CN110283101B - Cimaterol hapten, cimaterol antigen, chemiluminescence enzyme-linked immunoassay kit and application thereof - Google Patents
Cimaterol hapten, cimaterol antigen, chemiluminescence enzyme-linked immunoassay kit and application thereof Download PDFInfo
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- CN110283101B CN110283101B CN201910487861.XA CN201910487861A CN110283101B CN 110283101 B CN110283101 B CN 110283101B CN 201910487861 A CN201910487861 A CN 201910487861A CN 110283101 B CN110283101 B CN 110283101B
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- C07C255/00—Carboxylic acid nitriles
- C07C255/49—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
- C07C255/58—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the carbon skeleton
- C07C255/59—Carboxylic acid nitriles having cyano groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton containing cyano groups and singly-bound nitrogen atoms, not being further bound to other hetero atoms, bound to the carbon skeleton the carbon skeleton being further substituted by singly-bound oxygen atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
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Abstract
The invention discloses a cimaterol hapten, a cimaterol antigen, a chemiluminescent enzyme-linked immunoassay kit and application thereof. The invention discloses a cimaterol hapten, a corresponding artificial antigen, a chemiluminescence enzyme-linked immunoassay kit and application thereof. The cimaterol hapten provided by the invention is connected with carrier protein to obtain a cimaterol antigen. The cimaterol antigen can be applied to preparation of a cimaterol specific antibody. The preparation method is simple, convenient and feasible and has lower cost. The chemiluminescence enzyme-linked immunoassay kit has the detection range of 0.10-8.10 mug/L, the sensitivity of 0.51 mug/L, the detection limit of 0.10 mug/L, the recovery rate of 80.81-127.80%, low detection limit, high sensitivity, good stability, low cost and high speed, is very suitable for trace analysis and batch detection of a large number of samples, and has important practical application and popularization significance.
Description
Technical Field
The invention belongs to the technical field of biological detection. More particularly, relates to a cimaterol hapten, a cimaterol antigen, a chemiluminescence enzyme-linked immunoassay kit and application thereof.
Background
Cimaterol (Cimaterol), formula C 12 H 17 N 3 O, is a β -receptor agonist. In medicine, cimaterol modulates sympathetic nerve excitation, relaxes airway smooth muscle and is therefore commonly used in the treatment of asthma. In addition, studies have shown that cimaterol redistributes nutrients in animals, promotes protein deposition and inhibits adipogenesis, thereby significantly increasing lean meat percentage in animals. In animal production, the phenomenon of illegal addition of cimaterol exists. The cimaterol is slow in metabolism, long in acting time, stable in property and high in drug residue rate in the animal body, and can cause great harm to the health of an eater and even threaten life safety by long-term accumulation and organization in the animal body. As a novel clenbuterol substance, the detection means is lagged, and particularly, a rapid detection product is lacked.
The main methods for detecting cimaterol in the national standard are High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC). The detection is carried out by adopting a chromatography, the cost is high, the time consumption is long, the sample weighing amount is generally required to be increased to achieve higher sensitivity, the pretreatment of the chloramphenicol sample mostly adopts an organic reagent extraction method, and a redissolution method is carried out after evaporation to dryness, and the increase of the sample amount means that the dosage of an extracting agent is correspondingly increased, so that the pretreatment operation is more complicated, the consumed time is prolonged, the equipment and maintenance cost is high, the technical requirement on an instrument operator is higher, and the method is not suitable for field detection and monitoring, such as CN 103675145A. Although the chromatography can meet most detection requirements, the chromatography cannot meet the requirement of on-site rapid detection of a large number of samples, and is generally used as a confirmation method. The immunoassay method has the characteristics of low cost, simple operation, high speed, large sample amount for one-time detection, low instrumentization degree and the like. Therefore, in food safety detection, an immunoassay method is often used for preliminary screening, and then HPLC or GC is used for confirming a positive sample.
