CN1300586C - Fabrication method and application for patulin immune chromatography detection test paper - Google Patents
Fabrication method and application for patulin immune chromatography detection test paper Download PDFInfo
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- CN1300586C CN1300586C CNB031602991A CN03160299A CN1300586C CN 1300586 C CN1300586 C CN 1300586C CN B031602991 A CNB031602991 A CN B031602991A CN 03160299 A CN03160299 A CN 03160299A CN 1300586 C CN1300586 C CN 1300586C
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Abstract
The present invention discloses patulin immune chromatographic detection test paper, a making method thereof 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; patulin 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; patulin detecting antigens are positioned on the detection line, antiantibodies are positioned on the quality control line, wherein the antiantibodies are antibodies of the patulin 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. Patulin 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 patulin.
Description
Technical field
The invention belongs to biological technical field, disclose a kind of method for making and application of patulin immunity chromatography detection test paper.
Background technology
Patulin Patulin, PAT are by mycetogenetic a kind of toxic metabolite product, are at first found and separation and purification 1941 by Glister.Experiment finds that patulin is a kind of broad-spectrum antibiotic, but subsequently studies confirm that it has stronger toxicity to animal used as test.From then on the research of patulin is transferred in its toxicity and the pollution to food and feed.
In the fungi totally 3 belong to 16 kinds, as expand mould, Penicillium patulum, excellent type mould, soil mould etc., can produce patulin, investigation finds that the generation bacterium of some patulins is extensive in distributed in nature, often causes that fruit such as apple, hawthorn go rotten, the going mouldy of rice and feed etc.Song Jiayu etc. carry out patulin at 1995 pairs of Shandong Province's apples and hawthorn and goods thereof and detect, the pollution rate of rotten apple patulin is 40%, and the mildew and rot portion of distance 1cm place sampling patulin average content is 329 μ g/kg, apple goods pollution rate is 70%, and the positive average content is 80.6 μ g/kg.The half-finished Normal juice of Penicillium patulum toxin maximum limit water gaging fruit of China standard GB 14974-94 " in apple and the haw products patulin limit the quantity of hygienic standard " regulation, jam are 100 μ g/kg, and fruit juice, jam, fruit wine, can, hawthorn bar, cake are 50 μ g/kg.
Open up blue or green toxin and have strong neural poison, up neural paralysis, central nervous system oedema and focal hemorrhage appear in milk cow when poisoning; Subcutaneous dropsy, abdominal cavity and pleural effusion then appear in mouse, kidney hemostasis and sex change, pulmonary edema, expiratory dyspnea, hypourocrinia; Mouse hypodermic injection patulin repeatedly can produce local sarcoma; And teratogenesis, mutagenesis are arranged.
The method of mycotoxin detection at present can be divided into two classes.One class is a red, orange, green, blue, yellow (ROGBY), comprises thin-layered chromatography TLC, vapor-phase chromatography GC, high performance liquid chromatography HPLC etc.At first set up thin layer liquid phase chromatography TLC the seventies and detected patulin.Patulin GB detection method adopts thin-layered chromatography TLC in China's GB 14974-1994 apple and the haw products at present.But TLC is bad for the separating effect of patulin and hydroxymethylfurfural HMF, can only sxemiquantitative, and the not high 20 μ g/L of sensitivity, replaced therefore very soon by high performance liquid chromatography HPLC.Vapor-phase chromatography GC, gas chromatography-mass spectrometry machine method GC-MS also set up in succession afterwards.But these methods all need expensive equipment, therefore promote the use of and are subjected to certain restriction.Another kind of is immuno-chemical method.This method is not high to the purity requirement of sample, and has higher sensitivity and specificity, is suitable for the detection of sample in enormous quantities.Be widely used in the detection of various mycotoxins in recent years.These class methods comprise radio immunoassay RIA and enzyme-linked immunosorbent assay ELISA etc.Although the RIA method has higher sensitivity,, can't promote the use of owing to used harmful radiomaterial.The ELISA method is because simple to operate, safe in utilization and generally adopted.But do not examine plain report at present to patulin immunochemistry detection method.
Immunochromatography technique has been widely used in the clinical treatment detection at present.The test item of domestic and international existing supply of commodities and bibliographical information mainly comprises following aspect: forbidden drug detects, detect as amphetamine, ***e, hemp, morphine, heroin etc., infectious disease cause of disease, detect mark etc. as microspironema pallidum, gonococcus, genito-urinary system chlamydia trachomatis, helicobacter pylori etc., hormone detection, parasite, tumor marker, cardiovascular disease.Immunochromatography technique is used to detect patulin and has not yet to see report.
Summary of the invention
The object of the present invention is to provide a kind of method for making of patulin immunity chromatography detection test paper.
Another object of the present invention is to use this detection test paper.
Technical scheme of the present invention is:
A kind of patulin 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-patulin 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 patulin 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-patulin.
