CN105785011B - A kind of test strips for detecting maleic hydrazide and its preparation method and application - Google Patents
A kind of test strips for detecting maleic hydrazide and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of test strips for detecting maleic hydrazide and its preparation method and application, and the test strips include sample absorption pad, conjugate release pad, reaction film, adsorptive pads and bottom plate；It is characterized in that：Have to 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, the conjugate release pad on the reaction film and be coated with maleic hydrazide monoclonal antibody colloid gold label thing.The present invention also provides a kind of method for applying maleic hydrazide in above-mentioned ELISA test strip sample.The test strips and detection method that the present invention is provided have the advantages that simple to operate, sensitivity is high, detection speed is fast, cost is low, not examined equipment limit, can realize and maleic hydrazide in batch samples are used for quickly detecting and on-site supervision.
Description
Technical field
The present invention relates to the detection of maleic hydrazide, specifically a kind of colloidal gold strip for being used to detect maleic hydrazide, its is special
The detection that maleic hydrazide is remained suitable for tobacco leaf.
Background technology
Maleic hydrazide is also known as maleic acid hydrazide, and chemical name is maleic hydrazide（Maleic Hydrazide, MH）, it is a kind of
Plant growth regulator and selective herbicide.Maleic hydrazide can hinder plant cell division and reduction photosynthetic efficiency, therefore
It is commonly used for suppressing the vegetables such as storage period such as potato, onion and garlic germination, is also used in tobacco planting controlling tobacco leaf axillary bud
Growth.There is research to claim maleic hydrazide to be that cell chromosome can be caused to be broken under a kind of mutagenesis carcinogen, doses, so as to produce
Cytotoxicity, therefore Maleic Hydrazide Residues are of increasing concern.China provides that the maximum of maleic hydrazide is remained in garlic, onion, green onion
Limitation（MRL）For 15 mg/kg, the MRL of maleic hydrazide is 50 mg/kg in potato；International food code provide, garlic, onion,
The MRL of maleic hydrazide is that the MRL of maleic hydrazide in 15 mg/kg, potato is 50 mg/kg in green onion；Maleic hydrazide in European Union regulation garlic
MRL be 15 mg/kg；The U.S. provides that the MRL of maleic hydrazide is that the MRL of maleic hydrazide in 15 mg/kg, potato is 50 in onion
mg/kg；International tobacco scientific research Cooperation Centre（CORESTA）Provide the guiding residue limits of maleic hydrazide in tobacco（GRL）
For 80 mg/kg.
The residue analysis method of maleic hydrazide has a variety of：Distill AAS（AOAC）, HPLC- UV detection
Method（HPLC-UV）, HPLC MS（HPLC-MS）, gas chromatography, capillary electrophoresis, polarography etc., separately
Also there are the methods such as Flow Injection Analysis and pulse voltammetry outside, most common of which is preceding 3 kinds of analysis methods.But AOAC methods are to steaming
Distillation unit requires high, and sample pre-treatments are complicated, cumbersome, and alkali consumption is big, instrument seriously corroded；Maleic hydrazide in HPLC-UV methods
It is difficult to be separated with the interference impurity in sample, it is impossible to meet the detection requirement of actual sample；Maleic hydrazide is determined using HPLC-MS methods
Instrument cost it is then of a relatively high.Therefore, develop a kind of not examined equipment limit and can realize to enter batch samples
The problem of product and method of row quick detection turn into the urgent need to address.
The content of the invention
It is an object of the invention to overcome the high to device dependence of the existing method presence for detecting maleic hydrazide, and not
The problem of quick detection to batch samples can be realized there is provided a kind of simple to operate, sensitivity is high, detection speed is fast, into
Test strips of this low, not examined equipment limit and its preparation method and application, are entered with realizing to maleic hydrazide in batch samples
Row quick detection and on-site supervision.
In order to realize the purpose of the present invention, the invention provides a kind of test strips for detecting maleic hydrazide, the test strips include
Sample absorption pad, conjugate release pad, reaction film, adsorptive pads and bottom plate；Wherein, have on the reaction film and be coated with maleic hydrazide
In the detection line of hapten-carrier protein conjugate and the nature controlling line for being coated with sheep anti mouse antiantibody, the conjugate release pad
It is coated with maleic hydrazide monoclonal antibody-colloid gold label thing；The maleic hydrazide hapten-carrier protein conjugate is by maleic hydrazide half
Antigen is obtained with carrier protein couplet；The maleic hydrazide monoclonal antibody is made with maleic hydrazide hapten-carrier protein conjugate
Prepared for immunogene.