Chinese patent document CN 103630689A discloses an enzyme linked immunosorbent assay kit for detecting cimaterol drug residues, and a preparation method and application thereof, wherein a diazotization method is adopted to synthesize the cimaterol artificial complete antigen. Therefore, the establishment of an economic, convenient, reliable, specific, sensitive, rapid and accurate method for quantitatively detecting the cimaterol in the animal food has considerable necessity. The chemiluminescence immunoassay method is a labeling immunoassay technology which combines a chemical reflecting system with immunoreaction and is used for detecting trace antigens or antibodies. The detection principle is as follows: the antigen on the surface of the solid phase carrier and the corresponding antigen in the sample are combined with the antibody competitively, then an enzyme-labeled secondary antibody is added to form an antigen-antibody enzyme-labeled compound, finally, a luminescent substrate is added for enzymatic luminescence after washing, and the luminescence intensity value is detected, wherein the antigen concentration is in negative correlation with the luminescence intensity value. The method has the outstanding advantages of high sensitivity, wide linear dynamic range, long optical signal duration, simple and quick analysis method, stable result, small error, good safety and long service life. Compared with an instrumental analysis method, the method has the advantages that: can realize large-batch field detection, and is more economical and time-saving.
Disclosure of Invention
The primary object of the present invention is to overcome the above-mentioned drawbacks and deficiencies of the prior art and to provide a cimaterol hapten. The hapten has the advantages of accuracy, sensitivity, rapidness and capability of detecting the cimaterol in a high-throughput manner, provides a certain theoretical guidance for development of a cimaterol rapid detection product, and has an important significance for food safety monitoring.
The second purpose of the invention is to provide a cimaterol antigen prepared by the cimaterol hapten.
The third purpose of the invention is to provide the application of the cimaterol antigen in preparing a cimaterol specific antibody.
The fourth purpose of the invention is to provide a cimaterol specific antibody prepared by applying the cimaterol antigen and a preparation method thereof.
A fifth object of the present invention is to provide the use of the above-mentioned cimaterol-specific antibody for the detection of cimaterol.
The sixth purpose of the invention is to provide a chemiluminescent enzyme-linked immunoassay kit prepared by applying the cimaterol antigen and the cimaterol specific antibody.
The above purpose of the invention is realized by the following technical scheme:
a cimaterol hapten which is a compound represented by formula (I):
The invention also discloses a preparation method of the compound shown in the formula (I), which is characterized by comprising the following steps:
adding cimaterol and 4-bromobutenoic acid into a N, N-Dimethylformamide (DMF) solvent, stirring for dissolving, adding anhydrous potassium carbonate, and magnetically stirring at 50-60 ℃ for reacting for 2-4 hours; separating and purifying to obtain the cimaterol hapten.
Further, in a preferred embodiment of the present invention, the mass ratio of the cimaterol, the 4-bromobutenic acid and the anhydrous potassium carbonate is 2-4: 2-3: 4 to 6.
Further, in a preferred embodiment of the present invention, the mass ratio of the cimaterol, 4-bromobutenic acid and anhydrous potassium carbonate is 3.5: 2.86: 5.
the cimaterol antigen provided by the invention is a conjugate obtained by coupling a compound shown in a formula (I) and a carrier protein.
As the carrier protein, there can be used, for example, Bovine Serum Albumin (BSA), Ovalbumin (OVA), Human Serum Albumin (HSA), Murine Serum Albumin (MSA), thyroid protein (TG) or hemocyanin (KLH), etc. Among them, Bovine Serum Albumin (BSA) is most effective.
The invention also discloses a preparation method of the cimaterol antigen, which comprises the following steps:
s1, dissolving 0.1-0.5 mmoL of cimaterol hapten in N, N-dimethylformamide, adding Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) 0.2-1.0 mmoL respectively while stirring, stirring and reacting for 12-24 h at 4-8 ℃, and centrifuging to obtain a supernatant to obtain a cimaterol hapten reaction solution;
S2, dissolving carrier protein in 0.1-0.3 mol/L PBS to obtain a carrier protein solution;
s3, dropwise adding the cimaterol hapten reaction liquid into a carrier protein solution under the stirring condition, and stirring and reacting at 4-8 ℃ overnight;
s4, centrifuging to take supernatant, dialyzing for 2-3 d with PBS at 4 ℃, and changing the solution 2-3 times every day during the dialysis to obtain the cimaterol antigen.
The cimaterol antigen can be used as immunogen to prepare cimaterol specific antibody, and can also be used as coating antigen to prepare chemiluminescence ELISA plate.
The specific antibody prepared by using the cimaterol antigen can be a monoclonal antibody or a polyclonal antibody.
The cimaterol antigen and the specific antibody can be applied to detection of cimaterol.
The invention also discloses a chemiluminescent enzyme-linked immunoassay kit prepared by applying the cimaterol antigen and the specific antibody.
