CN102495212A - Method and test paper for rapidly detecting microcystins - Google Patents
Method and test paper for rapidly detecting microcystins Download PDFInfo
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- CN102495212A CN102495212A CN2011104254075A CN201110425407A CN102495212A CN 102495212 A CN102495212 A CN 102495212A CN 2011104254075 A CN2011104254075 A CN 2011104254075A CN 201110425407 A CN201110425407 A CN 201110425407A CN 102495212 A CN102495212 A CN 102495212A
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Abstract
The invention relates to the detection of a water sample and establishes a rapid colloidal gold immunochromatography detection method and test paper of microcystins MC-LR. The method comprises the following steps of: firstly, preparing a colloidal gold with the average diameter of about 30 nm and utilizing the colloidal gold to mark an anti-microcystins MC-LR monoclonal antibody; covering a conjugate of a microcystins MC-LR hapten and keyhole limpet hemocyanin on a nitrocellulose membrane as a detection belt; taking a goat anti-mouse second antibody as a control belt; and establishing an immunochromatography test paper method for rapidly detecting the microcystins MC-LR. According to the method, the sensitivity can reach to 3 ng/mL and a judged result can be observed by naked eyes only after 3-5 min; and the method has the characteristics of rapidness, direct observation, simplicity in operation, convenience for use and the like, so that the method can be used as an effective way for screening whether a lot of field freshwater and freshwater products are infected by the microcystins MC-LR.
Description
Technical field
The present invention relates to water sample detection method and reagent, be specially a kind of method and test paper of fast detecting Microcystin.
Background technology
Along with chemical fertilizer is used in industrialization, development of urbanization and agricultural production in a large number; A large amount of nutriments injects water body, and lake eutrophication is serious day by day, causes algae rapidly to grow; The excessive multiplication of blue-green algae; Can cause that water taste is stench, transparency descends, consume the water body dissolved oxygen DO, algae self such as microcystis kutz wherein, Oscillatoria, Anabaena, Nostoc and metabolic product is toxic and carcinogenesis is detrimental to health.Investigation shows that the wawter bloom that takes place in China's freshwater lake 80% is poisonous, and in the toxin that poisonous blue-green alga bloom produces, (Microcystin MC) is one of the widest, that toxicity the is maximum kind that distributes to Microcystin.
Microcystin (Microcystins) is a kind of cyclic peptide hepatotoxin; Found that at present MCs reaches more than 70 kind of isomers; That wherein common and commercialization is extracted is MC-LR, MC-YR, MC-RR, MC-LF, MC-LW, and L, R, Y, F, W represent leucine, arginine, tyrosine, phenylalanine, tryptophane respectively.Microcystin can suppress the activity of serine and serine/threonine protein phosphatase I (PP1) and 2A (PP2A); When low concentration, just there are proprietary hepatotoxicity wind agitation and cancer to bring out activity to liver; The acute hepatotoxicity symptom of MCs shows as hepatonecrosis; Liver function forfeiture and hepatorrhagia are 40～8001a g/kg to the LD50 of the lumbar injection of mouse, MC-LR be research at present the most in detail and toxicity maximum.In order to reduce toxicity and the potential hazard property of MCs to people and aquatic animal, the safely instruction value MC-LR of Microcystin is 1000ng/L in the world health organisation recommendations water.
The safety standard of relevant MCs; The safe concentration that advise various countries is slightly different; 1998, advise in " using water hygiene benchmark " that The World Health Organization (WHO) publishes that the MCs standard is 1.0 μ g/L in the potable water, the daily intake that tentative MC can tolerate temporarily is 0.04 μ g/kg.dBW; The MCs that state such as English, U.S.A limits in natural water body and the potable water is 1.0 μ g/L, and Canadian health tissues thinks that acceptable MCs standard is 0.5 μ g/L in the potable water.China does not all stipulate the MCs limit standard for a long time; Up to 2006; " water hygienic standard " in new promulgation (GB5749-2006) just increased microcapsule algae toxin at toxicity; Issued " microcystic toxins checking method " simultaneously (GB/T20466-2006), stipulated its limit value 0.001mg/Lmg/L.But limiting the quantity of of MC also do not formulated standard in the aquatic products such as fish, remains further to be studied.
