CN101930006A - High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof - Google Patents
High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof Download PDFInfo
- Publication number
- CN101930006A CN101930006A CN 201010245801 CN201010245801A CN101930006A CN 101930006 A CN101930006 A CN 101930006A CN 201010245801 CN201010245801 CN 201010245801 CN 201010245801 A CN201010245801 A CN 201010245801A CN 101930006 A CN101930006 A CN 101930006A
- Authority
- CN
- China
- Prior art keywords
- pad
- test strip
- aflatoxin
- sensitivity
- detection line
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention belongs to the field of biological detection and provides a high-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly, which is characterized by comprising a test strip, wherein a water-absorbing pad, a detection pad, a gold label pad and a sample pad are adhered on one side of the test strip from top down; the pads are overlapped at the connected parts; the detection pad uses a nitrocellulose film as a substrate pad; transverse quality control lines and detection lines are arranged on the nitrocellulose film from top down; the detection lines are coated with aflatoxin B1-bovine serum albumin (AFB1-BSA) coupling and the quality control lines are coated with rabbit anti-mouse polyclonal antibody; and a nano gold-labeled anti-aflatoxin common monoclonal antibody, which is generated by hybrid tumor cell strain 1C11 having a collection number of CCTCC No.C201013, is sprayed on the gold label pad transversely. The test strip is used for detecting total aflatoxin content and has the characteristics of quick detection, simple operation and high flexibility.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to high-sensitivity immunochromatographitest test strip of a kind of fast detecting total aflatoxin content and preparation method thereof.
Background technology
Aflatoxin mainly is the secondary metabolite that is produced by aspergillus flavus and aspergillus parasiticus secretion, is a kind of natural toxic compounds that can cause the various infringements of people and animals.Kind surplus aflatoxin (AFT) has found 20 at present, wherein most importantly aflatoxin B1, B2, G1 and G2.
Behind aflatoxin contamination food and the feed, can directly or indirectly enter human food's chain, threaten human beings'health and life security, its extent of injury is directly proportional with the intake of aflatoxin.Aflatoxin extensively is present in rice, corn, peanut, sesame, soybean, in the food such as the agricultural product such as vegetable seed and the flesh of fish, the link of its contaminated food products and feed is many, and multiple aflatoxin often exists simultaneously, total aflatoxin content (aflatoxin B1 in food and the feed has all been stipulated in countries in the world for this reason, B2, the total amount of G1 and G2) maximum allow content and with it as compulsory standard, therefore strengthen detection to total aflatoxin content in food and the feed, particularly speed is surveyed, so that in time understand and grasp the health information of food and feed, be an important step that strengthens foodsafety.
Aflatoxin detection technique commonly used in the prior art mainly contains thin layer chromatography, exact instrument analytic approach, immune analysis method.The immune analysis method has overcome the above two shortcoming, have high specificity, highly sensitive, sample pre-treatments is simple, cost is low, little to the contamination hazard of experimenter and environment, be suitable for advantages such as on-the-spot batch detection.Wherein the immunochromatography Fast Detection Technique based on nm of gold has easy, quick, sensitive, as to be suitable for on-the-spot detection advantage, has great using value and application prospect.The existing at present quick detection test paper bar at aflatoxin one-component such as aflatoxin B1 (AFB1) comes out, but because most aflatoxin-contaminated sample all contains AFB1 aflatoxin incessantly, disturb or cross pollution thereby easily testing result is caused, and the sensitivity of commercially available Aspergillus flavus toxin immuno chromatography detecting test paper strip is on the low side relatively, detection is limited to 5-200ng/g, can not satisfy the current detection demand of strict aflatoxin limit standard more well.Therefore, research is set up a kind of high-sensitivity immunochromatographitest test strip at total aflatoxin content and is had very important significance and using value for the aflatoxin tool in monitoring food and the agricultural product.
Summary of the invention
Technical matters to be solved by this invention is to provide high-sensitivity immunochromatographitest test strip of a kind of fast detecting total aflatoxin content and preparation method thereof at the deficiency of above-mentioned prior art existence.This test strips is used to detect total aflatoxin content (total amount of aflatoxin B1, B2, G1 and G2), has to detect fast characteristics simple to operate, highly sensitive.
The present invention solves the problems of the technologies described above the technical scheme that is adopted to be:
The high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content (seeing Fig. 1 and Fig. 2), comprise cardboard, the one side of cardboard is pasted adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively, adjacent each pad overlaps in the junction and connects, described detecting pad is base wad with the nitrocellulose filter, horizontal nature controlling line and detection line are set on the nitrocellulose filter from top to bottom, described detection line is coated with aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA), and nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit; Described gold mark pad transverse jet scribbles the aspergillus flavus resisting toxin general purpose single clonal antibody of nano gold mark, and described aspergillus flavus resisting toxin general purpose single clonal antibody is the hybridoma cell strain 1C11 generation of CCTCC NO.C201013 by preserving number.
Press such scheme, the long 16～18mm of described adsorptive pads, wide 2～4mm; Long 25～the 30mm of detecting pad, wide 2～4mm; Long 6～the 9mm of gold mark pad, wide 2～4mm; Long 12～the 18mm of sample pad, wide 2～4mm, the overlapping length of adjacent each pad is 1～3mm.
Press such scheme, described adsorptive pads is a thieving paper.
Press such scheme, the spacing of detection line on the described detecting pad and nitrocellulose filter upper edge is 15～20mm, and the spacing of nature controlling line and detection line is 5～10mm.
Press such scheme, the package amount of the required aflatoxin B1-bovine serum albumin(BSA) conjugate (AFB1-BSA) of every centimetre of detection line is 80～400ng on the described detecting pad; The package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 200～500ng.
Press such scheme, the particle diameter of used nm of gold is 15～20nm in the described gold mark pad; The consumption of the aspergillus flavus resisting toxin general purpose single clonal antibody of the nano gold mark that the last every centimetre of spraying length of described gold mark pad is required is 48～144ng.
The preparation method of the high-sensitivity immunochromatographitest test strip of aforesaid fast detecting total aflatoxin content may further comprise the steps:
(1) preparation of adsorptive pads
Thieving paper cut out promptly get adsorptive pads;
(2) preparation of detecting pad
The bag quilt of detection line:
The conjugate (AFB1-BSA) of aflatoxin B1-bovine serum albumin(BSA) is mixed with the coating buffer A of 0.1～0.5mg/mL; In distance nitrocellulose filter upper edge is the position of 15～20mm, with a spray mode coating buffer A is laterally wrapped and on nitrocellulose filter, to be obtained detection line, the package amount of the conjugate (AFB1-BSA) of required aflatoxin B1-bovine serum albumin(BSA) is 80～400ng on every centimetre of detection line, under 37～40 ℃ of conditions dry 8～20 minutes then;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is mixed with the coating buffer B of 0.5mg/mL; Position in distance detection line 5～10mm, with a spray mode coating buffer B is laterally wrapped by on nitrocellulose filter, obtain nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 200～500ng on every centimetre of nature controlling line, under 37～40 ℃ of conditions dry 8～20 minutes then;
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid A soak, take out, drying is 10～16 hours under 37～40 ℃ of conditions, gets sample pad, puts room temperature preservation in the exsiccator then.
(4) preparation of gold mark pad
Glass fibre membrane is put into confining liquid B to soak, take out, drying is 10～16 hours under 37～40 ℃ of conditions, on dry glass fibre membrane, laterally spray with the aspergillus flavus resisting toxin general purpose single clonal antibody solution of a spray mode nano gold mark, the aspergillus flavus resisting toxin general purpose single clonal antibody of every centimetre of required nano gold mark of spraying length is 48～144ng, and vacuum freeze drying 2～6h puts room temperature preservation in the exsiccator then;
Described aspergillus flavus resisting toxin general purpose single clonal antibody is the hybridoma cell strain 1C11 generation of CCTCC NO.C201013 by preserving number;
(5) assembling of test strips
Paste adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively in the one side of cardboard, adjacent each pad overlaps in the junction and connects, and overlapping length is 1～3mm, promptly gets the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content.