The chemiluminescence enzyme-linked immunoassay kit comprises: the kit comprises a chemiluminescent enzyme label plate coated with a cimaterol antigen, a cimaterol antibody, an enzyme-labeled secondary antibody, a cimaterol standard solution, a chemiluminescent solution and a cleaning solution.
Further, in a preferred embodiment of the invention, the cimaterol antigen is a conjugate of cimaterol and Bovine Serum Albumin (BSA), and the coating concentration of the cimaterol antigen is 10-46.67 μ g/L.
Further, in a preferred embodiment of the invention, the package of cimaterol antigenThe coating solution is composed of 1.69-2 g of Na 2 CO 3 And 2.95-4 g NaHCO 3 Dissolving in 1-1.5L deionized water to obtain the product.
Further, in the preferred embodiment of the present invention, the chemiluminescent elisa plate is a detachable opaque white luminescent plate.
The chemiluminescent ELISA plate is a 96-hole detachable opaque ELISA plate made of polystyrene, is coated with a cimaterol antigen capable of being specifically combined with an anti-cimaterol antibody, and seals sites on the surface of the micropores which do not adsorb the cimaterol antigen.
The preparation method of the chemiluminescent ELISA plate coated with the cimaterol antigen comprises the following steps: diluting cimaterol antigen with coating solution as required, adding coating solution into micropores of a luminescent plate, incubating overnight at 37 deg.C, removing coating solution, washing with washing solution, adding sealing solution into each pore, incubating at 37 deg.C for 2 hr, removing liquid from pores, drying, and vacuum sealing with aluminum film.
In addition, many substances can be used as the cimaterol antigen solid phase carrier, and for example, polystyrene, nitrocellulose, polyethylene, polypropylene, polyacrylamide, cross-linked glucose, silicone rubber, agarose gel, and the like can be used as the cimaterol antigen solid phase carrier. The carrier may be in the form of wells, paper sheets, beads, etc.
Further, in a preferred embodiment of the present invention, the chemiluminescent solution comprises solution A and solution B, wherein solution A is prepared by dissolving 20mg of p-iodophenol, 8mg of luminol and 1.21g of Tris in 100mL of deionized water, and adjusting pH to 8.4 with hydrochloric acid; the liquid B is prepared by mixing 0.40% H by volume 2 O 2 Dissolving 1.21g of Tris in 100mL of deionized water, and adjusting the pH value to 7.0 by hydrochloric acid to obtain the compound; when in use, the liquid A and the liquid B are mixed according to the volume ratio of 1: 1, and mixing.
Further, in a preferred embodiment of the present invention, the washing solution is a 20-fold concentrated washing solution, which is 0.4mol/L phosphate buffer solution with pH 7.4 containing 0.5% Tween20 by volume; when in use, the detergent is diluted to 1 time by deionized water.
Further, in the preferred embodiment of the present invention, the enzyme-labeled secondary antibody used is a goat anti-mouse IgG secondary antibody of 1.00mg/mL, and the working concentration is 0.67 g/mL.
Further, in a preferred embodiment of the present invention, the blocking solution used is prepared by dissolving 0.10g of Bovine Serum Albumin (BSA), 5.00g of glycine, and 5.00g of sucrose in 100mL of PBS (0.01mol/L pH 7.4).
Further, in the preferred embodiment of the invention, the cimaterol standard solution is diluted to a series of concentrations of 0.00. mu.g/L, 0.10. mu.g/L, 0.30. mu.g/L, 0.90. mu.g/L, 2.70. mu.g/L, 8.10. mu.g/L cimaterol standard solution with 0.01mol/L, pH 6.50.50 Phosphate Buffer (PB) at the time of use.
Further, in a preferred embodiment of the present invention, the formulation of the Phosphate Buffer (PB) is: 2.90g NaH was weighed out separately 2 PO 4 ·12H 2 O and 0.20g NaH 2 PO 4 And adding deionized water to the solution to make the volume of the solution reach 1L.
The detection range of the chemiluminescent enzyme-linked immunoassay kit for the cimaterol is 0.10-8.10 mug/L, the sensitivity is 0.51 mug/L, the detection limit is 0.10 mug/L, and the recovery rate is 80.81-127.80%.