The carrier protein is bovine serum albumin(BSA), ovalbumin, hemocyanin, thyroprotein or human seralbumin egg
In vain.
The maleic hydrazide haptens is to generate sulfonic group maleic hydrazide by sulfonic group maleic anhydride and sulfuric acid hydrazine reaction, then with 3-
Hydracrylic acid reaction is obtained, and its molecular structural formula is：
The sheep anti mouse antiantibody is to carry out immune prepare to goat by immunogene of mouse source antibody.
The sample absorption pad, conjugate release pad, reaction film, adsorptive pads are pasted onto on bottom plate successively, the conjugate
Release pad 1/3 ~ 1/2 is capped under sample absorption pad.
The bottom plate is the material that PVC bottom plates or other hard do not absorb water；The sample absorption pad is suction strainer paper or oil strain
Paper；The conjugate release pad is mineral wool or polyester material；The adsorptive pads are blotting paper；The reaction film is cellulose nitrate
Plain film or cellulose acetate film.
A kind of method for preparing above-mentioned test strips, comprises the following steps：
1）Prepare the conjugate release pad for being coated with maleic hydrazide monoclonal antibody-colloid gold label thing；
2）Prepare that there is the detection line for being coated with maleic hydrazide hapten-carrier protein conjugate and sheep anti mouse is coated with and resist
The reaction film of the nature controlling line of body；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, adsorptive pads and the bottom plate prepared is assembled into
Test strips.
Specifically, step includes：
1）Sulfonic group maleic anhydride and sulfuric acid hydrazine reaction are generated into sulfonic group maleic hydrazide, then reacted with 3- hydracrylic acids, system
Standby maleic hydrazide haptens；
2）By maleic hydrazide haptens and carrier protein couplet, maleic hydrazide hapten-carrier protein conjugate is prepared；
3）Mouse is immunized with maleic hydrazide hapten-carrier protein conjugate, mouse boosting cell and myeloma cell are passed through
Fusion, screening, obtain secreting the hybridoma cell strain of maleic hydrazide monoclonal antibody；
4）Mouse IgG immune health goat is extracted, sheep anti mouse antiantibody is obtained；
5）Maleic hydrazide hapten-carrier protein conjugate and sheep anti mouse antiantibody are coated in the detection line of reaction film respectively
（T）And nature controlling line（C）On；
6）Collaurum is prepared with trisodium citrate and gold chloride reaction；
7）The maleic hydrazide monoclonal antibody of preparation is added in the collaurum of preparation, obtain maleic hydrazide monoclonal antibody-
Colloid gold label thing；
8）Maleic hydrazide monoclonal antibody-colloid gold label thing is sprayed in conjugate release pad, taken after 37 DEG C of 1 h of baking
Go out, be placed in dry environment and save backup；
9）Sample absorption pad is used and contains 0.5% bovine serum albumin(BSA)（Mass fraction）, pH 7.2 0.1 mol/L phosphate
Buffer solution soaks dries 2 h at 2 h, 37 DEG C；
10）Sample absorption pad, conjugate release pad, reaction film, adsorptive pads on being pasted in order on bottom plate, conjugate are released
Put pad has 1/3 region to be absorbed by the sample pad covering from initiating terminal.The wide small bars of 3 mm, plus plastic casing are finally cut into, is vacuum-packed,
It can be preserved 12 months under the conditions of 4 ~ 30 DEG C.The 1/3 of conjugate release pad, which is absorbed by the sample pad covering, can extend testing result sight
The time is examined, sample absorption pad can be made to detect that liquid is fully absorbed and fully reacted with gold labeling antibody, so as to reduce error.
Using the method for maleic hydrazide in above-mentioned ELISA test strip sample, comprise the following steps：
（1）Pre-treatment is carried out to sample；
（2）Detected with test strips；
（3）Analyze testing result.
