CN106053788A - Cotinine homogeneous enzyme immune detection reagent and preparation and detection methods thereof - Google Patents

Cotinine homogeneous enzyme immune detection reagent and preparation and detection methods thereof Download PDF

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CN106053788A
CN106053788A CN201610574864.3A CN201610574864A CN106053788A CN 106053788 A CN106053788 A CN 106053788A CN 201610574864 A CN201610574864 A CN 201610574864A CN 106053788 A CN106053788 A CN 106053788A
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cotinine
mentioned
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glucose
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虞留明
周桓
汪兴周
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SUZHOU EVERMED CO Ltd
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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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

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Abstract

The invention relates to a cotinine detection reagent and preparation and detection methods thereof, in particular to a cotinine homogeneous enzyme immune detection reagent and preparation and detection methods thereof. The cotinine homogeneous enzyme immune detection reagent comprises an anti-cotinine specificity antibody and an indicating reagent used for detecting the anti-cotinine specificity antibody-cotinine compound; the anti-cotinine specificity antibody is obtained by a cotinine immunogen immune animal. The cotinine homogeneous enzyme immune detection reagent and the preparation and detection methods thereof, provided by the invention, have the benefits that a cotinine immunogen of the cotinine homogeneous enzyme immune detection reagent is strong in specificity and high in immunogenicity, and the prepared anti-cotinine specificity antibody is strong in specificity and high in titer and has no cross reaction with 62 common drugs; the homogeneous enzyme immune detection reagent containing the anti-cotinine specificity antibody can conveniently, quickly and accurately determine the content of cotinine in a sample and simultaneously can determine a plurality of samples on a fully-automatic biochemical analyzer, so that the rapid determination of high throughput of the cotinine is realized, the accurate rate is high, the specificity is strong, and the accuracy and the detection efficiency are greatly improved.

Description

A kind of cotinine homogeneous enzyme immunoassay detectable and preparation thereof and detection method
Technical field
The present invention relates to a kind of cotinine detectable and preparation thereof and detection method, be specifically related to a kind of cotinine homogeneous Enzyme immunologic function test reagent and preparation thereof and detection method.
Background technology
Cotinine (Cotinine) structural formula is as shown in formula III:
Formula III
Along with the development in epoch, the harm that smoking causes has become one of the most serious public health problem in the world today.Various countries Community health's problem caused by the exposure of smoking and second hand smoking is given and greatly pays close attention to and huge economic input by government.State In inside and outside existing numerous research reports Nicotiana tabacum L., various compositions are for the harm of human body, and Nicotiana tabacum L. discharges 4000 in combustion Multiple chemical composition, can cause multiple disease, serious harm health.2005, China signed " Nicotiana tabacum L. control as performing states Framework convention processed ", make Tobacco Control have legal basis；2012, Chinese Government issued " Chinese tobacco controlling planning (2012-2015 Year) ", propose to create smokeless environment, implement public place and ban on opium-smoking and the opium trade comprehensively.
Nicotine, is commonly called as nicotine (nicotine), is a kind of main alkaloid being present in plant of Solanaceae Nicotiana tabacum L., at cigarette In grass, average content reaches 4%, accounts in Nicotiana tabacum L. more than the 90% of alkaloid total amount, and nicotine is to cause smoking addiction or tobacco dependence Principal element.Nicotine in Nicotiana tabacum L. there are about 40% and is transferred directly in cigarette mainstream flue gas and side-stream smoke, enters in human body Nicotine direct oral cavity, throat, trachea and alveolar be absorbed into blood circulation, the most about 80% cytochrome in liver First it is metabolised to cotinine (cotinine) under the effect of oxidase P450-2A6 (CYP450-2A6), then enters one through liver Step metabolism, is formed multiple metabolite, is excreted by approach such as urine, saliva, perspiration afterwards.Nicotine main metabolic is produced Thing has cotinine and glucosides, 3-hydroxyl cotinine and glucosides thereof, nicotine glucosides, nornicotine, fall cotinine, nicotine nitrogen oxygen Compound, cotinine nitrogen oxides, these metabolites account for more than the 90% of absorption of human body nicotine total amount, and wherein cotinine accounts for The 70%-80% of all metabolite.Research proves: the physiological action of human body is mainly included maincenter god by nicotine and metabolite thereof Impact through system, cardiovascular system and hormonal system etc..Actively during smoking, nicotine absorbs in a large number can accelerate heart rate, raises Blood pressure, and reduce appetite, heavy dose of nicotine can cause nausea and vomiting, can induce cancer, cause death time serious；Passive suction During cigarette, though low content alkaloid does not has heavy dose of nicotine toxicity big, but also can have a negative impact to healthy, as caused exhaling The multiple diseases such as desorption system disease, cardiovascular disease.
Nicotine and major metabolite cotinine thereof can be distributed to each position of human body with blood, blood, urine, milk, The biological samples such as saliva, seminal fluid and hair all can detect that these materials.But, the nicotine half-life in vivo is only 2 ～3h, it is only capable of the absorption situation in several hours of past of reflection, and the half-life of cotinine is up to 70～100h, can reflect in the past The absorption situation of 3～4d, and owing to nicotine is the exclusive source of cotinine, and affected less by dietary factor, therefore Cotinine has become assessment person under inspection's fume exposure degree, smoking capacity and the smoking generally acknowledged at present to Health Impact degree Maximally effective biomarker.
