CN102495212B - Method and test paper for rapidly detecting microcystins - Google Patents
Method and test paper for rapidly detecting microcystins Download PDFInfo
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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 development and the agricultural production of industrialization, urbanization in a large number, a large amount of nutriments injects water body, lake eutrophication is day by day serious, cause algae rapidly to grow, the excessive multiplication of blue-green algae, can cause that water taste is stench, transparency declines, consume Dissolved Oxygen in Water, algae self such as microcystis kutz wherein, Oscillatoria, Anabaena, Nostoc and metabolic product is toxic and carcinogenesis, are detrimental to health.Investigation demonstration, the wawter bloom occurring in China's freshwater lake 80% is poisonous, in the toxin producing at poisonous blue-green alga bloom, Microcystin (Microcystin, MC) is one of the widest, toxicity the is maximum kind that distributes.
Microcystin (Microcystins) is a kind of cyclic peptide hepatotoxin, found that at present MCs reaches more than 70 kind of isomers, that wherein the most common and commercialization is extracted is MC-LR, MC-YR, MC-RR, MC-LF, MC-LW, and L, R, Y, F, W represent respectively leucine, arginine, tyrosine, phenylalanine, tryptophane.Microcystin can suppress the activity of serine and serine/threonine protein phosphatase I (PP1) and 2A (PP2A), when low concentration, to liver, just there are proprietary hepatotoxicity wind agitation and cancer to bring out activity, the Acute Hepatic toxicity symptom of MCs shows as hepatonecrosis, liver function is lost and hepatorrhagia, to the LD50 of the lumbar injection of mouse, be 40～8001a g/kg, MC-LR be at present research the most in detail and toxicity maximum.In order to reduce MCs to the toxicity of people and aquatic animal and potential hazard, in world health organisation recommendations water, the safely instruction value MC-LR of Microcystin is 1000ng/L.
The safety standard of relevant MCs, the safe concentration that advise various countries is slightly different, 1998, the < < that the World Health Organization (WHO) (WHO) publishes is with advising in water hygiene benchmark > > that in potable water, MCs standard is 1.0 μ g/L, and the daily intake that tentative MC can tolerate is temporarily 0.04 μ g/kg.dBW; The MCs that Ying,Mei Deng state limits in natural water body and potable water is 1.0 μ g/L, and Canadian health tissues thinks in potable water that acceptable MCs standard is 0.5 μ g/L.China does not all stipulate MCs limit standard for a long time, until 2006, at the new < < water hygienic standard > > (GB5749-2006) promulgating, just at toxicity, increased microcapsule algae toxin, promulgate < < microcystic toxins checking method > > (GB/T20466-2006) simultaneously, 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, need further to be studied.
Because fresh water is the drinking-water source of the mankind and animal, one of cause of disease of life and health ，MCYe Shi China three large liver cancer of the Microcystin serious harm mankind that fresh water bloom causes, so MC has been subject to Chinese scholars, the common people's attention.At present, the method for detection MC is mainly bioanalysis (mouse method), high performance liquid chromatography (HPLC) and immunoassay technology.Mouse method is simple and easy to do, but shortcoming is to distinguish kind and the structure of toxin, be disturbed larger, data out of true; HPLC method can accurate analysis toxin content and kind, 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, fast and convenient, accurate due to it, there is high degree of specificity and hypersensitivity, visual result is reliable, and reagent and amount of samples few, without valuable instrument and equipment, simplified loaded down with trivial details routine operation process, also reduced the error causing because of operation, therefore become a kind of composite immune technology with fastest developing speed at present, be subject to domestic and international researchist's extensive concern simultaneously.
For promoting Microcystis aeruginosa and toxin study thereof, need to set up a kind of simply, fast and accurately, the detection method of system.At present the detection of MC is had to a lot of methods, as high performance liquid chromatography (HPLC), thin layer chromatography (TLC), mass spectrum (MS), enzyme linked immunosorbent assay (ELISA) and phosphoprotein phosphatase inhibition test etc.In addition also have
Immune colloid gold quick diagnosis technology is to be based upon on the basis of Enzyme-linked Immunosorbent Assay, to using collaurum as tracer label substance markers thing, new technology that just can result of determination by direct observation.This technology has the advantages such as simple, quick, accurate and pollution-free, in clinical medicine detection, hormone test food safety detection, medicament residue and drugs fast detecting, and many diagnostic fields such as antigen-antibody develop rapidly, be one of four large immunolabelling techniques.At present international and domesticly about Microcystin, detect main enzyme linked immunosorbent detection technology (Enzyme-linked immunosorbent assay, ELISA), the existing kit of selling ELISA detection MC-LR produced in USA of China, domestic also have several development ELISA 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 also there are no sale.