Antibody antigen reaction and immunochromatographiassays assays of the test strips of the detection maleic hydrazide of the present invention using high degree of specificity
Technology, maleic hydrazide monoclonal antibody-colloid gold label thing is fixed in conjugate release pad, and the maleic hydrazide in sample is in flowing
During, combined with maleic hydrazide monoclonal antibody-colloid gold label thing in conjugate release pad, form maleic hydrazide-antibody-glue
Body gold label.Maleic hydrazide in sample is tied with the maleic hydrazide hapten-carrier protein conjugate competition in reaction film detection line
Close maleic hydrazide monoclonal antibody-colloid gold label thing, judged according to the detection line red stripes depth in analyte sample fluid whether
Contain maleic hydrazide residual.
During detection, sample instills sample absorption pad after processing, when the concentration of maleic hydrazide in the sample less than test limit or
When being zero, monoclonal antibody-colloid gold label thing meeting and the maleic hydrazide haptens-load being fixed on reaction film in chromatography process
Body protein conjugate is combined, in detection line（T）And nature controlling line（C）One red stripes of each appearance in place, and the colour developing of T lines is more aobvious than C line
Color depth is consistent with the colour developing of C lines；If the concentration of maleic hydrazide in the sample is equal to or higher than test limit, monoclonal antibody-colloid
Golden label can all be combined with maleic hydrazide, so that because competitive reaction will not be with maleic hydrazide hapten-carrier albumen at T lines
Conjugate combines and occurs without red stripes or developed the color than C line shallow.As shown in Figure 2.
It is negative：Work as nature controlling line（C）Show red stripes, detection line（T）Red stripes are also showed that simultaneously, and（T）Line
Color is close or is deeper than（C）During line, feminine gender is judged to.
It is positive：Work as nature controlling line（C）Show red stripes, and detection line（T）Do not develop the color or（T）Line color is shallower than（C）Line
When, it is judged to the positive.
It is invalid：Work as nature controlling line（C）Do not show red stripes, then no matter detection line（T）Whether show red stripes, should
It is invalid that test strips are judged to.
The test strips of the present invention have that sensitivity height, high specificity, cost are low, simple to operate, detection time is short, do not examined
Measurement equipment limitation, suitable various units are used, storage is 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, cost is low, easily promote the use of.
Brief description of the drawings
Fig. 1 is test strips cross-sectional view, in figure：1st, sample absorption pad；2nd, conjugate release pad；3rd, reaction film；
4th, adsorptive pads；5th, detection line；6th, nature controlling line；7th, bottom plate；
Fig. 2 is ELISA test strip result judgement figure；
Fig. 3 is maleic hydrazide hapten synthesis figure.
Embodiment
The present invention is expanded on further with reference to specific embodiment.It should be understood that these embodiments are merely to illustrate this
Invention, and it is not limited to the scope of the present invention.In addition, the scope that those skilled in the art limits in appended claims
Interior to carry out various changes or modification to the present invention, these are changed or modification should equally fall into the protection domain of invention.
The preparation of the test strips of the detection maleic hydrazide of embodiment 1
The preparation method of the test strips is mainly included the following steps that：
1）Prepare the conjugate release pad for being coated with maleic hydrazide monoclonal antibody-colloid gold label thing；
2）Prepare that there is the detection line for being coated with maleic hydrazide hapten-carrier protein conjugate and sheep anti mouse is coated with and resist
The reaction film of the nature controlling line of body；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, adsorptive pads and the assembling of PVC bottom plates prepared
Into test strips.
Substep narration in detail below：
1st, the synthesis of maleic hydrazide haptens（Synthetic route is shown in accompanying drawing 3）And identification
React a：Take the g of sulfonic group maleic anhydride 1.0, plus DMF（DMF）Dissolving, plus hydrazine sulfate 0.33
G, hydrogenation potassium oxide 0.2 g, 70 DEG C of h of stirring reaction 4.Stop reaction, add water, plus watery hydrochloric acid adjusts pH value to 6,1,2- dichloros
Ethane is extracted, and is divided and is gone aqueous phase, organic phase washing, concentration, upper silicagel column, 1:The elution separation of 1 chloroform-methanol, obtains sulfonic group suppression
Bud pellet 0.96 g, yield 89.7%.Nuclear-magnetism is identified1H NMR（Anhydrous Py, 300MHz）δ：8.10（t, 1H）, 8.0（t, 2H）,
8.326（13, 1H）, 2.02（t, 1H）；
React b：Take 0.96 g sulfonic group maleic hydrazides, plus pyridinium dissolution, plus the g of oxalyl chloride 0.62, plus DMF 0.2 mL, 60
DEG C 2 h of reaction, water on the rocks, ethyl acetate, extraction, anhydrous sodium sulfate drying, revolving is evaporated, plus acetonitrile dissolving, plus 3- hydroxyls third
0.77 g of acid, plus the mL of triethylamine 0.5, are stirred at room temperature 3 h.Stop reaction, be evaporated organic solvent, 1:2 n-hexanes-ether is tied again
Crystalline substance, obtains the g of sulfonylation hydracrylic acid maleic hydrazide haptens product 0.81, yield 60.1%.Nuclear-magnetism is identified1H NMR（CDCl3,
300MHz）δ：4.414（6, 2H, t, J=6.044）, 8.300（8, 1H）, 2.943（9, 2H, t, J=6.044）.