At present, the cotinine assay method of document report mainly has: euzymelinked immunosorbent assay (ELISA), thin layer chromatography, barbital acid system, hair Cons electrophoresis chemoluminescence method, gas chromatography, gas-liquid chromatography, high performance liquid chromatography, GC-MS, Liquid Chromatography-Mass Spectrometry, Liquid Chromatography-Tandem Mass Spectrometry, radio immunoassay etc..But these traditional detection methods Mostly having instrument and equipment expensive, the detection time is long, and complex operation needs to use a large amount of poisonous and hazardous organic solvent, Easily to environment, and experimenter is had many defects such as higher technology requirement, be therefore unfavorable for promoting the use of.Mesh Before, there is no nicotine metabolism thing method for quick and the products such as practical, cost performance is high cotinine both at home and abroad.Therefore, grind Study carefully exploitation quick, sensitive, high flux, the cotinine determination method of automatization and product, receive biomedicine, Nicotiana tabacum L. The extensive attention in multiple fields such as science and technology, medical treatment & health and environmental health.
Summary of the invention
For solving the deficiencies in the prior art, it is an object of the invention to provide one not only safety but also can be quick, efficient, clever Quick, cotinine homogeneous enzyme immunoassay detectable accurately detecting cotinine content in sample to be tested and preparation method thereof, and Can be combined with various types of automatic biochemistry analyzers, cotinine homogeneous enzyme immunoassay detection side less demanding to testing staff Method.
Another object of the present invention is for providing a kind of cotinine contributing to preparing rapid and accurate determination cotinine content The cotinine derivant of homogeneous enzyme immunoassay detectable.
In order to realize above-mentioned target, the technical solution used in the present invention is:
A kind of cotinine homogeneous enzyme immunoassay detectable, including: anti-cotinine specific antibody, to be used for detecting anti-cotinine special The indicator of property antibody-cotinine complex；Above-mentioned anti-cotinine specific antibody is obtained by cotinine immunogen immune animal Arriving, the immunogenic structural formula of cotinine is as shown in formula I:
Formula I
In formula, carrier is for having immunogenic protein；Above-mentioned indicator selected from enzyme reagent, radiosiotope reagent, Fluorometric reagent or chemical illuminating reagent.
Above-mentioned indicator is selected from enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes Portugal Glucose-6-phosphate dehydrogenase-hapten enzyme mark conjugate；The substrate of above-mentioned enzyme is G-6-P；
Described glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate is derived with cotinine by glucose-6-phosphate dehydrogenase (G6PD) Thing reaction is formed.
The structural formula of above-mentioned cotinine derivant is as shown in formula II:
Formula II
The synthetic method of cotinine derivant, it is characterised in that synthetic route is:
The preparation method of a kind of cotinine homogeneous enzyme immunoassay detectable, it is characterised in that comprise the steps:
(1) synthesis of cotinine derivant and purification, and carry out Structural Identification；
(2) the immunogenic synthesis of cotinine: make cotinine derivant and there is immunogenic protein carrier be connected；
(3) with cotinine immunogen immune animal, preparation purification anti-cotinine specific antibody；
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) solution, swashs Cotinine derivant alive, makes glucose-6-phosphate dehydrogenase (G6PD) be connected with cotinine derivant, and purification connects product；
(5) preparation of cotinine homogeneous enzyme immunoassay detectable:
The preparation of reagent A: mixed by anti-cotinine specific antibody and homogeneous zymolyte；
The preparation of reagent B: mixed with Tris buffer by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing.
The preparation method of aforesaid a kind of cotinine homogeneous enzyme immunoassay detectable, in described step (2), protein carries Body is BSA, and concrete synthesis step is as follows:
A. weigh 1.0～5.0 g potassium dihydrogen phosphates, 2.0～8.0 g disodium hydrogen phosphates, 5.0～15.0 g sodium chloride, 0.5～ 1.5 g magnesium chlorides, are dissolved in 1.0～3.0 L deionized waters jointly, regulate pH to 7.0～8.0, make buffer solution A；
B. weigh 2.0～5.0 mg BSA, be dissolved under room temperature in 2.0～5.0 mL above-mentioned buffer solution A, make BSA solution；
C. weighing 2.0～5.0 mg cotinine derivants, be dissolved in 200.0～500.0 μ l above-mentioned buffer solution A, making can For peaceful derivative solution；
D. when above-mentioned cotinine derivative solution just becomes clarification, it is added dropwise in above-mentioned BSA solution, then this is mixed Solution stirs 0.5～3.0 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution A by reacted above-mentioned mixed solution, after dialysis, gained solution is cotinine Immunogen solution, adds the NaN of mass fraction 0.05%～0.3% in cotinine immunogen solution3, store at-20 DEG C.
