CN105785011A - Test strip for detecting maleic hydrazide, as well as preparation method and application thereof - Google Patents

Test strip for detecting maleic hydrazide, as well as preparation method and application thereof Download PDF

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CN105785011A
CN105785011A CN201610145751.1A CN201610145751A CN105785011A CN 105785011 A CN105785011 A CN 105785011A CN 201610145751 A CN201610145751 A CN 201610145751A CN 105785011 A CN105785011 A CN 105785011A
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maleic hydrazide
test strips
sample
pad
coated
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CN105785011B (en
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陈黎
范子彦
冯静
吴小胜
潘立宁
胡斌
唐纲岭
刘惠民
曹东山
崔华鹏
王晓瑜
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Beijing Kwinbon Biotechnology Co Ltd
Zhengzhou Tobacco Research Institute of CNTC
National Tobacco Quality Supervision and Inspection Center
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Beijing Kwinbon Biotechnology Co Ltd
Zhengzhou Tobacco Research Institute of CNTC
National Tobacco Quality Supervision and Inspection Center
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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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
    • 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
    • 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
    • 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
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody

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Abstract

The invention provides a test strip for detecting maleic hydrazide, as well as a preparation method and application thereof. The test strip comprises a sample absorption pad, a conjugate release pad, a reaction film, an absorbent pad and a bottom plate, and is characterized in that a detection line coated with a maleic hydrazide hapten-carrier protein conjugate and a quality control line coated with an anti-goat and anti-mouse antibody are arranged on the reaction film, and a maleic hydrazide monoclonal antibody-colloidal gold marker is sprayed on the conjugate release pad. The invention also provides a method for detecting the maleic hydrazide in a sample by virtue of the test strip. The test strip and the detection method have the advantages of being simple in operation, high in sensitivity and detection speed, low in cost and free of limits of detection equipment, and maleic hydrazide in a large batch of samples can be rapidly detected and monitored on the spot.

Description

A kind of test strips detecting maleic hydrazide and its preparation method and application
Technical field
The present invention relates to the detection of maleic hydrazide, a kind of colloidal gold strip for detecting maleic hydrazide, it is special The detection of maleic hydrazide residual be applicable to tobacco leaf.
Background technology
Maleic hydrazide is also known as maleic acid hydrazide, and chemical name is maleic hydrazide (Maleic Hydrazide, MH), is a kind of Plant growth regulator and selective herbicide.Maleic hydrazide can hinder plant cell division and reduce photosynthetic efficiency, therefore It is commonly used for suppressing the vegetables such as storage period such as potato, onion and garlic to germinate, is also used in tobacco planting controlling tobacco leaf axillalry bud Growth.There is research to claim maleic hydrazide to be a kind of mutagenesis carcinogen, cell chromosome can be caused under doses to rupture, thus produce Cytotoxicity, therefore Maleic Hydrazide Residues is of increasing concern.China specifies, the maximum residual of maleic hydrazide in garlic, onion, green onion Limitation (MRL) is 15 mg/kg, and in potato, the MRL of maleic hydrazide is 50 mg/kg；International food code specify, garlic, onion, In green onion, the MRL of maleic hydrazide is 15 mg/kg, and in potato, the MRL of maleic hydrazide is 50 mg/kg；Maleic hydrazide in European Union regulation garlic MRL be 15 mg/kg；The U.S. specifies, in onion, the MRL of maleic hydrazide is 15 mg/kg, and in potato, the MRL of maleic hydrazide is 50 mg/kg；The guiding residue limits (GRL) of maleic hydrazide in international tobacco scientific research Cooperation Centre (CORESTA) regulation tobacco It is 80 mg/kg.