Chemical shift δ=2.9,4.4 be spacerarm hydroxyacetic acid on methylene hydrogen resonance absorbing peak, both presence card
Bright spacerarm is coupled successfully, and maleic hydrazide haptens structure is correct.
2nd, the synthesis and identification of maleic hydrazide coupled antigen
It is prepared by immunogene --- maleic hydrazide haptens and bovine serum albumin(BSA)（BSA）Coupling obtains immunogene（Also it can select
Other albumen such as human albumin, rabbit serum proteins）.
18 mg haptens are taken, are dissolved in 1 mL DMF；Plus carbodiimides（EDC）11 mg, stir 2 h at room temperature,
Plus n-hydroxysuccinimide（NHS）9 mg, continue to react 4 h, obtain reaction solution A；The mg of BSA 30 are weighed, are allowed to fully molten
Solution is in the mol/L phosphate buffers of 4 mL 0.1（PB, pH 7.0）In, reaction solution A is slowly dropped to protein solution dropwise
In, and 24 h are stirred at room temperature, with 0.01 mol/L phosphate buffers（PBS）4 DEG C of 3 d of dialysis, change 3 dialysis daily
Liquid, to remove unreacted small-molecule substance, obtains immunogene.
It is prepared by coating antigen --- maleic hydrazide haptens and ovalbumin（OVA）Coupling obtains coating antigen（Also it can select other
Albumen such as hemocyanin, fibrin）.
12 mg haptens are taken, are dissolved in 1 mL DMF；Plus the mg of EDC 7,24 h are stirred at room temperature, obtain reaction solution A；
The mg of OVA 40 are weighed, are allowed to be substantially dissolved in the mol/L PB of 6 mL 0.1（pH7.0）In, reaction solution A is slowly added dropwise dropwise
Into protein solution, and 24 h are stirred at room temperature, with 0.01 mol/L PBS, 4 DEG C of 3 d of dialysis, 3 dialyzates are changed daily,
To remove unreacted small-molecule substance, coating antigen is obtained.
In the ratio of synthesis maleic hydrazide coupled antigen reaction haptens, carrier protein and coupled product used, carry out ultraviolet
（200 ~ 400 nm）Sweep measuring, by compare three respectively 260 nm and 280 nm light absorption value calculate its combine than.
The maximum absorption band phase of the maximum absorption band and maleic hydrazide haptens, carrier protein of conjugate maleic hydrazide hapten-carrier albumen
Than there occurs obvious change, the synthesis for showing maleic hydrazide hapten-carrier albumen is successful.It is computed, haptens and BSA
Combination ratio be 15:1, and OVA combination ratio is 11:1.
3rd, the preparation of maleic hydrazide monoclonal antibody
（1）The acquisition of hybridoma
1）First immunisation：By maleic hydrazide haptens-BSA conjugate（Immunogene）Freund's complete adjuvant with equivalent is fully newborn
Change, the Balb/c mouse of 6 week old, every 0.2 mL is subcutaneously injected；
2）Booster immunization is twice：Since first immunisation, booster immunization once, is replaced with not formula Freund's incomplete adjuvant every two weeks
Freund's complete adjuvant, method and the same first immunisation of dosage；
3）Last time booster immunization eyeground vein blood sampling survey potency and suppression after one week, have suppression and potency reach 1:
Following final immunization is carried out when more than 10000：Intraperitoneal injection is not added with the mL of immunogen solution 0.1 of any adjuvant, is put to death after three days
Mouse, takes its spleen to be merged with myeloma cell；
4）Cell supernatant, the positive hole of screening are determined using indirect competitive enzyme-linked immunosorbent analysis method.Utilize limiting dilution
Method carries out cloning to positive hole, obtains and sets up the hybridoma cell strain of stably excreting maleic hydrazide monoclonal antibody, take and be in
Cell suspension is made with frozen stock solution in the hybridoma of exponential phase, is sub-packed in cryopreservation tube, is preserved for a long time in liquid nitrogen.