The preparation method of aforesaid a kind of cotinine homogeneous enzyme immunoassay detectable, described step (4) detailed process is:
A. weigh 1.0～5.0 g potassium dihydrogen phosphates, 1.0～5.0 g disodium hydrogen phosphates, 5.0～15.0 g sodium chloride, 0.5～ 1.5 g magnesium chlorides, are dissolved in 1.0～3.0 L deionized waters jointly, regulate pH to 7.0～8.0, make buffer solution B；
B. weigh 5.0～20.0 mg glucose-6-phosphate dehydrogenase (G6PD)s, be dissolved in the 2.5～15.0 above-mentioned bufferings of mL under room temperature molten In liquid B, regulate pH to 8.5, make glucose-6-phosphate dehydrogenase (G6PD) solution；
C. weigh 2.0～5.0 mg cotinine derivants, be dissolved in the dimethylformamide that 100.0～500.0 μ l are dried (DMF), in, concussion is dissolved, and makes cotinine derivative solution；
D., when above-mentioned cotinine derivative solution just becomes clarification, it is added dropwise over above-mentioned glucose-6-phosphate dehydrogenase (G6PD) molten In liquid, complete dropping in 30 seconds, then this mixed solution is stirred 0.5～3.0 hour at 2-8 DEG C；
E. reacted above-mentioned mixed solution is purified by G-25 gel chromatography column, it is thus achieved that end product be Fructus Vitis viniferae Sugar-6-phosphate dehydrogenase-hapten conjugation thing, adds quality in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution The BSA and the NaN of mass fraction 0.05%～0.3% of mark 0.3%～0.8%3, store at 2-8 DEG C.
The preparation method of aforesaid a kind of cotinine homogeneous enzyme immunoassay detectable, the detailed process of step (5) is as follows:
The preparation of reagent A: by the nicotinamide adenine dinucleotide (NAD) of 2.0～8.0 g oxidation state, 1.0～3.0 g Fructus Vitis viniferaes Sugar-6-phosphoric acid (G-6-P) the Tris buffer solution that 0.5～3.0 L concentration are 55 mM, pH=8.0 makes homogeneous zymolyte； The anti-cotinine specific antibody of preparation being added in above-mentioned homogeneous zymolyte, antibody is 1 with the volume ratio of homogeneous zymolyte: 100～1:10000；
The preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing is added to concentration is 120 mM, pH= In the Tris buffer of 8.2, above-mentioned conjugate is 1:100～1:10000 with the volume ratio of Tris buffer.
Utilize the detection method of cotinine homogeneous enzyme immunoassay detectable, it is characterised in that comprise the following steps:
1) sample to be tested is contacted with anti-cotinine specific antibody；
2) according to the combination situation of cotinine in sample to be tested Yu anti-cotinine specific antibody, indicator judgment sample is utilized The content of middle cotinine；
Described sample to be tested is serum, blood plasma, saliva, urine, seminal fluid or milk；
Described indicator is selected from enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes Fructus Vitis viniferae Sugar-6-phosphate dehydrogenase-hapten enzyme mark conjugate；The substrate of above-mentioned enzyme is G-6-P；Described glucose-6-phosphorus Acidohydrogenase-hapten enzyme mark conjugate is formed by glucose-6-phosphate dehydrogenase (G6PD) and cotinine derivatives reaction.
The invention have benefit that: the cotinine immunogens of the present invention is strong, immunogenicity is high, prepares Anti-cotinine specific antibody high specificity, titer are high, and with 62 kinds of common medicines without any cross reaction；Containing above-mentioned The homogeneous enzyme immunoassay detectable of anti-cotinine specific antibody can easily and fast, the cotinine accurately determined in sample Content, and multiple sample can be measured on automatic clinical chemistry analyzer simultaneously, it is achieved the rapid survey of high flux of cotinine Fixed, accuracy is high, and high specificity, degree of accuracy is all enhanced before comparing with detection efficiency, is simultaneously achieved detection The full-automation of process, less demanding to testing staff, it is easy to accomplish and promote the use of.
Accompanying drawing explanation
Fig. 1 is cotinine homogeneous enzyme immunoassay reaction normal curve chart；
Fig. 2 is cotinine homogeneous enzyme immunoassay correlation analysis figure.
Detailed description of the invention
The technical solution used in the present invention is:
Cotinine immunogen, its structural formula is as shown in formula I:
Formula I
In formula, carrier has immunogenicity, it is preferred that carrier is for having immunogenic protein.Although other are sufficiently large Possessing immunogenic material as carrier, but can also select protein as carrier under normal circumstances.The most frequently used immunity Immunogenic carrier includes serum albumin, hemocyanin (KLH) and Elityran.Carrier in the present invention is preferably serum egg In vain.
A kind of anti-cotinine specific antibody, is obtained by the cotinine immunogen immune animal shown in formula I.
In the present invention, " antibody " of indication refers not only to complete antibody molecule, also includes retaining complete antibody specificity knot The antibody fragment of conjunction ability or derivant.The antibody of the present invention can be polyclonal antibody can also be monoclonal antibody, excellent Elect polyclonal antibody as.
The method obtaining polyclonal antibody is to use the cotinine immunogen shown in formula I, after adding or being not added with adjuvant, Carrying out immunity at one or more position of animal, host animal includes: rabbit, goat, mice, sheep, Cavia porcellus or horse.Continue Immunity is carried out always, until antibody titer reaches the highest.Animal timing blood sampling obtains appropriate specific corrosioning anteserum, and antiserum is permissible Purification.
Monoclonal antibody can be made by somatocyte hybriding technology.
A kind of cotinine homogeneous enzyme immunoassay detectable, including: above-mentioned anti-cotinine specific antibody, be used for detecting anti-can Indicator for peaceful specific antibody-cotinine complex.Indicator is selected from enzyme reagent, radiosiotope reagent, glimmering Light reagent and chemical illuminating reagent.Preferably, indicator is enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme.Wherein, Enzyme mark conjugate includes glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate, and it can be obtained by chemical synthesis process.
