CN102661946B - Malachite chemiluminescence ELISA detection method and kit - Google Patents
Malachite chemiluminescence ELISA detection method and kit Download PDFInfo
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- CN102661946B CN102661946B CN201210106196.3A CN201210106196A CN102661946B CN 102661946 B CN102661946 B CN 102661946B CN 201210106196 A CN201210106196 A CN 201210106196A CN 102661946 B CN102661946 B CN 102661946B
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Abstract
The invention discloses a malachite chemiluminescence ELISA detection method and a kit. According to the invention, it is the first time to discuss chemiluminescence ELISA mechanism of malachite, a malachitechemiluminescence ELISA detection system and a detection method are successively established, and perfect combination of high sensitivity and high specificity is realized. The kit provided by the invention has high sensitivity, accuracy, precision and stability, is used to simplify operation steps and reaction time and reduce errors caused by complex operation, is very suitable for trace analysis and batch detection of malachite residues, and is of great practical application significance.
Description
Technical field
The present invention relates to chemiluminescent enzyme-linked immunosorbent technical field of immunoassay, be specifically related to a kind of chemiluminescence enzyme linked immune detection method of malachite green and the kit that using said method detects malachite green.
Background technology
Malachite green (malachite, MG), has another name called Viride Nitens, salt matrix is green, belongs to Synthesis of diaminodiphenyl, has the spinoffs such as high toxin, high residue, high carcinogenic and high teratogenesis, mutagenesis, and human health and environment are caused to serious harm.FDA forbids that malachite green is used in fishery already, the U.S., Canada, European Union are all defined in the aquatic products such as food fish and must not detect malachite green, and China was listed in the veterinary drug and compound inventory > > (No. 193, Ministry of Agriculture's bulletin) thereof of < < food animal forbidding in May, 2002.Yet, due to the cheap price of malachite green with at saprolegniasis, there is no good substitute aspect preventing and treating, some illegal retailers, still in illegal use, cause malachite green event to happen occasionally.
At present, for the more conventional detection method of malachite green vestigial, be instrument analytical method, comprise liquid phase chromatography, liquid chromatography-mass spectrography and Liquid Chromatography-Tandem Mass Spectrometry.Instrumental method have accuracy high, reproducible, be suitable for the advantages such as quantitatively detection, but need to carry out post purifying (as aluminium oxide solid-phase extraction column) to sample processes, complicated operation, makes this method be difficult to be widely used in extensive high flux detection.The applicant has set up the enzyme-linked immune detection method of malachite green, made up well the deficiency of instrument detection method, there is high specific and the advantage such as low detectability extremely, in fields such as clinical, bio-pharmaceuticals and environmental chemistries, be used widely, be especially applicable to on-the-spot screening and the analysis of batch samples rapid screening.
Chemiluminescent enzyme-linked immunosorbent immunoassay technology is the technology of chemoluminescence method detection and immunodetection combination, there is the high sensitivity of chemoluminescence method and the high specific of immunoassay simultaneously, chemiluminescent enzyme-linked immunosorbent immune detection can be applicable to the detection in large concentration range, the chemical substance that participates in reaction is stable, cheap, and is applicable to various types of immunoassays.
But, remarkable difference due to compound structure and character, in practical application chemiluminescent enzyme-linked immunosorbent immunoassay technology, specific to a certain compound, set up chemiluminescence reaction system targetedly, search out the simple and rapid sample-pretreating method that adapts to described detection method, thereby set up the chemiluminescence enzyme linked immune detection method of holonomic system, and therefore provide stability high, simple to operate, equipment requirement is low, cheap malachite green CLEIA kit, successfully realize the detection of high sensitivity and high specific, this still belongs to blank in the art, belong to the technical barrier of not yet capturing.Therefore, there is no at present the bibliographical information of any chemiluminescent enzyme-linked immunosorbent immune detecting system about malachite green, also without commercial malachite green chemiluminescence enzyme linked immunoassay reagent kit product, greatly affected malachite green restriction and used and strict monitoring work.
Summary of the invention
The object of the invention is to detect for existing malachite green the deficiency of the kit using, a kind of method that high specific and high sensitivity have concurrently, specific aim solves malachite green chemiluminescent enzyme-linked immunosorbent immune detection is provided.
Another object of the present invention is to provide the kit that utilizes described method, cheap, simple to operate, can realize in enormous quantities, detect rapidly and accurately malachite green.