Summary of the invention
The present invention aims to provide a kind of method for 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.First the collaurum of having prepared mean grain size 30nm left and right, 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 nitrocellulose filter as detecting band, sheep anti mouse two is anti-as controlling band, according to Immune competition method principle, set up the immuno-chromatographic test paper strip method of fast detecting microcapsule phycotoxin MC-LR.The method sensitivity can reach 3ng/mL, only need 3～5min can estimate judged result, there is the features 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 for fast detecting Microcystin, comprises successively point sample hole, detects band (T line) and control band (C line); On point sample hole, spray mouse monoclone antibody against MC-LR-BSA compound of colloid gold label, detect band for the conjugate of microcapsule phycotoxin MC-LR haptens and key hole azurin, control and be with spraying sheep anti mouse two anti-.
The base material of test paper is nitrocellulose filter or qualitative filter paper.
The preparation method of mouse monoclone antibody against MC-LR-BSA compound of colloid gold label is: (1) is by collaurum and mouse-anti MC-LR monoclonal antibody solution mix and blend 0.5～2hr, add calf serum BSA to continue to stir 20～45min, the amount ratio 1ml of collaurum and mouse monoclone antibody against MC-LR 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 and be precipitated and dissolved in pH=7.3～7.45 damping fluid, mouse monoclone antibody against MC-LR and damping fluid amount ratio 100～600 μ g/ml, be preferably 400 μ g/ml, then to add BSA to BSA concentration be 8～12mg/ml.
Collaurum preparation method is: the chloric acid gold solution that is 0.005%～0.015% by concentration is heated to boiling, adds citric acid to mix and continues to boil 4～8min; Chloric acid gold is 1: 0.012～0.0016 with the amount ratio of citric acid.
The preparation method of mouse monoclone antibody against MC-LR is to get MC-LR haptens and mix the rear immune mouse of hatching with complete freund adjuvant with the conjugate of key hole azurin; After 10～15 days, get MC-LR haptens and mix after hatching with incomplete freund adjuvant with the conjugate of key hole azurin, every 10～15 days immune mouses, totally three times.
The conjugate preparation method of MC-LR haptens and key hole azurin is: Microcystin activates rear and key hole azurin (KLH) hybrid reaction preparation, and both weight ratios are 1: 6～1: 7; And with phosphate buffer PBS dialysis, get supernatant.
The conjugate of monoclonal antibody, MC-LR haptens and key hole azurin in mouse monoclone antibody against MC-LR-BSA compound of colloid gold label and the anti-weight ratio of sheep anti mouse two 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 point sample hole to observation colour developing result in 3～5min.Mouse monoclone antibody against MC-LR-BSA compound amount ratio 100 μ l of water sample to be measured and colloid gold label: 0.25～1.5 μ g, being preferably is 100 μ l: 1 μ g.
MC-LR is polypeptide, without special group, for preparation monoclonal antibody, has certain difficulty.This research has obtained high-quality MC-LR monoclonal antibody on the basis of successfully synthesizing MC-LR comlete antigen, for successfully preparing MC-LR colloidal gold colloidal gold detection test paper strip, lays the foundation.