A kind of method for making of patulin 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 patulin is arranged in the sample, then patulin moves to trace particle bond pad through the chromatography effect, antibodies with the anti-patulin of limiting the quantity of, when moving to detection line, the antibody of the anti-patulin 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-patulin 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 do not have patulin in the sample, the then antibody of the anti-patulin 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 patulin immunity chromatography detection test paper: comprise the steps that (A) preparation of patulin antigen comprises the immunizing antigen of polyclonal antibody or monoclonal antibody and the preparation that patulin 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 patulin immunity chromatography detection test paper: the preparation of patulin antigen comprises the steps: that (A) patulin makes its hydroxyl link to each other with ester bond with the glutaric acid carboxyl by the glutaric anhydride method, 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 patulin antigen as immunizing antigen.(D) select a kind of of patulin antigen as detecting antigen.
A kind of method for making of patulin 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 patulin; (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 patulin 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 patulin 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 patulin immunity chromatography detection test paper: the preparation of trace particle bond pad comprises the steps: (A) antibody with the anti-patulin of trace particle mark, and antibody comprises IgG type antibody, IgM type antibody, IgA antibody; (B) antibody of the good anti-patulin of trace particle mark is dispersed on the nitrocellulose filter.
A kind of method for making of patulin 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 patulin 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 patulin immunity chromatography detection test paper in detecting patulin.
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 etc., wherein the most successful label of utilization is a collaurum.
Trace particle is selected colour developing mark commonly used for use, and when detection line, nature controlling line such as normal colour developing, naked eyes can be easy to differentiate, but instrument is differentiated the sensitivity of magnetic-particle.Higher.
Immunochromatography technique of the present invention detects patulin, the principle of measuring is similar to the indirect competitive ELISA method, the antibody of the anti-patulin of coating finite concentration trace particle mark on glass fibre, on nitrocellulose membrane, adsorb detection antigen (because patulin is not directly fixed on nitrocellulose membrane, so need to prepare the conjugate of patulin 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.
Patulin 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 Penicillium patulum is have the polyclonal antibody or the monoclonal antibody of high affinity, high degree of specificity, the preparation of artificial antigen (immunizing antigen) is particularly important, need be according to careful selection coupling methods such as the molecular size of patulin, space structure picture, chemical property.Coupling process will keep the integrality of patulin 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, patulin 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.Because have hydroxyl on the patulin molecule, the present invention designs with the glutaric anhydride method its hydroxyl is linked to each other with ester bond with the glutaric acid carboxyl, forms new compound.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 patulin 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 patulin.(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 patulin 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 patulin 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 patulin immunity chromatography detection test paper of the present invention.
Fig. 3 is the detection line partial enlarged drawing of patulin immunity chromatography detection test paper of the present invention.
Fig. 4 is the nature controlling line partial enlarged drawing of patulin immunity chromatography detection test paper of the present invention.
Fig. 5 is a patulin immunity chromatography detection test paper principle of work synoptic diagram of the present invention.
Embodiment
Embodiment 1
A kind of patulin 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-patulin of trace particle 9 marks 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 patulin 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-patulin.
It is such detecting principle: drip sample 12 on sample pad 2, if in the sample 12 patulin is arranged, then patulin moves to trace particle bond pad 3 through the chromatography effect, combine with the antibody 10 of the anti-patulin of limiting the quantity of, when moving to detection line 6, the antibody 10 of the anti-patulin 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-patulin 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 patulin 8 in the sample 12, the then antibody 10 of the anti-patulin 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 patulin immunity chromatography detection test paper comprises the steps, (A) preparation of patulin antigen comprises the immunizing antigen of polyclonal antibody or monoclonal antibody and the preparation that patulin 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.
Embodiment 2
In embodiment 2-7:
Embodiment 8
A kind of method for making of patulin immunity chromatography detection test paper, the preparation of patulin antigen comprises the steps: that (A) patulin makes its hydroxyl link to each other with ester bond with the glutaric acid carboxyl by the glutaric anhydride method, 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 patulin antigen as immunizing antigen.(D) select a kind of of patulin 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 patulin; (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 patulin 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 patulin 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.
A kind of method for making of patulin immunity chromatography detection test paper, the preparation of trace particle bond pad 3 comprise the steps: (A) antibody 10 with the anti-patulin of trace particle mark, and antibody 10 comprises IgG type antibody, IgM type antibody, IgA antibody; (B) antibody 10 of the good anti-patulin 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 patulin 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 patulin test paper, and it specifically is applied to concrete detection.
Comprise the steps:
(1). the preparation of patulin artificial antigen comprises being used for the immunizing antigen and the detection antigen that is used to prepare detection line of immune animal with preparation polyclonal antibody or monoclonal antibody.
(2). the preparation of nitrocellulose filter detection line 6 and nature controlling line 7.
1. the conjugate of using patulin and carrier protein is as detecting the antigen linear spotting as detection line 6.
2. the antibody linear spotting of antibody 10 of using patulin is as nature controlling line 7.