Above-mentioned purpose of the present invention is achieved by following technical solution:
The chemiluminescence enzyme linked immune detection method that a kind of malachite green is provided, comprises the following steps:
(1) testing sample pre-treatment;
(2) malachite green envelope antigen is coated in to luminous solid phase carrier, adds respectively standard specimen or the testing sample after pre-treatment, then add respectively anti-malachite green antibody, reaction;
(3) after step (2) reaction, add respectively enzyme labeling thing to react, then add luminous substrate carry out luminous and measure the luminous value of testing sample;
(4) inhibiting rate of the mean value calculation sample to be checked of the luminous value of measuring with step (3), according to the inhibiting rate of the typical curve of malachite green and sample to be checked, calculates the concentration of testing sample Malachite Green.
Wherein, the described testing sample of step (1) can be water sample or organize sample, if water sample, described pre-treatment is for filtering or the limpid water sample of centrifugal acquisition;
If testing sample is when organizing sample, the pre-treatment of testing sample comprises the following steps:
(a) extract and organize sample; By organizing sample to smash to pieces, add extract, 10 seconds of high-speed homogenization; The extract that described extraction adopts is prepared according to following proportioning: 10mL 0.36mol/L oxammonium hydrochloride solution, 15mL1.0mol/L p-toluenesulfonic acid solution and 25mL 0.05mol/L ammonium acetate form;
(b) in step (a) system, add acetonitrile, rear centrifugal treating is extracted in concussion, collects respectively supernatant and residue;
(c) step (b) adds acetonitrile in residue obtained, concussion extract after centrifugal treating, collect supernatant;
(d) combining step (b) and step (c) gained supernatant, add methylene chloride, and stratification after concuss is collected dichloromethane layer; Repeat to add methylene chloride, concussion, layering, collection dichloromethane layer, merges vacuum by methylene chloride and is spin-dried for, and with acetonitrile, redissolves and to obtain redissolution liquid;
(e) get step (d) gained redissolution liquid and add PBS buffer solution to mix, described PBS damping fluid is by 0.4g KH
2pO
4, 5.8g Na
2hPO
412H
2o, 16g NaCl and 0.4g KCl, adding distil water is settled to the PBS damping fluid that pH value that 2000mL is configured to is 7.4, obtains testing sample.
As a kind of preferred version, testing sample is when organizing sample, and the pre-treatment of testing sample is more specifically for comprising the following steps:
(a) will organize sample to smash to pieces, accurately take 5.0g, add extract 5mL, 10 seconds of high-speed homogenization; Described extract is prepared according to following proportioning: 10mL 0.36mol/L oxammonium hydrochloride solution, 15mL 1.0mol/L p-toluenesulfonic acid solution and 25mL 0.05mol/L ammonium acetate form.
(b) in step (a) system, add 5mL acetonitrile, rear centrifugal treating is extracted in concussion, collects respectively supernatant and residue;
Described concussion preferred 10min of extraction time, centrifugal treating is preferably processed 5min at rotating speed 4000r/min.
(c) step (b) adds 5mL acetonitrile in residue obtained, concussion extract after centrifugal treating, collect supernatant;
Described concussion preferred 5min of extraction time, centrifugal treating is preferably processed 5min at rotating speed 4000r/min.
(d) combining step (b) and step (c) gained supernatant, add 10mL methylene chloride, and stratification after concuss is got dichloromethane layer and steamed in bottle to revolving; Again add 5mL methylene chloride, concussion, the standing dichloromethane layer of getting, mixes twice dichloromethane layer and is spin-dried for, and with 0.5mL acetonitrile, redissolves and to obtain redissolution liquid;
Described concussion is 5 seconds preferably.
(e) get step (d) gained redissolution liquid and add 4.5mL PBS buffer solution to mix, obtain testing sample.
The present invention is especially applicable to being applied to the tissue of aquatic product product, preferably aquatic product product is removed after head, tail, fin and shell, remaining edible carry out follow-up pre-treatment step after partly smashing to pieces, when being applied to the tissue detection of aquatic product product, more preferably adopt musculature.
Described smashing to pieces comprise and adopt cutter chopping, or mortar grinds, or the method such as tissue mincer's homogenate.In processing procedure, avoid as far as possible occurring emulsion layer, if occur, emulsion layer is again centrifugal after can adding appropriate deionized water again to mix.Sample extracting solution can adopt that common normal temperature is centrifugal or cryogenic freezing is centrifugal.
The described anti-malachite green antibody of step (2) is preferably anti-malachite green polyclonal antibody, monoclonal antibody or genetic engineering antibody.Described malachite green envelope antigen and anti-malachite green antibody can adopt existing conventional products, or synthetic voluntarily, and synthetic method is conventional with reference to the art.First synthetic malachite green haptens, synthesizes envelope antigen by malachite green haptens and carrier protein covalent coupling; By described malachite green haptens and the synthetic immunizing antigen of carrier protein covalent coupling, with immune antigen immune rabbit or mouse, prepare malachite green polyclonal antibody, utilize hybridoma technology prepare malachite green monoclonal antibody or utilize gene engineering method to prepare genetic engineering antibody.The preferred bovine serum albumin of described carrier protein or ovalbumin.