The residue analysis method of maleic hydrazide has multiple: distillation AAS (AOAC), HPLC-UV detection Method (HPLC-UV), HPLC MS (HPLC-MS), gas chromatography, capillary electrophoresis, polarography etc., separately Also having the method such as Flow Injection Analysis and pulse voltammetry outward, most common of which is front 3 kinds of analysis methods.But AOAC method is to steaming Distillation unit requires height, and sample pre-treatments is complicated, complex operation, and alkali consumption is big, instrument seriously corroded；Maleic hydrazide in HPLC-UV method It is difficult to separate with the interference impurity in sample, it is impossible to meet the testing requirement of actual sample；HPLC-MS method is used to measure maleic hydrazide Instrument cost the most of a relatively high.Therefore, the exploitation the most examined equipment of one limits and is capable of entering batch samples The product of row quickly detection and method become problem in the urgent need to address.
Summary of the invention
It is an object of the invention to overcome that the method for existing detection maleic hydrazide exists is high to device dependence, and not The problem being capable of the quick detection to batch samples, it is provided that one is simple to operate, highly sensitive, detection speed is fast, one-tenth Test strips that this equipment low, the most examined limits and its preparation method and application, to realize entering maleic hydrazide in batch samples Row quickly detection and on-site supervision.
In order to realize the purpose of the present invention, the invention provides a kind of test strips detecting maleic hydrazide, this test strips includes Sample absorption pad, bond release pad, reaction film, adsorptive pads and base plate；Wherein, described reaction film has it is coated with maleic hydrazide The detection line of hapten-carrier protein conjugate and the nature controlling line being coated with sheep anti mouse antiantibody, in described bond release pad It is coated with maleic hydrazide monoclonal antibody-colloid gold label thing；Described maleic hydrazide hapten-carrier protein conjugate is by maleic hydrazide half Antigen obtains with carrier protein couplet；Described maleic hydrazide monoclonal antibody is to make with maleic hydrazide hapten-carrier protein conjugate Prepare for immunogene.
Described carrier protein is bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein or human seralbumin egg In vain.
Described maleic hydrazide haptens be by sulfonic group maleic anhydride and hydrazine sulfate react generation sulfonic group maleic hydrazide, then with 3- Hydracrylic acid reaction obtains, and its molecular structural formula is:
Described sheep anti mouse antiantibody is for immunogene, goat to be carried out immunity with mouse source antibody to prepare.
Described sample absorption pad, bond release pad, reaction film, adsorptive pads are pasted onto on base plate successively, described bond Release pad 1/3 ~ 1/2 is capped under sample absorption pad.
The material that described base plate is PVC base plate or other hard do not absorb water；Described sample absorption pad is suction strainer paper or oil strain Paper；Described bond release pad is mineral wool or polyester material；Described adsorptive pads is blotting paper；Described reaction film is cellulose nitrate Element film or CAM.
A kind of method preparing above-mentioned test strips, comprises the following steps:
1) preparation is coated with the bond release pad of maleic hydrazide monoclonal antibody-colloid gold label thing；
2) preparation has the detection line being coated with maleic hydrazide hapten-carrier protein conjugate and is coated with sheep anti mouse antiantibody The reaction film of nature controlling line；
3) by 1) and 2) the bond release pad for preparing, reaction film be assembled into test paper with sample absorption pad, adsorptive pads and base plate Bar.
