CN203178269U - Imidacloprid ELISA (Enzyme Linked ImmunoSorbent Assay) detection kit - Google Patents

Abstract

The utility model relates to an imidacloprid ELISA (Enzyme Linked ImmunoSorbent Assay) detection kit. The kit comprises a 96-hole ELISA plate, an enzyme-labeled secondary antibody, an antibody working fluid, a standard substance solution with six concentrations, a high-concentration standard substance, a substrate fluid A, a substrate fluid B, a stop solution, a concentrated scrubbing solution and a concentrated redissolved solution. With the adoption of an indirect competitive ELISA method, a coupled antigen is pre-wrapped on an ELISA plate microporous strip, and residue imidacloprid in a sample and the coupled antigen pre-wrapped on the microporous strip compete for an imidacloprid antibody; after the enzyme-labeled secondary antibody is added, through color development with the substrate fluid, a sample light absorption value is negatively related to the content of the residue imidacloprid, and is compared with a standard curve and then multiplied by a corresponding dilution ratio, so that the residual quantity of imidacloprid in the sample can be obtained. Compared with an instrument analysis technology, the detection kit has the characteristics of being simple and easy to operate, low in cost, high in sensibility and the like, and can play an important role of residual quantity detection of imidacloprid.

Description

Imidacloprid ELISA detection kit

Technical field

The utility model relates to a kind of kit that detects Imidacloprid, particularly detects enzyme linked immunological (ELISA) detection kit of Imidacloprid.

Background technology

Imidacloprid (IMI) is nicotinoids ultra high efficiency pesticide, has wide spectrum, efficient, low toxicity, low-residual, and insect is difficult for producing resistance, to characteristics such as people, animal, plant and natural enemies securities, and have tag, multiple actions such as stomach toxicity and interior suction.

At present, the Pesticidal products that contains IMI more than 120 countries 140 surplus kind of a crops registration use, range of application relates to kind of crop surplus grain, economy, fruit tree, tealeaves and the vegetables etc. 60, and controlling object relates to 7 order 50 various pests such as Homoptera, Semiptera, Thysanoptera, Diptera, coleoptera, Lepidoptera.

A large amount of uses of IMI have not only caused environment and have had a strong impact on, and livestock and poultry make its enrichment in vivo because long-term eating contains the residual plant of IMI simultaneously, enter human body finally by food chain, are detrimental to health.Worked up the maximum residue limit standard (MRLs) of IMI in the multiple agricultural product at present both at home and abroad.European Union's rehearing in 2008 and revised the MRLs of IMI in nearly 400 kinds of agricultural product, wherein the MRL in most of cereal, fruit, the vegetables is 0.05 mg/kg.The MRL of IMI is 0.05 mg/kg in U.S.'s regulation cereal, the vegetables, and the MRL of IMI is 0.3 mg/kg in the poultry meat product, is 0.05 mg/kg in the poultry prod.Japan's agriculture chemical residues " positive list system " (Positive List System) in the food of execution on the 29th May in 2006 is more harsh to the regulation of the MRLs of IMI in poultry, the poultry product, wherein the different tissues of different animals all has corresponding MRLs, be 0.02 mg/kg in most of musculature, be 0.05 mg/kg in the liver, be 0.3 mg/kg in the fat, to not working out the agricultural chemicals of MRLs, its content in food is carried out " standard without exception ", namely can not surpass 0.01 mg/kg without exception.Domestic MRL to IMI in the agricultural product has only regulation roughly, namely is no more than 0.5 mg/kg in cereal, vegetables, see on the whole also lag behind aspect the MRLs formulation of China IMI in food external.