Because fresh water is the drinking-water source of the mankind and animal, the life and health that the Microcystin serious harm that fresh water bloom causes is human, MC also is one of cause of disease of China's three big liver cancer, so MC has received Chinese scholars, the common people's attention.At present, the method for detection MC mainly is bioanalysis (mouse method), high performance liquid chromatography (HPLC) and immunoassay technology.The mouse method is simple and easy to do, but shortcoming is to distinguish the kind and the structure of toxin, receives interference bigger, the data out of true; The HPLC method can accurately be analyzed the content and the kind of toxin, and detectability can be low to moderate ng/g, but sample pretreatment process is complicated, and instrument is expensive, needs special analytical technology personnel; Immunochromatographic method is the immunoassay mode that comes across a kind of uniqueness of phase early 1980s, because its is fast and convenient, accurate, has high degree of specificity and hypersensitivity; Visual result is reliable, and reagent and amount of samples are few, need not valuable instrument and equipment; Simplified loaded down with trivial details routine operation process; Also reduce simultaneously the error that causes because of operation, therefore become a kind of composite immune at present with fastest developing speed technology, received domestic and international researchist's extensive concern.
Be to promote Microcystis aeruginosa and toxin study thereof, need set up a kind of simply, fast and accurately, the detection method of system.Detection to MC at present has a lot of methods, like high performance liquid chromatography (HPLC), TLC (TLC), mass spectrum (MS), EUSA (ELISA) and phosphoprotein phosphatase inhibition test etc.Also have in addition
Immune colloid gold quick diagnosis technology is that being based upon on the basis of enzyme linked immunological absorption, with collaurum is as tracer label substance markers thing, through Direct observation just can result of determination new technology.This technology has advantages such as simple, quick, accurate and pollution-free; Detect food safety detection, medicament residue and drugs fast detecting at clinical medicine detection, hormone; And many diagnostic fields such as antigen-antibody develop rapidly, are one of four big immunolabelling techniques.At present international and domestic about main enzyme linked immunosorbent detection technology (the Enzyme-linked immunosorbent assay of Microcystin detection; ELISA); The existing ELISA that sells produced in usa of China detects the kit of MC-LR; Domestic also have a few development ELISA of family detection kit, but quality is not too high.And about the colloidal gold colloidal gold detection test paper strip technology of MC-LR understatement road still, China market is not seen yet has sale.
Summary of the invention
The present invention aims to provide a kind of method that is used for the fast detecting Microcystin.
The present invention also provides the test paper of fast detecting Microcystin.
The present invention has set up the quick colloidal gold immunochromatographimethod detection method of microcapsule phycotoxin MC-LR.At first prepared the collaurum about mean grain size 30nm, in order to mark microcapsule algae toxin resistant MC-LR monoclonal antibody; The conjugate of microcapsule phycotoxin MC-LR haptens and key hole azurin is coated on the nitrocellulose filter as detecting band; Sheep anti mouse two is anti-as the control band; According to immune competition law principle, set up the immuno-chromatographic test paper strip method of fast detecting microcapsule phycotoxin MC-LR.This method sensitivity can reach 3ng/mL; Only need 3～5min can estimate judged result; Have characteristics such as quick, directly perceived, simple to operate, easy to use, can be used as the effective means whether on-the-spot a large amount of examination fresh water and freshwater product infect microcapsule phycotoxin MC-LR.
Technical scheme is that a kind of test paper that is used for the fast detecting Microcystin comprises the point sample hole successively, detects band (T line) and control band (C line); The mouse-anti MC-LR monoclonal antibody-BSA compound of spraying colloid gold label on the point sample hole detects the conjugate of band for microcapsule phycotoxin MC-LR haptens and key hole azurin, and control is with spraying sheep anti mouse two anti-.
The base material of test paper is nitrocellulose filter or qualitative filter paper.