Press such scheme, described coating buffer A obtains according to following method preparation: with the commercially available aflatoxin B1 of 10～50mg-bovine serum albumin(BSA) conjugate (AFB1-BSA), 1～2g bovine serum albumin(BSA), 1～2g sucrose, 0.02～0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
Described coating buffer B obtains according to following method preparation: with the anti-mouse polyclonal antibody of 50mg rabbit, 0.02～0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained.
Press such scheme, described confining liquid A obtains according to following method configuration: with 1～2g bovine serum albumin(BSA), 2～5g sucrose, 0.02～0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
Described confining liquid B obtains according to following method preparation: with 1～2g bovine serum albumin(BSA), 0.1～0.2mL triton x-100,0.3～05g polyvinylpyrrolidone, 2～5g sucrose, 0.02～0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained.
Press such scheme, the aspergillus flavus resisting toxin general purpose single clonal antibody solution of described nano gold mark is to adopt unsaturated labelling method preparation, and its concrete grammar is: get the commercially available mass concentration of 50.0mL and be 0.01% nano-Au solution, regulator solution pH value is 5.5; The aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL 0.1mg/mL under the state that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 50.0mL mark and preserves liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant will precipitate that to preserve liquid with mark washing resuspended, obtain the 5.0mL concentrate, it is standby to put 4 ℃ of refrigerators, and wherein the mass concentration of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of nano gold mark is 0.04mg/mL;
Described mark washing is preserved liquid and is obtained according to following method preparation: 2.0g polyglycol-20000, and the 0.2g Sodium azide, 0.1235 gram boric acid, pure water is settled to 1000mL, 0.22 μ m membrane filtration gained.
The application of the high-sensitivity immunochromatographitest test strip of aforesaid fast detecting total aflatoxin content, method is as follows: take by weighing levigate testing sample, the adding volumetric concentration is 60～80% methanol aqueous solution, the mass volume ratio of testing sample and methanol aqueous solution is 3g/mL, mixing, ultrasonic Extraction is 5～10 minutes under 50～60 ℃ of water-baths, left standstill 5～10 minutes, with supernatant liquor is 2.5 times of extract dilute with waters, the final concentration that makes methyl alcohol in the dilution is 24～32%, obtain sample solution, get 100 μ L again and dilute good sample solution as detecting the sample pad that liquid dropwise adds the high-sensitivity immunochromatographitest test strip of a fast detecting total aflatoxin content, it is as test strip, get 100 μ L water simultaneously as negative controls, dropwise add the sample pad of the high-sensitivity immunochromatographitest test strip of another fast detecting total aflatoxin content, it is test strips in contrast, reads the result after 15 minutes;
Testing result: (1) positive: when the nature controlling line of test strip demonstrates red lines, and detection line is not when developing the color, perhaps nature controlling line demonstrates red lines, and detection line color comparison is according to the test strips detection line when of light color, be judged to the positive, show that total aflatoxin content in the testing sample is that the total content of aflatoxin B1, B2, G1 and G2 is greater than or equal to 0.5ng/g.(2) feminine gender: when the nature controlling line of test strip demonstrates red lines, and detection line color and control stripes bar detection line color near the time, be judged to negative findings, show that total aflatoxin content in the testing sample is that the total content of aflatoxin B1, B2, G1 and G2 is lower than 0.5ng/g; (3) invalid: when nature controlling line did not develop the color, no matter the detection line of test strip showed or does not show red lines, and it is invalid that this test strips is judged to.
The principle of work of the high-sensitivity immunochromatographitest test strip of this fast detecting total aflatoxin content: when testing sample solution joins on the sample pad of test strips lower end, testing sample solution moves to the adsorptive pads direction along test strips by capillary action, when it moved to gold mark pad, the aspergillus flavus resisting toxin general purpose single clonal antibody of nano gold mark was dissolved.In sample, contain aflatoxin B1, B2, G1, during among the G2 one or more, the aspergillus flavus resisting toxin general purpose single clonal antibody of the nano gold mark that aflatoxin will fill up with the gold mark is in conjunction with also together upwards swimming, when the detection of antigens line is being fixed in its arrival, antigen will be competed limited antigen binding site on the aspergillus flavus resisting toxin general purpose single clonal antibody of combining nano gold mark with aflatoxin, aflatoxin content is high more in the sample, antigen on the detection line can in conjunction with the aspergillus flavus resisting toxin general purpose single clonal antibody of nano gold mark will be few more, the colour developing band color of formation is shallow more.When the nano gold mark of antigen institute combination be less than certain quantity the time, the detection line place will not have red lines and occur.No matter whether contain aflatoxin in the sample, aspergillus flavus resisting toxin general purpose single clonal antibody nano gold mark or nano gold mark that antigen on the not tested survey line is intercepted and captured and the bond of aflatoxin will continue to move to nature controlling line and combine with second antibody on the nature controlling line and developed the color by enrichment, so do not contain aflatoxin in the sample, be two red stripes when being negative, promptly nature controlling line and detection line are redness; Contain a certain amount of aflatoxin, two kinds of situations are arranged when promptly positive: a red nature controlling line 1, only occurs, detection line does not develop the color; 2, a red nature controlling line and a light red detection line; Nature controlling line does not have colour band to occur then showing that test strips lost efficacy.
Beneficial effect of the present invention is:
(1) detects total aflatoxin content.The antibody that the immuno-chromatographic test paper strip of fast detecting total aflatoxin content provided by the invention uses is aspergillus flavus resisting toxin general purpose single clonal antibody, therefore is used to detect total aflatoxin content, and actual application value is big.
(2) sample-pretreating method is simple.Sample pre-treatments only need add extract in the sample ultrasonic Extraction 5～10 minutes, left standstill then 5～10 minutes, got the supernatant dilute with water and can detect for 2.5 times, and whole sample pre-treatment process is simple, fast.
(3) simple to operate.Only need when detecting sample extracting solution dropwise is added on the sample pad of test strips and get final product, operate, do not need the professional for single step with the immuno-chromatographic test paper strip of this fast detecting total aflatoxin content, simple, convenient.
(4) testing process does not need the aflatoxin standard solution as positive control.Do not need to use the aflatoxin standard solution as positive control during the immuno-chromatographic test paper strip test sample of fast detecting total aflatoxin content provided by the invention, and only need water to get final product, thereby avoided the secondary pollution of aflatoxin as negative control.
(5) highly sensitive.The immuno-chromatographic test paper strip of fast detecting total aflatoxin content provided by the invention is limited to 0.5ng/g to the lowest detection of total aflatoxin content in the sample, and this detectability is less than minimum the limit the quantity of required value of European Union to total aflatoxin content in the food.
Description of drawings
Fig. 1 is the front elevation of the immuno-chromatographic test paper strip of fast detecting total aflatoxin content of the present invention.Among the figure: 1 cardboard, 2 adsorptive pads; 3 detecting pads; 4 gold medals mark pad; 5 sample pad; 6 nature controlling lines; 7 detection lines.
Fig. 2 is the side-looking structural representation of the immuno-chromatographic test paper strip of fast detecting total aflatoxin content of the present invention.Among the figure: 1 cardboard; 2 adsorptive pads; 3 detecting pads; 4 gold medals mark pad; 5 sample pad.
Fig. 3 be embodiment 2 process decision chart as a result.Among the figure: 1 control stripes bar; 2 test strip; 3 nature controlling lines; 4 detection lines.
