CN100405063C - Fabrication method and application for citrinin immune chromatography detection test paper - Google Patents
Fabrication method and application for citrinin immune chromatography detection test paper Download PDFInfo
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- CN100405063C CN100405063C CNB031350119A CN03135011A CN100405063C CN 100405063 C CN100405063 C CN 100405063C CN B031350119 A CNB031350119 A CN B031350119A CN 03135011 A CN03135011 A CN 03135011A CN 100405063 C CN100405063 C CN 100405063C
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Abstract
The present invention discloses citrinin immune chromatographic detection test paper, a making method and the application thereof. The detecting test paper comprises a gasket, a sample pad, a trace particle compound pad, a cellulose nitrate film, a water absorbing pad, a detection line and a quality control line, wherein the sample pad, the trace particle compound pad, the cellulose nitrate film and the water absorbing pad are orderly arranged on the gasket; citrinin antibodies labeled by trace particles are positioned on the trace particle compound pad, and the detection line and the quality control line are arranged on the cellulose nitrate film; citrinin detecting antigens are positioned on the detection line, and antiantibodies are positioned on the quality control line, wherein the antiantibodies are antibodies of the citrinin antibodies. The present invention has the advantages of radioimmunoassay and enzyme linked immunosorbent assay ELISA. Simultaneously, the present invention has the advantages of simplicity, rapidness, storage at normal temperature and single detection. Citrinin can be detected once, and no additional instrument is needed besides commercial reagents. The test paper is especially suitable for the rapid field detection of citrinin.
Description
Technical field
The invention belongs to biological technical field, disclose a kind of citrinin immunity chromatography detection test paper and preparation method thereof and application.
Background technology
Citrinin is the mycotoxin that some bacterial strain of Penicillium and aspergillus produces, the separated first purifying in 1931.It can cause significant Toxicity of Kidney, and carcinogenicity is arranged.Investigation finds that the generation bacterium of some citrinins is extensive in distributed in nature, often causes going mouldy of crops such as corn, rice.In recent years, the pollution problem of citrinin more and more causes people's attention.On the mycotoxin and local ephrosis and the symposial of the urinary tract tumour of holding Lyons, France in 1991, the effect of citrinin in the local ephrosis of Balcan takes place has been discussed, caused the great attention of international cancer research association.Then, citrinin is detected Europe branch of the council by international school of life and health sciences nature toxin and classifies one of toxin that must detect as.Nineteen ninety-five, France scholar Blanc confirmed that some monascus strain also can produce citrinin.This discovery has caused no small vibrations at food circle.Before this, monascus is to use as the bacterial classification that food production is processed always.In China, utilize the history of the existing more than one thousand years of monascus fermenting and producing food and medicine, many traditional foods such as red yeast rice, wine of rice fermented with red yeast and fermented bean curd etc. are very popular.Modern study also find to have in the metabolic product of monascus many in food, medicine and chemical industry of great value tunning.The natural monascorubin good as excellent quality, coloring, that tone is abundant; Can significantly suppress cholesterol is synthetic, reduce lipids contents Monacolin Monacollin K and Lovastain Lovastain; Also have the profuse ergosterol of content, long-chain fatty acid and multiple antibiotic active material.At the beginning of the nineties, states such as America and Europe, Japan have brought considerable economic for the red colouring agent for food, also used as a Chinese medicine manufacturer of China to the edible red colouring agent for food, also used as a Chinese medicine of China, medicinal red colouring agent for food, also used as a Chinese medicine and the surge of Related product demand thereof.The citrinin problem not only makes the monascus product outlet of China be subjected to loss, also seriously threatens people's health.Countries such as Japan, Germany have formulated the new standard at the red colouring agent for food, also used as a Chinese medicine Related product of China's outlet, and the content of regulation citrinin must be lower than setting, otherwise forbid import to be sold.And the monascus product of China does not reach these standards mostly.In the face of this severe situation, should set up the method for citrinin fast detecting as early as possible, could ensure people ' s health, and save this ancient and young food production industry.
The technology that detects citrinin at present can be divided into two classes, one class is a red, orange, green, blue, yellow (ROGBY), comprise thin-layer chromatography, gas chromatography, liquid chromatography etc., it is the chemical analysis method of most important mycotoxin always, thin-layer chromatography just was used later on owing to problems such as method sensitivity and separation efficiencies in 1985 very much.The analytical approach or the liquid phase chromatography of now commonplace mycotoxin, comprise the liquid chromatograph mass spectrography technology, aspect method applicability, separation efficiency and sensitivity, liquid phase chromatography and liquid chromatograph mass spectrography technology all are that additive method institute is irreplaceable.In recent years, Capillary Electrophoresis and capillary electrophoresis-mass spectrometry coupling technique also begin to be applied in the pathogenic eukaryotes field and have caused extensive concern with its high separating efficiency.But, thereby use and be restricted because its apparatus expensive, complicated operation and the purity of sample had higher requirement be not suitable for large batch of sample is detected.Another kind of is immuno-chemical method, since not high to the purity requirement of sample, and have higher sensitivity and specificity.Be particularly suitable for the detection of sample in enormous quantities, be widely used in the detection of various mycotoxins in recent years.These class methods generally include radio immunoassay RIA and enzyme-linked immunosorbent assay ELISA.Although the RIA method has higher sensitivity, owing to used harmful radiomaterial, so can't promote the use of.The ELISA method is because simple to operate, safe in utilization and generally adopted.
Immunochromatographic method is the qualitative determination method that grows up on the immunochemistry basis, it is a solid phase with the fibre strip chromatographic material often, make sample solution swimming on chromatography strip by capillary action, and make on determinand in the sample and the chromatographic material acceptor simultaneously at determinand, as antibody or antigen, the immune response of high special high-affinity takes place, in the chromatography process immune complex by enrichment or certain zone of being trapped in chromatographic material as detecting band, by enzyme reaction or the direct label that can estimate of utilization, as collaurum, and obtain experimental phenomena intuitively, as colour developing.Free label is then crossed and is detected band, reaches the purpose of separating automatically with binding label.The common trace particle of immunochromatography technique has collaurum, latex, and electroselenium, gelatin, magnetic-particle etc., wherein the most successful label of utilization is a collaurum.
Trace particle is if select colour developing mark commonly used for use, and when detection line, nature controlling line normally develop the color, naked eyes can directly be differentiated, if adopt magnetic-particle as trace particle, then need the instrument interpretation.