Specifically, step includes:
1) sulfonic group maleic anhydride and hydrazine sulfate reacting generation sulfonic group maleic hydrazide, then reacts with 3-hydracrylic acid, preparation presses down Bud pellet haptens；
2) by maleic hydrazide haptens and carrier protein couplet, maleic hydrazide hapten-carrier protein conjugate is prepared；
3) with maleic hydrazide hapten-carrier protein conjugate immune mouse, by mouse boosting cell and myeloma cell by merging, Screening, obtains secreting the hybridoma cell strain of maleic hydrazide monoclonal antibody；
4) extract mouse IgG immune health goat, obtain sheep anti mouse antiantibody；
5) respectively maleic hydrazide hapten-carrier protein conjugate and sheep anti mouse antiantibody are coated in the detection line (T) of reaction film With on nature controlling line (C)；
6) collaurum is prepared with trisodium citrate and gold chloride reaction；
7) the maleic hydrazide monoclonal antibody of preparation is joined in the collaurum of preparation, obtain maleic hydrazide monoclonal antibody-colloid Gold label；
8) being sprayed in bond release pad by maleic hydrazide monoclonal antibody-colloid gold label thing, 37 DEG C are dried taking-up after 1 h, put Save backup in dry environment；
9) by sample absorption pad with containing 0.5% bovine serum albumin(BSA) (mass fraction), the 0.1 mol/L phosphate-buffered of pH 7.2 Immersion steeps 2 h, dries 2 h at 37 DEG C；
10) on base plate, sample absorption pad, bond release pad, reaction film, adsorptive pads, bond release pad are pasted in order Have 1/3 region to be absorbed by the sample pad from initiating terminal to cover.Finally it is cut into the wide little bar of 3 mm, adds plastic casing, vacuum packaging, 4 ~ 30 Can preserve 12 months under the conditions of DEG C.The 1/3 of bond release pad be absorbed by the sample pad covering can extend testing result observe time Between, sample absorption pad can be made to be fully absorbed by detection liquid and fully react with gold labeling antibody, thus reducing error.
Apply the method for maleic hydrazide in above-mentioned ELISA test strip sample, comprise the steps:
(1) sample is carried out pre-treatment；
(2) detect by test strips；
(3) testing result is analyzed.
The test strips of the detection maleic hydrazide of the present invention uses antibody antigen reaction and the immunochromatographiassays assays of high degree of specificity Technology, is fixed on maleic hydrazide monoclonal antibody-colloid gold label thing in bond release pad, and the maleic hydrazide in sample is in flowing During, the maleic hydrazide monoclonal antibody-colloid gold label thing in bond release pad is combined, and forms maleic hydrazide-antibody-glue Body gold label.Maleic hydrazide in sample and the maleic hydrazide hapten-carrier protein conjugate competition knot on reaction film detection line Close maleic hydrazide monoclonal antibody-colloid gold label thing, whether judge in analyte sample fluid according to the detection line red stripes depth Remain containing maleic hydrazide.
During detection, sample instills sample absorption pad after treatment, when maleic hydrazide concentration in the sample less than detection limit or When being zero, monoclonal antibody-colloid gold label thing can be with the maleic hydrazide haptens-load being fixed on reaction film in chromatography process Body protein conjugate combines, and a red stripes respectively occurs in detection line (T) and nature controlling line (C) place, and the colour developing of T line shows than C line Color depth or consistent with the colour developing of C line；If the concentration that maleic hydrazide is in the sample is equal to or higher than detection limit, monoclonal antibody-colloid Gold label all can be combined with maleic hydrazide, thus because competitive reaction will not be with maleic hydrazide hapten-carrier albumen at T line Conjugate combines and occurs without red stripes or more shallow than the colour developing of C line.As shown in Figure 2.
Negative: when nature controlling line (C) demonstrates that red stripes, detection line (T) also show that red stripes, and (T) line simultaneously When color is close or is deeper than (C) line, it is judged to feminine gender.
Positive: when nature controlling line (C) demonstrates red stripes, and detect line (T) and do not develop the color or (T) line color is shallower than (C) line Time, it is judged to the positive.
Invalid: when nature controlling line (C) does not demonstrate red stripes, the most no matter to detect whether line (T) demonstrates red stripes, should It is invalid that test strips is all judged to.
The test strips of the present invention has highly sensitive, high specificity, low cost, simple to operate, the detection time is short, do not examined Measurement equipment limits, is suitable for the use of various units, stores simple, the advantage of long shelf-life.With ELISA test strip maleic hydrazide of the present invention Method, easy, quick, directly perceived, accurate, applied widely, low cost, easily promote the use of.
Accompanying drawing explanation
Fig. 1 is test strips cross-sectional view, in figure: 1, sample absorption pad；2, bond release pad；3, reaction film； 4, adsorptive pads；5, detection line；6, nature controlling line；7, base plate；
Fig. 2 is ELISA test strip result process decision chart；
Fig. 3 is maleic hydrazide hapten synthesis figure.