The preparation method of the mouse-anti MC-LR monoclonal antibody-BSA compound of colloid gold label is: stirring 0.5～2hr is mixed with collaurum in (1) with mouse-anti MC-LR monoclonal anti liquid solution; Add calf serum BSA and continue to stir 20～45min; The amount ratio 1ml of collaurum and mouse-anti MC-LR monoclonal antibody and BSA wherein: 5～40 μ g: 8～12mg are preferably 1ml: 20 μ g: 10mg; Described collaurum particle diameter is at 28～32nm;
(2) get resolution of precipitate in pH=7.3～7.45 damping fluids, mouse-anti MC-LR monoclonal antibody and damping fluid amount ratio 100～600 μ g/ml are preferably 400 μ g/ml, and adding BSA to BSA concentration again is 8～12mg/ml.
The Preparation of Colloidal Gold method is: with concentration is that 0.005%～0.015% chloric acid gold solution is heated to boiling, adds the citric acid mixing and continues to boil 4～8min; The chloric acid gold is 1: 0.012～0.0016 with the amount ratio of citric acid.
Mouse-anti MC-LR MONOCLONAL ANTIBODIES SPECIFIC FOR method is to get the MC-LR haptens and mix hatching back immune mouse with complete freund adjuvant with the conjugate of key hole azurin; Get after 10～15 days the conjugate of MC-LR haptens and key hole azurin mixes with incomplete freund adjuvant hatch after, per 10～15 days immune mouses, totally three times.
The conjugate preparation method of MC-LR haptens and key hole azurin is: with the preparation of key hole azurin (KLH) hybrid reaction, both weight ratios are 1: 6～1: 7 after the Microcystin activation; And, get supernatant with phosphate buffer PBS dialysis.
The conjugate of the monoclonal antibody in the mouse-anti MC-LR monoclonal antibody-BSA compound of colloid gold label, MC-LR haptens and key hole azurin and sheep anti mouse two anti-weight ratios are 1: 1.8～2: 2～3; Be preferably 1: 18.75: 2.5.
Detection method is water sample to be measured to be dripped in the point sample hole observation colour developing result in 3～5min.The mouse-anti MC-LR monoclonal antibody of water sample to be measured and colloid gold label-BSA compound amount ratio 100 μ l: 0.25～1.5 μ g, being preferably is 100 μ l: 1 μ g.
MC-LR is a polypeptide, and no special group has certain difficulty for the preparation monoclonal antibody.This research has obtained high-quality MC-LR monoclonal antibody on the basis of successfully synthesizing the MC-LR comlete antigen, lay the foundation for successfully preparing the MC-LR colloidal gold colloidal gold detection test paper strip.
Along with people's deepens continuously to MCs harm understanding; " the Drinking Water hygienic practice " of the new promulgation of China Ministry of Public Health listed in the standard value of MC-LR, and the foundation of the detection method of quick, easy, economic Microcystin has become an instant job.Recently; 31 laboratories in country variant and area adopt distinct methods such as high performance liquid chromatography, enzyme linked immunological method and protein phosphatase enzyme method mensuration with Microcystin in a kind of sample, and its measured value had notable difference when the result was presented at and carries out the quantitative measurement Microcystin with commercialization MCLR as standard items.At present, have PDAD or be connected with the Microcystin detection means that mass spectral high performance liquid chromatograph is still the most extensively employing.High performance liquid chromatography is a kind of effectively detection method, and it can not only be qualitative with toxin, but also can provide accurately quantitatively.But its equipment needed thereby is expensive, and high to operating personnel's professional technique requirement, the foundation of method depends on a series of standard items, and testing process needs complicated pre-treatment toward contact, compares elapsed time.With biology is the detection method that basic method has been represented quick, simple MCs.ELISA to sample pre-treatments require low, operation of equipment simple, detection sensitivity and selectivity are high, but the kit cross reaction of commercialization MC S changes greatly, generally can only come quantitative toxin with the form of MCLR equivalent.Specificity takes place again with it with the thing to be checked and the bond of golden labelled antibody and combines and be trapped in the immunity technology for gold, can be through being observed visually the colour developing result.Have volume little, easy to carry, do not need instrument and equipment, simple to operate, can on-the-spot detect, 3～5min goes out the result and the result can be by naked eyes according to plurality of advantages such as T line color depth judgements; Be fit to very much batch samples is carried out on-the-spot primary dcreening operation, in the residue of veterinary drug fast detecting, quite paid close attention to.This research on the basis of the immunity colloidal gold test paper strip detection method of having set up Microcystin, to its technical parameter with detect index and evaluate, and Preliminary Applications is in actual sample detects, its sensitivity, repeatability, batch between stability all fine; Still do not have like product at present both at home and abroad and sell, have only ELISA to detect paper box and be born, compare with the ELISA kit; Our test strips of development is cheap, simple to operate, 3-5 minute detection time; The user of service there is not any technical requirement; Under the general not enough national conditions of China's professional knowledge, this product has very big promotional value, helps to provide the safety detection of China's aquatic products.