Summary of the invention
The object of the present invention is to provide a kind of citrinin immunity chromatography detection test paper and preparation method thereof.
Another object of the present invention is to use this detection test paper.
Technical scheme of the present invention is:
A kind of citrinin immunity chromatography detection test paper comprises liner, sample pad, trace particle bond pad, nitrocellulose filter, adsorptive pads, detection line, nature controlling line; Sample pad is arranged in order, trace particle bond pad, nitrocellulose filter, adsorptive pads on liner; The antibody that the anti-citrinin of trace particle mark is arranged on the trace particle bond pad, also be provided with detection line and nature controlling line on the nitrocellulose filter, have citrinin to detect antigen on the detection line, antiantibody is arranged on the nature controlling line, wherein antiantibody is the antibody of the antibody of anti-citrinin.
A kind of method for making of citrinin immunity chromatography detection test paper: trace particle be labeled as following any one: colloid gold particle; Latex particle; The electroselenium particle; Gelatin particle; Magnetic-particle.
It is such detecting principle: drip sample on sample pad, if citrinin is arranged in the sample, then citrinin moves to trace particle bond pad through the chromatography effect, antibodies with the anti-citrinin of limiting the quantity of, when moving to detection line, the antibody of the anti-citrinin of trace particle mark has not had unnecessary binding site and has combined with detection antigen on the detection line, and detection line does not develop the color or magnetic do not occur and strengthens.The antibody of the anti-citrinin of trace particle mark continues to move forward to nature controlling line, and with the antiantibody reaction on the nature controlling line, nature controlling line develops the color or magnetic occurs and strengthens.If no citrinin in the sample, the then antibody of the anti-citrinin of the trace particle mark on the trace particle bond pad and the detection antigen combination on the detection line, detection line develops the color or magnetic occurs and strengthens, and nature controlling line develops the color or magnetic occurs and strengthens.
A kind of method for making of citrinin immunity chromatography detection test paper: comprise the steps that (A) preparation of citrinin antigen comprises the immunizing antigen of monoclonal antibody and the preparation that citrinin detects antigen; (B) preparation of the detection line of nitrocellulose filter and nature controlling line; (C) preparation of trace particle bond pad; (D) assembling test strips.
A kind of method for making of citrinin immunity chromatography detection test paper: the preparation of citrinin antigen, comprise the steps: that (A) citrinin links to each other with peptide bond by the amino that active ester method makes its carboxyl and have the carboxyl and the micromolecular compound of amino simultaneously, forms new compound.(B) this compound links to each other with the high molecular weight material of protein, polysaccharide, polynucleotide, poly-D-lysine, lustrex or other synthetic of high molecular carrier mass by active ester method once more and forms artificial antigen; (C) select a kind of of citrinin antigen as immunizing antigen.(D) select a kind of of citrinin antigen as detecting antigen.
A kind of method for making of citrinin immunity chromatography detection test paper: the detection line of nitrocellulose filter and the preparation of nature controlling line comprise the steps: that (A) detects antigen detection line of linear spotting preparation on nitrocellulose filter with citrinin; (B) prepare a nature controlling line with antiantibody at the enterprising line shape of nitrocellulose filter point sample.
A kind of method for making of citrinin immunity chromatography detection test paper: detection line and nature controlling line are preparations like this: nitrocellulose filter is cut out by the wide size of 10-35mm; To fully dialyse through normal saline buffer solution, the citrinin that concentration is adjusted into 0.01-5mg/mL detect antigen on film linear spotting as detection line, detection line point sample position is from film base 4-16mm, be adjusted into the antiantibody spraying nature controlling line of 0.01-5mg/mL then with concentration on the top of film, nitrocellulose filter 4 drying at room temperature 30 minutes place the physiological saline of 0.1-5% skimmed milk power to soak 30 minutes, take out suck dry moisture, hatched 30 minutes in 37 ℃, put dry place hermetically storing under the room temperature.
A kind of method for making of citrinin immunity chromatography detection test paper: the preparation of trace particle bond pad comprises the steps: (A) antibody with the anti-citrinin of trace particle mark, and antibody comprises IgG type antibody, IgM type antibody, IgA antibody; (B) antibody of the good anti-citrinin of trace particle mark is dispersed on the nitrocellulose filter.
A kind of method for making of citrinin immunity chromatography detection test paper: the trace particle of preparation trace particle bond pad be following any one: colloid gold particle; Latex particle; The electroselenium particle; Gelatin particle; Magnetic-particle.
A kind of method for making of citrinin immunity chromatography detection test paper: the assembling test strips, step is as follows: the nitrocellulose filter that adds detection line and nature controlling line on liner 1; On liner, add trace particle bond pad; On liner, add sample pad; On liner, add adsorptive pads; Be assembled into test strip.
The application of a kind of citrinin immunity chromatography detection test paper in detecting citrinin.
Immunochromatography technique of the present invention detects citrinin, the principle of measuring is similar to the indirect competitive ELISA method, the antibody of the anti-citrinin of coating finite concentration trace particle mark on glass fibre, on nitrocellulose membrane, adsorb detection antigen (because citrinin is not directly fixed on nitrocellulose membrane, so need to prepare the conjugate of citrinin and carrier protein) and antiantibody respectively as detection line and nature controlling line as detection antigen.Form detection test paper folder with plastic bottom board, absorbent filter, glass fibre and nitrocellulose membrane.After sample adds, liquid is along upwards infiltration of filter paper, if contain toxin to be checked in the sample, then can with the labelled antibody on the detection antigenic competition glass fibre on the nitrocellulose membrane, when content of toxins is high, they can occupy the antigen-binding site of most labelled antibodies, thereby stop labelled antibody to combine with detection antigen on the detection line, thereby do not develop the color on detection line.On the contrary, if do not contain toxin to be checked in the sample, labelled antibody will combine with the antigen on the detection line and develop the color.Antiantibody on the nature controlling line then develops the color by the enrichment labelled antibody, with proof result's reliability.