The invention also provides a using method of the cimaterol chemiluminescence enzyme-linked immunoassay kit, which comprises the following steps:
s1, taking the kit out of a refrigeration environment, and balancing for 30-45 min at 15-35 ℃;
s2, taking out the chemiluminescent ELISA plate, adding cimaterol standard solutions with different concentrations into standard holes, adding a sample to be detected into a sample hole, adding an enzyme-labeled secondary antibody into each hole, adding a cimaterol antibody, covering a cover plate film, shaking and uniformly mixing, and incubating for 15min at 37 ℃;
s3, absorbing the reaction liquid in the plate holes, washing for 5 times by using 20 times of diluted washing liquid, and drying the enzyme-labeled plate; the washing liquid is obtained by diluting the concentrated washing liquid by 20 times;
s4, adding chemiluminescent liquid into each hole, patting and uniformly mixing, covering a cover plate film, and measuring the luminescence intensity value (RLU) of each hole after 3 min;
S5, calculation and analysis of detection results: and determining the content of the cimaterol in the sample.
Inhibition ratio (%) ═ B/B 0 X 100 (%), wherein: b is RLU of standard substance solution holes or sample holes to be detected of the cimaterol with different concentrations; b is 0 RLU which is a 0 concentration cimaterol standard solution;
and drawing a standard curve by taking the inhibition rate as an ordinate and taking the logarithm of the concentration of the cimaterol as an abscissa, thereby determining the content of the cimaterol in the sample.
The sample to be tested in S2 may be a tissue sample such as chicken/liver, pork/liver, shrimp, fish, etc., or a sample such as feed or urine.
Preferably, the sample to be tested in S2 is an animal urine sample.
When the sample to be detected is an animal urine sample, the pretreatment method comprises the following steps:
taking a clear and transparent animal urine sample to be tested, centrifuging the mixed urine sample at 4000-5000 r/min for 5-10 min, and taking supernatant to be tested.
Compared with the prior art, the invention has the following beneficial effects:
1. the chemiluminescent enzyme-linked immunoassay kit for the cimaterol has the detection range of 0.10-8.10 mug/L, the sensitivity of 0.51 mug/L, the detection limit of 0.10 mug/L and the recovery rate of 80.81-127.80%. The kit has the advantages of rapid detection, greatly shortened detection time, no consideration of the influence of the technical level of detection personnel, realization of the whole detection process only within about 20min, lower detection limit and higher sensitivity. Meanwhile, the stability and precision of the kit are greatly improved by adopting the coated plate coated with the antigen, and the detection is more stable and accurate.
2. High sensitivity, high specificity: compared with the existing ELISA kit for cimaterol, the ELISA kit overcomes the defects that the detection process of the kit is easily interfered by endogenous enzymes, and the detection of absorbance is also easily influenced by various external factors, and the chemiluminescence enzyme immunoassay kit for detecting cimaterol has higher sensitivity which can reach 0.51 mug/L by adopting the antibody with high specificity and high affinity.
3. In addition, the kit adopts a chemiluminescence method, and reagents with strong corrosivity such as concentrated sulfuric acid and the like and substrates with strong carcinogenicity in the ELISA kit are not used, so that the kit is more environment-friendly and safer; the method is simple to operate, low in cost, suitable for trace analysis and batch detection of a large number of samples or field screening, and has important practical application and popularization significance.
Drawings
FIG. 1 is a standard curve of a cimaterol chemiluminescent enzyme-linked immunoassay kit.
FIG. 2 is a scheme for the synthesis of cimaterol hapten.
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.
EXAMPLE 1 preparation of cimaterol hapten
Weighing 3.50mg of cimaterol and 2.86mg of 4-bromobutenic acid, adding into 10mL of DMF solvent, stirring for dissolving, adding 5mg of anhydrous potassium carbonate, magnetically stirring for reacting for 2h at 60 ℃, placing the solution after reaction in a rotary evaporator for evaporation, and purifying by column chromatography to obtain cimaterol hapten CIM-CA.
Performing mass spectrum and nuclear magnetic resonance hydrogen spectrum identification on the obtained cimaterol hapten, [ M + H] + 304.2; the result shows that the cimaterol hapten is successfully synthesized and is a compound shown as a formula (I):
example 2 preparation of artificial antigen, coatingen and specific antibody of cimaterol
(1) Preparation of artificial antigen of cimaterol
Dissolving the 0.10mmoL of the cimaterol hapten in 0.50mL of DMF, adding 0.20mmoL of DCC and NHS respectively while stirring, magnetically stirring at 4 ℃ for reaction for 12 hours, centrifuging and taking supernatant to obtain a cimaterol hapten reaction solution; weighing carrier protein BSA and dissolving in 4.00mL of 0.10mol/L PBS (pH8.00) to obtain a carrier protein solution; slowly dropwise adding the cimaterol hapten reaction solution into the carrier protein solution under the stirring condition, and stirring overnight at 4 ℃; centrifuging to obtain supernatant, dialyzing with PBS at 4 deg.C for 3d, changing liquid 2 times per day to obtain artificial antigen (immunogen), and freezing at-20 deg.C.