From bibliographical information, about the residual detection method of IMI mainly concentrates on high performance liquid chromatography (HPLC), vapor-phase chromatography (GC), electrochemical methods (EA) and four aspects of immunoassay (ELISA), be more prone to chromatographic technique and immunoassay technology.Although chromatography is the medicament residue detection method of generally acknowledging at present, this class methods ubiquity required instrument costliness, instrumentation technical requirement height, sample pre-treatments is loaded down with trivial details and analyze length consuming time, detect the cost height, be unsuitable for problem such as on-the-spot detection in real time.And the complexity of medicament residue immunoassay technology in residual pattern detection process, speed, sensitivity, accuracy, specificity, sample analysis capacity in the unit interval, detect aspect such as cost and give prominence to the comparative advantages of instrumental method, all deficiencies that instrumental method shows have almost been overcome, in the food quality safety detection, obtained application more and more widely in recent years, and approved by people gradually.

In today that environmental protection and agricultural product security cry are improved day by day, in the agricultural product effective detection means of residues of pesticides cause close important.To the regulation increasingly stringent of IMI MRLs in agricultural product, for adapting to foreign trade and the protection people's health better, the residual Fast Detection Technique of IMI is imperative in the developing food products along with in the world.Not only detectability is low for the immunological detection method of residue, high specificity, and easy and simple to handle, detection speed is fast, to detect cost low, is very easy to promote, and is the research focus that current medicament residue detects.Although the external bibliographical information of the existing residual immunology detection of IMI, it is higher all to exist residual sample pre-treatment program complexity, expense height, detectability to some extent, can not satisfy the problems such as MRLs of IMI in food in the world.Therefore, finding a more efficient methods to address these problems, is the major issue that pendulum needs to be resolved hurrily in face of us.

The utility model content

Problem to be solved in the utility model provides a kind of Imidacloprid reagent box for detecting content of small volume and less weight, it is easy and simple to handle, detect quick, accurate, highly sensitive, cost is low, good stability, the technology content that needs is not high, can carry out Imidacloprid Determination on content in a plurality of samples of one-time continuous, has reduced the needed time of detection sample.

The technical solution of the utility model is: a kind of Imidacloprid ELISA detection kit, the polystyrene ELISA Plate that comprises 96 holes in box body and the box, a cover plate film, 14 bottles of reagent and the recessed bottle position of putting reagent, a valve bag, it is characterized in that: ELISA Plate is to adopt the polystyrene agent plate in 96 holes as solid phase carrier, the check-out console that pre-bag is made by the Imidacloprid coupled antigen on the kit capillary strip, 14 bottles of reagent are respectively 6 bottles of standard solutions, the high concentration standard items, ELIAS secondary antibody, the antibody working fluid, substrate solution A liquid, substrate solution B liquid, stop buffer, concentrated cleaning solution, concentrate redissolution liquid, totally 15 of recessed bottle positions.

The standard solution Brown Glass Brown glass bottles and jars only of black caps, the high concentration standard items are with the Brown Glass Brown glass bottles and jars only of blue cap, ELIAS secondary antibody is with the white PE plastic bottle of red cap, the antibody working fluid is with the white PE plastic bottle of green cap, substrate solution A liquid is with the white PE plastic bottle of white cap, and substrate solution B liquid is with the black PE plastic bottle of red cap, and stop buffer is with the white PE plastic bottle of yellow cap, the concentrated cleaning solution translucent PE plastic bottle of hyaline cap, the concentrated translucent PE plastic bottle of liquid with blue cap that redissolve.

ELISA Plate is made up of outer frame support and removable 12 enzymes mark reaction capillary strips of being placed on it, each removable enzyme mark reaction capillary strip has 8 reacting holes, put into the vacuum aluminium foil bag, box body is carton box, recessed bottle position is made by plastic foam, be the cover plate film with the plastics dura mater, cover plate film size is consistent with ELISA Plate square section size, with 14 bottles of reagent with the reagent bottle splendid attire of different sizes, different colours and be fixed in the recessed bottle position, together be encapsulated in the carton box with ELISA Plate, cover plate film, valve bag, so that carry and transport.14 bottles of concentrated liquid that redissolve of standard solution, 1ml high concentration standard items, 12ml ELIAS secondary antibody, 7ml antibody working fluid, 7ml substrate solution A liquid, 7ml substrate solution B liquid, 7ml stop buffer, 40ml concentrated cleaning solution, 45ml that reagent is respectively 6 bottles of 1ml gradient concentrations.Reagent in each kit enough carries out 96 times and measures (comprising the standard analysis hole), both can carry out the detection of a plurality of samples of one-time continuous, also can take plate hole apart repeated detection.Put back in the aluminium foil bag with the valve bag sealing accuracy that like this can guarantee reagent box testing result with remaining.During detection, Imidacloprid in the sample will be competed the antibody of anti-Imidacloprid with the Imidacloprid coupled antigen of pre-bag quilt on the ELISA Plate capillary strip, after adding ELIAS secondary antibody, develop the color with substrate solution, the sample absorbance becomes negative correlation with the content of contained Imidacloprid, multiply by its corresponding extension rate more again with typical curve, can draw the content of Imidacloprid in the sample, it is easy to operation.