（2）The preparation of monoclonal antibody
1）Cell recovery：Maleic hydrazide monoclonal antibody hybridoma cell strain cryopreservation tube is taken out, is immediately placed in 37 DEG C of water-baths
Speed is melted, and centrifugation is removed after frozen stock solution, moves into culture culture in glassware；
2）Prepare ascites and antibody purification：Using method is induced in vivo, by Balb/c mouse（8 week old）Intraperitoneal injection sterilizing stone
Only, pneumoretroperitoneum injects hybridoma 5 × 10 to the mL/ of wax oil 0.5 within 7 days5Individual/only, gather ascites after 7 days.With octanoic acid-saturation sulphur
Sour ammonium method is purified, and obtains maleic hydrazide monoclonal antibody solution（- 20 DEG C of preservations）.
（3）The measure of antibody titer
The potency for determining antibody with indirect competitive ELISA method is 1:(200000~500000).
Indirect competitive ELISA method：With maleic hydrazide haptens-OVA conjugate coated elisa plates, maleic hydrazide standard items are added
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, is washed 3 ~ 5 times, is patted dry with blotting paper with cleaning solution；Add substrate nitrite ion, 25 DEG C of 15 min of reaction
Afterwards, terminate liquid terminating reaction is added；Setting ELIASA is determined at the nm of wavelength 450 per hole absorbance.
4th, the preparation of sheep anti mouse antiantibody
Using sheep as immune animal, pathogen-free domestic sheep is immunized by immunogene of mouse source antibody, sheep anti mouse is obtained and resists
Antibody.
5th, the preparation of maleic hydrazide monoclonal antibody-colloid gold label thing
（1）The preparation of collaurum
Mass fraction is diluted to 0.01% for 1% chlorauric acid solution with double distilled deionized water, takes 100 mL to be placed in taper
In bottle, boiling is heated to thermostatic electromagnetic agitator, it is 1% to add 1.5 mL mass fractions under continuous high temperature, lasting stirring
Citric acid three sodium solution, continuation be at the uniform velocity heated with stirring to solution in bright claret when stops, being cooled to after room temperature spend from
Sub- water returns to original volume, 4 DEG C of preservations.It is limpid transparent to prepare good collaurum and detect by an unaided eye, without muddiness, liquid
Surface is claret without floating object, the in the sunlight color of observing colloid gold.
（2）The preparation of maleic hydrazide monoclonal antibody-colloid gold label thing
Under magnetic stirring, the pH to 7.2 of collaurum is adjusted with 0.2 mol/L solution of potassium carbonate（The pH marks of different antibodies
Scope can change between 7 ~ 8）, it is molten to collaurum by the standard that 20 ~ 50 μ g antibody are added in every milliliter of colloidal gold solution
Above-mentioned maleic hydrazide monoclonal antibody is added in liquid, is stirred and evenly mixed, 10 min are stored at room temperature, adding 10% BSA makes it in collaurum
Whole mass fraction in solution is 1%, stands 10 min.12000 r/min, 4 DEG C of 40 min of centrifugation, abandon supernatant, precipitation is with again
Molten buffer solution is washed twice, and will be precipitated and is resuspended with the redissolution buffer solution that volume is the golden volume 1/10 of initial colloid, put 4 DEG C it is standby.
Redissolve buffer solution：Mass fraction containing BSA be 0.1% ~ 0.3%, Tween-80 mass fraction be 0.05% ~ 0.2%,
PH7.2 0.02 mol/L phosphate buffers.
6th, the preparation of conjugate release pad
Conjugate release pad is soaked in containing 0.5% BSA（Mass fraction）, pH 7.2 0.5 mol/L phosphate-buffereds
In liquid, 1 h is uniformly soaked, 37 DEG C of 3 h of baking are standby.Film instrument is sprayed by the maleic hydrazide prepared monoclonal antibody-colloid with Isoflow
Golden label even application is in conjugate release pad, and every 1 cm conjugates release pad sprays 0.01 mL maleic hydrazides monoclonal and resisted
After body-colloid gold label thing, it is placed in 37 DEG C of environment（Humidity ＜ 20%）Taken out after 60 min, be placed in dry environment（Humidity ＜
20%）In save backup.