Citrinin belongs to micromolecular compound, has only reactionogenicity and non-immunogenicity, can not stimulate body to produce immune response, must with the macromolecular carrier coupling after make artificial antigen and just can excite effective immune response.Can successfully obtain citrinin is had the monoclonal antibody of high affinity, high degree of specificity, the preparation of people's immunizing antigen be particularly important, need be according to careful selection coupling methods such as the molecular size of citrinin, space structure picture, chemical property.Coupling process will keep the integrality of citrinin structure as far as possible, especially can not destroy its molecular configurations, since its special three-D space structure just, antigenic determinant, match with the acceptor of corresponding lymphocytic cell surface, could start immune response at it.If its structure has looked like to take place trickle variation, just might cause its antigen to change.In addition, have only the corresponding acceptor of antigenic determinant and lymphocytic cell surface to contact, could start immune response, citrinin belongs to micromolecular compound, when it and the direct coupling of carrier protein, and not accessible acceptor, if can add a connection side chain by between, to help haptens to be exposed to the outside, and form the easily nearly property in desirable space, then effect may be better.Owing to have carboxyl on the citrinin molecule, the present invention design makes it have carboxyl and the micromolecular compound of amino such as the amino of EACA or δ-aminovaleric acid simultaneously and link to each other with peptide bond with one with active ester method, by active ester method it is linked to each other with carrier protein again, form a coupling bridge, obtained more satisfactory effect.
Advantage of the present invention and range of application:
The present invention with detected food, feed and red rice products in the past in the technology of citrinin compare and have following advantage: (1) is easy and simple to handle, quick, only needs 5-10 minute; (2) highly sensitive; (3) need not special instruments and equipment, except that commercially available reagent, do not need any instrument and equipment, spent low; Be particularly suitable for the field quick detection of citrinin.(4) need not to add again in addition substrate colour developing indication, and directly by the color judged result; (5) room temperature preservation, single part of mensuration can be carried.
The invention belongs to immune biological technical field, be mainly used in citrinin and detect.The applying unit of test strips of the present invention is mainly processing department, inspection for food hygiene department, the departments for supervision over product quality of food, feed etc.
Description of drawings
Fig. 1 is a citrinin immunity chromatography detection test paper visual texture synoptic diagram of the present invention.
Fig. 2 is the trace particle bond pad partial enlarged drawing of citrinin immunity chromatography detection test paper of the present invention.
Fig. 3 is the detection line partial enlarged drawing of citrinin immunity chromatography detection test paper of the present invention.
Fig. 4 is the nature controlling line partial enlarged drawing of citrinin immunity chromatography detection test paper of the present invention.
Fig. 5 is a citrinin immunity chromatography detection test paper principle of work synoptic diagram of the present invention.
Embodiment
A kind of citrinin immunity chromatography detection test paper comprises liner 1, sample pad 2, trace particle bond pad 3, nitrocellulose filter 4, adsorptive pads 5, detection line 6, nature controlling line 7, sample pad 2 is arranged in order, trace particle bond pad 3, nitrocellulose filter 4, adsorptive pads 5 on liner 1; The antibody 10 that the anti-citrinin of trace particle 9 marks is arranged on the trace particle bond pad 3 also is provided with detection line 6 and nature controlling line 7 on the nitrocellulose filter 4, have citrinin to detect antigen 8 on the detection line 6, and antiantibody 1 is arranged on the nature controlling line 7
1, wherein antiantibody 11 is the antibody of the antibody 10 of anti-citrinin.
It is such detecting principle: drip sample 12 on sample pad 2, if in the sample 12 citrinin is arranged, then citrinin moves to trace particle bond pad 3 through the chromatography effect, combine with the antibody 10 of the anti-citrinin of limiting the quantity of, when moving to detection line 6, the antibody 10 of the anti-citrinin of trace particle 9 marks has not had unnecessary binding site and has combined with detection antigen 8 on the detection line 6, and detection line 6 does not develop the color or magnetic do not occur and strengthens.The antibody 10 of the anti-citrinin of trace particle 9 marks continues to move forward to nature controlling line 7, and with antiantibody 11 reactions on the nature controlling line 7, nature controlling line 7 develops the color or magnetic occurs and strengthens.If no citrinin 8 in the sample 12, the then antibody 10 of the anti-citrinin of trace particle 9 marks on the trace particle bond pad 3 and detection antigen 8 combinations on the detection line 6, detection line 6 develops the color or magnetic occurs and strengthens, and nature controlling line 7 develops the color or magnetic occurs and strengthens.
The method for making of citrinin immunity chromatography detection test paper comprises the steps, (A) preparation of citrinin antigen comprises the immunizing antigen of monoclonal antibody and the preparation that citrinin detects antigen 8; (B) preparation of the detection line 6 of nitrocellulose filter 4 and nature controlling line 7; (C) preparation of trace particle bond pad 3; (D) assembling test strips.
In embodiment 2-7:
Trace particle is if select colour developing mark commonly used for use, and when detection line, nature controlling line normally develop the color, naked eyes can directly be differentiated, if adopt magnetic-particle as trace particle, then need the instrument interpretation.It is higher that instrument is differentiated the sensitivity of magnetic-particle.
Embodiment 8
A kind of method for making of citrinin immunity chromatography detection test paper, the preparation of citrinin antigen, comprise the steps: that (A) citrinin links to each other with peptide bond by the amino that active ester method makes its carboxyl and have the carboxyl and the micromolecular compound of amino simultaneously, forms new compound.(B) this compound links to each other with the high molecular weight material of protein, polysaccharide, polynucleotide, poly-D-lysine, lustrex or other synthetic of high molecular carrier mass by active ester method once more and forms artificial antigen; (C) select a kind of of citrinin antigen as immunizing antigen.(D) select a kind of of citrinin antigen as detecting antigen.All the other are with embodiment 1.
The detection line 6 of nitrocellulose filter 4 and the preparation of nature controlling line 7 comprise the steps: that (A) detects antigen 8 detection line 6 of linear spotting preparation on nitrocellulose filter 4 with citrinin; (B) prepare a nature controlling line 7 with antiantibody 11 at nitrocellulose filter 4 enterprising line shape point samples.All the other are with embodiment 1.
A kind of method for making of citrinin immunity chromatography detection test paper, detection line 6 and nature controlling line 7 are preparations like this: nitrocellulose filter 4 is cut out by the wide size of 10-35mm; To fully dialyse through normal saline buffer solution, the citrinin that concentration is adjusted into 0.01-5mg/mL detect antigen 8 on film linear spotting as detection line 6, detection line 6 point sample positions are from film base 4-16mm, be adjusted into the antiantibody 11 spraying nature controlling lines 7 of 0.01-5mg/mL then with concentration on the top of film, nitrocellulose filter 4 drying at room temperature 30 minutes, place the physiological saline of 0.1-5% skimmed milk power to soak 30 minutes, take out suck dry moisture, hatched 30 minutes in 37 ℃, put dry place hermetically storing under the room temperature.All the other are with embodiment 1.