The synthetic of the described malachite green envelope antigen of step (2) and anti-malachite green antibody also can be with reference to following methods:
Haptenic synthetic:
With the first synthesizing nitryl leucomalachite green of chemical method (NO
2-LMG), be then reduced to amino leucomalachite green (NH
2-LMG), by silicon chromatography, analyze both purity, and reclaim.
Coating antigen and immunogenic synthetic:
By carrier proteins such as malachite green haptens and bovine serum albumin or ovalbumins, by diazotising method, carry out coupling and obtain envelope antigen and immunogene.Immunogene and coating antigen carry out purifying by column chromatography, and purity is identified through SDS-PAGE electrophoresis.
The preparation of malachite green monoclonal antibody:
Animal immune: take haptens and carrier protein couplet thing carries out Immunity at intervals to Balb/c mouse as immunogene, indirect ELISA detects and obtains the mouse spleen that contains malachite green specific antibody in blood.
Fusion of Cells and clone: get the Balb/c mouse boosting cell and the myeloma cell SP20 fusion that produce specific antibody, adopt indirect competitive enzyme-linked immunosorbent method to measure cell conditioned medium liquid, screen positive hole.Utilize limiting dilution assay or microclone method to carry out cloning to positive hole, obtain and set up the hybridoma cell strain that produces monoclonal antibody.
Cell cryopreservation and recovery: get in the hybridoma of exponential phase and make cell suspension with cryopreserving liquid, be sub-packed in cryopreservation tube, in liquid nitrogen, preserve for a long time.During recovery, take out cryopreservation tube, put into immediately 37 ℃ of water-bath middling speeds and melt, after centrifugal removal cryopreserving liquid, move in culture flask and cultivate.
The preparation and purification of monoclonal antibody: adopt in body and induce method, by Balb/c mouse (8 week age) Intraperitoneal injection sterilizing paraffin oil, 7～14 days pneumoretroperitoneum injection hybridomas, gathered ascites after 7～10 days.Through sad-saturated sulfuric acid amine method or affinity chromatography, carry out ascites purifying, purity is identified through SDS-PAGE electrophoresis, bottle packing ,-20 ℃ of preservations.
The preparation of malachite green rabbit polyclonal antibody:
Adopt new zealand white rabbit as immune animal, take malachite green and carrier protein couplet thing carries out immunity to new zealand white rabbit as immunogene, repeatedly after immunity, measure serum antibody titer, heart is taken a blood sample, and obtains the polyclonal antibody of purifying through sulfuric acid amine fractional precipitation.
The preparation of malachite green genetic engineering antibody:
Genetic engineering antibody mainly refers to small molecular antibody, comprise: Fab (being formed by complete light chain and Fd), Fv (being formed by VH and VL), ScFv (single-chain antibody, between VH and VL, by one, connecting peptide is formed by connecting), single domain antibodies (being only comprised of VH) etc. are through the improved antibody of technique for gene engineering.Preparation method is: the RNA that extracts malachite green monoclonal cell or the mouse boosting cell after malachite green immunogen immune, reverse transcription is cDNA, designerantibodies weight chain amplimer, utilize round pcr to amplify the weight chain gene of antibody, insert suitable expression plasmid, at expression in escherichia coli, utilize immune affine method to carry out purifying, purity is identified by SDS-PAGE electrophoresis.
The drafting of the described typical curve of step (4) is the mean value calculation inhibiting rate with malachite green standard items luminous value, take inhibiting rate as ordinate, the semilog of malachite green concentration is horizontal ordinate drawing standard curve, obtain straight-line equation, according to equation, obtain the malachite green concentration of counter sample; The calculating formula of described inhibiting rate is:
Wherein, RLU
0for malachite green drug concentration is the luminous value of 0 o'clock, RLUx is the luminous value of malachite green drug concentration while being x, RLU
minluminous value for blank hole.
The present invention provides a kind of kit that detects the chemiluminescent enzyme-linked immunosorbent immunity of malachite green simultaneously, comprise box body, luminous plaque, also comprise anti-malachite green antibody-solutions, horseradish peroxidase-labeled goat anti-rabbit antibody solution, malachite green standard items storing solution and standard solution, luminous substrate liquid and substrate buffer solution, organize sample extract, washing lotion; Described luminous plaque is microwell plate, hole endoperidium have can with the envelope antigen of anti-malachite green antibody specific bond;
Described luminous substrate liquid is to contain luminol and the Tris-HCl buffer solution that is 9.0 to the pH value of iodophenol; As a kind of most preferably scheme, described luminous substrate liquid most preferably is and 2.0mg luminol, 0.8mg are dissolved in to the Tris-HCl damping fluid that the pH value of 10mL is 9.0 to iodophenol obtain.