The using method of above-mentioned cotinine homogeneous enzyme immunoassay detectable, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-cotinine specific antibody；
2) according to the combination situation of cotinine in sample to be tested Yu above-mentioned anti-cotinine specific antibody, indicator is utilized to judge The content of cotinine in sample.
Sample to be tested is various physiology samples, such as serum, blood plasma, urine, saliva, seminal fluid, milk etc..Preferably, treat Test sample is originally serum or blood plasma.
Below in conjunction with specific embodiment, further illustrate the present invention.
Embodiment one: the synthesis of cotinine derivant and structural confirmation thereof
The cotinine derivatives chemical structure used in following example is as shown in formula II:
Formula II
The synthetic route of this cotinine derivant is as follows:
Concrete synthesis step is as follows:
Weigh 1.4 g(6.35 mmol) compound 1, it is dissolved in the dimethylformamide (DMF) of 20 mL, is subsequently adding 0.73 G(6.35 mmol) N-hydroxy-succinamide and 1.46 g(7.62 mmol) 1-(3-dimethylamino-propyl)-3-ethyl- Carbon Asia diamidogen (EDCI), makes reaction mixture, is then stirred at room temperature overnight by this reaction mixture.Reaction completes After, the solvent of mixed solution to be removed, remaining solid matter is redissolved in the ethyl acrylate (EA) of 50 mL, then With purified water and brine, the organic facies after washing passes through Na2SO4It is dried process, finally carries out concentrating to obtain The cotinine derivant (Trans-4 Cotinine Derivative) of 1.1 g bright yellow solid.
The cotinine derivant (Trans-4 Cotinine Derivative) of above-mentioned bright yellow solid is carried out structure Identify: utilize Bruker Avance III plus 400 MHz and VARIAN MERCURY plus 300M to above-mentioned bright orange Color solid chemical compound carries out NMR (Nuclear Magnetic Resonance) spectrum scanning, uses TMS as internal standard.Result is as follows: 1H-NMR (400 MHz, DMSO_d6): δ 2.67-2.741 (m, 4H), 1.33-1.43 (m, 2H), 2.82-2.90 (m, 4H), 2.98- 3.04 (m, 1H), 3.31-3.39 (m, 1H), 4.88 (d, 1H), 7.39-7.46 (m, 1H), 7.66-7.73 (m, 1H), 8.61-8.69 (m, 2H).It is characterized as the cotinine derivant shown in formula II.
The immunogenic synthesis of embodiment two: BSA-cotinine
BSA-cotinine immunogen is formed by connecting with the cotinine derivant shown in formula II by bovine serum albumin (BSA), should Immunogenic concrete synthesis step is as follows:
A. weigh 2.72 g potassium dihydrogen phosphates, 4.26 g disodium hydrogen phosphates, 8.5 g sodium chloride, 0.95g magnesium chloride, jointly dissolve In 1L deionized water, regulate pH to 7.4, make buffer solution A；
B. weigh 3 mg BSA, be dissolved under room temperature in 3 mL above-mentioned buffer solution A, make BSA solution；
C. weigh 3 mg cotinine derivants, be dissolved in 300 μ l above-mentioned buffer solution A, make cotinine derivative solution；
D. when above-mentioned cotinine derivative solution just becomes clarification, it is added dropwise in above-mentioned BSA solution, then this is mixed Solution stirs 1 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution A by reacted above-mentioned mixed solution, after dialysis, gained solution is cotinine Immunogen solution, adds the NaN of 0.1% in cotinine immunogen solution3, store at-20 DEG C.The NaN of 0.1%3Refer to add Amount accounts for the mass percent of the immunogen solution of final gained, and concrete addition is according to the immunogen solution of gained after dialysis Depending on concrete quality.
Embodiment three: the preparation of anti-cotinine specific antibody
Above-mentioned prepared BSA-cotinine immunogen is used conventional method inoculation experiments animal rabbit, after booster immunization, takes anti-blood Clearly, specifically comprise the following steps that
With PBS, the BSA-cotinine immunogen of above-mentioned synthesis is diluted to 1.0 mg/ml, obtains antigenic solution, then with 1.0 Ml antigenic solution mixes with Freund's complete adjuvant, injects experimental animal rabbit.
After 2～3 weeks, more above-mentioned experimental animal rabbit is noted with incomplete Freund's adjuvant with antigenic solution identical for 1.0 ml Penetrate once, inject once every surrounding afterwards, amount to injection 4 times.
Above-mentioned experimental animal rabbit takes blood, and isolated and purified to obtain the anti-cotinine that titer is 1: 30000～1: 50000 special Property antibody.
Embodiment four: the preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing
A. weigh 1.09 g potassium dihydrogen phosphates, 1.70 g disodium hydrogen phosphates, 8.5 g sodium chloride, 0.95g magnesium chloride, jointly dissolve In 1L deionized water, regulate pH to 7.4, make buffer solution B；
B. weighing 10 mg glucose-6-phosphate dehydrogenase (G6PD)s, be dissolved in 7.5 mL above-mentioned buffer solution B under room temperature, regulation pH is extremely 8.5, make glucose-6-phosphate dehydrogenase (G6PD) solution；
C. weighing 3 mg cotinine derivants, be dissolved in the dimethylformamide (DMF) that 200 μ l are dried, concussion is dissolved, system Become cotinine derivative solution；
D., when above-mentioned cotinine derivative solution just becomes clarification, it is added dropwise over above-mentioned glucose-6-phosphate dehydrogenase (G6PD) molten In liquid, complete dropping in 30 seconds, then this mixed solution is stirred 2 hours at 2-8 DEG C；
E. reacted above-mentioned mixed solution is purified by G-25 gel chromatography column, it is thus achieved that end product be Fructus Vitis viniferae Sugar-6-phosphate dehydrogenase-hapten conjugation thing, adds 0.5% in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution The NaN of BSA and 0.1%3, store at 2-8 DEG C.The BSA of the 0.5% and NaN of 0.1%3Refer to that addition accounts for the idol of final gained The mass percent of connection thing solution, depending on concrete addition is according to the concrete quality of the conjugate solution of gained after dialysis.