Embodiment 11
A kind of method for making of citrinin immunity chromatography detection test paper, the preparation of trace particle bond pad 3 comprise the steps: (A) antibody 10 with the anti-citrinin of trace particle mark, and antibody 10 comprises IgG type antibody, IgM type antibody, IgA antibody; (B) antibody 10 of the good anti-citrinin of trace particle 9 marks is dispersed on the nitrocellulose filter 4.All the other are with embodiment 1.
Embodiment 12
A kind of method for making of citrinin immunity chromatography detection test paper, the assembling test strips, step is as follows: the nitrocellulose filter 4 that adds detection line 6 and nature controlling line 7 on liner 1; On liner 1, add trace particle bond pad 3; On liner 1, add sample pad 2; On liner 1, add adsorptive pads 5; Be assembled into test strip.All the other are with embodiment 1.
Embodiment 13
A kind of immunochromatography detects the preparation of citrinin test paper, and it specifically is applied to concrete detection.
Comprise the steps:
(1). the preparation of citrinin artificial antigen comprises being used for the immunizing antigen and the detection antigen that is used to prepare detection line of immune animal with the preparation monoclonal antibody.
(2). the preparation of nitrocellulose filter detection line 6 and nature controlling line 7.
1. the conjugate of using citrinin and carrier protein is as detecting the antigen linear spotting as detection line 6.
2. the antibody linear spotting of antibody 10 of using anti-citrinin is as nature controlling line 7.
(3). the preparation of trace particle bond pad 3:
The antibody 10 of the anti-citrinin of trace particle 9 marks is dispersed in makes trace particle bond pad 3 on the glass fibre.
(4). the assembling of test strips:
1. on liner 1, add the nitrocellulose filter 4 of detection line 6 and nature controlling line 7;
2. on liner 1, add trace particle bond pad 3;
3. on liner 1, add sample pad 2;
4. on liner 1, add adsorptive pads 5; Be assembled into test strip.
(5). the processing of sample to be checked.
Scheme specifically can be segmented quinquepartite:
(1). the preparation of citrinin artificial antigen comprises being used for the immunizing antigen and the detection antigen that is used to prepare detection line of immune animal with the preparation monoclonal antibody; (2). the preparation of nitrocellulose filter detection line 6 and nature controlling line 7; (3). the preparation of trace particle bond pad 3; (4). the assembling of test strips; (5). the processing of sample to be checked.
Wherein
The preparation of 1 citrinin artificial antigen:
1. citrinin 1.25mg (5 μ mol) is dissolved in the 0.3mL tetrahydrofuran (THF), nitrogen hydroxy succinic acid (NHS) 0.9mg (7.5 μ mol) and dicyclohexyl carbodiimide (DCC) 2mg (10 μ mol) are dissolved among the 0.18mL THF, the two mixing is put room temperature lucifuge oscillating reactions 24 hours.
2. after reacting completely, 10000rpm went precipitation in centrifugal 10 minutes.The supernatant vacuum is drained, and adds 600 μ L nitrogen, nitrogen-dimethyl formamide (DMF) dissolving, slowly splashes in 10 μ mol EACAs or δ-aminovaleric acid solution, at 400 μ L 1M Na
2CO
In 3Dissolving, oscillating reactions 2 hours (lucifuge) under the room temperature.
3. after reaction finishes, with 2mL chloroform extracting three times.The organic phase evaporated under reduced pressure.Heavily be dissolved among the 0.1-3mLTHF, repeat above-mentioned steps, the citrinin that connects aminocaproic acid is further activated.
4. 10000rpm removed post precipitation in centrifugal 10 minutes, the organic phase vacuum is drained, be dissolved in the 0.3-3mL dimethyl sulfoxide (DMSO), add again that (0.5mg is dissolved in 0.13MNaHCO in 0.9mL carrier mass (keyhole shellfish hemocyanin, bovine serum albumin(BSA), thyroglobulin, ovalbumin, skimmed milk power, polylysine or other synthetic have a kind of in the amino polymer substance) solution
3In), oscillating reactions 2 hours (lucifuge) under the room temperature.After reaction finishes, 4 ℃ of dialysis of 0.01M PBS (pH7.2) 72 hours, with Sephadex G-25 purifying, the mol ratio of UV scanning assay determination coupled product, vacuum freeze-drying is stand-by.
5. a kind of as immunizing antigen, another kind of in the artificial antigen of selecting to form as detecting antigen with the coupling of different carriers material.
2. the preparation of nitrocellulose filter detection line 6 and nature controlling line 7 comprises the steps:
3. the preparation of trace particle bond pad 3
(1) preparation of trace particle 9 and mark
1. collaurum colloidal sol prepares mark:
A) collaurum colloidal sol preparation: get 0.01% tetrachloro Jinsui River solution 200mL, be heated to boiling, add 1-20mL 1% sodium citrate aqueous solution, boiled 5 minutes, occur orange redly, the colloid gold particle diameter is determined as 10-80nm by Electronic Speculum;
B) colloid gold label antibody: get the 50mL collaurum, transfer pH (8.0) with 0.1mol/L sal tartari, stir down collaurum colloidal sol is mixed with antibody, add the polyglycol aqueous solution again, making ultimate density is 0.05%.With centrifugal 45 minutes of semifinished product 6000rpm, precipitation was suspended to 1.5mL with physiological saline, 4 ℃ of preservations.
2. electroselenium colloidal sol prepares and antibody labeling:
A) electroselenium colloidal sol preparation: add deionized water 550mL and polyacrylic acid 10g in four neck round-bottomed flasks of 1 liter of capacity, logical nitrogen stirring at room also adds hydrazine hydrate 69.5mL, continues to stir 20 minutes.Selenous acid 9.63g is dissolved in 280mL water, stirs under the room temperature and splashes in the reaction mixture, obtains the red selenium sol of 120nm.
B) electroselenium antibody labeling: the antibody 10 usefulness 20mmol/L pH7.3 phosphate buffers of anti-citrinin are made into 4.6mg/mL concentration solution, add 25 μ L in the 25mL of pH7.3 selenium sol, stirring at room added 1%PEG80001mL after 10 minutes, mixing, in 4 ℃ with 5000rpm centrifugal 5 minutes, obtain soft red precipitate, with containing 0.05%NaN
3Phosphate buffer be made into the 1mL suspension.