(3). the preparation of trace particle bond pad 3:
The antibody 10 of the anti-patulin 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 patulin artificial antigen (comprise immunizing antigen and detect antigen) comprises the steps:
1. 0.5mg patulin, 7.4mg glutaric anhydride and 1.6mg4-dimethylamino naphthyridine are dissolved in the 200 μ L anhydrous tetrahydro furans 37 ℃ of oscillating reactionss 1 hour; Add acid distilled water (pH2-3) cessation reaction of 100 μ L;
2. 12000rpm went precipitation in centrifugal 5 minutes, and supernatant vacuumizes the removal tetrahydrofuran; With 300 μ L chloroform extractings three times; Be purified into reaction product patulin-half glutaric acid with thin layer chromatography.
3. patulin-half glutaric acid with purifying is dissolved in the 100 μ L tetrahydrofurans, and 0.6mg nitrogen hydroxy succinic acid (NHS) and 3.2mg dicyclohexyl carbodiimide (DCC) are dissolved in the 200 μ L tetrahydrofurans.The two mixing is put room temperature lucifuge oscillating reactions 24 hours.
4. after reacting completely, 12000rpm went precipitation in centrifugal 5 minutes.The supernatant vacuum is drained, be dissolved in the 300 μ L dimethyl sulfoxide (DMSO)s, add again that (4mg is dissolved in 600 μ L0.13M NaHCO in 600 μ L carriers (key hole shellfish hemocyanin, bovine serum albumin(BSA), oralbumin, thyroglobulin, human serum albumins, poly-D-lysine, polyglutamic acid or the poly mixing propylhomoserin a kind of) solution
3In), vibrate lucifuge reaction 2 hours under the room temperature.
5. after reaction finishes, 4 ℃ of dialysis of 0.01M PBS (pH7.4) 72 hours, the mol ratio of UV scanning assay determination coupled product, vacuum freeze-drying is stand-by.
2. the preparation of nitrocellulose filter detection line 6 and nature controlling line 7
(1) patulin of preparation detection line 6 detects the selection of antigen 8
That selects detection antigen that bovine serum albumin(BSA), key eye shellfish hemocyanin, oralbumin, thyroglobulin or human serum albumins and the coupling of patulin-half glutaric acid prepare a kind ofly is used to prepare a detection line 6;
(2) selection of the antiantibody 11 of preparation nature controlling line 7
1. select antiantibody a kind of of the penicillin-fast IgG type of exhibition, IgM type, IgA type monoclonal antibody to be used to prepare a nature controlling line;
2. select antiantibody 11 a kind of of the polyclonal antibody of patulin to be used to prepare a nature controlling line 7;
(3) preparation method of nitrocellulose filter detection line 6 and nature controlling line 7
3. the preparation of trace particle bond pad 3
(1) trace particle of preparation trace particle bond pad is following a kind of
1. colloid gold particle
2. latex particle
3. electroselenium particle
4. gelatin particle
5. magnetic-particle
(2) preparation of tracer particles 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 and antibody are mixed, 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. 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, splashes in the reaction mixture under the stirring at room, obtains the red selenium sol of 120nm.
B) electroselenium antibody labeling: IgG antibody is made into 4.6mg/mL concentration solution with 20mmol/L pH7.3 phosphate buffer, add 25ul in the 25mL of pH7.3 selenium sol, stirring at room adds 1%PEG8000 1mL after 10 minutes, mixing, in 4 ℃ with 5000rpm centrifugal 5 minutes, obtain soft red precipitate, be made into the 1mL suspension with the phosphate buffer that contains 0.05%NaN3.
3. the preparation of latex and antibody labeling:
The color latex particle is available from Bangs Laboratories company.The antibody labeling program is: the phosphate buffer with pH7.1 is diluted to 1% concentration with latex, stirs to add a certain amount of IgG solution down, 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 an amount of phosphate buffer.
4. the 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 respectively, stirs down it is mixed with anti-patulin antibody, 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.
(3) preparation of trace particle bond pad 3
The antibody 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 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
(1) fluid sample disposal route: measure and wait for sample 25mL, place separating funnel, add isopyknic ethyl acetate, jolting 2 minutes, standing demix, repeat above step twice, merge organic phase, add 2.5mL1.5% sodium carbonate jolting 1 minute, behind the standing demix, discard the sodium carbonate layer, the same step is handled once with sodium carbonate again.Extract is filtered in the 100mL pyriform bottle, be concentrated near doing with vacuum decompression in 40 ℃ of water-baths, with a little chloroform washer bottle wall, concentrate and do, adding distil water 0.4mL constant volume is standby.
(2) solid and semi-solid sample treatment: take by weighing sample 25g and place mortar, after adding an amount of anhydrous sodium sulfate grinding, add in the triangular flask, adding 80mL ethyl acetate soaked 30 minutes, vibrated 30 minutes, and filtered, get filtrate 50mL, place separating funnel, add isopyknic ethyl acetate, jolting 2 minutes, standing demix, repeat above step twice, merge organic phase, add 2.5mL1.5% sodium carbonate jolting 1 minute, behind the standing demix, discard the sodium carbonate layer, the same step is handled once with sodium carbonate again.Extract is filtered in the 100mL pyriform bottle, be concentrated near doing with vacuum decompression in 40 ℃ of water-baths, with a little chloroform washer bottle wall, concentrate and do, adding distil water 0.4mL constant volume is standby.