7th, the preparation of reaction film
Detection line will be constituted in maleic hydrazide haptens-ovalbumin conjugate coating to reaction film, by sheep anti mouse antiantibody
It is coated on reaction film and constitutes nature controlling line.
Coating process：Maleic hydrazide haptens-ovalbumin conjugate is diluted to 1 mg/mL with phosphate buffer, used
Isoflow point film instruments are coated in the detection line on nitrocellulose filter（T）, package amount is 1.0 μ L/cm；With 0.01
Sheep anti mouse antiantibody is diluted to 200 μ g/mL by mol/L, pH7.4 phosphate buffer, is wrapped with Isoflow point film instruments
By in the nature controlling line on nitrocellulose filter（C）, package amount is 1.0 μ L/cm.The reaction film being coated with is placed in 37 DEG C of conditions
The lower h of drying 2, it is standby.
8th, the preparation of sample absorption pad
Sample absorption pad is used and contains 0.5% bovine serum albumin(BSA)（Mass fraction）, pH 7.2 0.1 mol/L phosphate delay
Fliud flushing soaks 2 h, and it is standby to dry 2 h at 37 DEG C.
9th, the assembling of test strips
Sample absorption pad, conjugate release pad, reaction film, adsorptive pads are pasted onto on PVC bottom plates in order successively；With reference to
Thing release pad has 1/3 region to be absorbed by the sample pad covering from initiating terminal, and the end of conjugate release pad and the top of reaction film connect
Connect, the end of reaction film is connected with the top of adsorptive pads, the top of sample absorption pad aligns with the top of PVC bottom plates, adsorptive pads
End alignd with the end of PVC bottom plates；There are detection line and nature controlling line, detection line on the reaction film（T）And nature controlling line（C）
In the strip tape perpendicular with the length of the test strips；Detection line is located at the side close to the end of conjugate release pad；Quality Control
Line is located remotely from the side of the end of conjugate release pad；Test strips are cut into the wide small bars of 3 mm with machine, mounted in special
In plastics fabrication, it can be preserved 12 months under the conditions of 4 ~ 30 DEG C.
The detection of maleic hydrazide in the sample of embodiment 2
1st, 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 from
In heart pipe, 6 mL acetonitriles are added, with the min of vortex instrument whirling motion 5；More than the rpm of room temperature 3000, centrifuges 5 min；Take upper organic phase
2 mL are into 10 mL polystyrene centrifuge tubes, in drying under 50~60 DEG C of water-bath nitrogen streams；100 μ L n-hexanes are added, whirlpool is used
The s of instrument whirling motion 30 is revolved, 0.5 mL samples is added and redissolves liquid（0.02mol/L PBS）, fully mixed with the s of vortex instrument whirling motion 30；
3000 rpm room temperatures（20-25 ℃）Centrifuge 5 min；Upper organic phase is removed, removing the μ L of layer aqueous phase 100 is used to analyze.
2nd, detected with test strips
The drop of sample solution 2 ~ 3 to be checked is drawn with Dispette vertically to drip in well, liquid starts timing when flowing,
React 10 min, result of determination.
3rd, testing result is analyzed
It is negative（-）：The colour developing of T lines develops the color deep or developed the color unanimously with C lines than C line, represents that maleic hydrazide concentration is less than inspection in sample
Survey limit, such as Fig. 2 a, 2b.
It is positive（+）：The colour developing of T lines does not develop the color than the shallow or T lines of C line colour developing, represents that maleic hydrazide concentration is equal to or higher than in sample
Test limit, such as Fig. 2 c, 2d.
It is invalid：Do not occur C lines, show the deterioration failure of incorrect operating process or test strips, such as Fig. 2 e, 2f.Herein
In the case of, specification should be read over again, and is retested with new test strips.
The sample detection example of embodiment 3
1st, test limit is tested
Take blank tobacco sample, wherein respectively addition maleic hydrazide to final concentration of 5,10,20 mg/kg, take test strips to enter
Row detection, each sample is repeated three times.