3. the preparation of latex and gelatin and antibody labeling:
Color latex particle and gelatin particle color are blue look.Phosphate buffer with pH7.1 is diluted to 1% concentration with latex particle or gelatin particle; get 50mL respectively; antibody 10 with latex or gelatin solution and anti-citrinin under stirring mixes, and stirring at room in 37 ℃ of water-baths hatchings 60 minutes, was placed 4-5 hour for 4 ℃ after 30 minutes.Centrifugal 30 minutes of 15000rpm, resuspended with the 2mL phosphate buffer.
4. magnetic-particle antibody labeling:
Magnetic-particle is available from Bangs Laboratories company.Phosphate buffer with pH7.1 is diluted to 1% concentration with magnetic-particle, gets 50mL, and stir down it is mixed with the antibody 10 of anti-citrinin, stirring at room 30 minutes, 37 ℃ of water-baths were hatched 60 minutes, placed 4 hours for 4 ℃.Centrifugal 30 minutes of 15000rpm, resuspended with the 2mL phosphate buffer.
(2) preparation of trace particle bond pad
The antibody 10 of the anti-citrinin of mark trace particle 9 mixed in proportion being dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.
4. the assembling of paper slip:
1. on liner 1, add the nitrocellulose filter 4 of detection line 6 and nature controlling line 7;
2. on liner 1, add trace particle bond pad 3;
3. on liner 1, add sample pad 2;
4. on liner 1, add adsorptive pads 5; Be assembled into test strip.
5. the processing of sample to be checked
1. solid sample disposal route: in the 50mL triangular flask, be weighed into red yeast rice 1-5g, divide three extractions, when extracting, under magnetic agitation, continue 1h, combining extraction liquid at every turn with 15mL methyl alcohol-chloroformic solution (1: 1)., dissolve with the PBS (pH7.6) that contains 10% methyl alcohol again at 40 ℃ of following evaporates to dryness with rotary evaporator.
2. contain fat testing sample disposal route: take by weighing sample 1-5g, soak 12h, use twice of isooctane degreasing then with 10-20mL methyl alcohol; Add distilled water again, use H
2SO
4Transfer pH to 4.5, behind chloroform extraction, water bath method is settled to 1mL with the PBS (pH7.6) that contains 10% methyl alcohol, and is standby.
3. the disposal route of feed sample: take by weighing sample 3.0-5.0g, add acetonitrile (or methyl alcohol): the mixed solution 20-30mL of sulfuric acid (20%)=99: 1, after the ultrasonic Treatment 15 minutes, centrifuging and taking supernatant (3000rpm/ minute, 20 minutes) repeats 3 times, extract merges, it is dried that 60 ℃ of water-baths are concentrated into, and adds PBS (pH7.6) dissolving that contains 10% methyl alcohol, is directly used in mensuration.
4. the disposal route of liquid sample: get the sample of certain volume, use filter paper filtering, add isopyknic PBS (pH7.6) that contains 10% methyl alcohol, standby.
Embodiment 14:
The method of colloidal gold immunochromatographimethod detection citrinin and the preparation of test paper also are used to detect the red yeast rice sample.
Present embodiment is divided into 5 parts: the processing of (1) sample to be checked; (2) preparation of artificial antigen; (3) preparation of nitrocellulose filter 4; (4) preparation of collaurum bond pad; (5) assembling of test strips; (6) detection and result judge
(1) processing of sample to be checked
In the 50mL triangular flask, be weighed into red yeast rice 5g, divide three extractions with 10mL methyl alcohol-chloroformic solution (1: 1), when extracting, under magnetic agitation, continue 1h, combining extraction liquid at every turn., dissolve with the PBS (pH7.6) that contains 10% methyl alcohol again at 40 degrees centigrade of following evaporates to dryness with rotary evaporator.
(2) preparation of artificial antigen
1. citrinin 1.25mg (5 μ mol) is dissolved in the 0.3mL tetrahydrofuran (THF).Nitrogen hydroxy succinic acid (NHS) 0.9mg (7.5 μ mol) and dicyclohexyl carbodiimide (DCC) 2mg (10 μ mol) are dissolved among the 0.2mL THF.The two mixing is put room temperature lucifuge oscillating reactions 24 hours.
2. after reacting completely, 10000rpm went precipitation in centrifugal 10 minutes.The supernatant vacuum is drained, and adds 200 μ L nitrogen, nitrogen-dimethyl formamide (DMF) dissolving, slowly splashes into (400 μ L 1M Na in the 10 μ mol EACA solution
2CO
3Dissolving), oscillating reactions 2 hours (lucifuge) under the room temperature.
3. after reaction finishes, with 0.2mL chloroform extracting three times.The organic phase evaporated under reduced pressure.Heavily be dissolved among the 0.4mLTHF, repeat above-mentioned steps, the citrinin that connects aminocaproic acid is further activated.
4. 10000rpm removed post precipitation in centrifugal 10 minutes, and the organic phase vacuum is drained, and was dissolved in the 0.2mL dimethyl sulfoxide (DMSO), added that (0.5mg is dissolved in 0.13MNaHCO in the 400 μ L keyhole shellfish hemocyanin solution again
3In), oscillating reactions 2 minutes (lucifuge) under the room temperature.
5. after reaction finishes, 0.01M PBS (pH7.2) 4C dialysis 72 hours, with Sephadex G-25 purifying, the mol ratio of UV scanning assay determination coupled product, vacuum freeze-drying is stand-by.
(3) preparation of nitrocellulose filter 4
(4) preparation of collaurum bond pad
1. collaurum colloidal sol prepares mark: get 0.01% tetrachloro Jinsui River solution 200mL, be heated to boiling, add 10mL 1% sodium citrate aqueous solution, boiled 5 minutes, occur orange redly, the colloid gold particle diameter is determined as 10-80nm by Electronic Speculum; Get the 50mL collaurum, transfer pH (8.0) with 0.1mol/L sal tartari, stir down collaurum colloidal sol is mixed with the antibody 10 of anti-citrinin, add the polyglycol aqueous solution again, making ultimate density is 0.05%.With centrifugal 45 minutes of semifinished product 6000g, precipitation was suspended to 1.5mL with physiological saline, 4 ℃ of preservations.
2. the antibody 10 of the anti-citrinin of colloid gold label is mixed in proportion being dispersed on the all-glass paper that thickness is 1m, the freeze-drying hermetically drying is preserved.