Described substrate buffer solution is the Tris-HCl buffer solution that the pH value that contains hydrogen peroxide is 7.0.As a kind of most preferably scheme, the hydrogen peroxide that it is 30% that described substrate buffer solution most preferably is 20uL volume by volume concentration is dissolved in the Tris-HCl damping fluid that the pH value of 10mL is 7.0 and obtains.
Described luminous plaque is preferably the detachable luminous plaque in 96 holes or 40 holes, hole endoperidium have can with the envelope antigen of anti-malachite green antibody specific bond, and closed porosity surface adsorption site not; It is that confining liquid seals that described sealing adopts 1.0～5.0% skim milk powder aqueous solution.
Further, described luminous plaque preferably adopts polystyrene micropore plate.
As a kind of preferred version, described anti-malachite green antibody is preferably anti-malachite green polyclonal antibody, monoclonal antibody or genetic engineering antibody.
Described washing lotion is by 0.4g KH
2pO
4, 5.8g Na
2hPO
412H
2the Tween-20 1mL that O, 16g NaCl, KCl 0.4g, concentration of volume percent are 0.05%, the PBST phosphate buffer that the pH value that adding distil water obtains to 2000mL configuration is 7.4.
Described malachite green standard items storing solution, for getting malachite green standard specimen 5.0mg, is dissolved in 50mL acetonitrile, is mixed with the standard items storing solution of 0.1mg/mL.
Described malachite green standard solution is the malachite green solution of 6 gradient concentrations, and concentration is followed successively by 0ng/mL, 0.02ng/mL, 0.1ng/mL, 0.5ng/mL, 2.5ng/mL, 12.5ng/mL.For standard solution storing solution is obtained with the dilution of PBS damping fluid.Described PBS damping fluid is by 0.4g KH
2pO
4, 5.8gNa
2hPO
412H
2o, 16g NaCl and 0.4g KCl, adding distil water is settled to the damping fluid that pH value that 2000mL configuration obtains is 7.4.
The preparation of the goat anti-rabbit antibody solution of described horseradish peroxidase-labeled is with reference to existing routine techniques.
The present invention also provides the using method of described kit, comprises the following steps:
(1) do the Parallel testing of standard and sample: in the standard items hole of luminous plaque, add standard items, add testing sample in sample well, then in every hole, add respectively anti-malachite green antibody-solutions, pat and mix, room temperature concussion is hatched;
(2) liquid in pouring out in the luminous plate hole of step (1), and clean up luminous plate hole by washing lotion;
(3) in the hole after step (2) is processed, add enzyme labeling thing solution, pat and mix, lucifuge incubated at room;
(4) liquid in pouring out in luminous plate hole, and clean up luminous plate hole by washing lotion;
(5) every hole adds the equal-volume mixed liquor of substrate buffer solution and luminous substrate liquid, pats and mixes, and under wavelength 425nm, measures each hole luminous value, reads light absorption value after adding luminescent solution in 5min;
(6) with the mean value calculation inhibiting rate of obtained sample luminous value, take inhibiting rate as ordinate, the semilog of malachite green concentration is horizontal ordinate drawing standard curve, obtains straight-line equation to be: y=-18.169x+100.13, R
2=0.9956.According to equation, obtain the malachite green concentration of counter sample; The calculating formula of described inhibiting rate is:
Wherein, RLU
0for malachite green drug concentration is the luminous value of 0 o'clock, RLUx is the luminous value of malachite green drug concentration while being x, and RLUmin is the luminous value in blank hole.
In step in the using method of described kit (1) and step (3), less demanding to incubation temperature, can be chosen under 37 ℃ of conditions and hatch with reference to existing Enzyme-multiplied immune technique, more preferably hatch at ambient temperature.