Detection of the present invention is limited to 2～3ng/ml, and 1.0ng/ml still has certain distance with the Drinking Water in China standard, can not detect whether National standard of water sample, but can be used for the preliminary examination of a large amount of water samples; Also can be used for a large amount of examinations of aquatic products such as fish.Because the colloidal gold strip detection method is simple, visual result, low price, be easy to carry; Therefore; In a large amount of examinations of water sample and aquatic products, certain use value is arranged, be used in combination, must reduce a lot of workloads and save a large amount of expenses with other complicated detection methods.Expectation will have wider application prospect in inspection and quarantine, food security department and sanitation monitoring department, and certain economic and social benefit are arranged.Simultaneously, can be the aquatic products edible safety detects the revision of international method or augments scientific basis is provided.
Embodiment
Below in conjunction with embodiment the present invention is done further in detail, intactly explains:
Reagent and instrument
Instrument: carbon dioxide constant incubator, superclean bench, ELIASA, 37 ℃ of constant incubators, constant temperature magnetic stirrer (state China electrical equipment), Biodot Membrane jetter (genome company), cutting cutter (Shanghai Korea Spro feels electronics technology).Electro-heating standing-temperature cultivator (encircling in the Tianjin).
Gold chloride, chemical reagent such as trisodium citrate are given birth to the worker available from Shanghai; Microcystin is available from her Jim Press company of Beijing; DMSO, water-soluble carbodiimide-NHS (EDC), N-hydroxy-succinamide (NHS), dicyclohexylcarbodiimide (DCC) and various cross reaction medicine all give birth to the worker available from Shanghai; Bovine serum albumin (BSA) is available from Chinese holly biotech firm; Fu Shi Freund's complete adjuvant, freund 's incomplete adjuvant, o-phenylenediamine (OPD) etc. are the Sigma Company products; Hyclone and NBCS are Chinese holly biotech firm product, and HAT, HTIMDM and soup-stock nutrient culture media are available from hyclone company, and PEG2000 is the Roche Company products, GAM-HPR, and DMSO etc. are available from Merk company.
The preparation of embodiment 1 comlete antigen
The 3mg Microcystin is dissolved among the 1.2mlDMF, adds 100 microlitre activating solutions (12mg EDC, HCL and 7mg NHS are dissolved among the 1ml DMF), and 4 ℃ of priming reactions spend the night.Get 1h in the carbonic acid buffer that 20mg KLH key hole azurin is dissolved in 5ml pH=9.6,4 ℃ are spent the night.
Mix the back with 0.01M PBS dialysis 3 days, the centrifuging and taking supernatant, with the concentration 1.4mg/ml of lorry method mensuration MCLR-KLH conjugate, dilution is different concentration as required then.