Embodiment five: the preparation of cotinine homogeneous enzyme immunoassay detectable
Cotinine homogeneous enzyme immunoassay detectable, including: above-mentioned anti-cotinine specific antibody, it is used for detecting anti-cotinine special The indicator of property antibody-cotinine complex.Indicator selected from enzyme reagent, radiosiotope reagent, fluorometric reagent and Chemical illuminating reagent.Preferably, indicator is enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme.Wherein, enzyme mark coupling Thing includes glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark conjugate, and it is obtained by above-mentioned chemical synthesis process.
Cotinine homogeneous enzyme immunoassay detectable before the use, in order to avoid the enzyme mark conjugate in indicator and enzyme Substrate react, the substrate of enzyme mark conjugate and enzyme is separated, does not mixes, so the substrate of enzyme is anti-with above-mentioned Cotinine specific antibody mixes.It is to say, cotinine homogeneous enzyme immunoassay detectable includes that two kinds are provided separately Reagent, specific as follows:
1. the preparation of reagent A: by 4.036g(11.25 mM) nicotinamide adenine dinucleotide (NAD) of oxidation state, 1.711g (11.25 mM) G-6-P (G-6-P) is placed in beaker D, by the Tris buffer solution system of 1L 55 mM, pH=8.0 Become homogeneous zymolyte；The anti-cotinine specific antibody of above-mentioned preparation is added in above-mentioned homogeneous zymolyte, antibody and homogeneous enzyme The volume ratio of substrate can be 1:100～1:10000, and the most concrete ratio is 1:500.
2. the preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of above-mentioned preparation-hapten conjugation thing is added to 120mM, In the Tris buffer of pH=8.2, above-mentioned conjugate can be 1:100～1:10000 with the volume ratio of Tris buffer, at this Ratio concrete in embodiment is 1:1250.
The using method of above-mentioned cotinine homogeneous enzyme immunoassay detectable, comprises the following steps:
1) sample to be tested is contacted with above-mentioned anti-cotinine specific antibody；
2) according to the combination situation of cotinine in sample to be tested Yu above-mentioned anti-cotinine specific antibody, indicator is utilized to judge The content of cotinine in sample to be tested.
Concrete, during detection, sample to be tested being added in reagent A, resisting in the cotinine in sample to be tested and reagent A can Occur specific binding for peaceful specific antibody, generate anti-cotinine specific antibody-cotinine complex；Add reagent B, Now the glucose-6-phosphate dehydrogenase (G6PD) in reagent B-hapten conjugation thing mixes with the substrate of the enzyme in reagent A, contacts, and sends out Raw enzymatic reaction, constitutes the indicator detecting anti-cotinine specific antibody-cotinine complex, and indicator is according to be measured In sample, cotinine judges the content of cotinine in sample to be tested with the combination situation of above-mentioned anti-cotinine specific antibody.
Owing to glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing resists with the cotinine competitive binding in sample to be tested Cotinine specific antibody, so, in sample to be tested, the amount of cotinine is the most, glucose-6-phosphorus free in homogeneous enzymatic solution The amount of acidohydrogenase-hapten conjugation thing is the most, and enzymatic reaction is the fastest, causes OD340Rise.
Above-mentioned sample to be tested is physiology sample, such as serum, blood plasma, urine, saliva, seminal fluid, milk etc..
As the preferred scheme of one, above-mentioned sample to be tested is serum or blood plasma.
Embodiment six: cotinine homogeneous enzyme immunoassay is checked
1, obtain standard curve: Beckman AU480 automatic clinical chemistry analyzer response parameter (see Table 1), operating process are set For: first reagent adding A, add standard substance, be eventually adding reagent B.After adding reagent B, measure the OD of different time points340Extinction Value, calculates reaction rate during various criterion product concentration, needs constantly to adjust reagent A and the volume of reagent B in actual mechanical process Ratio, adjusts light-metering point simultaneously, finally draws comparatively ideal reaction normal curve chart, as shown in Figure 1.
Table 1 Beckman AU480 automatic clinical chemistry analyzer response parameter
The standard curve obtained by the homogeneous enzyme immunoassay detectable of the present invention, replication basic, normal, high concentration Quality Control sample 10 times, above-mentioned Quality Control sample is: be dissolved in human serum by cotinine standard substance, to concentration be respectively 1.00,30.00, 150.00 ng/ml.Detection data and data analysis are shown in Table 2.