Our experiments show that this kit has very high sensitivity: the lowest detection of vegetables, fruit sample is limited to 20 μ g/kg, and the recovery is 80%-120%.With respect to other Imidacloprid detection methods, the required instrument of this kit is less, only need microplate reader, vortex instrument, homogenizer, balance, hydro-extractor, glass test tube, graduated pipette, ear washing bulb, polystyrene centrifuge tube and micropipettor etc., generally all there is outfit in similar laboratory, and required cost is lower.

The beneficial effects of the utility model are: can be used for the detection of Imidacloprid content quick, easy, sensitive, exactly.

Description of drawings

Fig. 1 is the lateral longitudinal sectional view (the long 8.55cm of being) of the utility model ELISA Plate;

Fig. 2 is the side drawing in side sectional elevation (the long 12.8cm of being) of the utility model ELISA Plate;

Fig. 3 is the vertical view of the utility model ELISA Plate;

Fig. 4 is the utility model cover plate membrane plane figure;

Fig. 5 is the utility model reagent bottle longitudinal profile planimetric map;

Fig. 6 is the vertical view of the utility model fixed foam mould;

Fig. 7 is the side view of the utility model box body and fixed foam mould.

Embodiment

One, the assembling of Imidacloprid ELISA detection kit

The pre-bag of enzyme mark reaction capillary strip (2) is fixed on the outer frame support (1) of ELISA Plate by Imidacloprid coupled antigen (3), and enzyme mark reaction capillary strip (2) can be with requiring dismounting; Capping enzyme mark reaction capillary strip (2) when cover plate film (4) is put water-bath or constant temperature oven internal reaction for ELISA Plate, cover plate film size is consistent with ELISA Plate square section size; The translucent PE plastics reagent bottle (5) of hyaline cap is used for encapsulation 40ml concentrated cleaning solution; The translucent PE plastics reagent bottle (5) of blue cap is used for encapsulation 45ml and concentrates redissolution liquid; The white PE plastics reagent bottle (6) of white cap is used for encapsulation 7ml substrate solution A liquid, the black PE plastics reagent bottles (6) of red cap are used for encapsulation 7ml substrate solution B liquid, the white PE plastics reagent bottle (6) of yellow cap is used for encapsulation 7ml stop buffer, the white PE plastics reagent bottle (7) of red cap is used for encapsulation 12ml ELIAS secondary antibody, the white PE plastics reagent bottle (7) of green cap is used for encapsulation 7ml antibody working fluid, the Brown Glass Brown glass bottles and jars only of black caps (8) is used for the standard solution of encapsulation 1ml/ bottle, and the Brown Glass Brown glass bottles and jars only of blue cap (8) is used for the high concentration standard solution of encapsulation 1ml/ bottle; Polyfoam mould (10) has 15 recessed bottle positions, placement location is followed successively by: 40ml concentrated cleaning solution bottle position (18), 45ml concentrates redissolution liquid bottle position (11), 7ml substrate solution A liquid bottle position (14), 7ml substrate solution B liquid bottle position (13), 7ml stop buffer bottle position (12), 7ml antibody working fluid bottle position (16), 12ml ELIAS secondary antibody bottle position (15), the standard solution of 6 various concentration of 1ml/ bottle and 1 1ml/ bottle high concentration standard solution bottle position (17), box body (9) is carton box.