During with ELISA test strip tobacco sample, when wherein maleic hydrazide addition concentration is 5 mg/kg, shown in test strips
The colour developing of T lines develops the color deep or developed the color unanimously with C lines than C line, is negative；When wherein maleic hydrazide addition concentration is 10,20 mg/kg,
Show that the colour developing of T lines does not develop the color than the shallow or T lines of C line colour developing in test strips, be positive, show this test strips to maleic hydrazide in tobacco leaf
The mg/kg of test limit 10.
2nd, false positive rate, false negative rate experiment
The tobacco leaf positive and known maleic hydrazide content for taking known maleic hydrazide content to be more than 10 mg/kg are less than 10 mg/
Each 20 parts of kg tobacco leaf negative sample, is detected with three batches of test strips, calculates its yin and yang attribute rate.
As a result show：During the ELISA test strip positive produced with 3 batches, as a result it is all positive, it is known that positive sample
Product coincidence rate is 100%, and false negative rate is 0；When detecting negative sample, as a result it is all negative, it is known that negative sample coincidence rate is
100%, false positive rate is 0.Illustrating the test strips of the detection maleic hydrazide of the present invention can quickly be examined to the maleic hydrazide in tobacco leaf
Survey.
3rd, specific test
Daminozide, chrysanthemum esters medicine etc. are diluted to 500 mg/L with pH7.2,0.2 mol/L phosphate buffer, used
Maleic hydrazide test strips are detected.As a result show, during with the mg/L daminozides of ELISA test strip 500, the chrysanthemum esters medicine, test paper
The colour developing of bar T lines develops the color deep or developed the color unanimously with C lines than C line, is negative.Illustrate this test strips to daminozide, chrysanthemum esters medicine without
Cross reaction.
Claims (5)
1. a kind of test strips for detecting maleic hydrazide, including sample absorption pad, conjugate release pad, reaction film, adsorptive pads and bottom plate；
Characterized in that, there is the detection line for being coated with maleic hydrazide hapten-carrier protein conjugate on the reaction film and be coated with
Maleic hydrazide monoclonal antibody-colloid gold label thing is coated with the nature controlling line of sheep anti mouse antiantibody, the conjugate release pad；Institute
It is to be prepared using maleic hydrazide hapten-carrier protein conjugate as immunogene to state maleic hydrazide monoclonal antibody；The suckering
Red hapten-carrier protein conjugate is obtained by maleic hydrazide haptens and carrier protein couplet, and the carrier protein is cow's serum
Albumin, ovalbumin, hemocyanin, thyroprotein or human serum albumins, the maleic hydrazide haptens is by sulfonic group
Maleic anhydride generates sulfonic group maleic hydrazide with sulfuric acid hydrazine reaction, then is obtained with the reaction of 3- hydracrylic acids, and its molecular structural formula is：
。
2. test strips according to claim 1, it is characterised in that the sample absorption pad, conjugate release pad, reaction
Film, adsorptive pads are pasted onto on bottom plate successively, and conjugate release pad 1/3 ~ 1/2 is capped under sample absorption pad.
3. test strips according to claim 1, it is characterised in that it using mouse source antibody is immune that the sheep anti mouse antiantibody, which is,
It is former that immune prepare is carried out to goat.
4. a kind of method for preparing any one of the claim 1-3 test strips, it is characterised in that comprise the following steps：
1）Prepare the conjugate release pad for being coated with maleic hydrazide monoclonal antibody-colloid gold label thing；
2）Prepare with the detection line for being coated with maleic hydrazide hapten-carrier protein conjugate and be coated with sheep anti mouse antiantibody
The reaction film of nature controlling line；
3）By 1）With 2）Conjugate release pad, reaction film and sample absorption pad, adsorptive pads and the bottom plate prepared is assembled into test paper
Bar.
5. the method for maleic hydrazide in a kind of any one of the application claim 1-3 ELISA test strip tobacco sample, its feature exists
In this method comprises the following steps：
1）Pre-treatment is carried out to sample；
2）Detected with test strips；
3）Analyze testing result.
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| CN108169481B (en) * | 2017-07-27 | 2023-07-07 | 河北省中药材质量检验检测研究中心有限公司 | Test strip for detecting paeoniflorin and preparation method and application thereof |
| CN109061149B (en) * | 2018-09-21 | 2021-04-16 | 中国烟草总公司郑州烟草研究院 | Time-resolved fluorescence immunochromatographic test strip for detecting butralin and preparation method and application thereof |
| 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 |
| 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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