Detailed description of the invention
The present invention is expanded on further below in conjunction with specific embodiment.Should be understood that these embodiments are merely to illustrate this Invention, and it is not limited to the scope of the present invention.It addition, the scope that those skilled in the art limits at appended claims In the present invention may be carried out various change or modification, these are changed or modify and should fall into the protection domain of invention equally.
Embodiment 1 detects the preparation of the test strips of maleic hydrazide
The preparation method of this test strips mainly comprises the steps that
1) preparation is coated with the bond release pad of maleic hydrazide monoclonal antibody-colloid gold label thing；
2) preparation has the detection line being coated with maleic hydrazide hapten-carrier protein conjugate and is coated with sheep anti mouse antiantibody The reaction film of nature controlling line；
3) by 1) and 2) the bond release pad for preparing, reaction film be assembled into examination with sample absorption pad, adsorptive pads and PVC base plate Paper slip.
Substep narration in detail below:
1, the haptenic synthesis of maleic hydrazide (synthetic route is shown in accompanying drawing 3) and qualification
Reaction a: take sulfonic group maleic anhydride 1.0 g, adds DMF (DMF) and dissolves, add hydrazine sulfate 0.33 g, add Potassium hydroxide 0.2 g, 70 DEG C of stirring reaction 4 h.Stop reaction, add water, add watery hydrochloric acid regulation pH value to 6,1,2-dichloroethanes Extraction, divides and removes aqueous phase, and organic phase is washed, and concentrates, upper silicagel column, and 1:1 chloroform-methanol wash-out separates, and obtains sulfonic group maleic hydrazide 0.96 g, yield 89.7%.Nuclear-magnetism is identified1The anhydrous Py of H NMR(, 300MHz) δ: 8.10(t, 1H), 8.0(t, 2H) and, 8.326 (13,1H), 2.02(t, 1H)；
Reaction b: take 0.96 g sulfonic group maleic hydrazide, add pyridinium dissolution, add oxalyl chloride 0.62 g, adds DMF 0.2 mL, and 60 DEG C anti- Answering 2 h, with frozen water, ethyl acetate, extraction, anhydrous sodium sulfate is dried, and rotation steaming is evaporated, and adds acetonitrile and dissolves, adds 3-hydracrylic acid 0.77 g, adds triethylamine 0.5 mL, 3 hs is stirred at room temperature.Stop reaction, be evaporated organic solvent, 1:2 n-hexane-Diethyl ether recrystallization, Obtain sulfonylation hydracrylic acid maleic hydrazide haptens product 0.81 g, yield 60.1%.Nuclear-magnetism is identified1H NMR(CDCl3, 300MHz) δ: 4.414(6,2H, t, J=6.044), 8.300(8,1H) and, 2.943(9,2H, t, J=6.044).
Chemical shift δ=2.9,4.4 it is the resonance absorbing peak of methylene hydrogen on spacerarm hydroxyacetic acid, both existence cards Bright spacerarm coupling success, maleic hydrazide haptens structure is correct.
2, the synthesis of maleic hydrazide coupled antigen and qualification
Immunogene prepares maleic hydrazide haptens and bovine serum albumin(BSA) (BSA) coupling obtains immunogene and (also can be selected for other Albumen such as human albumin, rabbit serum proteins).
Take 18 mg haptens, be dissolved in 1 mL DMF；Add carbodiimides (EDC) 11 mg, under room temperature, stir 2 h, Add N-hydroxy-succinamide (NHS) 9 mg, continue reaction 4 h, obtain reactant liquor A；Weigh BSA 30 mg, be allowed to the most molten Reactant liquor A, in 4 mL 0.1 mol/L phosphate buffers (PB, pH 7.0), is dropwise slowly dropped to protein solution by solution In, and stir 24 h at room temperature, with 0.01 mol/L phosphate buffer (PBS) 4 DEG C dialysis 3 d, change 3 dialysis every day Liquid, to remove unreacted small-molecule substance, obtains immunogene.