Fig. 4 be embodiment 3 process decision chart as a result.Among the figure: 1 control stripes bar; 2 test strip; 3 nature controlling lines; 4 detection lines.
Fig. 5 be embodiment 4 process decision chart as a result.Among the figure: 1 control stripes bar; 2 test strip; 3 nature controlling lines; 4 detection lines.
Embodiment
Embodiment 1: the preparation method of aspergillus flavus resisting toxin general purpose single clonal antibody
Aspergillus flavus resisting toxin general purpose single clonal antibody is the hybridoma cell strain 1C11 generation of CCTCC NO.C201013 by preserving number, and its preparation method may further comprise the steps:
1. animal immune
6 of purchase BALB/c mouse in 6 age in week, the aflatoxin complete A antigen FB1-BSA that immunity is commercially available.Immunity is with after aflatoxin comlete antigen and the emulsification of equivalent Fu Shi Freund's complete adjuvant, in the subcutaneous multi-point injection in mouse carotid back for the first time.Carry out after for the second time being immune to for 4 weeks, adopt freund 's incomplete adjuvant and the emulsification of equivalent aflatoxin comlete antigen, inject in mouse peritoneal.Immunity for the third time and immunity for the second time be 4 weeks at interval, and immunization ways is identical with it, carry out after being immune to immune for the third time 3 weeks the 4th time, and immunization ways is immune identical with the second time, is similarly lumbar injection.4 times immunizing dose is identical, is every mouse 40 μ g/mL.Back 8 days of 3 times each immunity, tail vein blood, separation of serum adopts indirect elisa method monitoring mice serum to tire.Back 8 days of the 4th immunity, tail vein blood, separation of serum, adopt indirect elisa method monitoring mice serum to tire, and measure mice serum sensitivity with the indirect competitive ELISA method, the mouse of the serum correspondence that selection is tired, sensitivity is all higher relatively carries out last booster immunization, and immunizing dose is before 2 times.
Aflatoxin complete A antigen FB1-BSA purchases the company in Sigma-Aldrich.
2. Fusion of Cells
In last booster immunization after 3 days, adopting 50% polyglycol is that PEG (molecular weight is 1450) makes fusion agent, carry out Fusion of Cells according to a conventional method, concrete steps: kill immune mouse under the aseptic condition, separating Morr. cell, with mouse source myeloma cell SP2/0 with 5: 1 number than mixing, wash cell mixing with the RPMI-1640 basic culture solution, merge with 50%PEG, merge and filled it up with the RPMI-1640 basic culture solution in back 1 minute, centrifugal, remove supernatant, the fused cell that mouse boosting cell and mouse source myeloma cell SP2/0 form is resuspended with the 72mLRPMI-1640 basic culture solution, resuspended cell is added drop-wise in the 96 porocyte culture plates, 2/hole, puts 37 ℃ of CO2gas incubator and cultivate, described RPMI-1640 basic culture solution is for containing 20% hyclone, and 2% growth factor and 1% hypoxanthine-aminopterin-thymidine is HAT.
Above-mentioned SP2/0 purchases in last ingression Ke bio tech ltd; The RPMI-1640 basic culture solution is purchased the company in Hyclone; 1% hypoxanthine-aminopterin-thymidine is that HAT purchases the company in Sigma-Aldrich.
3. the screening of cell line and clone
Treated after the Fusion of Cells that cell colony was long to accounting for 1/2 size at the bottom of the hole about the 12nd day, the nutrient solution flavescence can be carried out antibody test.Adopt the ELISA method that the culture hole that the hybridoma growth is arranged is screened, screening is carried out in two steps, and the first step adopts indirect elisa method to filter out the aspergillus flavus resisting toxin and the positive hole of not anti-carrier protein BSA; Second step adopted the indirect competitive ELISA method that the positive hole that the first step filters out is detected, use aflatoxin B1, B2, G1 and G2 totally 4 kinds of conduct competitions are former, (the higher finger competition of light absorption value was that 0 hole is that the final measured value in positive control hole is higher originally, and the higher finger inhibiting rate of sensitivity is 50% o'clock competition original content that is IC to select all higher hole of light absorption value and sensitivity
50Be worth less), adopt limiting dilution assay to clone, clone and adopted same two-step approach to detect in back about 10 days, so behind repeated cloning 2-3 time, acquisition hybridoma cell strain 1C11.
4. the general body MONOCLONAL ANTIBODIES SPECIFIC FOR of aspergillus flavus resisting toxin, purifying
The BALB/c mouse that the 1C11 injection was handled with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt sad-ammonium sulfate method antibody purification, concrete operations are: with double-deck filter paper filtering mouse ascites, 4 ℃, the centrifugal 15min of 12000r/min, draw supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, stir and slowly add caprylic acid down, every milliliter of required caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min, 4 ℃ leave standstill 2h, 4 ℃ then, the centrifugal 30min of 12000r/min, abandon precipitation, with the supernatant that obtains with double-deck filter paper filtering after, the volumetric molar concentration that adds 1/10 filtrate volume is that 0.1mol/L and pH value are 7.4 phosphate buffer, with the pH value to 7.4 that the sodium hydroxide solution of 2mol/L is regulated this mixed liquor, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL, 4 ℃ leave standstill 2h, 4 ℃ then, the centrifugal 30min of 12000r/min abandons supernatant, the gained precipitation is resuspended with the 0.01mol/L phosphate buffer of former ascites volume 1/10, the bag filter of packing into, to the pure water dialysis, it is freezing that the protein solution that fully dialysis is good is put-70 ℃ of refrigerators, use the freeze drier freeze-drying afterwards, collect freeze-dried powder, promptly get the good aspergillus flavus resisting toxin general purpose single clonal antibody of purifying, antibody is put in-20 ℃ of refrigerators standby;
Described acetate buffer is the 0.29g sodium acetate, and 0.141mL acetic acid adds water and is settled to the 100mL gained; The phosphate buffer of described 0.1mol/L is a 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, and 0.02g potassium chloride, potassium dihydrogen phosphate 0.02g add water and are settled to the 100mL gained.
5. the hypotype and the CHARACTERISTICS IDENTIFICATION of aspergillus flavus resisting toxin general purpose single clonal antibody
The hypotype of identifying the aspergillus flavus resisting toxin general purpose single clonal antibody of hybridoma cell strain 1C11 secretion with commercially available hypotype identification kit is IgG2a.Identify it to aflatoxin B1 with the indirect competitive ELISA method, B2, the sensitivity of G1 and G2 is respectively 1.2,1.3,2.2 and 18.0pg/mL, cross reacting rate be respectively 100.0%, 94.3%, 54.5% and 6.7%.
Embodiment 2-4: the preparation of the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content and application
The aspergillus flavus resisting toxin general purpose single clonal antibody that the aspergillus flavus resisting toxin general purpose single clonal antibody that following embodiment 2-4 uses prepares as embodiment 1.
The preparation method of the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content may further comprise the steps:
(1) preparation of adsorptive pads
Specification with thieving paper is cut out the wide 3mm of growth 16mm promptly gets adsorptive pads;
(the preparation of 2 detecting pads
The bag quilt of detection line:
The conjugate AFB1-BSA of aflatoxin B1-bovine serum albumin(BSA) is mixed with the coating buffer A of 0.1mg/mL; Position in distance nitrocellulose filter upper edge 15mm, with a spray mode coating buffer A is laterally wrapped by on nitrocellulose filter, obtain detection line, the package amount of the conjugate of every centimetre of required aflatoxin B1-bovine serum albumin(BSA) of detection line is 80ng, under 37 ℃ of conditions dry 15 minutes then;
Described coating buffer A is: the commercially available aflatoxin B1 of 10mg-bovine serum albumin(BSA) conjugate AFB1-BSA, the 1g bovine serum albumin(BSA), 1g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained.