Embodiment 14:
The method of colloidal gold immunochromatographimethod detection patulin and the preparation of test paper also are used to detect solid sample
Present embodiment is divided into 6 parts: the preparation of (1) patulin artificial antigen 8; (2) preparation of nitrocellulose filter 4; (3) preparation of collaurum bond pad; (4) assembling of test strips; (5) processing of sample to be checked; (6) detection and result judge.
(1) patulin detects the preparation of antigen 8, comprises the steps:
1. 0.5mg patulin, 7.4mg glutaric anhydride and 1.6mg 4-dimethylamino naphthyridine are dissolved in the 200 μ L anhydrous tetrahydro furans 37 ℃ of oscillating reactionss 1 hour; Add acid distilled water (pH2-3) cessation reaction of 100 μ L;
2. 12000rpm went precipitation in centrifugal 5 minutes, and supernatant vacuumizes the removal tetrahydrofuran; With 300 μ L chloroform extractings three times, merge extract; Be purified into reaction product (patulin-half glutaric acid) with thin layer chromatography.
3. patulin-half glutaric acid with purifying is dissolved in the 100 μ L tetrahydrofurans, and 0.6mg nitrogen hydroxy succinic acid (NHS) and 3.2mg dicyclohexyl carbodiimide (DCC) are dissolved in the 200 μ L tetrahydrofurans; The two mixing is put room temperature lucifuge oscillating reactions 24 hours.
4. after reacting completely, 12000rpm went precipitation in centrifugal 5 minutes.The supernatant vacuum is drained, and is dissolved in the 300 μ L dimethyl sulfoxide (DMSO)s, adds that (4mg is dissolved in 600 μ L0.13MNaHCO in the solution of 600 μ L skimmed milk powers 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, the mol ratio of UV scanning assay determination coupled product, vacuum freeze-drying is stand-by.
(2) preparation of nitrocellulose filter 4
1. select the detection antigen of preparation detection line 6 and the antiantibody 11 of preparation nature controlling line 7
A. select the detection antigen of skimmed milk power and patulin-half glutaric acid coupling preparation to be used to prepare detection line 6;
B. select the antiantibody 11 of the IgG type monoclonal antibody of patulin to be used to prepare nature controlling line 7.
2. prepare detection line 6 and nature controlling line 7
(3) preparation of collaurum bond pad
Getting the 50mL particle diameter respectively is the 30-50nm collaurum, transfers pH to 8.0 with 0.1mol/L sal tartari, stirs down collaurum colloidal sol and patulin monoclonal antibody are mixed, and adds the polyglycol aqueous solution again, and 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.The antibody of mark colloid gold particle is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.
(4) 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 collaurum bond pad; 3. on liner 1, add sample pad 2; 4. on liner 1, add adsorptive pads 5; Be assembled into test strip.
(5) processing of sample
Take by weighing solid sample 25g and place mortar, after adding an amount of anhydrous sodium sulfate and grinding, claim to triangular flask, add 80mL ethyl acetate and soaked 30 minutes, vibrated 30 minutes, filter, get filtrate 50mL, place separating funnel, add isopyknic ethyl acetate, jolting 2 minutes, standing demix repeats above step twice, merge organic phase, add 2.5mL1.5% sodium carbonate jolting 1 minute, behind the standing demix, discard the sodium carbonate layer, the same step is handled once with sodium carbonate again.Extract is filtered in the 100mL pyriform bottle, be concentrated near doing with vacuum decompression in 40 ℃ of water-baths, with a little chloroform washer bottle wall, concentrate and do, adding distil water 0.4mL constant volume is standby.
(6) detection and result judge
Detect: get the sample 0.4mL that handles well, vertically insert test strips centrifugal a moment, insertion depth is no more than sample pad, stops about 5 seconds in sample, vertically takes out test paper, observes after 5 minutes.
The result judges: 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.
| Detection line | Nature controlling line | The result judges |
| +/- | - | The result is unreliable |
| - | + | Contain patulin in the sample |
| + | + | In the sample in no patulin or the sample patulin content be lower than the detection lowest limit |
Embodiment 15:
The electroselenium immunochromatography detects the preparation of patulin test paper and is used for the tracer liquid sample
Present embodiment is divided into 6 parts: (1) patulin detects the preparation of antigen 8; (2) preparation of nitrocellulose filter 4; (3) preparation of electroselenium bond pad; (4) assembling of test strips; (5) processing of sample to be checked; (6) detection and result judge.