Along with people's deepening continuously to MCs harm understanding, the new < < Drinking Water hygienic practice > > promulgating of China Ministry of Public Health lists 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 as Microcystin in high performance liquid chromatography, enzyme linked immunological method and protein phosphatase enzyme method mensuration same sample, result be presented at using commercialization MCLR while carrying out quantitative measurement Microcystin as standard items its measured value have notable difference.At present, with diode array detector or be connected with mass spectrographic high performance liquid chromatograph and be still the most extensively the Microcystin detection means adopting.High performance liquid chromatography is a kind of effectively detection method, and it can not only be qualitative by toxin, but also accurate quantitative analysis can be provided.But its equipment needed thereby is expensive, 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, relatively elapsed time.Take biology as basic method has represented fast, the detection method of simple MCs.Euzymelinked immunosorbent assay (ELISA) to sample pre-treatments require low, equipment operating simple, detection sensitivity and selectivity high, but the kit cross reaction of commercialization MC S changes greatly, generally can only carry out quantitative toxin with the form of MCLR equivalent.There is specific binding with it again by the bond of thing to be checked and golden labelled antibody and be trapped in Immuno gold technology, can be by being observed visually colour developing result.Have volume little, easy to carry, do not need instrument and equipment, simple to operate, can Site Detection, 3～5min goes out result and result can be by naked eyes according to plurality of advantages such as T line color depth judgements, be applicable to very much batch samples to carry out on-the-spot primary dcreening operation, quite concerned in residue of veterinary drug fast detecting.This research is on the basis of immunity colloidal gold test paper strip detection method of having set up Microcystin, and 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; There is no at present like product both at home and abroad sells, only have ELISA Test paper box to be born, compare with ELISA kit, we development test strips cheap, simple to operate, detection time 3-5 minute, to user of service without any technical requirement, under the general not enough national conditions of China's professional knowledge, this product has very large promotional value, contributes 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 Drinking Water in China standard, and can not detect water sample and whether meet national standard, but can be for the preliminary examination of a large amount of water samples; Also can be for a large amount of examinations of the aquatic products such as fish.Because 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, have certain use value, the detection method complicated with other is combined with, and must reduce a lot of workloads and save a large amount of expenses.Expectation, by having wider application prospect in inspection and quarantine, food security department and sanitation monitoring department, has certain economic and social benefit.Meanwhile, can be 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 described in further detail and completely:
Reagent and instrument
Instrument: carbon dioxide constant incubator, superclean bench, microplate reader, 37 ℃ of constant incubators, thermostatic electromagnetic 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 Tianjin).
Gold chloride, the chemical reagent such as trisodium citrate are purchased from the raw work in Shanghai; Microcystin is purchased from her Jim Press company of Beijing; DMSO, water-soluble carbodiimide-NHS (EDC), N-hydroxy-succinamide (NHS), dicyclohexylcarbodiimide (DCC) and various cross reaction medicine are all purchased from the raw work in Shanghai; Bovine serum albumin (BSA) is purchased from Chinese holly biotech firm; Fu Shi Freund's complete adjuvant, freund 's incomplete adjuvant, o-phenylenediamine (OPD) Deng Wei Sigma company product; Hyclone and NBCS are Chinese holly biotech firm product, and HAT, HTIMDM and soup-stock nutrient culture media are purchased from hyclone company, and PEG2000 is Roche company product, GAM-HPR, and DMSO etc. are purchased from Merk company.
Embodiment 1 comlete antigen preparation
3mg Microcystin is dissolved in 1.2mlDMF, adds 100 microlitre activating solutions (12mg EDC, HCL and 7mg NHS are dissolved in 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.
After mixing, with 0.01M PBS dialysis 3 days, centrifuging and taking supernatant, used lorry method to measure the concentration 1.4mg/ml of MCLR-KLH conjugate, and then dilution is different concentration as required.
The preparation of embodiment 2 monoclonal antibodies (mouse-anti MC-LR)
Animal immune: appropriate MCLR-KLH mixes with the complete freund adjuvant of equal-volume, hatching completely, get 58 week age female BALB/C mice, subcutaneous 3 points of nape portion, four limbs armpit (popliteal) subcutaneous, and lumbar injection, antigen 500 μ l (being equivalent to every mouse immune antigen 1 00 μ g) after every injected in mice hatching.After 14 days, appropriate MCLH-KLH mixes with the incomplete freund adjuvant of equal-volume, subcutaneous multiple spot and lumbar injection antigen after hatching completely, by the immunity in every 14 days of this method once.Fundamental immunity is after 7 days for the third time, and tail vein blood detects and tires.
MCLH-K is coated with enzyme sample, and indirect Determination is 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 in exponential phase, merges according to a 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 cell conditioned medium indirect ELISA detects.26, strong positive hole, 138, weak positive hole.
Specific detection: choose positive cell hole supernatant and do indirect competitive ELISA experiment, detect the specificity of antibody in supernatant.Through 3 subclones, 8A9 and 6E12 cell Economical Purification, cell 100% positive of subclone.