The preparation of embodiment 2 monoclonal antibodies (mouse-anti MC-LR)
Animal immune: an amount of MCLR-KLH mixes with the complete freund adjuvant of equal-volume; Hatching fully; Get 58 the week age female BALB/C mice, subcutaneous 3 points of nape portion, four limbs armpit! popliteal) subcutaneous; And lumbar injection, every injected in mice hatching back antigen 500 μ l (being equivalent to every mouse immune antigen 1 00 μ g).After 14 days, an amount of MCLH-KLH mixes with the incomplete freund adjuvant of equal-volume, and hatching subcutaneous multiple spot in back and lumbar injection antigen are pressed the immunity in per 14 days of this method once fully.Fundamental immunity is after 7 days for the third time, and tail vein blood detects and tires.
MCLH-K encapsulates the enzyme sample, and indirect method is measured and tired.The clear gradient dilution of mice serum (1: 1000,1: 2000,1: 4000,1: 8000,1: 16000), blank BALB/C mice serum is done negative control.
Fusion of Cells: the aseptic immune mouse spleen cell of getting mixes with 5: 1 volume ratios with the SP2/0 myeloma cell who is in exponential phase, merges by conventional method, and fusion agent is PEG.
Cell screening: merge after 7 days, under the selectivity of HAT is cultivated, SP2/0, all death of splenocyte of not merging, hybridoma is the growth of colony shape, fusion rate 100%.Getting the cell conditioned medium indirect ELISA detects.26 in strong positive hole, 138 in weak positive hole.
Specific detection: choose positive cell hole supernatant and do the indirect competitive ELISA experiment, detect the specificity of antibody in the supernatant.Through 3 subclones, 8A9 and the complete purifying of 6E12 cell, cell 100% positive of subclone.
Antibody assessment: adopt indirect elisa method to measure.AC is adjusted to 1mg/ml, again with PBS dilution (1: 10000,1: 20000,1: 40000,1: 80000,1: 160000,1: 320000,1: 640000).It is 1: 6.4 * 10 that the highest ascites is tired
5
Antibody sensitivity detects: adopt the indirect competitive ELISA method to measure.IC50 is 0.81ng/ml.
The preparation of embodiment 3 collaurums
The preparation method of the colloid gold particle of introducing with reference to Frens1973 gets 1ml 1% gold chloride (HAuCl
4) solution, join in the 100ml water, be heated to and boil, add 0.5～4ml, 1% trisodium citrate again, mix and boil 5min, do not change up to color.This method can prepare the gold grain of 15～60nm different-diameter size, and grain size depends on the amount of the trisodium citrate of adding.
0.01% gold chloride (HAuCl
4) WS 100ml is heated to boiling, adds 1% trisodium citrate 1.5ml, mixing boiled 5 minutes, treat the colloidal gold solution color by indigo plant after purple stain is purplish red, cool off subsequent usely, obtain particle diameter 30nm (± 2nm) collaurum.
Embodiment 4 collaurums are to the mark of monoclonal antibody
Get the collaurum 2ml of embodiment 3 gained 30nm, add the monoclonal antibody 0.2ml of 200 μ g/ml embodiment, 2 preparations, stirring and evenly mixing 1 hour; Add 20mg BSA again, continue to stir centrifugal 30 minutes of 10000rpm 30 minutes; Remove supernatant, deposition is dissolved with PH7.420mM Tris-HCL 100 μ l, add BSA to final concentration be 10mg/ml; Put in 4 ℃ subsequent usely, obtain the mouse-anti MC-LR monoclonal antibody-BSA compound (being called down gold mark mouse-anti MC-LR) of colloid gold label.
The detection of embodiment 5 sensitivity
Gold mark mouse-anti MC-LR to variant monoclonal antibody concentration mark among the embodiment 4 encapsulates with the metal spraying machine, and the material of test paper is a nitrocellulose filter; Gold mark mouse-anti MC-LR (embodiment 4 preparations) is sprayed on the point sample hole, and detection line (T line) is gone up the mouse-anti MC-LR that spraying concentration is 0.75mg/ml (embodiment 2 preparations), and control line (C line) is gone up the commercially available sheep anti-mouse igg antibody (concentration 1.0mg/ml) of spraying; Quantity for spray is 2.5 μ l/cm (length of spraying is 1cm), drying, assembling test paper.That is, the monoclonal antibody consumption 1 μ g in the point sample hole among the gold mark mouse-anti MC-LR, T line MCLR-KLH consumption 1.875 μ g, C line 2.5 μ g sheep anti-mouse igg antibodies.During test, drip the MC-LR standard solution of 100 μ l variable concentrations on the point sample hole respectively.