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is made into the coating buffer B of 0.5mg/mL; In the position of distance detection line 5mm, with a some spray mode coating buffer B is laterally wrapped by on nitrocellulose filter, nature controlling line, the package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 200ng, under 37 ℃ of conditions dry 15 minutes then;
Described coating buffer B is with the anti-mouse polyclonal antibody of 50mg rabbit, the 0.02g sodium azide, and 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained.
The long 25mm of described nitrocellulose filter, wide 3mm.
(3) preparation of sample pad:
With the specification that glass fibre membrane is cut out the wide 3mm of growth 13mm, put into confining liquid A and soak, take out, drying is 16 hours under 37 ℃ of conditions, gets sample pad, puts room temperature preservation in the exsiccator then;
Described confining liquid A is the 1g bovine serum albumin(BSA), 2g sucrose, and the 0.02g sodium azide, 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained.
(4) preparation of gold mark pad:
Glass fibre membrane is cut out the specification of the wide 3mm of growth 9mm, putting into confining liquid B soaks, take out, drying is 16 hours under 37 ℃ of conditions, on the good glass fibre membrane of drying, laterally spray with the aspergillus flavus resisting toxin total amount general purpose single clonal antibody solution of some spray mode nano gold mark, the aspergillus flavus resisting toxin general purpose single clonal antibody of every centimetre of required nano gold mark of spraying length is 144ng, vacuum freeze drying 6h puts room temperature preservation in the exsiccator then;
Described confining liquid B is the 1g bovine serum albumin(BSA), 0.1mL triton x-100,0.3g polyvinylpyrrolidone, 2g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
The aspergillus flavus resisting toxin general purpose single clonal antibody solution of described nano gold mark is to adopt unsaturated labelling method preparation, its concrete grammar is: measuring the 50.0mL mass concentration and be 0.01% nano-Au solution, is 5.5 with 0.1mol/L wet chemical regulator solution pH value; The aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL 0.1mg/mL under the state that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 50.0mL mark and preserves liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant will precipitate that to preserve liquid with mark washing resuspended, obtain the 5.0mL concentrate, it is standby to put 4 ℃ of refrigerators, and wherein the mass concentration of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of nano gold mark is 0.04mg/mL;
The particle diameter of nm of gold is 15nm in the described nano-Au solution;
Described 0.1mol/L wet chemical is: 13.8g sal tartari is dissolved in pure water and is settled to 1000mL, 0.22 μ m membrane filtration gained; Described 0.1mg/mL aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution is that 1mg aspergillus flavus resisting toxin general purpose single clonal antibody is dissolved in the 10mL pure water and makes; Described 10% Bovine Serum Albumin in Aqueous Solution is dissolved in the 100mL pure water for the 10g bovine serum albumin(BSA), 0.22 μ m membrane filtration gained; Described mark washing is preserved liquid and is: 2.0g polyglycol-20000, and the 0.2g Sodium azide, 0.1235 gram boric acid, pure water is settled to 1000mL, 0.22 μ m membrane filtration gained;
(5) assembling of test strips: paste adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively in the one side of cardboard, adjacent each pad overlaps in the junction and connects, overlapping length is 1mm, promptly gets the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content, sees Fig. 1 and Fig. 2.
The application of the high-sensitivity immunochromatographitest test strip of above-mentioned fast detecting total aflatoxin content:
Take by weighing levigate 1# and 2# testing sample, the adding volumetric concentration is 80% methanol aqueous solution, the mass volume ratio of testing sample and methanol aqueous solution is 3g/mL, mixing, ultrasonic Extraction is 8 minutes under 50 ℃ of water-baths, left standstill 10 minutes, with supernatant liquor is 2.5 times of extract dilute with waters, the final concentration that makes methyl alcohol in the dilution is 32%, get sample solution, get 100 μ L again and dilute good sample solution as detecting the sample pad that liquid dropwise adds the high-sensitivity immunochromatographitest test strip (making test strip) of a fast detecting total aflatoxin content, get 100 μ L water simultaneously as negative controls, dropwise add the sample pad of the high-sensitivity immunochromatographitest test strip (comparing test strips) of another fast detecting total aflatoxin content, read the result after 15 minutes.
Testing result: the nature controlling line of 1# test strip demonstrates red lines, and detection line does not develop the color, then be judged to positive findings, show that total aflatoxin content in the testing sample is that the total content of aflatoxin B1, B2, G1 and G2 is higher than 0.5ng/g, sees Fig. 3-1; The nature controlling line of 2# test strip demonstrates red lines, and the comparison of detection line color is of light color according to the test strips detection line, then be judged to positive findings, show that total aflatoxin content in the testing sample is that the total content of aflatoxin B1, B2, G1 and G2 is greater than or equal to 0.5ng/g, sees Fig. 3-2.
The preparation method of the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content may further comprise the steps:
(1) preparation of adsorptive pads
Specification with thieving paper is cut out the wide 4mm of growth 18mm promptly gets adsorptive pads;
(2) preparation of detecting pad
The bag quilt of detection line:
The conjugate AFB1-BSA of aflatoxin B1-bovine serum albumin(BSA) is mixed with the coating buffer A of 0.25mg/mL; Position in distance nitrocellulose filter upper edge 18mm, with a spray mode coating buffer A is laterally wrapped by on nitrocellulose filter, obtain detection line, the package amount of the conjugate of aflatoxin B1-bovine serum albumin(BSA) that every centimetre of detection line is required is 200ng, under 38 ℃ of conditions dry 10 minutes then;
Described coating buffer A is: the commercially available aflatoxin B1 of 25mg-bovine serum albumin(BSA) conjugate AFB1-BSA, 1.5g bovine serum albumin(BSA), 1.5g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is made into the coating buffer B of 0.5mg/mL; In the position of distance detection line 8mm, with a some spray mode coating buffer B is laterally wrapped by on nitrocellulose filter, obtain nature controlling line, the package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 350ng, under 38 ℃ of conditions dry 10 minutes then;
Described coating buffer B is with the anti-mouse polyclonal antibody of 50mg rabbit, the 0.02g sodium azide, and 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
The long 28mm of described nitrocellulose filter, wide 4mm.