Antibody assessment: adopt indirect elisa method to measure.Antibody concentration is adjusted to 1mg/ml, then with PBS dilution (1: 10000,1: 20000,1: 40000,1: 80000,1: 160000,1: 320000,1: 640000).The highest titer of ascites is 1: 6.4 * 10
5.
Antibody sensitivity detects: adopt 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 100ml water, be heated to boil, then add 0.5～4ml, 1% trisodium citrate, mix and boil 5min, until color does not change.The method can be prepared the gold grain of 15～60nm different-diameter size, and grain size depends on the amount of the trisodium citrate adding.
0.01% gold chloride (HAuCl
4) aqueous solution 100ml is heated to boiling, adds 1% trisodium citrate 1.5ml, mixes, boil 5 minutes, until colloidal gold solution color by indigo plant after purple stain is purplish red, cooling standby, obtain the collaurum of particle diameter 30nm (± 2nm).
The mark of embodiment 4 collaurums to monoclonal antibody
Get the collaurum 2ml of embodiment 3 gained 30nm, the monoclonal antibody 0.2ml that adds 200 μ g/ml embodiment 2 preparations, stir and evenly mix 1 hour, then add 20mg BSA, continue to stir 30 minutes, centrifugal 30 minutes of 10000rpm, remove supernatant, precipitation is dissolved with PH7.420mM Tris-HCL 100 μ l, add BSA to final concentration be 10mg/ml, put in 4 ℃ standbyly, obtain mouse monoclone antibody against MC-LR-BSA compound (under be called gold mark mouse-anti MC-LR) of colloid gold label.
The detection of embodiment 5 sensitivity
With metal spraying machine, the gold mark mouse-anti MC-LR of variant monoclonal antibody concentration mark in embodiment 4 is coated with, the material of test paper is nitrocellulose filter; Gold mark mouse-anti MC-LR (embodiment 4 preparations) is sprayed on point sample hole, the mouse-anti MC-LR that the upper spraying of detection line (T line) concentration is 0.75mg/ml (embodiment 2 preparations), the commercially available sheep anti-mouse igg antibody (concentration 1.0mg/ml) of the upper spraying of control line (C line); Quantity for spray is 2.5 μ l/cm (length of spraying is 1cm), dry, assembling test paper.That is, the monoclonal antibody consumption 1 μ g in point sample hole in 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, on point sample hole, drip respectively the MC-LR standard solution of 100 μ l variable concentrations.
With each top condition, prepare gold label test strip, each concentration MC-LR standard items are done to sensitivity and detect, result is as follows:
The sensitivity effect comparison of the different MC-LR standard items of table 1 concentration
* note: 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 preserved in refrigerator, after taking out in refrigerator, need to place at room temperature 30 minutes, can break seal.
Determining 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, in stirring, mix 1 hour, add again 20mgBSA, continue to stir 30 minutes, centrifugal 30 minutes of 10000rpm, removes supernatant, precipitation is dissolved with PH7.420mM Tris-HCl 100 μ l, add BSA to final concentration be 10mg/ml.
With metal spraying machine, the gold mark mouse-anti MC-LR of variant monoclonal antibody concentration mark in embodiment 4 is coated with, all spray 2.5 μ l/cm (length of spraying is 1cm), dry, assembling test paper, with 50ng/ml, the impact of MC-LR positive sample and PBS application of sample (100 μ l) more different monoclonal antibody amounts on the sensitivity of gold mark monoclonal antibody.All the other conditions are with embodiment 5, and result is as follows.
The effect comparison of the different golden mark monoclonal antibody concentration of table 2
* note: refer to 5 minutes testing results
Result shows, 200 μ g/ml for monoclonal antibody concentration during golden mark are positive the most satisfied with the testing result of ' negative ' specimens.
Determining of embodiment 7 gold medal mark mouse monoclone antibody against MC-LR package amounts
With metal spraying machine, the gold mark mouse-anti MC-LR of embodiment 5 is coated with, sprays different discharge rates, dry, by technique assembling test paper, with MC-LR positive sample and the PBS application of sample (100 μ l) of 50ng/ml more coated concentration sensitivity just.The spraying length of T line and C line is with gold mark mouse-anti MC-LR, and result is as follows.