Coating antigen prepares maleic hydrazide haptens and ovalbumin (OVA) coupling obtains coating antigen and (also can be selected for other albumen Such as hemocyanin, fibrin).
Take 12 mg haptens, be dissolved in 1 mL DMF；Add EDC 7 mg, under room temperature, stir 24 h, obtain reactant liquor A； Weigh OVA 40 mg, be allowed to be substantially dissolved in 6 mL 0.1 mol/L PB(pH7.0) in, reactant liquor A is dropwise slowly added dropwise In protein solution, and stir 24 h at room temperature, with 0.01 mol/L PBS 4 DEG C dialysis 3 d, change 3 dislysates every day, To remove unreacted small-molecule substance, obtain coating antigen.
In synthesis maleic hydrazide coupled antigen reaction haptens used, carrier protein and the ratio of coupled product, carry out ultraviolet (200 ~ 400 nm) sweep measuring, calculate its combination ratio at the light absorption value of 260 nm and 280 nm respectively by comparing three.Conjugate The maximum absorption band of maleic hydrazide hapten-carrier albumen and maleic hydrazide haptens, carrier protein maximum absorption band compared with generation Obvious change, shows that the synthesis of maleic hydrazide hapten-carrier albumen is successful.It is computed, haptens and the combination of BSA Than being 15:1, the ratio of the combination with OVA is for 11:1.
3, the preparation of maleic hydrazide monoclonal antibody
(1) acquisition of hybridoma
1) first immunisation: by fully emulsified with the Freund's complete adjuvant of equivalent for maleic hydrazide haptens-BSA conjugate (immunogene), The Balb/c mouse of hypodermic injection 6 week old, every 0.2 mL；
2) booster immunization twice: from the beginning of first immunisation, booster immunization is once every two weeks, replaces Freund with not formula Freund's incomplete adjuvant The same first immunisation of Freund's complete adjuvant, method and dosage；
3) eyeground vein blood sampling survey titer and the suppression after a week of last booster immunization, has suppression and titer to reach 1:10000 Carry out following final immunization time above: lumbar injection is not added with immunogen solution 0.1 mL of any adjuvant, after three days, put to death mouse, Take its spleen and myeloma cell fusion；
4) use indirect competitive enzyme-linked immunosorbent to analyze method and measure cell supernatant, the positive hole of screening.Utilize limiting dilution assay pair Positive hole carries out cloning, obtains and sets up the hybridoma cell strain of stably excreting maleic hydrazide monoclonal antibody, take and be in logarithm The hybridoma frozen stock solution in growth period makes cell suspension, is sub-packed in cryopreservation tube, preserves for a long time in liquid nitrogen.
(2) preparation of monoclonal antibody
1) cell recovery: take out maleic hydrazide monoclonal antibody hybridoma cell strain cryopreservation tube, be immediately placed in 37 DEG C of water-bath middling speeds and melt, After centrifugal segregation frozen stock solution, move into and cultivate culture in glassware；
2) ascites and antibody purification are prepared: use and internal induce method, by Balb/c mouse (8 week old) Intraperitoneal injection sterilizing paraffin oil Only, within 7 days, pneumoretroperitoneum injects hybridoma 5 × 10 to 0.5 mL/5Individual/only, gather ascites after 7 days.With octanoic acid-saturated ammonium sulfate Method is purified, and obtains maleic hydrazide monoclonal antibody solution (-20 DEG C of preservations).
(3) mensuration of antibody titer
The titer measuring antibody with indirect competitive ELISA method is 1:(200000 ~ 500000).
Indirect competitive ELISA method: with maleic hydrazide haptens-OVA conjugate coated elisa plate, add maleic hydrazide standard items The sheep anti mouse antiantibody solution of solution, maleic hydrazide monoclonal antibody solution and horseradish peroxidase-labeled, 25 DEG C of reactions 30 Min, pours out liquid in hole, washs 3 ~ 5 times with cleaning solution, pats dry with blotting paper；Add substrate nitrite ion, 25 DEG C of reaction 15 min After, add stop buffer and terminate reaction；Set ELIASA at wavelength 450 nm, measure every hole absorbance.
4, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, for immunogene, pathogen-free domestic sheep is carried out immunity with mouse source antibody, obtain sheep anti mouse antiantibody.
5, the preparation of maleic hydrazide monoclonal antibody-colloid gold label thing
(1) preparation of collaurum
By double distilled deionized water, the chlorauric acid solution that mass fraction is 1% is diluted to 0.01%, takes 100 mL and be placed in conical flask, It is heated to boiling with thermostatic electromagnetic agitator, is the lemon of 1% at continuous high temperature, continuously stirred lower addition 1.5 mL mass fraction Acid three sodium solutions, continue at the uniform velocity to be heated with stirring to when solution is bright claret stop, using deionized water after being cooled to room temperature Return to original volume, 4 DEG C of preservations.It is limpid transparent for prepare good collaurum detecting by an unaided eye, and does not has muddiness, liquid surface Without floating thing, the color of observing colloid gold is claret in the sunlight.
(2) preparation of maleic hydrazide monoclonal antibody-colloid gold label thing
Under magnetic stirring, the pH label range of pH to the 7.2(different antibodies of collaurum is adjusted with 0.2 mol/L solution of potassium carbonate Between 7 ~ 8, can change), by every milliliter of colloidal gold solution adding the standard of 20 ~ 50 μ g antibody in colloidal gold solution Adding above-mentioned maleic hydrazide monoclonal antibody, stir and evenly mix, room temperature stands 10 min, adds 10% BSA and makes it at colloidal gold solution In whole mass fraction be 1%, stand 10 min.12000 r/min, 4 DEG C of centrifugal 40 min, abandon supernatant, and precipitation is slow with redissolving Rush liquid to wash twice, will precipitate resuspended with the redissolution buffer solution that volume is initial colloid gold volume 1/10, put 4 DEG C standby.
Redissolve buffer solution: the mass fraction containing BSA is 0.1% ~ 0.3%, the mass fraction of Tween-80 is 0.05% ~ 0.2%, The 0.02 mol/L phosphate buffer of pH7.2.
6, the preparation of bond release pad
Bond release pad is soaked in containing 0.5% BSA(mass fraction), in the 0.5 mol/L phosphate buffer of pH 7.2, Uniformly soaking 1 h, 37 DEG C of baking 3 h are standby.Maleic hydrazide monoclonal antibody-collaurum the mark that will prepare with Isoflow spray film instrument Note thing even application is in bond release pad, and every 1 cm bond release pad sprays 0.01 mL maleic hydrazide monoclonal antibody-glue After body gold label, it is placed in 37 DEG C of environment and takes out after (humidity ＜ 20%) 60 min, be placed in dry environment (humidity ＜ 20%) Save backup.
7, the preparation of reaction film
Maleic hydrazide haptens-ovalbumin conjugate is coated on reaction film composition detection line, sheep anti mouse antiantibody is coated Reaction film constitutes nature controlling line.
It is coated process: with phosphate buffer, maleic hydrazide haptens-ovalbumin conjugate is diluted to 1 mg/mL, uses The detection line (T) that Isoflow point film instrument is coated on nitrocellulose filter, package amount is 1.0 μ L/cm；With 0.01 Sheep anti mouse antiantibody is diluted to 200 μ g/mL by the phosphate buffer of mol/L, pH7.4, is wrapped with Isoflow point film instrument By the nature controlling line (C) on nitrocellulose filter, package amount is 1.0 μ L/cm.The reaction film being coated is placed in 37 DEG C of conditions Under be dried 2 h, standby.
8, the preparation of sample absorption pad
By sample absorption pad with containing 0.5% bovine serum albumin(BSA) (mass fraction), the 0.1 mol/L phosphate buffer of pH 7.2 Soak 2 h, dry 2 h at 37 DEG C standby.