Prepare gold label test strip with each top condition, each concentration MC-LR standard items is done sensitivity detect, the result is following:
The sensitivity effect of the different MC-LR standard items of table 1 concentration relatively
* annotate: refer to 5 minutes testing results
The T line positive result that do not develop the color, negative result develops the color.
The colour developing of C line is effective for this test paper, and the explanation test paper that do not develop the color is invalid.
Test paper is put in 2 ℃～30 ℃ and is preserved, away from humidity and illumination; The term of validity is 2 years.As in refrigerator, preserving, after taking out in the refrigerator, need place at room temperature 30 minutes, can break seal.
Confirming of embodiment 6 gold medal timestamp monoclonal antibody concentration
Get the collaurum 2ml of the 30nm of embodiment 3 preparations, add concentration 50～400 μ g/ml monoclonal antibody 0.2ml, mixing is 1 hour in the stirring; Add 20mgBSA again; Continue to stir 30 minutes, centrifugal 30 minutes of 10000rpm removes supernatant; Deposition is dissolved with PH7.420mM Tris-HCl 100 μ l, add BSA to final concentration be 10mg/ml.
Gold mark mouse-anti MC-LR to variant monoclonal antibody concentration mark among the embodiment 4 encapsulates with the metal spraying machine; All spray 2.5 μ l/cm (length of spraying is 1cm); Dry; The assembling test paper, with 50ng/ml, MC-LR positive sample and of the influence of the more different monoclonal antibody amounts of PBS application of sample (100 μ l) to the sensitivity of gold mark monoclonal antibody.All the other conditions are with embodiment 5, and the result is following.
The effect of the different golden mark monoclonal antibody concentration of table 2 relatively
* annotate: refer to 5 minutes testing results
The result shows, monoclonal antibody concentration is with 200 μ g/ml during golden mark, and testing result positive and negative sample is the most satisfied.
Confirming of embodiment 7 gold medals mark mouse-anti MC-LR monoclonal antibody package amount
Gold mark mouse-anti MC-LR to embodiment 5 encapsulates with the metal spraying machine, sprays different discharge rates, and drying is by technology assembling test paper, with the more different sensitivity height that encapsulate concentration of MC-LR positive sample and the PBS application of sample (100 μ l) of 50ng/ml.The spraying length of T line and C line is with gold mark mouse-anti MC-LR, and the result is following.
Positive sample and PBS's detects the result under the different discharge rates of table 3
* annotate: refer to 5 minutes testing results
When discharge rate reaches 2.5 μ l/cm, the result that detects of positive sample and PBS sample promptly adheres to specification, takes all factors into consideration the final package amount of confirming gold mark monoclonal antibody, and promptly metal spraying machine discharge rate is 2.5 μ l/cm.
Confirming of embodiment 8 detection lines (T) working fluid concentration
Adjust the concentration that encapsulates of MCLR-KLH, by technology assembling reagent strip, with the more different sensitivity height that encapsulate concentration with the PBS application of sample of positive sample, all the other are with embodiment 5, and the result sees table 4:
The effect that the different MC-LR-BSA of table 4 encapsulate concentration compares
* annotate: refer to 5 minutes testing results
Reach 0.75mg/ml when encapsulating concentration, can satisfy the requirement that detects of positive sample, can reach the requirement of PBS sample again, further raising encapsulates concentration and has then satisfied not the requirement of positive sample, reduces the requirement that detects that concentration then can not reach the PBS sample.Take all factors into consideration and confirm that finally it is 0.75mg/ml that mouse-anti MC-LR-BSA detection line encapsulates concentration.
Confirming of embodiment 9 control lines (C) working fluid concentration
Adjustment sheep anti-mouse igg antibody concentration is assembled reagent strip by normal preparation technology, with the PBS application of sample relatively difference encapsulate concentration control line developing time, all the other are with embodiment 5, the result sees table 5.