Under the different discharge rates of table 3, positive sample and PBS's detects result
* note: 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 has met designing requirement, considers the final package amount of determining gold mark monoclonal antibody, and metal spraying machine discharge rate is 2.5 μ l/cm.
Determining of embodiment 8 detection lines (T) working fluid concentration
Adjust the coated concentration of MCLR-KLH, by technique assembling reagent strip, with positive sample and PBS application of sample more coated concentration sensitivity just, all the other,, with embodiment 5, the results are shown in Table 4:
The effect comparison of the coated concentration of the different MC-LR-BSA of table 4
* note: refer to 5 minutes testing results
When coated concentration reaches 0.75mg/ml, can meet the requirement that detects of positive sample, can reach again the requirement of PBS sample, further improve the requirement that coated concentration can not meet positive sample, reduce the requirement that detects that concentration can not reach PBS sample.Considering the coated concentration of final definite mouse-anti MC-LR-BSA detection line is 0.75mg/ml.
Determining of embodiment 9 control lines (C) working fluid concentration
Adjust sheep anti-mouse igg antibody concentration, by normal preparation technology, assemble reagent strip, with the more different coated concentration control line developing times of PBS application of sample, all the other,, with embodiment 5, the results are shown in Table 5.
The effect comparison of table 5 different two anti-coated concentration
Coated concentration reaches 1.0mg/ml, and control line i.e. clear debating in 1min, meets designing requirement.The coated concentration 1.0mg/ml of final definite control line.
Determining 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: select the MC-LR standard items that detectable concentration is 5ng/ml, detection zone in 2 minutes (T) without band, a little less than PBS colour developing, test failure.
B scheme result: select the MC-LR standard items that detectable concentration is 10ng/ml, detection zone in 3 minutes (T), without band, PBS colour developing is strong, test passes.
C scheme result: select the MC-LR standard items that detectable concentration is 20ng/ml, detection zone in 5 minutes (T), without band, PBS colour developing is strong, test passes.
D scheme result: select the MC-LR standard items that detectable concentration is 30ng/ml, detection zone in 10 minutes (T) has band, PBS colour developing is strong, test failure.
According to result, selecting the reaction time is 3-5 minute observations, and within 10 minutes, result is invalid.
Determining of embodiment 11 colloid gold particles
Make by this method the gold grain of different-diameter size, mark mouse monoclone antibody against MC-LR, by technique 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 not occur detection line in 5min, PBS requires colour developing strong.)
The results are shown in Table 6:
* note: refer to 5 minutes testing results
The result of comprehensive relatively said method and invention, only have the collaurum of 30nm can meet positive detection time requires and the requirement of PBS result simultaneously, therefore select the collaurum of 30nm for mark, determine the preparation technology of colloid gold particle: 0.01% gold chloride (HAuCl
4) aqueous solution 100ml be heated to boiling, add 1% trisodium citrate 1.5ml, mix, boil 5 minutes, until colloidal gold solution color by indigo plant after purple stain is purplish red, cooling standby.The colloid gold particle that accordingly prepared by technique meets the testing requirement of test paper.
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, by capillary action, upwards divide a word with a hyphen at the end of a line, prior to the mouse-anti (anti-MC-IgG) of colloid gold label, there is specific immune response in the MC in sample, formed immune complex moves to calibration tape (being sprayed with MC-LR-KLH, calibration tape T band) on continuing; If the MC in sample is abundant, all combine the site of golden labeling antibody, immune complex does not stop in test section, test strip does not develop the color, immune complex moves to controls band (being sprayed with the second antibody of sheep anti mouse, contrast band C band), and the second immunity association reaction occurs, immune complex is trapped, and contrast band is aobvious red.In sample, not containing the amount of MC or MC during lower than detection limit, two bands are all aobvious red, according to the colored depth of test can judgement sample in determinand MC number.
The factor that affects colloid gold label is a lot, especially should be considerable mainly contain the size of colloid gold particle, the pH value of the purity of labelled antibody, solution and the pure degree of container used etc., and the present invention has paid special attention to above problem, and mark effect is better.The present invention reacts with gold chloride by groping the trisodium citrate of different amounts, different gold mark monoclonal antibody concentration, different gold mark monoclonal antibody package amounts, the coated concentration of different antigen conjugates, the effect that two different anti-coated concentration and different reaction time are studied 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 greatly improved detection efficiency.