On the chemiluminescent enzyme-linked immunosorbent immune system basis of the malachite green of setting up in the present invention, the measuring principle of realizing is: first envelope antigen is coated on solid phase carrier (luminous plaque), then add standard specimen or testing sample, add again anti-malachite green antibody, malachite green in envelope antigen and testing sample is competed anti-malachite green antibody, when testing sample malachite green content is high, the anti-malachite green antibody of being combined with solid phase antigen is just few, otherwise the anti-malachite green antibody being combined on solid phase antigen is just many, after reaction, add enzyme labeling thing to react, and then add luminous substrate carry out luminous and measured.When anti-malachite green antibody amount one timing, the testing sample adding is more containing malachite green, the malachite green antibody of being combined with solid phase antigen is just fewer, and luminescence-producing reaction weakens, and inhibiting rate increases, otherwise, luminescence-producing reaction strengthens, and inhibiting rate lowers, thereby maps and obtain typical curve according to the semilog relation between inhibiting rate and malachite green concentration, according to the inhibiting rate of the typical curve of malachite green and sample to be checked, can extrapolate the concentration of testing sample Malachite Green again.
The invention has the beneficial effects as follows:
The present invention is first for malachite green, inquired into its chemiluminescent enzyme-linked immunosorbent immune mechanism, chemiluminescent enzyme-linked immunosorbent immune detecting system and the detection method of malachite green have successfully been set up, realized the perfect adaptation of high sensitivity and high specific, this kit of the inventive method is 0.02～12.5ng/mL to malachite green linear detection range, detection is limited to 0.01ng/mL, and the chemical substance that participates in reaction is stable, cheap, and is applicable to various types of immunoassays.
Further, application the inventive method detects while organizing sample, the invention provides preferred pre-treating method, pre-treating method has been made following contribution to overall technical architecture of the present invention: 1. easy and simple to handle, technical requirement is low, organize that sample only need be smashed to pieces, lixiviate, centrifugal, evaporate to dryness, redissolution, operation is simple for overall process, guaranteed the simple of whole detection scheme; 2. extraction effect is good, adds ion pair solution to extract, and is conducive to malachite green stripping, adds the effect of concussion homogeneous, easier stripping is complete, and operation steps is simple in addition, has reduced process loss, after measured, extraction rate reached more than 80%, has guaranteed the accuracy of overall technical architecture testing result; 3. cost is low, and owing to not needing with separating column, extraction cost is low, according to the preresearch estimates of the art experience, with additive method, compares, and each sample can be saved sample preparation cost more than 30 yuan.
The inventive method precision is good, and relative standard deviation < 8.0%, easier, economical than ELISA method, save time, about 2 hours consuming time of ELISA method, the inventive method 50min consuming time), simultaneously sensitivity has improved approximately 10 times, accuracy, precision, stability; Reaction is less demanding to incubation temperature, room temperature.
Kit of the present invention has been simplified operation steps (not needing chromogenic reaction step) and has been shortened the reaction time (whole process only needs 50min), reduced the error causing because of complicated operation, based on above this kit of advantage, be highly suitable for trace analysis and the batch detection of malachite green vestigial, there is important practical application meaning.
Accompanying drawing explanation
Fig. 1 is the schematic diagram directly perceived of kit; Wherein, the 1 micropore luminous plaque for coated good envelope antigen, 2 is antibody-solutions, 3 is enzyme labelled antibody solution, 4 is luminous substrate liquid, and 5 is substrate buffer solution, and 6 is malachite green standard solution storing solution and the standard solution that posts solubility label, 7 is extract, and 8 is washing lotion, and 9 is operation instructions;
Fig. 2 is typical curve.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.Unless stated otherwise, in embodiment, reagent used is commercially available conventional reagent.
The preparation of embodiment 1 chemiluminescent enzyme-linked immunosorbent immunity epidemic disease kit sample
(1) preparation of washing lotion: by KH
2pO
40.4g, Na
2hPO
412H
2o 5.8g, NaCl 16g, KCl0.4g, Tween-20 0.05 volume %1mL, the PBST phosphate buffer that the pH value that adding distil water obtains to 2000mL configuration is 7.4.
(2) preparation of confining liquid: skimmed milk power 1.0～5.0g is dissolved in 100mL distilled water.
(3) preparation of luminous substrate liquid: 2.0mg luminol, 0.8mg are dissolved in iodophenol in the Tris-HCl damping fluid (pH value is 9.0) of 10mL, 4 ℃ of preservations.
(4) preparation of substrate buffer solution: 20 μ L hydrogen peroxide (30%) are dissolved in 10mL Tris-HCl damping fluid (pH value is 7.0), 4 ℃ of preservations.
(5) preparation of extract: 10mL 0.36mol/L oxammonium hydrochloride solution, 15mL 1.0mol/L p-toluenesulfonic acid solution and 25mL 0.05mol/L ammonium acetate damping fluid, mix 4 ℃ of preservations.