Two, the pre-treatment of sample

Vegetables, fruit sample pre-treating method

With homogenizer homogeneous sample; Take by weighing the equal pledge of 2.0 ± 0.05g to 50ml polystyrene centrifuge tube, add 2ml 2M sodium hydroxide solution respectively, 10ml ethyl acetate, with vortex instrument vortex 5min, more than the 3000g, the centrifugal 5min of room temperature (20-25 ℃); Pipette the 1ml upper organic phase to the clean glass test tube of 15ml, flow down and dry up in (50-60 ℃) water-bath nitrogen/air; Add 1ml redissolution working fluid, whirling motion 3min; Getting 50 μ l is used for analyzing.

Three, the operation steps of kit

1, required reagent is taken out from cold storage environment, place room temperature (20-25 ℃) more than the balance 30min, note all need shaking up before every kind of liquid reagent uses.

2, take out the microwell plate that needs quantity, no microwell plate is put into valve bag, be stored in (2-8 ℃).

3, the washing working fluid also need be risen again before use.

4, the corresponding micropore of sample and standard items working fluid is numbered according to the order of sequence, it is parallel that each sample and standard items are done 2 holes, and the position at record standard hole and sample aperture place.

5, add standard items/sample and antibody working fluid: add standard items/sample 50 μ l in the micropore of correspondence, add antibody working fluid 50 μ l/ holes again, the mixing that vibrates gently is with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.

6, wash plate: carefully open the cover plate film, liquid in the hole is dried, with washing working fluid 250 μ l/ holes, fully wash 4-5 time, each 10s at interval pats dry (bubble that is not eliminated after patting dry can be poked with original rifle head) with thieving paper.

7, add ELIAS secondary antibody 100 μ l/ holes, the mixing that vibrates gently with reacting 30min in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate, takes out and repeats to wash plate step 6.

8, colour developing: add substrate solution A liquid 50 μ l/ holes, add substrate solution B liquid 50 μ l/ holes again, the mixing that vibrates gently is with the 15min that develops the color in the rearmounted 25 ℃ of lucifuge environment of cover plate membrane cover plate.

9, measure: add stop buffer 50 μ l/ holes, the mixing that vibrates is gently set microplate reader in the 450nm place (suggestion detects with dual wavelength 450/630nm, please runs through data in 5min), measures every hole OD value.(if no microplate reader does not then add stop buffer and can judge with ocular estimate).

Four, the result judges

The result judges two kinds of methods, judges available the 1st kind of method roughly, quantitatively judges with the 2nd kind of method.Notice that the sample absorbance becomes negative correlation with the content of its contained Imidacloprid.

1, mean light absorbency value and the standard absorbance value with sample relatively can draw its concentration range (μ g/L).The absorbance of supposing sample 1 is 0.520, and the absorbance of sample 2 is 1. 210, and the titer absorbance is respectively: 0 μ g/L is 1.984; 4 μ g/L are 1.582; 12 μ g/L are 1.132; 36 μ g/L are 0.690; 108 μ g/L are 0.380; 324 μ g/L are 0.119.Then the concentration range of sample 1 is that 36 μ g/L-108 μ g/L multiply by its corresponding extension rate again and can draw the residual concentration range of Imidacloprid in the sample; The concentration range of sample 2 is 4 μ g/L-12 μ g/L, multiply by its corresponding extension rate again and can draw the residual concentration range of Imidacloprid in the sample.

2, quantitative test

(1) calculating of percentage absorptance, the percentage absorptance of standard items or sample equal the mean light absorbency value (diplopore) of standard items or sample divided by the mean light absorbency value of first standard items (0 standard), multiply by 100% again, that is:

The mean light absorbency value of B-standard items or sample solution

B ₀The mean light absorbency value of-0 μ g/L standard solution

(2) drafting of typical curve and calculating

Being ordinate with standard items percentage absorptance, is horizontal ordinate with the logarithm of Imidacloprid standard items concentration (μ g/L), the drawing standard curve map.In the percentage absorptance substitution typical curve with sample, read the corresponding concentration of sample from typical curve, multiply by the actual concentrations that its corresponding extension rate is Imidacloprid in the sample.