(1) patulin detects the preparation of antigen 8, comprises the steps:
1. 0.5mg patulin, 7.4mg glutaric anhydride and 1.6mg 4-dimethylamino naphthyridine are dissolved in the 200 μ L anhydrous tetrahydro furans 37 ℃ of oscillating reactionss 1 hour; Add acid distilled water (PH2-3) cessation reaction of 100 μ L;
2. 12000rpm went precipitation in centrifugal 5 minutes, and supernatant vacuumizes the removal tetrahydrofuran; With 300 μ L chloroform extractings three times; Be purified into reaction product (patulin-half glutaric acid) with thin layer chromatography.
3. patulin-half glutaric acid with purifying is dissolved in the 100 μ L tetrahydrofurans, and 0.6mg nitrogen hydroxy succinic acid (NHS) and 3.2mg dicyclohexyl carbodiimide (DCC) are dissolved in the 200 μ L tetrahydrofurans; The two mixing is put room temperature lucifuge oscillating reactions 24 hours.
4. after reacting completely, 12000rpm went precipitation in centrifugal 5 minutes.The supernatant vacuum is drained, and is dissolved in the 300 μ L dimethyl sulfoxide (DMSO)s, adds that (4mg is dissolved in 600 μ L 0.13M NaHCO in 600 μ L key hole shellfish hemocyanin (KLH) 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, the mol ratio of UV scanning assay determination coupled product, vacuum freeze-drying is stand-by.
(2) preparation of nitrocellulose filter 4:
1. select the detection antigen of preparation detection line 6 and the antiantibody 11 of preparation nature controlling line 7
A. select the key eye shellfish hemocyanin (KLH) and the detection antigen of patulin-half glutaric acid coupling preparation to be used to prepare detection line 6.
B. select the antiantibody of the IgG type monoclonal antibody of patulin to be used to prepare nature controlling line.
2. prepare detection line 6 and nature controlling line 7
(3) preparation of electroselenium bond pad:
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, splashes in the reaction mixture under the stirring at room, obtains the red selenium sol of 120nm.
B. electroselenium antibody labeling: patulin monoclonal anti body and function 20mmol/L pH7.3 phosphate buffer is made into 4.6mg/mL concentration solution, add 25 μ L in the 25mL of pH7.3 selenium sol, stirring at room adds 1%PEG8000 1mL after 10 minutes, mixing, in 4 ℃ with 5000rpm centrifugal 5 minutes, obtain soft red precipitate, be made into the 1mL suspension with the phosphate buffer that contains 0.05%NaN3.
C. the antibody 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.
(4) 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 electroselenium bond pad; 3. on liner 1, add sample pad 2; 4. on liner 1, add adsorptive pads 5; Be assembled into test strip.
(5) processing of sample:
Sample product 25mL such as measure, place separating funnel, add isopyknic ethyl acetate, jolting 2 minutes, standing demix, repeat above step twice, merge organic phase, add 2.5mL1.5% sodium carbonate jolting 1 minute, behind the standing demix, discard the sodium carbonate layer, the same step is handled once with sodium carbonate again.Extract is filtered in the 100mL pyriform bottle, be concentrated near doing with vacuum decompression in 40 ℃ of water-baths, with a little chloroform washer bottle wall, concentrate and do, adding distil water 0.4mL constant volume is standby.
(6) detection and result judge
Detect: get the sample 0.4mL that handles well, vertically insert test strips centrifugal a moment, insertion depth is no more than sample pad, stops about 5 seconds in sample, vertically takes out test paper, observes after 5 minutes.
The result judges: 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 6 no red lines appear as inefficacy.
| Detection line | Nature controlling line | The result judges |
| +/- | - | The result is unreliable |
| - | + | Contain patulin in the sample |
| + | + | In the sample in no patulin or the sample patulin content be lower than the detection lowest limit |
Embodiment 16:
The latex particle immunochromatography detects the preparation of patulin test paper and is used to detect semi-solid sample
Present embodiment is divided into 6 parts: (1) patulin detects the preparation of antigen 8; (2) preparation of nitrocellulose filter 4; (3) preparation of latex particle bond pad; (4) assembling of test strips; (5) processing of sample to be checked; (6) detection and result judge.
(1) patulin detects the preparation of antigen 8, comprises the steps:
1. 0.5mg patulin, 7.4mg glutaric anhydride and 1.6mg 4-dimethylamino naphthyridine are dissolved in the 200 μ L anhydrous tetrahydro furans 37 ℃ of oscillating reactionss 1 hour; Add acid distilled water (PH2-3) cessation reaction of 100 μ L;
2. 12000rpm went precipitation in centrifugal 5 minutes, and supernatant vacuumizes the removal tetrahydrofuran; With 300 μ L chloroform extractings three times; Be purified into reaction product (patulin-half glutaric acid) with thin layer chromatography.
3. patulin-half glutaric acid with purifying is dissolved in the 100 μ L tetrahydrofurans, and 0.6mg nitrogen hydroxy succinic acid (NHS) and 3.2mg dicyclohexyl carbodiimide (DCC) are dissolved in the 200 μ L tetrahydrofurans.The two mixing is put room temperature lucifuge oscillating reactions 24 hours.