(6) luminous plaque microwell plate is coated: envelope antigen pH9.6, the carbonate buffer solution of 0.05mol/L is diluted to 0.1～5ug/mL, every hole at luminous plaque adds 100uL, coated spending the night or 37 ℃ of coated 2h at 4 ℃, and coating buffer inclines, with washing lotion washing 2 times, pat dry, then in every hole, add 200uL 1.0～5.0% skimmed milk powers, put into after 37 ℃ of incubator 1h with washing lotion washing 2 times, pat dry rear being dried, 4 ℃ of preservations.
(7) preparation of malachite green standard items storing solution and standard solution: accurately take malachite green standard specimen 5.0mg, be dissolved in and make standard items storing solution in 50mL acetonitrile.The malachite green solution of preparing respectively 12.5ng/mL, 2.5ng/mL, 0.5ng/mL, 0.1ng/mL, 0.02ng/mL with PBS damping fluid dilution standard product storing solution, PBS damping fluid is 0ng/mL control sample in addition, 4 ℃ of preservations; Described PBS damping fluid is by 0.4gKH
2pO
4, 5.8g Na
2hPO
412H
2o, 16g NaCl and 0.4g KCl, adding distil water is settled to the damping fluid that pH value that 2000mL configuration obtains is 7.4.
(8) reagent packing: various reagent is prepared on request, measures qualified rear aseptic subpackaged.Enzymic-labelled antibody 12mL/ bottle, malachite green standard items storing solution 0.1mL/ bottle, luminous substrate liquid 7mL/ bottle, substrate buffer solution 7mL/ bottle, antibody working fluid 7mL/ bottle, extract 50mL/ bottle, washing lotion 50mL/ bottle.After packing, label, indicate lot number and the term of validity, 4 ℃ of preservations.
(9) assembling of kit: respectively by 1 of the luminous plaque of detachable coated good envelope antigen, each 1 bottle of antibody working fluid, enzyme mark antibody solution, luminous substrate liquid, substrate buffer solution, malachite green standard items storing solution, extract, concentrated washing lotion, 6 bottles of malachite green standard items, 2 of cover plate films, 1 of valve bag, 1 part of operation instructions, put respectively assigned address in kit, see shown in accompanying drawing 1.Kit encapsulates after the assay was approved, 4 ℃ of preservations.
The detection method of embodiment 2 chemiluminescent enzyme-linked immunosorbent immunity epidemic disease kits
(1) kit is taken out from cold storage environment, be placed in room temperature (20～24 ℃) more than balance 30min, the batten of enough standards and sample quantity used is fixed on to support, standard and sample are done two parallel laboratory tests, number in order.
(2) in standard items hole, add 50 μ L standard items, sample well adds 50 μ L testing samples.Then every hole adds the anti-malachite green antibody-solutions of 50 μ L, pats and mixes.In room temperature concussion, hatch 30min.
(3) pour out the liquid in hole, micropore frame is upside down on thieving paper and pats (every wheel washed plate beating 3 times) to guarantee the removing liquid in hole completely.With 250 μ L cleansing solutions, be filled with in hole, again outwell the liquid in micropore, then repetitive operation 5 times.
(4) every hole adds 100 μ L enzyme labelled antibody solution, pats and mixes, incubated at room 20min.Repeating step (3).
(5) every hole adds 100 μ L luminescent solutions (substrate buffer solution is mixed with substrate solution equal-volume), pats and mixes.At wavelength 425nm (take luminescent solution background value as blank), measure each hole luminous value, must after adding luminescent solution, in 5min, read luminous value.
With the mean value calculation inhibiting rate of obtained sample luminous value, the computing formula of inhibiting rate is:
Wherein, RLU
0for malachite green drug concentration is the luminous value of 0 o'clock, RLUx is the luminous value of malachite green drug concentration while being x, RLU
minluminous value for blank hole.
Take inhibiting rate as ordinate, and the semilog of malachite green concentration (ng/mL) is horizontal ordinate drawing standard curve, and calibration curve is linear within the scope of 0.02～12.5ng/mL, obtains straight-line equation, and the concentration of counter sample can be obtained according to equation.