The table 5 different two anti-effects that encapsulate concentration compare
Encapsulate concentration and reach 1.0mg/ml, control line i.e. clear debating in 1min, adheres to specification.Final definite control line encapsulates concentration 1.0mg/ml.
Confirming of 10 reaction time of embodiment
Method of operating is with embodiment 5, and standing time is different.
A scheme: react 2 minutes observationss.
B scheme: react 3 minutes observationss.
C scheme: react 5 minutes observationss.
D scheme: react 10 minutes observationss.
A scheme result: selecting detectable concentration is the MC-LR standard items of 5ng/ml, the no band of detection zone in 2 minutes (T), and a little less than the PBS colour developing, test failure.
B scheme result: selecting detectable concentration is the MC-LR standard items of 10ng/ml, the no band of detection zone in 3 minutes (T), and the PBS colour developing is strong, test passes.
C scheme result: selecting detectable concentration is the MC-LR standard items of 20ng/ml, the no band of detection zone in 5 minutes (T), and the PBS colour developing is strong, test passes.
D scheme result: selecting detectable concentration is the MC-LR standard items of 30ng/ml, and detection zone in 10 minutes (T) has band, and the PBS colour developing is strong, test failure.
According to choice reaction time as a result be 3-5 minute observations, the result was invalid in 10 minutes.
Confirming of embodiment 11 colloid gold particles
Make the gold grain of different-diameter size by this method; Mark mouse-anti MC-LR monoclonal antibody; By technology assembling test strips, with positive sample and PBS application of sample, relatively (positive sample is 50ng/mlMC-LR to the biologically active of variable grain size gold mark bond; Require detection line not occur in the 5min, PBS requires colour developing strong.)
The result sees table 6:
* annotate: refer to 5 minutes testing results
The result of comprehensive relatively said method and invention has only the collaurum of 30nm can satisfy positive detection time simultaneously and requires the requirement as a result with PBS, therefore selects the collaurum of 30nm to be used for mark, confirms the preparation technology of colloid gold particle: 0.01% gold chloride (HAuCl
4) WS 100ml is heated to boiling, adds 1% trisodium citrate 1.5ml, mixing boiled 5 minutes, treat the colloidal gold solution color by indigo plant after purple stain is purplish red, cool off subsequent use.The colloid gold particle of prepared satisfies the detection requirement of test paper in view of the above.
The principle that immunochromatographic method is analyzed MC is; Sample solution to be checked is added in the point sample hole of test strips one end; Upwards divide a word with a hyphen at the end of a line through capillary action; Specific immune response takes place prior to the mouse-anti (anti-MC-IgG) of colloid gold label in the MC in the sample, moves to calibration tape (being sprayed with MC-LR-KLH, calibration tape T band) on formed immune complex continues; If the MC in the sample is abundant, all combined the site of golden labeling antibody, then immune complex does not stop in the test section; Test strip does not develop the color; Immune complex moves to control band (being sprayed with the SA of sheep anti mouse, contrast band C band), and second kind of immune association reaction takes place; Immune complex is trapped, and the contrast band shows red.When the amount that does not contain MC or MC in the sample was lower than detection limit, it is red that two bands all show, but according to what of determinand MC in the colored depth judgement sample of test.
The factor that influences colloid gold label is a lot, the size that mainly contains colloid gold particle that should note especially, the purity of labelled antibody, the pH value of solution and the pure degree of used container etc., and the present invention has paid special attention to above problem, and the mark effect is better.Trisodium citrate and the gold chloride reaction of the present invention through groping different amounts; Different gold mark monoclonal antibody concentration, different gold mark monoclonal antibody package amounts, the different antigens conjugate encapsulates concentration; Different two anti-ly encapsulate concentration and different reaction time and study the effect of comparison colloidal gold chromatography method; The gold label test strip sensitivity of preparation reaches 3ng/ml thus, only needs 3～5min can estimate judged result, has improved detection efficiency greatly.