Table 2 sample determination and precision and the response rate are assessed
Blood sample Low In High
Sample concentration (ng/ml) 1.00 30.00 150.00
1 0.98 31.34 149.00
2 0.94 30.98 152.95
3 1.03 29.66 150.86
4 1.00 32.07 151.71
5 0.93 31.45 155.32
6 1.05 30.99 148.50
7 0.96 28.92 146.25
8 1.02 31.73 151.03
9 1.04 30.64 150.36
10 0.99 32.03 153.41
Meansigma methods (ng/ml) 1.00 30.98 150.94
Standard deviation (SD) 0.0424 1.0168 2.6243
Precision (CV%) 4.24 3.28 1.74
Response rate % 100.0 103.3 100.6
Testing result: the accuracy that the homogeneous enzyme immunoassay detectable of the present invention measures is high, the response rate 95%-105% scope it In, precision is high, and CV is below 5%.
Embodiment seven: interfering effects of drug is tested
Choosing 62 kinds of Common drugs, adjustment concentration, to 100.0 ng/ml, carries out interference test mensuration.62 kinds of common medicines with And measurement result is referring specifically to table 3.
Table 3 common interference medicine and measurement result
ID# Compound name It is equivalent to the concentration (ng/ml) of cotinine ID# Compound name It is equivalent to the concentration (ng/ml) of cotinine
1 Aspirin 0.0 32 Phenylpropanolamine 0.0
2 β-phenyl-ethylamine 0.0 33 Procainamide 0.0
3 Amphetamines 0.0 34 Procaine 0.0
4 Ampicillin 0.0 35 Quinidine 0.0
5 Bent 0.0 36 Zomepirac 0.0
6 Chlorpromazine 0.0 37 Phyenlephrinium 0.0
7 Clorazepate 0.0 38 Cinnamyl Ai Kening 0.0
8 Gemfibrozil 0.0 39 Ecgonine 0.0
9 Fenoprofen 0.0 40 The West, ground 0.0
10 Methamphetamine 0.0 41 Cotinine 0.0
11 Gentisic acid 0.0 42 Atenolol 0.0
12 Gemfibrozil 0.0 43 Propranolol 0.0
13 Hydrocodone 0.0 44 Glutethimide 0.0
14 Ibuprofen 0.0 45 Bute 0.0
15 Imipramine 0.0 46 Lysergic acid diethylamine 0.0
16 DADPS 0.0 47 Cannabinol 0.0
17 Naproxen 0.0 48 Loperamide 0.0
18 Hydrochlorothiazide 0.0 49 Isoxsuprine 0.0
19 Pethidine 0.0 50 Phenylalanine 0.0
20 Naloxone 0.0 51 Fluoxetine Hydrochloride 0.0
21 Ephedrine 0.0 52 Salbutamol 0.0
22 Nicotiamide 0.0 53 Penicillin 0.0
23 Ranitidine 0.0 54 Methyl diethanolamine 0.0
24 Amobarbital 0.0 55 Dimethylene dioxygen amfetamine 0.0
25 Methylenedioxyamphetamine 0.0 56 Doxylamine succinate 0.0
26 Tetrahydrocannabinol 0.0 57 Nalbuphine 0.0
27 Nystatin 0.0 58 Normorphine 0.0
28 Acetylmorphine 0.0 59 Oxycodone 0.0
29 Benzfetamine 0.0 60 KET 0.0
30 Promethazine 0.0 61 Diphenhydramine 0.0
31 Aspartame 0.0 62 Duromine 0.0
Measurement result: the concentration that above-mentioned 62 kinds of Common drugs are equivalent to cotinine is respectively less than 0.1 ng/ml.Visible, the present invention's Antibody is the specific antibody of anti-cotinine.
Embodiment eight: correlation analysis
100 example clinical samples are used respectively that liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay method and the present invention's is homogeneous Enzyme immunoreagent carries out correlation analysis, and the data of mensuration see table 4.
Table 4 clinical sample measured value
Catalogue number(Cat.No.) Homogeneous enzyme immunoassay method measured value (ng/ml) Liquid chromatography tandom mass spectrometry determination value (ng/ml)
1 4.67 4.70
2 24.82 24.94
3 1.60 1.61
4 3.84 3.90
5 6.42 6.43
6 236.51 238.40
7 107.02 109.00
8 54.93 55.11
9 11.39 11.32
10 304.99 299.26
11 122.45 125.03
12 58.44 59.32
13 8.35 8.49
14 100.69 101.77
15 27.51 28.05
16 99.93 100.00
17 3.30 3.25
18 70.58 69.54
19 105.33 107.01
20 76.90 78.39
21 15.01 14.95
22 35.02 37.00
23 250.99 247.79
24 2.90 2.98
25 30.00 30.15
26 4.24 4.25
27 7.03 7.07
28 84.53 85.50
29 31.95 31.09
30 18.21 18.23
31 333.52 330.06
32 67.77 68.29
33 111.59 112.84
34 2.01 2.00
35 7.63 7.92
36 100.00 100.96
37 86.10 88.59
38 4.07 4.01
39 14.92 15.76
40 135.03 133.12
41 20.55 20.78
42 103.22 101.99
43 65.12 66.43
44 5.66 6.01
45 1.99 2.00
46 233.58 237.17
47 3.09 3.22
48 142.92 140.04
49 98.04 100.51
50 9.62 10.26
51 135.01 135.95
52 211.33 215.03
53 186.49 188.88
54 2.00 1.97
55 17.78 17.89
56 109.25 111.10
57 4.95 4.86
58 19.43 19.14
59 2.89 2.82
60 7.52 7.51
61 36.07 36.07
62 4.51 4.70
63 84.39 86.33
64 301.21 298.27
65 12.50 12.86
66 173.27 176.11
67 3.22 3.25
68 32.75 33.09
69 3.09 3.25
70 11.03 11.60
71 253.10 255.00
72 22.65 22.70
73 100.41 101.57
74 15.37 15.30
75 217.33 220.84
76 6.39 6.21
77 8.61 8.53
78 91.03 91.88
79 187.20 189.04
80 7.00 7.11
81 214.55 215.98
82 13.26 13.75
83 2.53 2.55
84 92.39 91.00
85 5.18 5.16
86 29.67 29.77
87 3.07 3.03
88 195.01 198.39
89 3.69 3.72
90 33.04 33.78
91 281.74 285.06
92 4.40 4.50
93 2.21 2.25
94 8.52 8.59
95 11.99 11.77
96 100.03 99.16
97 289.38 291.08
98 10.44 9.86
99 73.25 74.20
100 3.01 3.04
Mapping above-mentioned data, see Fig. 2, the linear equation obtained is: y=0.9987x+0.482, coefficient R2 = 0.9997, show that the detectable of the present invention measures the accuracy height of cotinine clinical samples.