(3) preparation of sample pad:
With the specification that glass fibre membrane is cut out the wide 4mm of growth 15mm, put into confining liquid A and soak, take out, drying is 12 hours under 38 ℃ of conditions, gets sample pad, puts room temperature preservation in the exsiccator then;
Described confining liquid A is the 1.5g bovine serum albumin(BSA), 2.5g sucrose, and the 0.02g sodium azide, 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
(4) preparation of gold mark pad: the specification of glass fibre membrane being cut out the wide 4mm of growth 8mm, putting into confining liquid B soaks, take out, drying is 12 hours under 38 ℃ of conditions, on dry glass fibre membrane, laterally spray with the aspergillus flavus resisting toxin general purpose single clonal antibody solution of some spray mode nano gold mark, the aspergillus flavus resisting toxin general purpose single clonal antibody of every centimetre of required nano gold mark of spraying length is 96ng, vacuum freeze drying 4h puts room temperature preservation in the exsiccator then;
Described confining liquid B is the 1.5g bovine serum albumin(BSA), 0.1mL triton x-100,0.4g polyvinylpyrrolidone, 2.5g sucrose, 0.02g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
The aspergillus flavus resisting toxin general purpose single clonal antibody solution of described nano gold mark is to adopt unsaturated labelling method preparation, its concrete grammar is: measuring the 50.0mL mass concentration and be 0.01% nano-Au solution, is 5.5 with 0.1mol/L wet chemical regulator solution pH value; The aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL 0.1mg/mL under the state that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 50.0mL mark and preserves liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant will precipitate that to preserve liquid with mark washing resuspended, obtain the 5.0mL concentrate, it is standby to put 4 ℃ of refrigerators, and wherein the mass concentration of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of nano gold mark is 0.04mg/mL;
The particle diameter of nm of gold is 17nm in the described nano-Au solution;
Described 0.1mol/L solution of potassium carbonate is: 13.8g sal tartari is dissolved in pure water and is settled to 1000mL, 0.22 μ m membrane filtration gained; Described 0.1mg/mL aspergillus flavus resisting toxin general purpose single clonal antibody solution is that 1mg aspergillus flavus resisting toxin general purpose single clonal antibody is dissolved in the 10mL pure water and makes; Described 10% bovine serum albumin solution is dissolved in the 100mL pure water for the 10g bovine serum albumin(BSA), 0.22 μ m membrane filtration gained; Described mark washing is preserved liquid and is: 2.0g polyglycol-20000, and the 0.2g Sodium azide, 0.1235 gram boric acid, pure water is settled to 1000mL, 0.22 μ m membrane filtration gained;
(5) assembling of test strips: paste adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively in the one side of cardboard, adjacent each pad overlaps in the junction and connects, and overlapping length is 2mm, promptly get the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content, see Fig. 1 and Fig. 2.
The application of the immuno-chromatographic test paper strip of aforesaid fast detecting total aflatoxin content:
Take by weighing levigate testing sample, the adding volumetric concentration is 70% methanol aqueous solution, the mass volume ratio of testing sample and methanol aqueous solution is 3g/mL, mixing, Extraction by Ultrasound is 8 minutes under 55 ℃ of water-baths, left standstill 8 minutes, with supernatant liquor is 2.5 times of extract dilute with waters, the final concentration that makes methyl alcohol in the dilution is 28%, get sample solution, get 100 μ L again and dilute good sample solution as detecting the sample pad that liquid dropwise adds the immuno-chromatographic test paper strip (making test strip) of a fast detecting total aflatoxin content, get 100 μ L water simultaneously as negative controls, dropwise add the immuno-chromatographic test paper strip (comparing test strips) of another fast detecting total aflatoxin content, read the result after 15 minutes.
Testing result: the nature controlling line of test strip demonstrates red lines, and detection line color and control stripes bar detection line color are approaching, be judged to negative findings, show that total aflatoxin content in the testing sample is that the total content of aflatoxin B1, B2, G1 and G2 is lower than 0.5ng/g, sees Fig. 4.
The preparation method of the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content may further comprise the steps:
(1) preparation of adsorptive pads
Specification with thieving paper is cut out the wide 2mm of growth 17mm promptly gets adsorptive pads;
(2) preparation of detecting pad
The bag quilt of detection line:
The conjugate AFB1-BSA of aflatoxin B1-bovine serum albumin(BSA) is mixed with the coating buffer A of 0.5mg/mL; Position in distance nitrocellulose filter upper edge 20mm, with a spray mode coating buffer A is laterally wrapped by on nitrocellulose filter, obtain detection line, the package amount of aflatoxin B1-bovine serum albumin(BSA) conjugate that every centimetre of detection line is required is 400ng, under 39 ℃ of conditions dry 8 minutes then;
Described coating buffer A is: the commercially available aflatoxin B1 of 50mg-bovine serum albumin(BSA) conjugate AFB1-BSA, the 2g bovine serum albumin(BSA), 2g sucrose, 0.04g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is made into the coating buffer B of 0.5mg/mL; In the position of distance detection line 10mm, with a some spray mode coating buffer B is laterally wrapped by on nitrocellulose filter, obtain nature controlling line, the package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 500ng, under 39 ℃ of conditions dry 8 minutes then;
Described coating buffer B is with the anti-mouse polyclonal antibody of 50mg rabbit, the 0.04g sodium azide, and 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
The long 30mm of described nitrocellulose filter, wide 2mm.
(3) preparation of sample pad
With the specification that glass fibre membrane is cut out the wide 2mm of growth 17mm, put into confining liquid A and soak, take out, drying is 10 hours under 39 ℃ of conditions, gets sample pad, puts room temperature preservation in the exsiccator then;
Described confining liquid A is the 2g bovine serum albumin(BSA), 5g sucrose, and the 0.04g sodium azide, 0.8g sodium chloride, the 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained.
(4) preparation of gold mark pad
Glass fibre membrane is cut out the specification of the wide 2mm of growth 6mm, putting into confining liquid B soaks, take out, drying is 10 hours under 39 ℃ of conditions, on dry glass fibre membrane, laterally spray with the aspergillus flavus resisting toxin general purpose single clonal antibody solution of some spray mode nano gold mark, the aspergillus flavus resisting toxin general purpose single clonal antibody of every centimetre of required nano gold mark of spraying length is 72ng, vacuum freeze drying 3h puts room temperature preservation in the exsiccator then;
Described confining liquid B is the 2g bovine serum albumin(BSA), 0.15mL triton x-100,05g polyvinylpyrrolidone, 5g sucrose, 0.04g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
The aspergillus flavus resisting toxin general purpose single clonal antibody solution of described nano gold mark is to adopt unsaturated labelling method preparation, its concrete grammar is: measuring the nano-Au solution that the 50.0mL mass concentration is 0.0.1%, is 5.5 with 0.1mol/L wet chemical regulator solution pH value; The aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL 0.1mg/mL under the state that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 50.0mL mark and preserves liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant will precipitate that to preserve liquid with mark washing resuspended, obtain the 5.0mL concentrate, it is standby to put 4 ℃ of refrigerators, and wherein the mass concentration of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of nano gold mark is 0.04mg/mL;
The particle diameter of nm of gold is 20nm in the described nano-Au solution;
Described 0.1mol/L wet chemical is: 13.8g sal tartari is dissolved in pure water and is settled to 1000mL, 0.22 μ m membrane filtration gained; Described 0.1mg/mL aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution is that 1mg aspergillus flavus resisting toxin general purpose single clonal antibody is dissolved in the 10mL pure water and makes; Described 10% Bovine Serum Albumin in Aqueous Solution is dissolved in the 100mL pure water for the 10g bovine serum albumin(BSA), 0.22 μ m membrane filtration gained; Described mark washing is preserved liquid and is: 2.0g polyglycol-20000, and the 0.2g Sodium azide, 0.1235 gram boric acid, pure water is settled to 1000mL, 0.22 μ m membrane filtration gained;
(5) assembling of test strips: paste adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively in the one side of cardboard, adjacent each pad overlaps in the junction and connects, overlapping length is 3mm, promptly gets the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content, sees Fig. 1 and Fig. 2.
The application of the immuno-chromatographic test paper strip of aforesaid fast detecting total aflatoxin content:
Take by weighing levigate 1# and 2# testing sample, the adding volumetric concentration is 60% methanol aqueous solution, the mass volume ratio of testing sample and methanol aqueous solution is 3g/mL, mixing, Extraction by Ultrasound is 10 minutes under 60 ℃ of water-baths, left standstill 10 minutes, with supernatant liquor is 2.5 times of extract dilute with waters, the final concentration that makes methyl alcohol in the dilution is 24%, obtain sample solution, get 100 μ L again and dilute good sample solution as detecting the sample pad that liquid dropwise adds the immuno-chromatographic test paper strip (making test strip) of a fast detecting total aflatoxin content, get 100 μ L water simultaneously as negative controls, dropwise add the sample pad of the immuno-chromatographic test paper strip (comparing test strips) of another fast detecting total aflatoxin content, read the result after 15 minutes.