9, the assembling of test strips
Sample absorption pad, bond release pad, reaction film, adsorptive pads are pasted onto on PVC base plate the most in order；Bond is released Putting pad has 1/3 region to be absorbed by the sample pad covering from initiating terminal, and the end of bond release pad is connected with the top of reaction film, instead Answering the end of film to be connected with the top of adsorptive pads, the top of sample absorption pad aligns with the top of PVC base plate, the end of adsorptive pads Align with the end of PVC base plate；Have detection line and nature controlling line on described reaction film, detection line (T) and nature controlling line (C) all in institute State the strip tape that the length of test strips is perpendicular；Detection line is located close to the side of the end of bond release pad；Nature controlling line is positioned at Side away from the end of bond release pad；Test strips machine is cut into the wide little bar of 3 mm, is contained in special plastics system In card, can preserve 12 months under the conditions of 4 ~ 30 DEG C.
The detection of maleic hydrazide in embodiment 2 sample
1, the pre-treatment of sample
With homogenizer homogeneous tobacco leaf sample；Weigh the tobacco leaf sample after 3.0 ± 0.05 g homogeneous to 15 mL polystyrene centrifuge tubes In, add 6 mL acetonitriles, with vortex instrument whirling motion 5 min；Room temperature 3000 more than rpm, centrifugal 5 min；Take upper organic phase 2 mL To 10 mL polystyrene centrifuge tubes, flow down in 50～60 DEG C of water-bath nitrogen and dry up；Add 100 L n-hexanes, use vortex instrument Whirling motion 30 s, adds 0.5 mL sample redissolution liquid (0.02mol/L PBS), fully mixes with vortex instrument whirling motion 30 s；3000 Rpm room temperature (20-25 DEG C) is centrifuged 5 min；Remove upper organic phase, take off layer aqueous phase 100 L for analyzing.
2, detect by test strips
Draw sample solution to be checked 2 ~ 3 with Dispette vertically to drip in well, start timing during liquid flowing, reaction 10 min, it is determined that result.
3, testing result is analyzed
Negative (-): the colour developing of T line is deeper than the colour developing of C line or consistent with the colour developing of C line, represents that in sample, maleic hydrazide concentration is less than detection limit, Such as Fig. 2 a, 2b.
Positive (+): the colour developing of T line is more shallow than the colour developing of C line or T line does not develops the color, and represents that in sample, maleic hydrazide concentration equals to or higher than Detection limit, such as Fig. 2 c, 2d.
Invalid: C line does not occurs, show the deterioration failure of incorrect operating process or test strips, such as Fig. 2 e, 2f.At this In the case of, should again read over specification, and retest by new test strips.
Embodiment 3 sample detection example
1, detection limit test
Take blank tobacco sample, add maleic hydrazide extremely final concentration of 5,10,20 mg/kg the most respectively, take test strips and examine Surveying, each sample is repeated three times.
During with ELISA test strip tobacco sample, when wherein maleic hydrazide interpolation concentration is 5 mg/kg, test strips demonstrates The colour developing of T line is deeper than the colour developing of C line or consistent with the colour developing of C line, is negative；Be 10 when wherein maleic hydrazide adds concentration, 20 mg/kg time, Demonstrate in test strips that the colour developing of T line is more shallow than the colour developing of C line or T line does not develops the color, be positive, show that this test strips is to maleic hydrazide in tobacco leaf Detection limit 10 mg/kg.
2, false positive rate, false negative rate test
Take the known maleic hydrazide content tobacco leaf positive more than 10 mg/kg and known maleic hydrazide content less than 10 mg/kg's Each 20 parts of tobacco leaf negative sample, detects by three batches of test strips, calculates its yin and yang attribute rate.
Result shows: during the ELISA test strip positive produced by 3 batches, result is all positive, it is known that positive sample Product coincidence rate is 100%, and false negative rate is 0；During detection negative sample, result is all negative, it is known that negative sample coincidence rate is 100%, false positive rate is 0.Illustrate that the maleic hydrazide in tobacco leaf can quickly be examined by the test strips of the detection maleic hydrazide of the present invention Survey.