4. after reacting completely, 12000rpm went precipitation in centrifugal 5 minutes.The supernatant vacuum is drained, and is dissolved in the 300 μ L dimethyl sulfoxide (DMSO)s, adds that (4mg is dissolved in 600 μ L 0.13M NaHCO in 600 μ L oralbumin (OVA) 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, the mol ratio of UV scanning assay determination coupled product, vacuum freeze-drying is stand-by.
(2) preparation of nitrocellulose filter 4:
1. select the detection antigen of preparation detection line 6 and the antiantibody 11 of preparation nature controlling line 7
A. select the detection antigen of oralbumin and patulin-half glutaric acid coupling preparation to be used to prepare detection line;
B. select the antiantibody of the IgM type monoclonal antibody of patulin to be used to prepare nature controlling line.
2. prepare detection line 6 and nature controlling line 7
(3) preparation of latex bond pad
The color latex particle is available from Bangs Laboratories company, and color is blue look.Phosphate buffer with pH7.1 is diluted to 1% concentration with latex particle, gets 50mL respectively, stirs down latex solution patulin monoclonal antibody is mixed, 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 is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.(4) 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 latex bond pad; 3. on liner 1, add sample pad 2; 4. on liner 1, add adsorptive pads 5; Be assembled into test strip.
(5) processing of sample
Take by weighing semi-solid sample 25g and place mortar, after adding an amount of anhydrous sodium sulfate and grinding, claim to triangular flask, add 80mL ethyl acetate and soaked 30 minutes, vibrated 30 minutes, filter, get filtrate 50mL, place separating funnel, add isopyknic ethyl acetate, jolting 2 minutes, standing demix repeats above step twice, merge organic phase, add 2.5mL1.5% sodium carbonate jolting 1 minute, behind the standing demix, discard the sodium carbonate layer, the same step is handled once with sodium carbonate again.Extract is filtered in the 100mL pyriform bottle, be concentrated near doing with vacuum decompression in 40 ℃ of water-baths, with a little chloroform washer bottle wall, concentrate and do, adding distil water 0.4mL constant volume is standby.
(6) detection and result judge
Detect: 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, vertically takes out test paper, observes after 5 minutes.
The result judges as follows:
| Detection line | Nature controlling line | The result judges |
| +/- | - | The result is unreliable |
| - | + | Contain patulin in the sample |
| + | + | In the sample in no patulin or the sample patulin content be lower than the detection lowest limit |
Embodiment 17:
The gelatin particle immunochromatography detects the preparation of patulin test paper and is used to detect semi-solid sample
Present embodiment is divided into 6 parts: (1) patulin detects the preparation of antigen 8; (2) preparation of nitrocellulose filter 4; (3) preparation of gelatin particle bond pad; (4) assembling of test strips; (5) processing of sample to be checked; (6) detection and result judge.
(1) patulin detects the preparation of antigen, comprises the steps:
1. 0.5mg patulin, 7.4mg glutaric anhydride and 1.6mg 4-dimethylamino naphthyridine are dissolved in the 200 μ L anhydrous tetrahydro furans 37 ℃ of oscillating reactionss 1 hour; Add acid distilled water (pH2-3) cessation reaction of 100 μ L;
2. 12000rpm went precipitation in centrifugal 5 minutes, and supernatant vacuumizes the removal tetrahydrofuran; With 300 μ L chloroform extractings three times; Be purified into reaction product (patulin-half glutaric acid) with thin layer chromatography.
3. patulin-half glutaric acid with purifying is dissolved in the 100 μ L tetrahydrofurans, and 0.6mg nitrogen hydroxy succinic acid (NHS) and 3.2mg dicyclohexyl carbodiimide (DCC) are dissolved in the 200 μ L tetrahydrofurans.The two mixing is put room temperature lucifuge oscillating reactions 24 hours.
4. after reacting completely, 12000rpm went precipitation in centrifugal 5 minutes.The supernatant vacuum is drained, and is dissolved in the 300 μ L dimethyl sulfoxide (DMSO)s, adds that (4mg is dissolved in 600 μ L0.13M NaHCO in the 600 μ L key hole shellfish hemocyanin 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, the mol ratio of UV scanning assay determination coupled product, vacuum freeze-drying is stand-by.
(2) preparation of nitrocellulose filter 4:
1. select the detection antigen of preparation detection line 6 and the antiantibody 11 of preparation nature controlling line 7
A. select the detection antigen of key hole shellfish hemocyanin and the coupling of patulin-half glutaric acid preparation to be used to prepare detection line;
B. select the antiantibody 11 of the IgG type monoclonal antibody of patulin to be used to prepare nature controlling line 7.
2. prepare detection line 6 and nature controlling line 7
(3) preparation of gelatin bond pad:
Gelatin particle is available from Zodolabs company, and color is blue look.Phosphate buffer with pH7.1 is diluted to 1% concentration with gelatin particle, gets 50mL respectively, stirs down gelatin solution patulin monoclonal antibody is mixed, 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.Mark gelatin particle antibody is mixed and be dispersed on the all-glass paper that thickness is 1mm, the freeze-drying hermetically drying is preserved.