Owing to the detection process of the present invention is to be completed by instrument full-automation, so less demanding, easily to testing staff In realizing and promoting the use of.
It should be noted that the foregoing is only embodiments of the invention, not thereby limit the scope of the claims of the present invention, Every equivalent structure utilizing description of the invention and accompanying drawing content to be done or equivalence flow process conversion, or be directly or indirectly used in Other correlative technology fields, are the most in like manner included in the scope of patent protection of the present invention.

Claims (10)

1. a cotinine homogeneous enzyme immunoassay detectable, including: anti-cotinine specific antibody, to be used for detecting anti-cotinine special The indicator of heterogenetic antibody-cotinine complex；Above-mentioned anti-cotinine specific antibody is by cotinine immunogen immune animal Obtaining, the immunogenic structural formula of cotinine is as shown in formula I:
Formula I
In formula, carrier is for having immunogenic protein；Above-mentioned indicator selected from enzyme reagent, radiosiotope reagent, Fluorometric reagent or chemical illuminating reagent.
A kind of cotinine homogeneous enzyme immunoassay detectable the most according to claim 1, it is characterised in that above-mentioned indicator Selected from enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes glucose-6-phosphate dehydrogenase (G6PD)-half Antigen enzyme mark conjugate；The substrate of above-mentioned enzyme is G-6-P；Described glucose-6-phosphate dehydrogenase (G6PD)-hapten enzyme mark Conjugate is formed by glucose-6-phosphate dehydrogenase (G6PD) and cotinine derivatives reaction.
Cotinine homogeneous enzyme immunoassay detectable the most according to claim 2, it is characterised in that above-mentioned cotinine derivant Structural formula as shown in formula II:
Formula II.
4. cotinine derivant, structural formula is as shown in formula II:
Formula II.
Cotinine derivant the most according to claim 4, it is characterised in that synthetic route is:
。
6. the preparation method of a cotinine homogeneous enzyme immunoassay detectable, it is characterised in that comprise the steps:
(1) synthesis of the cotinine derivant described in claim 3,4 or 5 and purification, and carry out Structural Identification；
(2) the immunogenic synthesis of cotinine: make cotinine derivant and there is immunogenic protein carrier be connected；
(3) with cotinine immunogen immune animal, preparation purification anti-cotinine specific antibody；
(4) preparation of glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing: prepare glucose-6-phosphate dehydrogenase (G6PD) solution, swashs Cotinine derivant alive, makes glucose-6-phosphate dehydrogenase (G6PD) be connected with cotinine derivant, and purification connects product；
(5) preparation of cotinine homogeneous enzyme immunoassay detectable:
The preparation of reagent A: mixed by anti-cotinine specific antibody and homogeneous zymolyte；
The preparation of reagent B: mixed with Tris buffer by glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing.
The preparation method of a kind of cotinine homogeneous enzyme immunoassay detectable the most according to claim 6, it is characterised in that institute In the step (2) stated, protein carrier is BSA, and concrete synthesis step is as follows:
A. weigh 1.0～5.0 g potassium dihydrogen phosphates, 2.0～8.0 g disodium hydrogen phosphates, 5.0～15.0 g sodium chloride, 0.5～ 1.5 g magnesium chlorides, are dissolved in 1.0～3.0 L deionized waters jointly, regulate pH to 7.0～8.0, make buffer solution A；
B. weigh 2.0～5.0 mg BSA, be dissolved under room temperature in 2.0～5.0 mL above-mentioned buffer solution A, make BSA solution；
C. weighing 2.0～5.0 mg cotinine derivants, be dissolved in 200.0～500.0 μ l above-mentioned buffer solution A, making can For peaceful derivative solution；
D. when above-mentioned cotinine derivative solution just becomes clarification, it is added dropwise in above-mentioned BSA solution, then this is mixed Solution stirs 0.5～3.0 hour at 2-8 DEG C；
E. being dialysed with above-mentioned buffer solution A by reacted above-mentioned mixed solution, after dialysis, gained solution is cotinine Immunogen solution, adds the NaN of mass fraction 0.05%～0.3% in cotinine immunogen solution3, store at-20 DEG C.