3, specific test
Daminozide, chrysanthemum esters medicine etc. are diluted to 500 mg/L with the phosphate buffer of pH7.2,0.2 mol/L, with pressing down bud Red test strips detects.Result shows, during with this ELISA test strip 500 mg/L daminozide, chrysanthemum esters medicine, and test strips T line Develop the color deeper than the colour developing of C line or consistent with the colour developing of C line, be negative.Illustrate that this test strips is anti-without intersecting to daminozide, chrysanthemum esters medicine Should.

Claims (7)

1. detect a test strips for maleic hydrazide, including sample absorption pad, bond release pad, reaction film, adsorptive pads and base plate； It is characterized in that having on described reaction film and be coated with the detection line of maleic hydrazide hapten-carrier protein conjugate and be coated with The nature controlling line of sheep anti mouse antiantibody, described bond release pad is coated with maleic hydrazide monoclonal antibody-colloid gold label thing；Institute State maleic hydrazide hapten-carrier protein conjugate to be obtained with carrier protein couplet by maleic hydrazide haptens；Described maleic hydrazide Dan Ke Grand antibody is to prepare using maleic hydrazide hapten-carrier protein conjugate as immunogene.
Test strips the most according to claim 1, it is characterised in that described sample absorption pad, bond release pad, reaction Film, adsorptive pads are pasted onto on base plate successively, and bond release pad 1/3 ~ 1/2 is capped under sample absorption pad.
Test strips the most according to claim 1, it is characterised in that described carrier protein is bovine serum albumin(BSA), egg white egg In vain, hemocyanin, thyroprotein or human serum albumins.
Test strips the most according to claim 1, it is characterised in that described maleic hydrazide haptens is by sulfonic group maleic anhydride Reacting generation sulfonic group maleic hydrazide with hydrazine sulfate, then react with 3-hydracrylic acid and obtain, its molecular structural formula is:
。
Test strips the most according to claim 1, it is characterised in that described sheep anti mouse antiantibody is with mouse source antibody as immunity Former goat is carried out immunity prepare.
6. prepare the method for test strips described in any one of claim 1-5 for one kind, it is characterised in that comprise the following steps:
1) preparation is coated with the bond release pad of maleic hydrazide monoclonal antibody-colloid gold label thing；
2) preparation has the detection line being coated with maleic hydrazide hapten-carrier protein conjugate and is coated with sheep anti mouse antiantibody The reaction film of nature controlling line；
3) by 1) and 2) the bond release pad for preparing, reaction film be assembled into test paper with sample absorption pad, adsorptive pads and base plate Bar.
7. apply the method for maleic hydrazide in ELISA test strip tobacco sample described in claim 1-5 for one kind, it is characterised in that the party Method comprises the following steps:
1) sample is carried out pre-treatment；
2) detect by test strips；
3) testing result is analyzed.
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CN109061156A (en) * 2018-09-21 2018-12-21 中国烟草总公司郑州烟草研究院 A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting pendimethalin
CN109061148A (en) * 2018-09-21 2018-12-21 中国烟草总公司郑州烟草研究院 A kind of test strips and its preparation method and application detecting butralin
CN109061149A (en) * 2018-09-21 2018-12-21 中国烟草总公司郑州烟草研究院 A kind of time-resolved fluoroimmunoassay chromatograph test strip and its preparation method and application detecting butralin
CN109061156B (en) * 2018-09-21 2021-07-13 中国烟草总公司郑州烟草研究院 Time-resolved fluorescence immunochromatographic test strip for detecting pendimethalin and preparation method and application thereof
CN109061148B (en) * 2018-09-21 2021-07-16 中国烟草总公司郑州烟草研究院 Test strip for detecting butralin and preparation method and application thereof
CN111856000A (en) * 2020-06-04 2020-10-30 北京勤邦生物技术有限公司 Test strip and method for detecting chlordane
CN111856000B (en) * 2020-06-04 2023-07-07 北京勤邦科技股份有限公司 Test strip and method for detecting chlordane

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