Testing result: the nature controlling line of 1# test strip does not develop the color, and the detection line of test strip shows red lines, and it is invalid then to be judged to, and sees Fig. 5-1; The nature controlling line of 2# test strip does not develop the color, and the detection line of test strip does not also show red lines, and it is invalid then to be judged to, and sees Fig. 5-2.
Claims (10)
1. the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content, comprise cardboard, the one side of cardboard is pasted adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively, adjacent each pad overlaps in the junction and connects, described detecting pad is base wad with the nitrocellulose filter, horizontal nature controlling line and detection line are set on the nitrocellulose filter from top to bottom, and described detection line is coated with aflatoxin B1-bovine serum albumin(BSA) conjugate, and nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit; Described gold mark pad transverse jet scribbles the aspergillus flavus resisting toxin general purpose single clonal antibody of nano gold mark, and described aspergillus flavus resisting toxin general purpose single clonal antibody is the hybridoma cell strain 1C11 generation of CCTCC NO.C201013 by preserving number.
2. the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content according to claim 1 and 2 is characterized in that: the long 16～18mm of described adsorptive pads, wide 2～4mm; Long 25～the 30mm of detecting pad, wide 2～4mm; Long 6～the 9mm of gold mark pad, wide 2～4mm; Long 12～the 18mm of sample pad, wide 2～4mm, the overlapping length of adjacent each pad is 1～3mm.
3. the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content according to claim 1 and 2, it is characterized in that: the spacing of detection line on the described detecting pad and nitrocellulose filter upper edge is 15～20mm, and the spacing of nature controlling line and detection line is 5～10mm.
4. the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content according to claim 1 and 2 is characterized in that: the package amount of the required aflatoxin B1-bovine serum albumin(BSA) conjugate of every centimetre of detection line is 80～400ng on the described detecting pad; The package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 200～500ng.
5. the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content according to claim 1 and 2 is characterized in that: the particle diameter of used nm of gold is 15～20nm in the described gold mark pad; The consumption of the aspergillus flavus resisting toxin general purpose single clonal antibody of the nano gold mark that the last every centimetre of spraying length of described gold mark pad is required is 48～144ng.
6. the preparation method of the high-sensitivity immunochromatographitest test strip of claim 1 or 2 described fast detecting total aflatoxin contents, it is characterized in that: it may further comprise the steps:
(1) preparation of adsorptive pads
Thieving paper cut out promptly get adsorptive pads;
(2) preparation of detecting pad
The bag quilt of detection line:
The conjugate of aflatoxin B1-bovine serum albumin(BSA) is mixed with the coating buffer A of 0.1～0.5mg/mL; In distance nitrocellulose filter upper edge is the position of 15～20mm, with a spray mode coating buffer A is laterally wrapped and on nitrocellulose filter, to be obtained detection line, the package amount of the conjugate of required aflatoxin B1-bovine serum albumin(BSA) is 80～400ng on every centimetre of detection line, under 37～40 ℃ of conditions dry 8～20 minutes then;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is mixed with the coating buffer B of 0.5mg/mL; Position in distance detection line 5～10mm, with a spray mode coating buffer B is laterally wrapped by on nitrocellulose filter, obtain nature controlling line, the package amount of the anti-mouse polyclonal antibody of required rabbit is 200～500ng on every centimetre of nature controlling line, under 37～40 ℃ of conditions dry 8～20 minutes then;
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid A soak, take out, drying is 10～16 hours under 37～40 ℃ of conditions, gets sample pad, puts room temperature preservation in the exsiccator then.
(4) preparation of gold mark pad
Glass fibre membrane is put into confining liquid B to soak, take out, drying is 10～16 hours under 37～40 ℃ of conditions, on dry glass fibre membrane, laterally spray with the aspergillus flavus resisting toxin general purpose single clonal antibody solution of a spray mode nano gold mark, the aspergillus flavus resisting toxin general purpose single clonal antibody of every centimetre of required nano gold mark of spraying length is 48～144ng, and vacuum freeze drying 2～6h puts room temperature preservation in the exsiccator then;
Described aspergillus flavus resisting toxin general purpose single clonal antibody is the hybridoma cell strain 1C11 generation of CCTCC NO.C201013 by preserving number;
(5) assembling of test strips
Paste adsorptive pads, detecting pad, gold mark pad and sample pad from top to bottom successively in the one side of cardboard, adjacent each pad overlaps in the junction and connects, and overlapping length is 1～3mm, promptly gets the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content.
7. the preparation method of the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content according to claim 6, it is characterized in that: described coating buffer A obtains according to following method preparation: with the commercially available aflatoxin B1 of 10～50mg-bovine serum albumin(BSA) conjugate, 1～2g bovine serum albumin(BSA), 1～2g sucrose, 0.02～0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
Described coating buffer B obtains according to following method preparation: with the anti-mouse polyclonal antibody of 50mg rabbit, 0.02～0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained.
8. the preparation method of the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content according to claim 6, it is characterized in that: described confining liquid A obtains according to following method configuration: with 1～2g bovine serum albumin(BSA), 2～5g sucrose, 0.02～0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained;
Described confining liquid B obtains according to following method preparation: with 1～2g bovine serum albumin(BSA), 0.1～0.2mL triton x-100,0.3～05g polyvinylpyrrolidone, 2～5g sucrose, 0.02～0.05g sodium azide, 0.8g sodium chloride, 0.29g disodium hydrogen phosphate, 0.02g potassium chloride, the 0.02g potassium dihydrogen phosphate adds water and is settled to the 100mL gained.
9. the preparation method of the high-sensitivity immunochromatographitest test strip of fast detecting total aflatoxin content according to claim 6, it is characterized in that: the aspergillus flavus resisting toxin general purpose single clonal antibody solution of described nano gold mark is to adopt unsaturated labelling method preparation, its concrete grammar is: get the commercially available mass concentration of 50.0mL and be 0.01% nano-Au solution, regulator solution pH value is 5.5; The aspergillus flavus resisting toxin general purpose single clonal antibody aqueous solution that slowly adds 2mL 0.1mg/mL under the state that stirs continues to stir 30min; Adding mass concentration and be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin(BSA) is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons precipitation; With the centrifugal 30min of supernatant 12000r/min, abandoning supernatant adds the washing of 50.0mL mark and preserves liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant will precipitate that to preserve liquid with mark washing resuspended, obtain the 5.0mL concentrate, it is standby to put 4 ℃ of refrigerators, and wherein the mass concentration of the aspergillus flavus resisting toxin general purpose single clonal antibody solution of nano gold mark is 0.04mg/mL;
Described mark washing is preserved liquid and is obtained according to following method preparation: 2.0g polyglycol-20000, and the 0.2g Sodium azide, 0.1235 gram boric acid, pure water is settled to 1000mL, 0.22 μ m membrane filtration gained.