(4) 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 gelatin particle bond pad; 3. on liner 1, add sample pad 2; 4. on liner 1, add adsorptive pads 5; Be assembled into test strip.
(5) processing of sample:
Take by weighing semi-solid sample 25g and place mortar, after adding an amount of anhydrous sodium sulfate and grinding, be added in the triangular flask, add 80mL ethyl acetate and soaked 30 minutes, vibrated 30 minutes, filter, get filtrate 50mL, place separating funnel, add isopyknic ethyl acetate, jolting 2 minutes, standing demix repeats above step twice, merge organic phase, add 2.5mL1.5% sodium carbonate jolting 1 minute, behind the standing demix, discard the sodium carbonate layer, the same step is handled once with sodium carbonate again.Extract is filtered in the 100mL pyriform bottle, be concentrated near doing with vacuum decompression in 40 ℃ of water-baths, with a little chloroform washer bottle wall, concentrate and do, adding distil water 0.4mL constant volume is standby.
(6) detection and result judge
Detect: 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, vertically takes out test paper, observes after 5 minutes.
The result judges as follows:
| Detection line | Nature controlling line | The result judges |
| +/- | - | The result is unreliable |
| - | + | Contain patulin in the sample |
| + | + | In the sample in no patulin or the sample patulin content be lower than the detection lowest limit |
Embodiment 18:
The magnetic-particle immunochromatography detects the preparation of patulin test paper and is used for the tracer liquid sample
Present embodiment is divided into 6 parts: (1) patulin detects the preparation of antigen 8; (2) preparation of nitrocellulose filter 4; (3) preparation of magnetic-particle bond pad; (4) assembling of test strips; (5) processing of sample to be checked; (6) detection and result judge.
(1) patulin detects the preparation of antigen 8, comprises the steps:
1. 0.5mg patulin, 7.4mg glutaric anhydride and 1.6mg 4-dimethylamino naphthyridine are dissolved in the 200 μ L anhydrous tetrahydro furans 37 ℃ of oscillating reactionss 1 hour; Add acid distilled water (PH2-3) cessation reaction of 100 μ L;
2. 12000rpm went precipitation in centrifugal 5 minutes, and supernatant vacuumizes the removal tetrahydrofuran; With 300 μ L chloroform extractings three times; Be purified into reaction product (patulin-half glutaric acid) with thin layer chromatography.
3. patulin-half glutaric acid with purifying is dissolved in the 100 μ L tetrahydrofurans, and 0.6mg nitrogen hydroxy succinic acid (NHS) and 3.2mg dicyclohexyl carbodiimide (DCC) are dissolved in the 200 μ L tetrahydrofurans.The two mixing is put room temperature lucifuge oscillating reactions 24 hours.
4. after reacting completely, 12000rpm went precipitation in centrifugal 5 minutes.The supernatant vacuum is drained, and is dissolved in the 300 μ L dimethyl sulfoxide (DMSO)s, adds that (4mg is dissolved in 600 μ L 0.13M NaHCO in 600 μ L bovine serum albumin(BSA) (BSA) 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, the mol ratio of UV scanning assay determination coupled product, vacuum freeze-drying is stand-by.
(2) preparation of nitrocellulose filter 4:
1. select the detection antigen of preparation detection line 6 and the antiantibody 11 of preparation nature controlling line 7
A. select the detection antigen of bovine serum albumin(BSA) and patulin-half glutaric acid coupling preparation to be used to prepare detection line;
B. select the antiantibody of the IgG type monoclonal antibody of patulin to be used to prepare nature controlling line.
2. prepare detection line 6 and nature controlling line 7
(3) 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 respectively, stirs down the patulin monoclonal antibody is mixed, 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.
(4) 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 magnetic-particle bond pad; 3. on liner 1, add sample pad 2; 4. on liner 1, add adsorptive pads 5; Be assembled into test strip.
(5) processing of sample:
Sample product 25mL such as measure, place separating funnel, add isopyknic ethyl acetate, jolting 2 minutes, standing demix, repeat above step twice, merge organic phase, add 2.5mL1.5% sodium carbonate jolting 1 minute, behind the standing demix, discard the sodium carbonate layer, the same step is handled once with sodium carbonate again.Extract is filtered in the 100mL pyriform bottle, be concentrated near doing with vacuum decompression in 40 ℃ of water-baths, with a little chloroform washer bottle wall, concentrate and do, adding distil water 0.4mL constant volume is standby.
(6) detection and result judge
Detect: 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, vertically takes out test paper, reads the instrument judged result with magnetic after 5 minutes.