The detection of embodiment 3 flesh of fish samples
Feminine gender and positive flesh of fish sample (confirming concentration through HPLC) are minced, get the homogeneous good flesh of fish of 5.0g, add the extract 5mL in kit, after high-speed homogenization 10s, add 5.0mL acetonitrile, mechanical shaking extraction 10min, then the centrifugal 5min of centrifugal 4000r/min, transfers to supernatant in a new centrifuge tube; In residue, add 5.0mL acetonitrile, concussion sample 5min, then under room temperature with the centrifugal 5min of rotating speed 4000r/min, get supernatant and merge to for the first time in supernatant pipe.After whole supernatants mix, add after 10mL methylene chloride thermal agitation 5s, take off layer solution and be transferred in rotary evaporation bottle, with 10mL methylene chloride, repeating aforesaid operations once; Lower floor's solution is incorporated in to 40 ℃ of rotary evaporated to dryness, with 0.5mL acetonitrile, redissolves, add 4.5mL PBS buffer solution to mix, described PBS damping fluid is by 0.4g KH
2pO
4, 5.8gNa
2hPO
412H
2o, 16g NaCl and 0.4g KCl, adding distil water is settled to the damping fluid that pH value that 2000mL configuration obtains is 7.4; This dilute sample can be for detection of.Then utilize CLEIA kit of the present invention to detect.Experimental result is shown in Table 1.
Table 1 flesh of fish sample preparation testing result
The detection of embodiment 4 water samples
By feminine gender and positive water sample (confirming concentration through HPLC), with middling speed Xinhua Filter paper filtering, filtrate is sample extracting solution, then utilizes CLEIA kit of the present invention to detect.Experimental result is shown in Table 2.
Table 2 water sample is processed testing result
Embodiment 5 storage life tests
Kit is positioned over to 4 ℃, gets respectively 0,2,4,6,8,9,10,11 and the kit of 12 months, malachite green standard model (0.1ng/mL) is detected, measurement result is as table 3.
Table 3 kit storage life test findings
From result, can find out, kit at least can be preserved 12 months at 4 ℃.
Embodiment 6 kit sensitivity determinations
Utilize malachite green standard solution to react, according to experimental result drawing standard curve, see shown in accompanying drawing 2, by accompanying drawing 2, can be obtained: straight-line equation is y=-18.169x+100.13, inhibiting rate (RLU/RLU
0) within the scope of concentration 0.02～12.5ng/mL, being significant linear relationship with the logarithm value of malachite green concentration, related coefficient is R
2=0.9956, and minimum detectability is 0.01ng/mL.
Table 4
Embodiment 7 accuracy tests
The malachite green standard model of getting 2 concentration (0.1ng/mL and 2.5ng/mL), adds in negative sample, and each concentration is established 6 repetitions, measures.
The result of the kit recovery is as follows, and water sample is 90%～104%, and organizing sample is 82%～109%.
Embodiment 8 Precision Experiments
Get the malachite green standard specimen of 2 concentration (0.1ng/mL and 2.5ng/mL), add in negative water sample, each concentration is established 6 repetitions, and at same reagent box, measures respectively from 3 different kits in 3 days respectively.Result is as follows, and variation within batch coefficient is 4.2%, and interassay coefficient of variation is 7.5%.
Claims (4)
1. a chemiluminescence enzyme linked immune detection method for malachite green, is characterized in that comprising the following steps:
(1) testing sample pre-treatment;
(2) malachite green envelope antigen is coated in to luminous solid phase carrier, adds respectively standard specimen or the testing sample after pre-treatment, then add respectively anti-malachite green antibody response; Described envelope antigen is the comlete antigen that malachite green haptens NH2-LMG and carrier protein couplet form;
(3) after step (2) reaction, add respectively enzyme labeling thing to react, then add luminous substrate carry out luminous and measure the luminous value of testing sample; Described luminous substrate liquid obtains for 2.0mg luminol, 0.8mg are dissolved in to the Tris-HCl damping fluid that the pH value of 10mL is 9.0 to iodophenol;
(4) inhibiting rate of the mean value calculation sample to be checked of the luminous value of measuring with step (3), according to the inhibiting rate of the typical curve of malachite green and sample to be checked, calculates the concentration of testing sample Malachite Green;
Wherein, the described testing sample of step (1) is for organizing sample, and described histioid pre-treatment comprises the following steps:
(a) extract and organize sample; By organizing sample to smash to pieces, add extract, 10 seconds of high-speed homogenization; The extract that described extraction adopts is prepared according to following proportioning: 10mL0.36mol/L oxammonium hydrochloride solution, 15mL1.0mol/L p-toluenesulfonic acid solution and 25mL0.05mol/L ammonium acetate form;
(b) in step (a) system, add acetonitrile, rear centrifugal treating is extracted in concussion, collects respectively supernatant and residue;
(c) step (b) adds acetonitrile in residue obtained, concussion extract after centrifugal treating, collect supernatant;
(d) combining step (b) and step (c) gained supernatant, add methylene chloride, and stratification after concuss is collected dichloromethane layer; Repeat to add methylene chloride, concussion, layering, collection dichloromethane layer, merges vacuum by methylene chloride and is spin-dried for, and with acetonitrile, redissolves and to obtain redissolution liquid;
(e) get step (d) gained redissolution liquid and add PBS buffer solution to mix, described PBS damping fluid is by 0.4gKH
2pO
4, 5.8gNa
2hPO
412H
2o, 16gNaCl and 0.4gKCl, adding distil water is settled to the PBS damping fluid that pH value that 2000mL is configured to is 7.4, obtains testing sample.