(5) assembling of paper slip:
1. on liner 1, add the nitrocellulose filter 4 of detection line 6 and nature controlling line 7;
2. on liner 1, add trace particle bond pad 3;
3. on liner 1, add sample pad 2;
4. on liner, 1 add adsorptive pads 2; Be assembled into test strip.
(6) detection and result judge:
Get the sample 0.5mL that handles well, vertically insert test strips centrifugal a moment, insertion depth is no more than sample pad 2, stops about 5 seconds in sample, and vertically take out test paper, judged result after 5 minutes: detection line and nature controlling line place have redness to appear as feminine gender; The detection line redfree occurs, and nature controlling line occurs red positive; The nature controlling line redfree appears as inefficacy.
Embodiment 15
The method of latex particle or gelatin particle immunochromatography detection citrinin and the preparation of test paper also are used to detect red colouring agent for food, also used as a Chinese medicine fermented bean curd sample.
Present embodiment is divided into 5 parts: the processing of (1) sample to be checked; (2) preparation of artificial antigen; (3) preparation of nitrocellulose filter; (4) preparation of latex particle or gelatin particle bond pad; (5) assembling of test strips; (6) detection and result judge
(1) red colouring agent for food, also used as a Chinese medicine fermented bean curd sample treatment
Take by weighing sample 2g, soak 1h, use twice of isooctane degreasing then with 10mL methyl alcohol; Add distilled water again, use H
2SO
4Transfer pH to 4.5, behind the chloroform extraction, water bath method, with PBS (pH7.6) dissolving that contains 10% methyl alcohol, standby.
(the preparation of 2 artificial antigens
1. citrinin 1.25mg (5 μ mol) is dissolved in the 0.3mL tetrahydrofuran (THF).Nitrogen hydroxy succinic acid (NHS) 0.9mg (7.5 μ mol) and dicyclohexyl carbodiimide (DCC) 2mg (10 μ mol) are dissolved among the 0.2mL THF.The two mixing is put room temperature lucifuge oscillating reactions 24 hours.
2. after reacting completely, 10000rpm went precipitation in centrifugal 10 minutes.The supernatant vacuum is drained, and adds the dissolving of 200 μ L nitrogen, nitrogen-dimethyl formamide (DMF), slowly splashes into (400 μ L 1M Na in the EACA solution of 10 μ mol
2CO
3Dissolving), oscillating reactions 2 hours (lucifuge) under the room temperature.
3. after reaction finishes, with 0.2mL chloroform extracting three times.The organic phase evaporated under reduced pressure.Heavily be dissolved among the 0.4mLTHF, repeat above-mentioned steps, the citrinin that connects aminocaproic acid is further activated.
4. 10000rpm removed post precipitation in centrifugal 10 minutes, and the organic phase vacuum is drained, and was dissolved in the 0.2mL dimethyl sulfoxide (DMSO), added that (0.5mg is dissolved in 0.13MNaHCO in the 400 μ L skimmed milk power solution again
3In), oscillating reactions 2 hours (lucifuge) under the room temperature.
5. after reaction finished, the SephadexG-25 purifying was used in 0.01M PBS (pH7.2) 4C dialysis 72 hours, the mol ratio of UV scanning assay determination coupled product, and vacuum freeze-drying is stand-by.
(3) preparation of nitrocellulose filter 4
(4) preparation of latex and gelatin bond pad:
Color latex particle and gelatin particle color are blue look.Phosphate buffer with pH7.1 is diluted to 1% concentration with latex particle or gelatin particle, gets 50mL respectively, stirs down solution is mixed with the antibody 10 of anti-citrinin, and stirring at room in 37 ℃ of water-bath hatchings 60 minutes, was placed 4-5 hour for 4 ℃ after 30 minutes.Centrifugal 30 minutes of 15000rpm, resuspended with the 2mL phosphate buffer.The antibody of mark latex particle or gelatin particle is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.
(5) assembling of paper slip:
1. on liner 1, add the nitrocellulose filter 4 of detection line 6 and nature controlling line 7;
2. on liner 1, add trace particle bond pad 3;
3. on liner 1, add sample pad 2;
4. on liner 1, add adsorptive pads 5; Be assembled into test strip.
(6) detection and result judge:
Get the sample 0.5mL that handles well, vertically insert test strips centrifugal a moment, insertion depth is no more than sample pad 2, stops about 5 seconds in sample, and vertically take out test paper, judged result after 5 minutes: detection line 6 and nature controlling line place 7 all have blue look to appear as feminine gender; Detection line 6 no blue looks occur, and it is positive that blue look appears in nature controlling line 7; Nature controlling line 7 no blue looks appear as inefficacy.
It is such detecting principle: drip sample on sample pad 2, if external citrinin is arranged in the sample, then the citrinin in the sample moves to trace particle bond pad 3 through the chromatography effect, combine with the antibody 10 of the anti-citrinin of limiting the quantity of, when moving to detection line 6, the antibody 10 of the anti-citrinin of trace particle 9 marks has not had the detection antigen 8 on unnecessary binding site and the detection line 6, detect antigen 8 and be the combining of coupled product of citrinin and carrier, detection line 6 does not develop the color or the magnetic enhancing do not occur.The antibody 10 of the anti-citrinin of trace particle 9 marks continues to move forward to nature controlling line 7, with antibody 11 reactions of the antibody 10 of anti-citrinin on the nature controlling line 7, and nature controlling line 7 colour developings or magnetic occurs and strengthen.If no citrinin 8 in the sample, then the antibody 10 of the anti-citrinin of trace particle 9 marks on the trace particle bond pad 3 and the detection antigen on the detection line 6 coupled product of carrier (citrinin with) 8 combines, detection line 6 colour developing or magnetic occurs and strengthen, nature controlling line 7 the same colour developings or magnetic occurs and strengthen.
Embodiment 16
The method of electroselenium immunochromatography detection citrinin and the preparation of test paper also are used to detect the feed sample
Present embodiment is divided into 5 parts: the processing of (1) sample to be checked; (2) preparation of artificial antigen; (3) preparation of nitrocellulose filter 4; (4) preparation of electroselenium bond pad; (5) assembling of test strips; (6) detection and result judge
(1) processing of sample to be checked
In the 50mL centrifuge tube, take by weighing feed sample 3.0g, add acetonitrile (or methyl alcohol): the mixed solution 20mL of sulfuric acid (20%)=99: 1, ultrasonic Treatment is after 15 minutes, centrifuging and taking supernatant (3000rpm/ minute, 20 minutes), repeat totally 3 times, extract merges, and 60 ℃ of water-baths are concentrated into dried, add PBS (pH7.6) dissolving that contains 10% methyl alcohol, be directly used in mensuration.