Claims (4)
1. a test paper for fast detecting Microcystin, is characterized in that, comprises successively point sample hole, detects band and controls band; On point sample hole, spray mouse monoclone antibody against MC-LR-BSA compound of colloid gold label, detect band for the conjugate of microcapsule phycotoxin MC-LR haptens and key hole azurin, control and be with spraying sheep anti mouse two anti-; The base material of test paper is nitrocellulose filter or qualitative filter paper;
The conjugate of monoclonal antibody, MC-LR haptens and key hole azurin in mouse monoclone antibody against MC-LR-BSA compound of described colloid gold label and the anti-weight ratio of sheep anti mouse two are 1:1.8～2:2～3;
The preparation method of mouse monoclone antibody against MC-LR-BSA compound of described colloid gold label is:
(1), by collaurum and mouse-anti MC-LR monoclonal antibody solution mix and blend 0.5～2hr, add calf serum BSA to continue to stir 20～45min, wherein amount ratio 1ml:5～40 μ g:8～12mg of collaurum and mouse monoclone antibody against MC-LR and BSA;
Described collaurum particle diameter is at 28～32nm, and its preparation method is: the chloric acid gold solution that is 0.005%～0.015% by concentration is heated to boiling, adds citric acid to mix and continues to boil 4～8min; Chloric acid gold is 1:0.012～0.0016 with the amount ratio of citric acid;
(2) get and be precipitated and dissolved in pH=7.3～7.45 damping fluid, mouse monoclone antibody against MC-LR and damping fluid amount ratio 100～600 μ g/ml, then to add BSA to BSA concentration be 8～12mg/ml;
The preparation method of the conjugate of described microcapsule phycotoxin MC-LR haptens and key hole azurin is: Microcystin activates rear and key hole azurin hybrid reaction preparation, and both weight ratios are 1:6～1:7; And with phosphate buffer PBS dialysis, get supernatant;
The preparation method of described mouse monoclone antibody against MC-LR is to get MC-LR haptens and mix the rear immune mouse of hatching with complete freund adjuvant with the conjugate of key hole azurin; After 10～15 days, get MC-LR haptens and mix after hatching with incomplete freund adjuvant with the conjugate of key hole azurin, every 10～15 days immune mouses, totally three times.
2. the test paper of fast detecting Microcystin described in claim 1, it is characterized in that, in preparation method's step (1) of mouse monoclone antibody against MC-LR-BSA compound of described colloid gold label, the amount ratio of collaurum and mouse monoclone antibody against MC-LR and BSA is 1ml:20 μ g:10mg; In step (2), mouse monoclone antibody against MC-LR and damping fluid amount ratio are 400 μ g/ml.
3. a method for fast detecting Microcystin, is characterized in that, water sample to be measured is dripped in the point sample hole of test paper described in claim 1 or 2 any one, places 3～5min; Mouse monoclone antibody against MC-LR-BSA compound amount ratio 100 μ l:0.25～1.5 μ g of water sample to be measured and colloid gold label.
4. a kind of method of fast detecting Microcystin described in claim 3, is characterized in that, mouse monoclone antibody against MC-LR-BSA compound amount ratio 100 μ l:1 μ g of water sample to be measured and colloid gold label.
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| CN103604926B (en) * | 2013-11-27 | 2015-03-18 | 云南师范大学 | Method for detecting microcystin-LR by immunochromatographic test strip |
| CN104330568A (en) * | 2014-10-15 | 2015-02-04 | 南京医科大学 | Colloidal gold immunity chromatography kit for detecting cyanophycean toxin |
| WO2017108115A1 (en) * | 2015-12-22 | 2017-06-29 | Centre National De La Recherche Scientifique - Cnrs - | Device for detecting neurotoxins and process for manufacture thereof |
| CN106990246A (en) * | 2017-04-01 | 2017-07-28 | 天津农学院 | A kind of microcysin LR enzyme-linked immunologic detecting kit |
| CN107167585B (en) * | 2017-04-07 | 2018-09-18 | 华南农业大学 | A kind of new small molecule structure and its application in terms of detecting blue-green alge hepatotoxin |
| CN112098652B (en) * | 2020-07-29 | 2022-04-22 | 西安交通大学 | Paper-based enzyme-linked immunosorbent assay for fixing and capturing antibody based on covalent bonding method |
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