2. a kit of realizing the malachite green chemiluminescent enzyme-linked immunosorbent immunity of method described in claim 1, it is characterized in that comprising box body, luminous plaque, also comprise anti-malachite green antibody-solutions, horseradish peroxidase-labeled goat anti-rabbit antibody solution, malachite green standard items storing solution and standard solution, luminous substrate liquid and substrate buffer solution, organize sample extract, washing lotion; Described luminous plaque is microwell plate, hole endoperidium have can with the envelope antigen of anti-malachite green antibody specific bond;
Described luminous substrate liquid obtains for 2.0mg luminol, 0.8mg are dissolved in to the Tris-HCl damping fluid that the pH value of 10mL is 9.0 to iodophenol;
Described substrate buffer solution obtains for the hydrogen peroxide that is 30% by 20 μ L volume by volume concentrations is dissolved in the Tris-HCl damping fluid that the pH value of 10mL is 7.0;
Described luminous plaque is 96 holes or 40 holes, hole endoperidium have can with the envelope antigen of anti-malachite green antibody specific bond, and closed porosity surface adsorption site not; It is confining liquid that described sealing adopts the skim milk powder aqueous solution that w/v is 1.0～5.0%;
Described washing lotion is by 0.4gKH
2pO
4, 5.8gNa
2hPO
412H
2o, 16gNaCl, 0.4gKCl, 1mLTween-20, adding distil water is settled to the PBST phosphate buffer that pH value that 2000mL is configured to is 7.4;
Described malachite green standard items storing solution, for getting malachite green standard specimen 5.0mg, is dissolved in 50mL acetonitrile, is mixed with the standard items storing solution of 0.1mg/mL;
Described malachite green standard solution is the malachite green solution of 6 gradient concentrations, and concentration is followed successively by 0ng/mL, 0.02ng/mL, and 0.1ng/mL, 0.5ng/mL, 2.5ng/mL, 12.5ng/mL, by obtaining standard solution storing solution with the dilution of PBS damping fluid; Described PBS damping fluid is by 0.4gKH
2pO
4, 5.8gNa
2hPO
412H
2o, 16gNaCl and 0.4gKCl, adding distil water is settled to the PBS damping fluid that pH value that 2000mL is configured to is 7.4.
3. kit according to claim 2, is characterized in that described luminous plaque is polystyrene micropore plate.
4. a using method for kit claimed in claim 2, is characterized in that comprising the following steps:
(1) do the Parallel testing of standard and sample: in the standard items hole of luminous plaque, add standard items, add testing sample in sample well, then in every hole, add respectively anti-malachite green antibody-solutions, pat and mix, room temperature concussion is hatched;
(2) liquid in pouring out in the luminous plate hole of step (1), and clean up luminous plate hole by washing lotion;
(3) in the hole after step (2) is processed, add enzyme labeling thing solution, pat and mix, lucifuge incubated at room;
(4) liquid in pouring out in luminous plate hole, and clean up luminous plate hole by washing lotion;
(5) every hole adds the equal-volume mixed liquor of substrate buffer solution and luminous substrate liquid, pats and mixes, and under wavelength 425nm, measures each hole luminous value, reads light absorption value after adding luminescent solution in 5min;
(6) with the mean value calculation inhibiting rate of obtained sample luminous value, take inhibiting rate as ordinate, the semilog of malachite green concentration is horizontal ordinate drawing standard curve, obtains straight-line equation, obtains the malachite green concentration of counter sample according to equation.
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| CN103630688B (en) * | 2013-11-11 | 2016-05-25 | 洛阳莱普生信息科技有限公司 | A kind of canine parvovirus chemiluminescence detection kit |
| CN105891190B (en) * | 2016-04-04 | 2019-01-04 | 广东工业大学 | One kind being based on chemiluminescent crystal violet detection method |
| CN107091834B (en) * | 2017-05-24 | 2019-07-02 | 青岛科技大学 | A method of DNA is detected based on hair fastener mispairing circulation amplifying technique |
| CN108715847A (en) * | 2018-05-31 | 2018-10-30 | 江南大学 | A kind of fast detection method and its application enhancing malachite green fluorescence based on DNA |
| CN110006880B (en) * | 2019-03-20 | 2021-05-25 | 浙江农林大学 | Compound for direct-reading chromogenic rapid detection of malachite green and application |
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