Five, the material preparation method of using in the kit

1, the preparation of antigen

The haptens preparation

Add Imidacloprid 0.25g in two mouthfuls of bottles of 100ml, the dimethyl formamide of 1ml (DMF), the ethanol of hydration tin chloride 0.75g and 10ml, nitrogen purge, 65 ℃ of reactions of temperature control 40min, reactant liquor shows brown, and thin-layer chromatography (TLC) detection reaction is complete.Handle: be cooled to room temperature, add ethyl acetate 50ml, add saturated sodium bicarbonate aqueous solution and be adjusted to alkalescent, salt out in a large number, ethyl acetate 40ml * 2 time extraction turbid solution merges organic phase, the salt water washing, anhydrous sodium sulfate drying, solvent evaporated gets solid product 0.22g.

Immunogen preparing---Imidacloprid and bovine serum albumin(BSA) (BSA) coupling obtains immunogene.

Get the 7mg haptens, be dissolved among the 1ml DMF; Get glutaraldehyde water solution 0.1ml and add in the haptens solution, stir 24h under the room temperature, can obtain reactant liquor A; Take by weighing bovine serum albumin(BSA) (BSA) 30mg, make it fully to be dissolved in the 2.7ml 0.1mol/L carbonate buffer solution (CB) (pH 9.6), dropwise slowly be added drop-wise in the protein solution reactant liquor A, and under room temperature, stir 24h, with the sodium borohydride aqueous solution 0.2ml reduction reaction of 5mol/L 4 hours, with 4 ℃ of dialysis of 0.01mol/L phosphate buffer (PBS) 3d, change dislysate every day 3 times, to remove unreacted small-molecule substance; Packing, standby in-20 ℃ of preservations.

The coating antigen preparation---Imidacloprid and ovalbumin (OVA) coupling obtains immunogene.

Get the 7mg haptens, be dissolved among the 1ml DMF; Get glutaraldehyde water solution 0.1ml and add in the haptens solution, stir 24h under the room temperature, can obtain reactant liquor A; Take by weighing OVA 30mg, make it fully to be dissolved in 2.7ml 0.1mol/L CB(pH 9.6) in, dropwise slowly be added drop-wise in the protein solution reactant liquor A, and under room temperature, stir 24h, with the sodium borohydride aqueous solution 0.2ml reduction reaction of 5M 4 hours, with 4 ℃ of dialysis of 0.01mol/L PBS 3d, change dislysate every day 3 times, to remove unreacted small-molecule substance; Packing, standby in-20 ℃ of preservations.

2, Imidacloprid MONOCLONAL ANTIBODIES SPECIFIC FOR

Animal immune: the immunogene that above-mentioned steps is obtained is injected in the Balb/c mouse body, and immunizing dose is 150 μ g/, makes it produce antiserum.

Fusion of Cells and cloning: after the mice serum measurement result is higher, get its splenocyte, in the 8:1(quantitative proportion) ratio and SP2/0 myeloma cell's fusion, adopt indirect competitive ELISA to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay that cloning is carried out in positive hole, up to the hybridoma cell strain that obtains secreting the Imidacloprid monoclonal antibody.

Cell cryopreservation and recovery: the monoclonal hybridoma strain is made 1 * 10 with cryopreserving liquid ⁶The cell suspension of individual/ml is in the medium-term and long-term preservation of liquid nitrogen.Take out frozen pipe during recovery, put into 37 ℃ of water-bath middling speeds immediately and melt, behind the centrifugal removal cryopreserving liquid, move in the culture flask and cultivate.

The production of monoclonal antibody and purifying: the Balb/c mouse peritoneal is only injected sterilization paraffin oil 0.5ml/, the stable monoclonal hybridoma strain 5 * 10 of pneumoretroperitoneum injection in 7 days ⁵Individual/as only, to gather ascites after 7 days.Carry out the ascites purifying with sad-saturated ammonium sulfate method ,-20 ℃ of preservations.