The result judges as follows: it is positive that the magnetic enhancing appears in the relevant position, and nonmagnetic enhancing is negative
| Detection line | Nature controlling line | The result judges |
| +/- | - | The result is unreliable |
| - | + | Contain patulin in the sample |
| + | + | In the sample in no patulin or the sample patulin content be lower than the detection lowest limit |
Claims (7)
1, a kind of method for making of patulin immunity chromatography detection test paper, the patulin 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) has sample pad (2) in order on liner (1), trace particle bond pad (3), nitrocellulose filter (4), adsorptive pads (5); The antibody (10) that the anti-patulin 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 patulin 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-patulin; It is characterized in that: comprise the steps that (A) preparation of patulin antigen comprises the immunizing antigen of monoclonal antibody and the preparation that patulin 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; Wherein: 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 patulin 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.
2, the method for making of a kind of patulin immunity chromatography detection test paper as claimed in claim 1 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.
3, the method for making of a kind of patulin immunity chromatography detection test paper as claimed in claim 1, it is characterized in that: the preparation of patulin antigen, comprise the steps: that (A) patulin makes its hydroxyl link to each other with ester bond with the glutaric acid carboxyl by the glutaric anhydride method, 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 patulin antigen as immunizing antigen; (D) select a kind of of patulin antigen as detecting antigen; The carrier that is used to prepare the patulin artificial antigen of detection line and is used to prepare the immunizing antigen of anti-patulin antibody is to select like this: that 1. selects bovine serum albumin(BSA), key eye shellfish hemocyanin, oralbumin, thyroglobulin, human serum albumins a kind ofly is used to prepare the patulin artificial antigen for carrier; That 2. selects poly-D-lysine, polyglutamic acid, poly mixing propylhomoserin a kind ofly is used to prepare the patulin artificial antigen for carrier; 3. the patulin immunizing antigen can be identical with the carrier that detects antigen, also can be different.
4, the method for making of a kind of patulin immunity chromatography detection test paper as claimed in claim 1, 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 patulin 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.
5, the method for making of a kind of patulin immunity chromatography detection test paper as claimed in claim 1, it is characterized in that: the preparation of trace particle bond pad (3) comprises the steps: (A) antibody (10) with the anti-patulin of trace particle mark, and antibody (10) comprises IgG type antibody, IgM type antibody, IgA antibody; (B) antibody (10) of the good anti-patulin of trace particle (9) mark is dispersed on the nitrocellulose filter (4).
6, the method for making of a kind of patulin immunity chromatography detection test paper as claimed in claim 1 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.
7, the application of patulin immunity chromatography detection test paper in detecting patulin of adopting the described method for making of claim 1 to make.
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Cited By (1)
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| CN101172081B (en) * | 2006-10-30 | 2010-09-15 | 三洋电机株式会社 | Chair massaging machine |
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| CN102081094B (en) * | 2009-11-27 | 2013-12-18 | 北京维德维康生物技术有限公司 | Method for detecting patulin and special enzyme linked immunosorbent assay kit thereof |
| CN102192980B (en) * | 2010-03-08 | 2014-04-09 | 苏州浩欧博生物医药有限公司 | Nonmetallic colloidal particle immune analysis reagent and method |
| CN110579606A (en) * | 2019-09-04 | 2019-12-17 | 武玉香 | detection card for rapidly and quantitatively detecting penicillin and application thereof |
| CN114716535B (en) * | 2022-06-10 | 2022-08-26 | 北京纳百生物科技有限公司 | Synthetic method of patulin artificial antigen and preparation and application of monoclonal antibody thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999018439A1 (en) * | 1997-10-07 | 1999-04-15 | Ucb Bioproducts, S.A. | Testing device for determining analytes in a liquid dairy product |
| CN1271860A (en) * | 1999-04-23 | 2000-11-01 | 郑州博赛生物技术研究所 | Test strip for detecting more antigens of typhoid bacillus |
| CN1438486A (en) * | 2002-12-31 | 2003-08-27 | 江西中德生物工程有限公司 | Method for detecting ABO blood type using immuno-chromatography, its prepared indicator paper and use |
-
2003
- 2003-09-30 CN CNB031602991A patent/CN1300586C/en not_active Expired - Lifetime
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999018439A1 (en) * | 1997-10-07 | 1999-04-15 | Ucb Bioproducts, S.A. | Testing device for determining analytes in a liquid dairy product |
| CN1271860A (en) * | 1999-04-23 | 2000-11-01 | 郑州博赛生物技术研究所 | Test strip for detecting more antigens of typhoid bacillus |
| CN1438486A (en) * | 2002-12-31 | 2003-08-27 | 江西中德生物工程有限公司 | Method for detecting ABO blood type using immuno-chromatography, its prepared indicator paper and use |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101172081B (en) * | 2006-10-30 | 2010-09-15 | 三洋电机株式会社 | Chair massaging machine |
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Effective date of registration: 20100727 Address after: 214174 No. 1608 Huishan Avenue, Huishan Economic Development Zone, Wuxi, 1301-1310 Patentee after: WUXI ZODOLABS BIOTECH Co.,Ltd. Address before: 330029 No. 210,, Gaoxin hi tech Overseas Building, Jiangxi, Nanchang Patentee before: Jiangxi Zodogenes Biotechnology Co.,Ltd. |
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