(2) Preparation of cimaterol coating antigen
The method is the same as the preparation method of the artificial antigen.
(3) Preparation of cimaterol monoclonal antibody
3 female Balb/c mice of 6-8 weeks old are immunized by adopting the artificial antigen. The immunogen is diluted to 1.00mg/mL by physiological saline, and each mouse is injected with 100mL of the immunogen for 4 times. Completely Freund's adjuvant is added for the first immunization, and subcutaneous multi-point injection is carried out; two weeks later, a second immunization was performed with immunogen plus incomplete freund's adjuvant, and boosting was performed by subcutaneous multiple injections as well. The boost was performed every two weeks thereafter, and the mice were subjected to tail-clipping to collect serum one week after the fourth immunization, and the antiserum titer was determined using ELISA. Mice producing the highest titers of antiserum were boosted 3d prior to cell fusion and were directly injected i.p. with 0.50mL of the corresponding antigen (without adjuvant). Spleen cells from immunized mice were fused with SP2/0 myeloma cells at a ratio of 10:1 and 5% CO at 37 ℃ 2 Culturing in HAT medium under the condition. After about 7 days after the fusion, positive hybridoma cells with high titer and high inhibition rate were selected and labeled by indirect competition ELISA. After the positive cells are subcloned for 3 times by a limiting dilution method and are all 100% positive, cell strains are established and frozen. And inoculating the hybridoma cells into the abdominal cavity of the mouse, preparing ascites, and purifying the monoclonal antibody for later use by using Protein G affinity chromatography.
(4) Preference of concentration of coating antigen to concentration of antibody
1) 7.00mg/mL of cimaterol antigen was diluted at different fold (e.g.1: 50000, 1:100000, 1:150000, 1:200000) and coated longitudinally at 150. mu.L/well to opaque white luminescent plate, incubated overnight at 4 ℃, washed twice with washing solution and blotted dry on absorbent paper.
2) Adding a sealing solution 170 mu L/hole for sealing, standing overnight at 37 ℃, drying and putting into an oven.
3) Different gradients of cimaterol standard solutions diluted in 50 μ L/well PBS were added.
4) Adding 50 μ L/well enzyme-labeled secondary antibody, adding 50 μ L/well Cimeterol monoclonal antibody (1:10000, 1:50000, 1:100000, 1:150000) diluted with PBS with different gradients, incubating at 37 deg.C for 15min, washing the plate for 5 times, and drying on absorbent paper.
5) Adding 50 μ L of chemiluminescence solution A and B into each well, tapping, mixing, covering with cover plate membrane, and measuring luminescence value RLU of each well with chemiluminescence immunoassay analyzer after 3 min. The specificity determination is carried out by taking the concentration of the coating antigen and the dilution concentration of the antibody, of which the luminous values have obvious gradient changes along with the concentration of the coating antigen, as the optimal concentration. The optimal concentration of the coating antigen is 46.67 mu g/L, and the dilution factor of the antibody is 100000 times.
(5) Determination of antibody sensitivity
The optimal concentration of the cimaterol monoclonal antibody is 46.67 mug/L by taking the coating concentration as the coating source, and the antibody sensitivity is measured after the cimaterol monoclonal antibody is diluted 100000 times.
1) Taking a 96-hole opaque white luminescent plate, diluting the coating source to 46.67 mu g/L with diluent, adding 150 mu L into each hole, incubating overnight at 4 ℃, washing twice with washing solution, and patting dry on absorbent paper
2) Adding 170 mu L/hole sealing solution for sealing, standing overnight at 37 ℃, drying and putting into an oven.
3) Different gradients of cimaterol standard solution diluted with 50 μ Ι/well PB were added.
4) Adding 50 mu L/hole enzyme-labeled secondary antibody and 50 mu L/hole 100000 times diluted cimaterol antibody in sequence, incubating for 15min at 37 ℃, washing the plate for 5 times, and drying on absorbent paper.