Claims (10)
1. the test paper of a fast detecting Microcystin is characterized in that, comprises the point sample hole successively, detects band and control band; The mouse-anti MC-LR monoclonal antibody-BSA compound of spraying colloid gold label on the point sample hole detects the conjugate of band for microcapsule phycotoxin MC-LR haptens and key hole azurin, and control is with spraying sheep anti mouse two anti-.
2. the test paper of the said fast detecting Microcystin of claim 1 is characterized in that, the base material of test paper is nitrocellulose filter or qualitative filter paper.
3. the test paper of the said fast detecting Microcystin of claim 1; It is characterized in that the conjugate of the monoclonal antibody in the mouse-anti MC-LR monoclonal antibody-BSA compound of said colloid gold label, MC-LR haptens and key hole azurin and sheep anti mouse two anti-weight ratios are 1: 1.8～2: 2～3.
4. the test paper of claim 1 or 3 said fast detecting Microcystins; It is characterized in that the conjugate of the monoclonal antibody in the mouse-anti MC-LR monoclonal antibody-BSA compound of said colloid gold label, MC-LR haptens and key hole azurin and sheep anti mouse two anti-weight ratios are 1: 18.75: 2.5.
5. the test paper of the said fast detecting Microcystin of claim 1; It is characterized in that; The preparation method of the mouse-anti MC-LR monoclonal antibody-BSA compound of said colloid gold label is: stirring 0.5～2hr is mixed with collaurum in (1) with mouse-anti MC-LR monoclonal anti liquid solution; Add calf serum BSA and continue to stir 20～45min, wherein the amount ratio 1ml of collaurum and mouse-anti MC-LR monoclonal antibody and BSA: 5～40 μ g: 8～12mg;
Described collaurum particle diameter is at 28～32nm, and its preparation method is: with concentration is that 0.005%～0.015% chloric acid gold solution is heated to boiling, adds the citric acid mixing and continues to boil 4～8min; The chloric acid gold is 1: 0.012～0.0016 with the amount ratio of citric acid;
(2) get resolution of precipitate in pH=7.3～7.45 damping fluids, mouse-anti MC-LR monoclonal antibody and damping fluid amount ratio 100～600 μ g/ml, adding BSA to BSA concentration again is 8～12mg/ml.
6. the test paper of the said fast detecting Microcystin of claim 5 is characterized in that, the amount ratio of collaurum and mouse-anti MC-LR monoclonal antibody and BSA is 1ml in the step (1): 20 μ g: 10mg; Mouse-anti MC-LR monoclonal antibody and damping fluid amount ratio are 400 μ g/ml in the step (2).
7. the test paper of the said fast detecting Microcystin of claim 1; It is characterized in that; The preparation method of the conjugate of said microcapsule phycotoxin MC-LR haptens and key hole azurin is: with the preparation of key hole azurin (KLH) hybrid reaction, both weight ratios are 1: 6～1: 7 after the Microcystin activation; And, get supernatant with phosphate buffer PBS dialysis.
8. the test paper of the said fast detecting Microcystin of claim 5 is characterized in that, said mouse-anti MC-LR MONOCLONAL ANTIBODIES SPECIFIC FOR method is to get the MC-LR haptens and mix hatching back immune mouse with complete freund adjuvant with the conjugate of key hole azurin; Get after 10～15 days the conjugate of MC-LR haptens and key hole azurin mixes with incomplete freund adjuvant hatch after, per 10～15 days immune mouses, totally three times.
9. the method for a fast detecting Microcystin is characterized in that, water sample to be measured is dripped in the point sample hole of each said test paper of claim 1～8, places 3～5min; The mouse-anti MC-LR monoclonal antibody of water sample to be measured and colloid gold label-BSA compound amount ratio 100 μ l: 0.25～1.5 μ g.
10. the method for the said a kind of fast detecting Microcystin of claim 9 is characterized in that, the mouse-anti MC-LR monoclonal antibody of water sample to be measured and colloid gold label-BSA compound amount ratio 100 μ l: 1 μ g.
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