10. the application of the high-sensitivity immunochromatographitest test strip of claim 1 or 2 described fast detecting total aflatoxin contents, it is characterized in that: take by weighing levigate testing sample, the adding volumetric concentration is 60～80% methanol aqueous solution, the mass volume ratio of testing sample and methanol aqueous solution is 3g/mL, mixing, ultrasonic Extraction is 5～10 minutes under 50～60 ℃ of water-baths, left standstill 5～10 minutes, with supernatant liquor is 2.5 times of extract dilute with waters, the final concentration that makes methyl alcohol in the dilution is 24～32%, obtain sample solution, get 100 μ L again and dilute good sample solution as detecting the sample pad that liquid dropwise adds the high-sensitivity immunochromatographitest test strip of a fast detecting total aflatoxin content, it is as test strip, get 100 μ L water simultaneously as negative controls, dropwise add the sample pad of the high-sensitivity immunochromatographitest test strip of another fast detecting total aflatoxin content, it is test strips in contrast, reads the result after 15 minutes;
Testing result: (1) positive: when the nature controlling line of test strip demonstrates red lines, and detection line is not when developing the color, perhaps nature controlling line demonstrates red lines, and detection line color comparison is according to the test strips detection line when of light color, be judged to the positive, show that total aflatoxin content in the testing sample is that the total content of aflatoxin B1, B2, G1 and G2 is greater than or equal to 0.5ng/g.(2) feminine gender: when the nature controlling line of test strip demonstrates red lines, and detection line color and control stripes bar detection line color near the time, be judged to negative findings, show that total aflatoxin content in the testing sample is that the total content of aflatoxin B1, B2, G1 and G2 is lower than 0.5ng/g; (3) invalid: when nature controlling line did not develop the color, no matter the detection line of test strip showed or does not show red lines, and it is invalid that this test strips is judged to.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102458016A CN101930006B (en) | 2010-08-05 | 2010-08-05 | High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102458016A CN101930006B (en) | 2010-08-05 | 2010-08-05 | High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101930006A true CN101930006A (en) | 2010-12-29 |
| CN101930006B CN101930006B (en) | 2012-08-22 |
Family
ID=43369300
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102458016A Active CN101930006B (en) | 2010-08-05 | 2010-08-05 | High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101930006B (en) |
Cited By (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102207504A (en) * | 2011-03-23 | 2011-10-05 | 北京华创远航科技有限公司 | Enzyme-linked immunosorbent assay kit, and preparation method thereof |
| US20120034711A1 (en) * | 2010-08-05 | 2012-02-09 | Oil Crops Research Institute, Chinese Academy of Agricultural Science | Digital immunochromatographic test strip for semi-quantitative detection of aflatoxin B1 and preparation method thereof |
| CN102455325A (en) * | 2011-03-23 | 2012-05-16 | 杨晓波 | High flux multipurpose liquid sample rapid ultrasonic analyzer |
| CN103278631A (en) * | 2013-04-03 | 2013-09-04 | 中国农业科学院油料作物研究所 | Aflatoxin B1 flow lag immunization time distinguishing fluorescence rapid-detection kit and application thereof |
| CN103278630A (en) * | 2013-04-03 | 2013-09-04 | 中国农业科学院油料作物研究所 | Immunity chromatography test strip for synchronously detecting aflatoxin and ochratoxin A mixed pollution, and preparation method and application thereof |
| CN103575887A (en) * | 2012-08-01 | 2014-02-12 | 北京勤邦生物技术有限公司 | Test paper card for detecting aflatoxin B1 and application of test paper card |
| CN105242047A (en) * | 2015-10-31 | 2016-01-13 | 中国农业科学院油料作物研究所 | Aflatoxin B1 graphene oxide immunochromatographic test strip and application thereof |
| CN105548553A (en) * | 2016-02-04 | 2016-05-04 | 中国农业科学院油料作物研究所 | Colloidal gold immunochromatography test strip for rapidly detecting capsaicinoids as well as preparation method and application thereof |
| CN106680481A (en) * | 2016-11-23 | 2017-05-17 | 百奥森（江苏）食品安全科技有限公司 | Detecting card for detecting aflatoxin in corns |
| CN107101991A (en) * | 2017-05-31 | 2017-08-29 | 东南大学 | A kind of high sensitivity multiplex detection chromatograph test strip |
| CN110108874A (en) * | 2019-04-30 | 2019-08-09 | 中国农业科学院油料作物研究所 | Quickly colloid gold immune analysis test paper item, preparation and its application of detection cyclopiazonic acid |
| CN111500546A (en) * | 2020-05-07 | 2020-08-07 | 上海中药制药技术有限公司 | Cell strain secreting four subtype antibodies against aflatoxin, antibody secreted by cell strain and immunochromatography detection card |
| WO2021093884A1 (en) * | 2019-11-15 | 2021-05-20 | 中国农业科学院油料作物研究所 | Immunochromatographic test strip for detecting diacetoxyscirpenol pollution, and preparation method therefor and application thereof |
| CN113655219A (en) * | 2021-08-20 | 2021-11-16 | 中国人民解放军军事科学院军事医学研究院 | Combined quantitative detection method for CRP and SAA based on up-conversion luminescence immunochromatography technology |
| CN113804887A (en) * | 2021-08-25 | 2021-12-17 | 中国农业科学院油料作物研究所 | Immunoassay kit with multiple detection scales and application thereof |
| CN113820492A (en) * | 2021-09-29 | 2021-12-21 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Detection preparation for detecting atrazine, kit and detection method thereof |
| CN113848317A (en) * | 2021-08-25 | 2021-12-28 | 福建农林大学 | Test strip for semi-quantitatively detecting aflatoxin based on gold nanoflower technology |
| US20220412966A1 (en) * | 2019-11-15 | 2022-12-29 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Time-resolved fluorescence kit for synchronously detecting 4,15-diacetoxyscirpenol, aflatoxin b1, and sterigmatocystin |
| US11971403B2 (en) * | 2019-11-15 | 2024-04-30 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Time-resolved fluorescence kit for synchronously detecting 4,15-diacetoxyscirpenol, aflatoxin B1, and sterigmatocystin |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410771A (en) * | 2002-01-17 | 2003-04-16 | 大连普瑞康生物技术有限公司 | Aflatoxin fast detecting apparatus and its making method |
| CN1673748A (en) * | 2005-04-12 | 2005-09-28 | 江苏省微生物研究所有限责任公司 | Aftatoxin B1 golden standard detection test paper box and producing process thereof |
| US20080160538A1 (en) * | 2005-02-18 | 2008-07-03 | Saul Steven J | Lateral Flow Test Kit and Method for Detecting an Analyte |
| WO2008141351A1 (en) * | 2007-05-21 | 2008-11-27 | Erber Aktiengesellschaft | Method for quantitatively determining analytes using a test element and test system and use thereof |
-
2010
- 2010-08-05 CN CN2010102458016A patent/CN101930006B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1410771A (en) * | 2002-01-17 | 2003-04-16 | 大连普瑞康生物技术有限公司 | Aflatoxin fast detecting apparatus and its making method |
| US20080160538A1 (en) * | 2005-02-18 | 2008-07-03 | Saul Steven J | Lateral Flow Test Kit and Method for Detecting an Analyte |
| CN1673748A (en) * | 2005-04-12 | 2005-09-28 | 江苏省微生物研究所有限责任公司 | Aftatoxin B1 golden standard detection test paper box and producing process thereof |
| WO2008141351A1 (en) * | 2007-05-21 | 2008-11-27 | Erber Aktiengesellschaft | Method for quantitatively determining analytes using a test element and test system and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| 《粮油食品科技》 20051121 赵晓联等 金标免疫层析法检测黄曲霉毒素B_1的方法 49-51 1-10 第13卷, 第06期 2 * |
| 《食品与生物技术学报》 20061130 孙秀兰等 黄曲霉毒素B_1金标检测体系建立过程中的影响因素 37-41 1-10 第25卷, 第06期 2 * |
Cited By (26)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120034711A1 (en) * | 2010-08-05 | 2012-02-09 | Oil Crops Research Institute, Chinese Academy of Agricultural Science | Digital immunochromatographic test strip for semi-quantitative detection of aflatoxin B1 and preparation method thereof |