5) Adding 50 μ L of chemiluminescence solution A and B into each well, tapping, mixing, covering with cover plate membrane, and measuring luminescence value RLU of each well with chemiluminescence immunoassay analyzer after 3 min.
6) The results were calculated as inhibition ratio (%) < B/B 0 X 100 (%) and B isLuminescence value in competition with a solution of standard concentration, B 0 For the luminescence without addition of standard, IC50 was calculated to be 0.51. mu.g/L, i.e., the sensitivity of the cimaterol monoclonal antibody.
EXAMPLE 3 preparation of a Chemiluminiscence ELISA kit for cimaterol
(1) Preparation of reagents
1) ELISA plate coated with cimaterol antigen: and a 96-hole detachable enzyme label plate is coated with the cimaterol antigen and the confining liquid, and the coating concentration is 0.05 mg/L. The cimaterol antigen is a conjugate of cimaterol hapten CIM-CA and BSA.
Diluting the coating antigen to 46.67. mu.g/L with the coating solution, adding 150. mu.L of the coating solution into each well, incubating overnight at 4 ℃, pouring out the liquid in the wells, washing 2 times with the washing solution, and patting dry. Then adding 170 mu L of confining liquid into each hole, incubating for 3h at 37 ℃, pouring out the liquid in the hole, placing in an oven at 37 ℃ for drying, and then storing at 4 ℃ by using an aluminum foil bag in a vacuum sealing manner.
2) Preparing a cimaterol standard solution: accurately weighing cimaterol standard solution, diluting with chromatographic grade methanol to 1.00mg/mL, and adding 0.01mol/L PB buffer solution (formula of 2.90g NaH) 2 PO 4 ·12H 2 O、0.20g NaH 2 PO 4 And fixing the volume to 1L) to respectively prepare 0.00 mu g/L, 0.10 mu g/L, 0.30 mu g/L, 0.90 mu g/L, 2.70 mu g/L and 8.10 mu g/L cimaterol solution, and storing at 4 ℃.
3) The concentration of the cimaterol monoclonal antibody is 6.00 mg/mL; the working concentration was 1:100000, diluted with PBS.
4) Chemical luminescent liquid: consists of a solution A and a solution B; when in use, the solution A and the solution B are mixed according to the volume ratio of 1: 1. The preparation method of the solution A comprises the following steps: 20.00mg of p-iodophenol, 8.00mg of luminol and 1.21g of Tris are dissolved in 100mL of deionized water, and the pH value is adjusted to 8.50 by hydrochloric acid; the preparation method of the solution B comprises the following steps: the volume fraction is 0.40% H 2 O 2 1.21g Tris was dissolved in 100mL deionized water and adjusted to pH 7.00 with hydrochloric acid.
5) Concentrating the washing solution by 20 times; when in use, the detergent is diluted to 1 time by deionized water.
6) Configuration of enzyme-labeled secondary antibody: the goat anti-mouse IgG secondary antibody at 1.00mg/mL was diluted to 0.67 g/mL.
(2) Reagent split charging
And (3) performing aseptic subpackage after the determination of each reagent is qualified, wherein each bottle contains 1 mL of diluted cimaterol standard strain line solution, 7mL of cimaterol antibody, 7mL of enzyme-labeled secondary antibody, 7mL of chemiluminescent liquid A, 7mL of chemiluminescent liquid B and 50mL of 20-time concentrated washing solution. Subpackaging, labeling, marking lot number and validity period, and storing at 4 deg.C.
(3) Assembly of the kit
Respectively placing 1 block of the chemiluminescent enzyme label plate coated with the cimaterol antigen, 1 bottle of each of the cimaterol antibody, the enzyme-labeled secondary antibody, the chemiluminescent liquid A, the chemiluminescent liquid B and the 20-time concentrated cleaning solution, 6 bottles of the cimaterol standard solution and 1 part of an instruction manual in a designated position in a reagent kit, packaging after the reagent kit is qualified, and storing at 4 ℃.
Example 4 application of cimaterol chemiluminescence enzyme-linked immunoassay kit
(1) Detection Using the kit of example 3 of the present invention
1) The kit of example 3 was taken out and left to equilibrate at room temperature (20-24 ℃) for more than 30min,
2) Taking out the chemiluminescent ELISA plate, adding 50 μ L of standard solutions with different concentrations into the standard holes, adding 50 μ L of sample to be detected into the sample holes, adding 50 μ L of enzyme-labeled secondary antibody into each hole, adding 50 μ L of cimaterol antibody, covering with a cover plate film, shaking for 10min on a micro-oscillator, and incubating at 37 deg.C for 15 min.
3) Sucking reaction liquid in the plate holes, adding about 300 mu L of washing liquid into each hole, standing for about 20s, removing liquid, washing for 5 times in the way, and finally beating the plate dry; the plate can also be washed 5 times with an automatic plate washer, and the microporous frame is inverted and patted on absorbent paper after washing (patting 3 times per round of plate washing) to ensure complete removal of liquid in the wells.
4) Adding 50 μ L of chemiluminescence solution A and B into each well, tapping, mixing, covering with cover plate membrane, measuring luminescence value RLU of each well with chemiluminescence immunoassay analyzer after 3min, and storing data.
(2) Calculation and analysis of detection results
Inhibition ratio (%) ═ B/B 0 X 100 (%), wherein: b is the luminous value of the standard substance solution hole or the sample hole to be detected of the cimaterol with different concentrations; b is 0 The luminescence value of the 0-concentration cimaterol standard solution is obtained; and drawing a standard curve by taking the inhibition rate as a vertical coordinate and the logarithm of the concentration of the cimaterol as a horizontal coordinate, and substituting the light absorption value of the sample hole into the standard curve to obtain the content of the cimaterol in the sample to be detected. The influence of the operation skill of the detection personnel is not considered, and the whole detection process can be completed within 20 min. The analysis of the detection result can also be calculated and analyzed by using computer professional software.
(3) Standard curve
A cimaterol standard curve chart (shown in figure 1) is obtained by analyzing the detection result of the standard solution, and shows that the kit has the linear detection range of 0.10-8.10 mug/L, the sensitivity of 0.51 mug/L and the detection limit of 0.10 mug/L for cimaterol.
Example 5 application evaluation of a cimaterol chemiluminescent enzyme-linked immunoassay kit
(1) Sensitivity and detection limit of kit
The sensitivity of the kit is determined according to a conventional method, the range of a standard curve is 0.00-8.10 mu g/L, and IC is 50 The floating range of (A) is 0.46-0.89 mu g/L; the 20 samples were tested, and the concentrations corresponding to the percent absorbance values were determined from the standard curve, and the detection limit was expressed as the mean of the 20 sample concentrations plus 3 standard deviations, which indicated that the detection limit of the method was 0.10. mu.g/L for the animal urine sample.
(2) Accuracy and precision test
And adding the cimaterol standard substance into an animal urine sample without cimaterol to ensure that the final concentrations of the cimaterol standard substance in the sample are respectively 0.50 mu g/L, 1.00 mu g/L and 3.00 mu g/L, and respectively pretreating the added sample according to the method to obtain a detection sample solution. 3 kits were extracted from each of the three different batches of kits for detection, and the detection method was repeated 4 times for each experiment as described above, and the coefficient of variation was calculated separately. Wherein:
1) The recovery rate was used as an index for evaluating the accuracy, and the calculation formula was a method of calculating the recovery rate of the added concentration of the sample, i.e., the average recovery Rate (RG) was 100% of the ratio of the measured value to the true value.
2) The Coefficient of Variation (CV) is (the ratio of the standard deviation of each parallel sample to the average value of each parallel sample) multiplied by 100 percent; the calculating method of the variation coefficient in the batch comprises the steps of obtaining the average value of the variation coefficients of all parallel samples in the same measurement, namely CV in the batch; the calculation method of the batch-to-batch coefficient of variation is that the average value of the batch CV is the coefficient of variation of the measurement results of the same sample in different batches.
3) The results are shown in Table 1. The experimental result shows that the average recovery rate of the kit is in the range; the relative standard deviation in each batch and between batches is less than 20 percent.
TABLE 1 accuracy and precision test results
(3) Testing of shelf life of kits
1) Placing the kit in the embodiment 3 at 2-8 ℃, respectively taking the kits placed for 0, 2, 4, 6, 8, 9, 10, 11 and 12 months, and obtaining the luminescence value and IC (integrated Circuit) of the cimaterol standard sample 50 And measuring the parameters of the addition recovery rate and the intra-batch variation coefficient.
2) The kit is stored at 37 ℃ for 12 days, and the luminescence value and IC of the cimaterol standard sample are measured every day 50 And measuring the parameters of the addition recovery rate and the intra-batch variation coefficient.