The preparation method of a kind of cotinine homogeneous enzyme immunoassay detectable the most according to claim 6, it is characterised in that institute Step (4) detailed process stated is:
A. weigh 1.0～5.0 g potassium dihydrogen phosphates, 1.0～5.0 g disodium hydrogen phosphates, 5.0～15.0 g sodium chloride, 0.5～ 1.5 g magnesium chlorides, are dissolved in 1.0～3.0 L deionized waters jointly, regulate pH to 7.0～8.0, make buffer solution B；
B. weigh 5.0～20.0 mg glucose-6-phosphate dehydrogenase (G6PD)s, be dissolved in the 2.5～15.0 above-mentioned bufferings of mL under room temperature molten In liquid B, regulate pH to 8.5, make glucose-6-phosphate dehydrogenase (G6PD) solution；
C. weigh 2.0～5.0 mg cotinine derivants, be dissolved in the dimethylformamide that 100.0～500.0 μ l are dried (DMF), in, concussion is dissolved, and makes cotinine derivative solution；
D., when above-mentioned cotinine derivative solution just becomes clarification, it is added dropwise over above-mentioned glucose-6-phosphate dehydrogenase (G6PD) molten In liquid, complete dropping in 30 seconds, then this mixed solution is stirred 0.5～3.0 hour at 2-8 DEG C；
E. reacted above-mentioned mixed solution is purified by G-25 gel chromatography column, it is thus achieved that end product be Fructus Vitis viniferae Sugar-6-phosphate dehydrogenase-hapten conjugation thing, adds quality in glucose-6-phosphate dehydrogenase (G6PD)-hapten conjugation thing solution The BSA and the NaN of mass fraction 0.05%～0.3% of mark 0.3%～0.8%3, store at 2-8 DEG C.
The preparation method of a kind of cotinine homogeneous enzyme immunoassay detectable the most according to claim 6, it is characterised in that step Suddenly the detailed process of (5) is as follows:
The preparation of reagent A: by the nicotinamide adenine dinucleotide of oxidation state of 2.0～8.0 g, 1.0～3.0 Fructus Vitis viniferaes of g Sugar-6-phosphoric acid Tris the buffer solution that 0.5～3.0 L concentration are 55 mM, pH=8.0 makes homogeneous zymolyte；Will preparation Anti-cotinine specific antibody be added in above-mentioned homogeneous zymolyte, the volume ratio of antibody and homogeneous zymolyte is 1:100～1: 10000；
The preparation of reagent B: the glucose-6-phosphate dehydrogenase (G6PD) of preparation-hapten conjugation thing is added to concentration is 120 mM, pH= In the Tris buffer of 8.2, above-mentioned conjugate is 1:100～1:10000 with the volume ratio of Tris buffer.
10. utilizing the detection method of cotinine homogeneous enzyme immunoassay detectable described in claim 1 to 3 any one, it is special Levy and be, comprise the following steps:
(1) sample to be tested is contacted with anti-cotinine specific antibody；
(2) according to the combination situation of cotinine in sample to be tested Yu anti-cotinine specific antibody, indicator is utilized to judge sample The content of cotinine in Ben；
Described sample to be tested is serum, blood plasma, saliva, urine, seminal fluid or milk；
Described indicator is selected from enzyme reagent, including: enzyme mark conjugate and the substrate of enzyme；Above-mentioned enzyme mark conjugate includes Fructus Vitis viniferae Sugar-6-phosphate dehydrogenase-hapten enzyme mark conjugate；The substrate of above-mentioned enzyme is G-6-P；Described glucose-6-phosphorus Acidohydrogenase-hapten enzyme mark conjugate is formed by glucose-6-phosphate dehydrogenase (G6PD) and cotinine derivatives reaction.
CN201610574864.3A 2016-07-21 2016-07-21 Cotinine homogeneous enzyme immune detection reagent and preparation and detection methods thereof Pending CN106053788A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106496309A (en) * 2016-11-24 2017-03-15 北京开景基因技术有限公司 Microballoon antigen and preparation method thereof and the preparation method of anti-cotinine antibody
US11630106B2 (en) 2017-05-19 2023-04-18 Philip Morris Products S.A. Diagnostic test for distinguishing the smoking status of a subject

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KR20130117988A (en) * 2012-04-19 2013-10-29 주식회사 아큐젠헬스케어 Linker for increasing the efficiency of hapten-carrier conjugation
CN105092831A (en) * 2015-08-14 2015-11-25 苏州博源医疗科技有限公司 17-hydroxycorticosteroid immunodetection reagent and preparation method thereof

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Publication number Priority date Publication date Assignee Title
US5527686A (en) * 1991-07-29 1996-06-18 Serex, Inc. Differential binding affinities and dissociation assays based thereon
US6455047B1 (en) * 1997-09-19 2002-09-24 Serex, Inc. Methods to improve immunogenicity of antigens and specificity of antibodies
US20090253219A1 (en) * 2000-04-14 2009-10-08 Quantrx Biomedial Corporation Positive detection lateral-flow apparatus and method for small and large analytes
KR20130117988A (en) * 2012-04-19 2013-10-29 주식회사 아큐젠헬스케어 Linker for increasing the efficiency of hapten-carrier conjugation
CN105092831A (en) * 2015-08-14 2015-11-25 苏州博源医疗科技有限公司 17-hydroxycorticosteroid immunodetection reagent and preparation method thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106496309A (en) * 2016-11-24 2017-03-15 北京开景基因技术有限公司 Microballoon antigen and preparation method thereof and the preparation method of anti-cotinine antibody
US11630106B2 (en) 2017-05-19 2023-04-18 Philip Morris Products S.A. Diagnostic test for distinguishing the smoking status of a subject

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