| CN102207504A (en) * | 2011-03-23 | 2011-10-05 | 北京华创远航科技有限公司 | Enzyme-linked immunosorbent assay kit, and preparation method thereof |
| CN102455325A (en) * | 2011-03-23 | 2012-05-16 | 杨晓波 | High flux multipurpose liquid sample rapid ultrasonic analyzer |
| CN102455325B (en) * | 2011-03-23 | 2013-04-03 | 杨晓波 | High flux multipurpose liquid sample rapid ultrasonic analyzer |
| CN103575887A (en) * | 2012-08-01 | 2014-02-12 | 北京勤邦生物技术有限公司 | Test paper card for detecting aflatoxin B1 and application of test paper card |
| CN103278631A (en) * | 2013-04-03 | 2013-09-04 | 中国农业科学院油料作物研究所 | Aflatoxin B1 flow lag immunization time distinguishing fluorescence rapid-detection kit and application thereof |
| CN103278630A (en) * | 2013-04-03 | 2013-09-04 | 中国农业科学院油料作物研究所 | Immunity chromatography test strip for synchronously detecting aflatoxin and ochratoxin A mixed pollution, and preparation method and application thereof |
| CN103278630B (en) * | 2013-04-03 | 2014-04-09 | 中国农业科学院油料作物研究所 | Immunity chromatography test strip for synchronously detecting aflatoxin and ochratoxin A mixed pollution, and preparation method and application thereof |
| CN103278631B (en) * | 2013-04-03 | 2014-04-09 | 中国农业科学院油料作物研究所 | Aflatoxin B1 flow lag immunization time distinguishing fluorescence rapid-detection kit and application thereof |
| CN105242047A (en) * | 2015-10-31 | 2016-01-13 | 中国农业科学院油料作物研究所 | Aflatoxin B1 graphene oxide immunochromatographic test strip and application thereof |
| CN105548553A (en) * | 2016-02-04 | 2016-05-04 | 中国农业科学院油料作物研究所 | Colloidal gold immunochromatography test strip for rapidly detecting capsaicinoids as well as preparation method and application thereof |
| CN106680481A (en) * | 2016-11-23 | 2017-05-17 | 百奥森（江苏）食品安全科技有限公司 | Detecting card for detecting aflatoxin in corns |
| CN107101991A (en) * | 2017-05-31 | 2017-08-29 | 东南大学 | A kind of high sensitivity multiplex detection chromatograph test strip |
| CN110108874A (en) * | 2019-04-30 | 2019-08-09 | 中国农业科学院油料作物研究所 | Quickly colloid gold immune analysis test paper item, preparation and its application of detection cyclopiazonic acid |
| US11635429B2 (en) | 2019-11-15 | 2023-04-25 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Immunochromatography test strip for detecting pollution of diacetoxyscirpenol, preparation method therefor, and application thereof |
| WO2021093884A1 (en) * | 2019-11-15 | 2021-05-20 | 中国农业科学院油料作物研究所 | Immunochromatographic test strip for detecting diacetoxyscirpenol pollution, and preparation method therefor and application thereof |
| US11971403B2 (en) * | 2019-11-15 | 2024-04-30 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Time-resolved fluorescence kit for synchronously detecting 4,15-diacetoxyscirpenol, aflatoxin B1, and sterigmatocystin |
| US20220404352A1 (en) * | 2019-11-15 | 2022-12-22 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Immunochromatography test strip for detecting pollution of diacetoxyscirpenol, preparation method therefor, and application thereof |
| US20220412966A1 (en) * | 2019-11-15 | 2022-12-29 | Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences | Time-resolved fluorescence kit for synchronously detecting 4,15-diacetoxyscirpenol, aflatoxin b1, and sterigmatocystin |
| CN111500546A (en) * | 2020-05-07 | 2020-08-07 | 上海中药制药技术有限公司 | Cell strain secreting four subtype antibodies against aflatoxin, antibody secreted by cell strain and immunochromatography detection card |
| CN113655219A (en) * | 2021-08-20 | 2021-11-16 | 中国人民解放军军事科学院军事医学研究院 | Combined quantitative detection method for CRP and SAA based on up-conversion luminescence immunochromatography technology |
| CN113655219B (en) * | 2021-08-20 | 2023-10-13 | 中国人民解放军军事科学院军事医学研究院 | Combined quantitative detection method for CRP and SAA based on upward forwarding optical immunochromatography |
| CN113848317A (en) * | 2021-08-25 | 2021-12-28 | 福建农林大学 | Test strip for semi-quantitatively detecting aflatoxin based on gold nanoflower technology |
| WO2023025074A1 (en) * | 2021-08-25 | 2023-03-02 | 中国农业科学院油料作物研究所 | Immunoassay kit having multiple test scales, and application thereof |
| CN113804887A (en) * | 2021-08-25 | 2021-12-17 | 中国农业科学院油料作物研究所 | Immunoassay kit with multiple detection scales and application thereof |
| CN113820492A (en) * | 2021-09-29 | 2021-12-21 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Detection preparation for detecting atrazine, kit and detection method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101930006B (en) | 2012-08-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101930006B (en) | High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof | |
| CN103217531B (en) | Immunochromatography test strip for synchronously detecting mixed pollution of aflatoxin, ochratoxin A and zearalenone, and preparation method | |
| CN102109518A (en) | Aflatoxin M1 immunochromatographic test strip and preparation method thereof | |
| CN102253209B (en) | Aflatoxin M1 (AFM1) immunity chromatography test paper strip and preparation method thereof | |
| CN101993855B (en) | Hybridoma cell line 1C11 and anti-aflatoxin general monoclonal antibody generated by same as well as applications thereof | |
| CN103257230B (en) | Immunochromatographic test strip for synchronously detecting mixed pollution of aflatoxin and zearalenone, preparation method and application thereof | |
| CN101900728A (en) | Multi-test-line immunochromatographic test strip for semi-quantitatively detecting aflatoxin B1 and preparation method thereof | |
| CN102747043B (en) | Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same | |
| CN103278630B (en) | Immunity chromatography test strip for synchronously detecting aflatoxin and ochratoxin A mixed pollution, and preparation method and application thereof | |
| CN108776219B (en) | Immunochromatographic test strip for rapidly detecting alternaria tenuipili acid | |
| CN103215229B (en) | Hybridoma cell strain AFB3G1, monoclonal antibody thereof and multi-detection line immunochromatography test strip for semi-quantitatively detecting aflatoxin B1 | |
| CN103215230A (en) | Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit | |
| CN101661043A (en) | Method for detecting fumonisin by colloidal gold immunochromatographic test | |
| CN105548553A (en) | Colloidal gold immunochromatography test strip for rapidly detecting capsaicinoids as well as preparation method and application thereof | |
| CN102798720A (en) | Immune chromatography test paper for quantitative determination of clenbuterol based on up-conversion fluorescent nanoparticle label and preparation method thereof | |
| CN103215231A (en) | Hybridoma cell strain 1H2, anti-ochratoxin A monoclonal antibody produced from same and application thereof | |
| CN103333864B (en) | Monoclonal antibody of toxoplasma gondii resistant MIC3 protein and application monoclonal antibody | |
| CN101915841A (en) | Ternary detection test bar of beta-stimulants albuterol, clenobuterol hydrochloride and ractopamine and preparation method thereof | |
| CN102879574A (en) | Bar/pat gene transfer plant and rapid test strip for derivative product of bar/pat transgenic plant | |
| CN101887061A (en) | Colloidal gold immuno-chromatography test paper strip used for detecting escherichia coil F5 pilus and preparation method thereof | |
| CN103255109B (en) | Hybridoma cell strain AFG1G6, its monoclonal antibody and aflatoxin G1 immunochromatographic test strip | |
| CN102876634A (en) | PMSG (pregnant mare serum gonadotropin) double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) kit | |
| CN103849604A (en) | Hybridoma cell strain ST03, anti-aflatoxin biosynthesis precursor ST monoclonal antibody and application thereof | |
| CN103232975A (en) | Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody | |
| CN112067811A (en) | Test strip for detecting alternaria alternata and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |