 2 times by using a washing solution, each time for 30 seconds, the mixture is patted dry, then 200 mug of sealing solution is added into each hole, the mixture is incubated for 2 hours at 25 ℃ in a dark place, the liquid in the hole is poured out, and the mixture is vacuum-sealed and stored by using an aluminum film after the mixture is dried; the coating buffer solution is carbonate buffer solution with the pH value of 9.6 and 0.05mol/L, the sealing solution is phosphate buffer solution with the pH value of 7.1-7.5 and contains 1-3 wt% of casein and 0.1-0.3 mol/L;
(2) Terazosin hydrochloride standard solution: 8 concentration gradients of 1000 μg/L,200 μg/L, 40 μg/L,8 μg/L,1.6 μg/L,0.32 μg/L,0.064 μg/L,0.0128 μg/L, respectively;
(3) The terazosin hydrochloride polyclonal antibody prepared in example 3;
(4) Enzyme conjugate: horseradish peroxidase-labeled terazosin hydrochloride polyclonal antibody prepared in example 3;
(5) Substrate color development liquid: the liquid A is carbamide peroxide, and the liquid B is tetramethyl benzidine;
(6) The termination liquid is 2mol/L H 2 SO 4 ；
(7) The washing liquid has pH value of 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.
2. 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 is diluted with an enzyme conjugate diluent in a 1:10 volume ratio (i.e., one portion of enzyme conjugate concentrate is added to 10 portions of enzyme conjugate diluent, ready for use) as desired. Adding 50 mu L of standard substance/sample into corresponding microwells, adding 50 mu L of enzyme conjugate working solution, gently shaking and mixing, and placing the mixture in a light-shielding environment at 25 ℃ for reaction for 30min after covering with a cover plate film. Spin-drying the liquid in the holes, and adding 250 mu L/hole of washing working solution; washing for 4-5 times, and at intervals of 10s each time, pouring out the washing liquid in the plate holes, and drying by using absorbent paper (after drying, the unused gun heads can be used for puncturing by the bubbles which are not clear). Adding 50 mu L/hole of substrate color development liquid A, adding 50 mu L/hole of substrate color development liquid B, gently shaking and uniformly mixing, and placing the mixture in a light-shielding environment at 25 ℃ for reaction for 10min after a cover plate film is used; adding 50 mu L of stop solution per hole, gently shaking and mixing, setting an enzyme label instrument and 450nm, and measuring the OD value of each hole.
3. 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 wells) divided by the average of the absorbance values of the first standard (0 μg/L) and multiplied by 100%. And drawing a standard curve chart by taking the percentage absorbance of the standard substance as an ordinate and taking the logarithm of the concentration (mug/L) of the terazosin hydrochloride 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 factor corresponding to the standard curve to obtain the actual concentration of the terazosin hydrochloride in the sample.
The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, 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 (2)

1. The terazosin hydrochloride artificial antigen combination is characterized by comprising an immunogen and a coating antigen, wherein the immunogen is obtained by coupling terazosin hydrochloride hapten with bovine serum albumin with a structural formula shown as a formula (I),
formula (I);
the coating antigen is obtained by coupling terazosin hydrochloride hapten with chicken ovalbumin with a structural formula shown as a formula (II),
formula (II).
2. A kit for detecting terazosin hydrochloride, comprising the terazosin hydrochloride artificial antigen combination of claim 1.
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