3, the preparation of sheep anti mouse antiantibody

Being immune animal with the sheep, is immunogen immune pathogen-free domestic sheep with mouse source antibody, obtains the sheep anti mouse antiantibody.

4, the preparation of enzyme labeling antiantibody

Adopt the sodium periodate method after improveing to carry out coupling sheep anti mouse antiantibody and horseradish peroxidase (HRP).The molar concentration rate of enzyme and antibody is 4:1 in traditional sodium periodate method requirement reaction system, because horseradish peroxidase produces many sites of being combined with antibody under strong oxidation, Huo Hua horseradish peroxidase molecule has served as the bridge that connects each molecule like this, reduced the enzymatic activity of enzyme labeling thing, made in the conjugate of preparation and be mixed with many condensates.In order to address this problem, we improve traditional method, that is:

(1) saved amino closed process, because can produce self amino amino reality that connects seldom;

(2) the volumetric molar concentration ratio that reduces horseradish peroxidase and antibody is to 2:1, and the method after the improvement is easier than traditional method, to the loss minimizing of enzymatic activity.

5, the preparation of ELISA Plate

Be cushioned liquid with bag coating antigen is diluted to 20 μ g/ml, every hole adds 100 μ l, 37 ℃ of lucifuges are hatched 2h, and liquid in the hole of inclining is with cleansing solution washing 2 times, each 30s, pat dry, add 200 μ l confining liquids then in every hole, 37 ℃ of lucifuges are hatched 2h, inclining, liquid pats dry in the hole, and preserve with the vacuum seal of aluminium film dry back.

Claims (3)

1. Imidacloprid ELISA detection kit, the polystyrene ELISA Plate that comprises 96 holes in box body and the box, a cover plate film, 14 bottles of reagent and the recessed bottle position of putting reagent, a valve bag, it is characterized in that: ELISA Plate is to adopt the polystyrene agent plate in 96 holes as solid phase carrier, the check-out console that pre-bag is made by the Imidacloprid coupled antigen on the kit capillary strip, 14 bottles of reagent are respectively 6 bottles of standard solutions, the high concentration standard items, ELIAS secondary antibody, the antibody working fluid, substrate solution A liquid, substrate solution B liquid, stop buffer, concentrated cleaning solution, concentrate redissolution liquid, totally 15 of recessed bottle positions.

2. kit according to claim 1, it is characterized in that: box body is carton box; The polystyrene ELISA Plate in 96 holes is put in the vacuum aluminium foil bag; The cover plate film is the plastics dura maters; The standard solution Brown Glass Brown glass bottles and jars only of black caps, the high concentration standard items are with the Brown Glass Brown glass bottles and jars only of blue cap, ELIAS secondary antibody is with the white PE plastic bottle of red cap, the antibody working fluid is with the white PE plastic bottle of green cap, substrate solution A liquid is with the white PE plastic bottle of white cap, substrate solution B liquid is with the black PE plastic bottle of red cap, stop buffer is with the white PE plastic bottle of yellow cap, the concentrated cleaning solution translucent PE plastic bottle of hyaline cap, concentrate redissolution liquid with the translucent PE plastic bottle of blue cap, made by plastic foam for recessed bottle.

3. kit according to claim 1 is characterized in that: ELISA Plate is made up of outer frame support and 12 removable enzymes mark reaction capillary strips of being placed on it, and each dismantled and assembled enzyme mark reacts capillary strip 8 reacting holes; Cover plate film size is consistent with ELISA Plate square section size; 6 bottles of standard solutions, the 1ml/ bottle; 1 bottle of high concentration standard items, the 1ml/ bottle; 1 bottle of ELIAS secondary antibody, 12ml; 1 bottle of antibody working fluid, 7ml; 1 bottle of substrate solution A liquid, 7ml; 1 bottle of substrate solution B liquid, 7ml; 1 bottle of stop buffer, 7ml; 1 bottle of concentrated cleaning solution, 40ml; Concentrate 1 bottle of redissolution liquid, 45ml.

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