(2) preparation of artificial antigen
1. citrinin 1.25mg (5 μ mol) is dissolved in the 0.3mL tetrahydrofuran (THF).Nitrogen hydroxy succinic acid (NHS) 0.9mg (7.5 μ mol) and dicyclohexyl carbodiimide (DCC) 2mg (10 μ mol) are dissolved among the 0.2mL THF.The two mixing is put room temperature lucifuge oscillating reactions 24 hours.
2. after reacting completely, 10000rpm went precipitation in centrifugal 10 minutes.The supernatant vacuum is drained, and adds 200 μ L nitrogen, nitrogen-dimethyl formamide (DMF) dissolving, slowly splashes into (400 μ L 1M Na in the 10 μ mol EACA solution
2CO
3Dissolving), oscillating reactions 2 hours (lucifuge) under the room temperature.
3. after reaction finishes, with 0.2mL chloroform extracting three times.The organic phase evaporated under reduced pressure.Heavily be dissolved among the 0.4mLTHF, repeat above-mentioned steps, the citrinin that connects aminocaproic acid is further activated.
4. 10000rpm removed post precipitation in centrifugal 10 minutes, and the organic phase vacuum is drained, and was dissolved in the 0.2mL dimethyl sulfoxide (DMSO), added that (0.5mg is dissolved in 0.13MNaHCO in the 400 μ L oralbumin solution again
3In), oscillating reactions 2 hours (lucifuge) under the room temperature.
5. after reaction finished, the SephadexG-25 purifying was used in 0.01M PBS (pH7.2) 4C dialysis 72 hours, the mol ratio of UV scanning assay determination coupled product, and vacuum freeze-drying is stand-by.
(3) preparation of nitrocellulose filter 4
(4) preparation of electroselenium bond pad:
The electroselenium antibody labeling: the antibody of anti-citrinin is made into 4.6mg/mL concentration solution with 20mmol/L pH7.3 phosphate buffer, getting 25 μ L respectively adds in the 25mL selenium sol, stirring at room added 1%PEG80001mL after 10 minutes, mixing, in 4 ℃ with 5000rpm centrifugal 5 minutes, obtain soft red precipitate, with containing 0.05%NaN
3Phosphate buffer be made into the 1mL suspension.The antibody 10 of the anti-citrinin of mark electroselenium particle is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.
(5) assembling of paper slip:
1. on liner 1, add the nitrocellulose filter 4 of detection line 6 and nature controlling line 7;
2. on liner 1, add trace particle bond pad 3;
3. on liner 1, add sample pad 2;
4. on liner 1, add adsorptive pads 5; Be assembled into test strip.
(6) detection and result judge:
Get the sample 0.5mL that handles well, vertically insert test strips centrifugal a moment, insertion depth is no more than sample pad, stops about 5 seconds in sample, and vertically take out test paper, judged result after 5 minutes: detection line 6 and nature controlling line 7 places have red line to appear as feminine gender; Detection line 6 no red lines occur, and nature controlling line 7 occurs red positive; Nature controlling line 7 no red lines appear as inefficacy.
Embodiment 17
Magnetic-particle is that the immunochromatography of trace particle detects the preparation of the method for citrinin and test paper and is used to detect the wine of rice fermented with red yeast sample
Present embodiment is divided into 5 parts: the processing of (1) sample to be checked; (2) preparation of artificial antigen; (3) preparation of nitrocellulose filter 4; (4) preparation of magnetic-particle bond pad; (5) assembling of test strips; (6) detection and result judge
(1) processing of sample to be checked
Get the sample of certain volume, use filter paper filtering, add isopyknic PBS (pH7.6) that contains 10% methyl alcohol, standby.
(2) preparation of artificial antigen
1. citrinin 1.25mg (5 μ mol) is dissolved in the 0.3mL tetrahydrofuran (THF).Nitrogen hydroxy succinic acid (NHS) 0.9mg (7.5 μ mol) and dicyclohexyl carbodiimide (DCC) 2mg (10 μ mol) are dissolved among the 0.2mL THF.The two mixing is put room temperature lucifuge oscillating reactions 24 hours.
2. after reacting completely, 10000rpm went precipitation in centrifugal 10 minutes.The supernatant vacuum is drained, and adds 200 μ L nitrogen, nitrogen-dimethyl formamide (DMF) dissolving, slowly splashes into (400 μ L 1M Na in the 10 μ mol EACA solution
2CO
3Dissolving), oscillating reactions 2 hours (lucifuge) under the room temperature.
3. after reaction finishes, with 0.2mL chloroform extracting three times.The organic phase evaporated under reduced pressure.Heavily be dissolved among the 0.4mLTHF, repeat above-mentioned steps, the citrinin that connects aminocaproic acid is further activated.
4. 10000rpm removed post precipitation in centrifugal 10 minutes, and the organic phase vacuum is drained, and was dissolved in the 0.2mL dimethyl sulfoxide (DMSO), added that (0.5mg is dissolved in 0.13M NaHCO in the 400 μ L thyroglobulin solution again
3In), oscillating reactions 2 hours (lucifuge) under the room temperature.
5. after reaction finishes, 4 ℃ of dialysis of 0.01M PBS (pH7.2) 72 hours, with Sephadex G-25 purifying, the mol ratio of UV scanning assay determination coupled product, vacuum freeze-drying is stand-by.
(3) preparation of nitrocellulose filter 4
(4) preparation of magnetic-particle bond pad:
Magnetic-particle is available from Bangs Laboratories company.Phosphate buffer with pH7.1 is diluted to 1% concentration with magnetic-particle, gets 50mL, stirs down it is mixed with the antibody 10 of anti-citrinin, and stirring at room in 37 ℃ of water-bath hatchings 60 minutes, was placed 4-5 hour for 4 ℃ after 30 minutes.Centrifugal 30 minutes of 15000rpm, resuspended with the 2mL phosphate buffer.The antibody of mark magnetic-particle is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.
(5) assembling of paper slip:
1. on liner 1, add the nitrocellulose filter 4 of detection line 6 and nature controlling line 7;
2. on liner 1, add trace particle bond pad 3;
3. on liner 1, add sample pad 2;
4. on liner 1, add adsorptive pads 5; Be assembled into test strip.