3) Storing the kit in a refrigerator at-20 deg.C for 12 days, and measuring luminescence value and IC of cimaterol standard sample every day 50 And measuring the parameters of the addition recovery rate and the intra-batch variation coefficient.
The results show that all indexes of the kit completely meet the requirements through three condition storage tests, so that the kit can be stored for 12 months at the temperature of 2-8 ℃ according to the results.
From the above results, the kit of the present invention can be stored at 2 to 8 ℃ for at least one year.
(4) Cross reaction rate test
Selecting other drugs with similar structure or function to cimaterol to carry out cross reaction test, and obtaining IC respectively according to standard curves of various drugs 50 And cross-reactivity. The smaller the cross-reaction rate with other medicines is, the better the detection specificity of the cimaterol chemiluminescence detection kit to cimaterol is. The results are shown in Table 2.
TABLE 2 Cross-reactivity ratio of the cimaterol kit
2) The experimental results show that: the cross reaction rate with the clenbuterol hydrochloride and the phenylethanolamine A is high, so that the method can be applied to screening and detecting the cimaterol residue in an actual sample, and can also be applied to detecting the clenbuterol hydrochloride and the phenylethanolamine A; the antibody established chemiluminescence enzyme-linked immunoassay kit obtained by the invention has important practical significance for the rapid detection of stimulant drug residues in animal urine samples.
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.
Claims (12)
1. A cimaterol hapten, which is a compound of formula (I):
2. a process for the preparation of a compound of formula (I) according to claim 1, comprising the steps of:
adding cimaterol and 4-bromobutenoic acid into an N, N-dimethylformamide solvent, stirring for dissolving, adding anhydrous potassium carbonate, and reacting for 2-4 hours at 50-60 ℃ by magnetic stirring; separating and purifying to obtain the cimaterol hapten.
3. The preparation method according to claim 2, wherein the mass ratio of the cimaterol, the 4-bromobutenic acid and the anhydrous potassium carbonate is 2-4: 2-3: 4 to 6.
4. A cimaterol antigen which is a conjugate obtained by conjugating a compound of formula (I) according to claim 1 to a carrier protein.
5. The cimaterol antigen of claim 4 wherein the carrier protein is human serum albumin, bovine serum albumin, ovalbumin, murine serum protein or rabbit serum protein.
6. A method of preparing a cimaterol antigen as claimed in claim 4 or claim 5 which comprises the steps of:
s1, dissolving 0.1-0.5 mmoL of cimaterol hapten in N, N-dimethylformamide, adding dicyclohexylcarbodiimide and N-hydroxysuccinimide by 0.2-1.0 mmoL respectively while stirring, stirring and reacting for 12-24 h at 4-8 ℃, centrifuging and taking supernatant to obtain a cimaterol hapten reaction solution;
s2, dissolving carrier protein in 0.1-0.3 mol/L PBS to obtain a carrier protein solution;
s3, dropwise adding the cimaterol hapten reaction liquid into a carrier protein solution under the stirring condition, and stirring and reacting at 4-8 ℃ overnight;
s4, centrifuging to take supernatant, dialyzing for 2-3 d with PBS at 4 ℃, and changing the solution 2-3 times every day during the dialysis to obtain the cimaterol antigen.
7. Use of a cimaterol antigen according to claim 4 or 5 in the preparation of a cimaterol specific antibody.
8. Use of the cimaterol antigen according to claim 4 or 5, and the specific antibody according to claim 7 for the detection of cimaterol.
9. A chemiluminescent enzyme-linked immunoassay kit prepared by using the cimaterol antigen of claim 4 or 5 and the specific antibody of claim 7.
10. The chemiluminescent enzyme-linked immunoassay kit of claim 9, comprising: the kit comprises a chemiluminescent enzyme label plate coated with a cimaterol antigen, a cimaterol antibody, an enzyme-labeled secondary antibody, a cimaterol standard solution, a chemiluminescent solution and a cleaning solution.
11. The chemiluminescent enzyme-linked immunoassay kit according to claim 10, wherein the cimaterol antigen is a conjugate of cimaterol and bovine serum albumin, and the coating concentration of the cimaterol antigen is 10-46.67 μ g/L.
12. The chemiluminescent enzyme-linked immunoassay kit according to claim 11, wherein the coating solution of the cimaterol antigen is composed of 1.69-2 g Na 2 CO 3 And 2.95-4 g NaHCO 3 Dissolving in 1-1.5L deionized water to obtain the product.
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