(6) detection and result judge:
Get the sample 0.5mL that handles well, in centrifugal a moment, vertically insert test strips, insertion depth is no more than sample pad 2, stopped about 5 seconds in sample, vertically take out test paper, read the instrument judged result with magnetic after 5 minutes: detection line and nature controlling line place magnetic occurs and strengthen negative; The nonmagnetic enhancing of detection line, nature controlling line be magnetic strengthen positive; The nonmagnetic enhancing of nature controlling line appears as inefficacy.
Embodiment 18
Use 1% bovine serum albumin(BSA) to replace the 0.1-5% skimmed milk power, all the other methods of pressing embodiment 15 detect.
Use 1% skimmed milk power to compare with 1% bovine serum albumin(BSA) through the contrast discovery, use 1% skimmed milk power cost lower, background absorption can reduce by 20%, and the sensitivity that background reduces by 20% meaning detection improves 20%.
Embodiment 19
Method according to embodiment 15 detects, to a plurality of sample detection, the content of the citrinin in the red yeast rice is 3.2mg/kg-1500mg/kg, and the content of citrinin is 193mg/kg-17452mg/kg in the monascorubin, and the citrinin content in the feed is 5mg/kg-632mg/kg.
Rolling off the production line of the sample detection of the foregoing description is highly sensitive.
Claims (9)
1. citrinin immunity chromatography detection test paper, comprise liner (1), sample pad (2), trace particle bond pad (3), nitrocellulose filter (4), adsorptive pads (5), detection line (6), nature controlling line (7), sample pad (2) is arranged on liner (1) in order, trace particle bond pad (3), nitrocellulose filter (4), adsorptive pads (5); The antibody (10) that the anti-citrinin of trace particle (9) mark is arranged on the trace particle bond pad (3), also be provided with detection line (6) and nature controlling line (7) on the nitrocellulose filter (4), there is citrinin to detect antigen (8) on the detection line (6), antiantibody (11) is arranged on the nature controlling line (7), and wherein antiantibody (11) is the antibody of the antibody (10) of anti-citrinin.
2. as a kind of method for making of citrinin immunity chromatography detection test paper, it is characterized in that: comprise the steps that (A) preparation of citrinin antigen comprises the immunizing antigen of monoclonal antibody and the preparation that citrinin detects antigen (8); (B) preparation of the detection line (6) of nitrocellulose filter (4) and nature controlling line (7); (C) preparation of trace particle bond pad (3); (D) assembling test strips.
3. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 2 is characterized in that: trace particle (9) be labeled as following any one: colloid gold particle; Latex particle; The electroselenium particle; Gelatin particle; Magnetic-particle.
4. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 2, it is characterized in that: the preparation of citrinin antigen, comprise the steps: that (A) citrinin links to each other with peptide bond by the amino that active ester method makes its carboxyl and have the carboxyl and the micromolecular compound of amino simultaneously, forms new compound; (B) this compound links to each other with the high molecular weight material of protein, polysaccharide, polynucleotide, poly-D-lysine, lustrex or other synthetic of high molecular carrier mass by active ester method once more and forms artificial antigen; (C) select a kind of of citrinin antigen as immunizing antigen; (D) select a kind of of citrinin antigen as detecting antigen.
5. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 2, it is characterized in that: the detection line (6) of nitrocellulose filter (4) and the preparation of nature controlling line (7) comprise the steps: that (A) detects antigen (8) with citrinin and go up a linear spotting preparation detection line (6) at nitrocellulose filter (4); (B) prepare a nature controlling line (7) with antiantibody (11) at the enterprising line shape of nitrocellulose filter (4) point sample.
6. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 5 is characterized in that: detection line (6) and nature controlling line (7) are to prepare like this: nitrocellulose filter (4) is cut out by the wide size of 10-35mm; To fully dialyse through normal saline buffer solution, the citrinin that concentration is adjusted into 0.01-5mg/mL detect antigen (8) on film linear spotting as detection line (6), detection line (6) point sample position is from film base 4-16mm, be adjusted into antiantibody (11) the spraying nature controlling line (6) of 0.01-5mg/mL then with concentration on the top of film, nitrocellulose filter (4) drying at room temperature 30 minutes, place the physiological saline of 0.1-5% skimmed milk power to soak 30 minutes, take out suck dry moisture, hatched 30 minutes in 37 ℃, put dry place hermetically storing under the room temperature.
7. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 2, it is characterized in that: the preparation of trace particle bond pad (3) comprises the steps: (A) antibody (10) with the anti-citrinin of trace particle mark, and antibody (10) comprises IgG type antibody, IgM type antibody, IgA antibody; (B) antibody (10) of the good anti-citrinin of trace particle (9) mark is dispersed on the nitrocellulose filter (4).
8. the method for making of a kind of citrinin immunity chromatography detection test paper as claimed in claim 2 is characterized in that: the assembling test strips, and step is as follows: the nitrocellulose filter (4) that adds detection line (6) and nature controlling line (7) on liner (1); On liner (1), add trace particle bond pad (3); On liner (1), add sample pad (2); On liner (1), add adsorptive pads (5); Be assembled into test strip.
9. the application of citrinin immunity chromatography detection test paper in detecting citrinin of adopting the described method for making of claim 2 to make.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101434655B (en) * | 2008-12-12 | 2011-11-09 | 广东工业大学 | Preparation of egg yolk antibody against citrinin |
| CN106749653A (en) * | 2016-12-07 | 2017-05-31 | 普菲特益斯生物科技（北京）有限公司 | The preparation method of anti-citrinin monoclonal antibody, citrinin kit and its detection method |
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| EP1687628B1 (en) | 2003-11-14 | 2011-01-26 | Alere Switzerland GmbH | Fluid sample analysis device with sealable sample storage reservoir |
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| JP3157356U (en) * | 2006-07-26 | 2010-02-18 | インバーネス・メデイカル・スウイツツアーランド・ゲゼルシヤフト・ミツト・ベシユレンクテル・ハフツング | Analytical equipment for biological samples |
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| CN105295053A (en) * | 2015-11-23 | 2016-02-03 | 天津科技大学 | Citrinin molecular imprinting material and preparation method thereof |
| CN115166231A (en) * | 2021-11-26 | 2022-10-11 | 国家食品安全风险评估中心 | Side-stream immunochromatographic method for citrinin |
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| CN106749653A (en) * | 2016-12-07 | 2017-05-31 | 普菲特益斯生物科技（北京）有限公司 | The preparation method of anti-citrinin monoclonal antibody, citrinin kit and its detection method |
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