CN102253209A - Aflatoxin M1 (AFM1) immunity chromatography test paper strip and preparation method thereof - Google Patents
Aflatoxin M1 (AFM1) immunity chromatography test paper strip and preparation method thereof Download PDFInfo
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- MJBWDEQAUQTVKK-IAGOWNOFSA-N aflatoxin M1 Chemical compound C=1([C@]2(O)C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O MJBWDEQAUQTVKK-IAGOWNOFSA-N 0.000 title claims abstract description 109
- 238000012360 testing method Methods 0.000 title claims abstract description 109
- 238000002360 preparation method Methods 0.000 title claims description 42
- 230000036039 immunity Effects 0.000 title abstract description 9
- 238000004587 chromatography analysis Methods 0.000 title abstract 2
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- 239000002108 aflatoxin M1 Substances 0.000 title 2
- 238000001514 detection method Methods 0.000 claims abstract description 76
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 64
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Abstract
The invention belongs to the field of biological detection. An AFM1 immunity chromatography test paper strip is characterized in that: the test paper strip comprises a paperboard, an absorbent pad, a detection pad, a colloidal gold pad and a sample pad are sequentially pasted on one side of the paperboard, and adjacent pads overlappingly connect at a joint; the detection pad treats a cellulose nitrate membrane as a base pad, a quality control wire and a detection wire are arranged on the cellulose nitrate membrane in a top-down manner, the detection wire is coated with an AFM1-bovine serum albumin (AFM1-BSA) conjugate, and the quality control wire is coated with a rabbit-anti-mouse polyclonal antibody; and the colloidal gold pad is transversely sprayed with a nanogold labeled AFM1 monoclonal antibody which is generated by a hybridoma cell strain 2C9 with the accession number of CCTCCNO.C201018. The test paper strip which is used for AFM1 detection has the characteristics of rapid detection, simple operation and high sensitivity.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of aflatoxin M 1 immuno-chromatographic test paper strip 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 aflatoxin B1 is toxicity and the strongest material of carcinogenicity.Aflatoxin M 1 is the hydroxylation metabolism product of aflatoxin B1, also is a kind of strong carcinogen.Take in the feed of AFB1 pollution when mammal after, AFM1 can be secreted in the middle of milk through hydroxylation in vivo.Just contain aflatoxin M 1 in the milk of output behind the feed of milk cow through taking in the aflatoxin B1 pollution.Because aflatoxin M 1 quite stable, pasteurization also can't be killed it, not only will detect the animal feed raw material so detect aflatoxin M 1, and will detect final products.For this reason countries in the world stipulated all that the maximum of aflatoxin M 1 in cow's milk and the goods thereof allows content and with it as compulsory standard, be 0.5 ng/mL as the highest permission content of aflatoxin M 1 in Chinese Government's regulation cow's milk and the goods (sterilized milk, fresh raw milk, full-fat cow milk, evaporated milk, condensed milk, cream) thereof.
Aflatoxin detection technique commonly used in the prior art mainly contains thin layer chromatography (TLC), high performance liquid chromatography (HPLC), immune analysis method.The above two have the sample pre-treatments complex steps, time and effort consuming, instrument costs an arm and a leg, the shortcoming that needs the professional to operate etc., and the immune analysis method has high specificity, sample pre-treatments is simple, cost is low, to advantage such as the contamination hazard of experimenter and environment is little.The most frequently used immune analysis method has enzyme linked immunosorbent assay, nm of gold immunochromatographic method etc.The ELISA kit of at present existing aflatoxin M 1 comes out, and visits the aflatoxin M 1 ELISA detection kit of research and development such as sending out R-Biopharm company and U.S. Abraxis company as Germany.And demonstrate bigger using value and application prospect because of its shorter detection time and simpler sample pre-treatments based on the immunochromatography Fast Detection Technique of nm of gold.But also do not come out in the world at present at aflatoxin M 1 immuno-chromatographic test paper strip.Therefore, research set up a kind of at aflatoxin M 1 immuno-chromatographic test paper strip for the monitoring aflatoxin M 1 the content tool have very important significance and using value.
Summary of the invention
Technical matters to be solved by this invention provides a kind of aflatoxin M 1 immuno-chromatographic test paper strip and preparation method thereof.This test strips is used to detect aflatoxin M 1, has quick, simple to operate, the highly sensitive characteristics that detect.
The present invention solves the problems of the technologies described above the technical scheme that is adopted to be:
Aflatoxin M 1 immuno-chromatographic test paper strip (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, and described detection line is coated with aflatoxin M 1-bovine serum albumin(BSA) conjugate (AFM1-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 M1 monoclonal antibody of nano gold mark, and described aspergillus flavus resisting toxin M1 monoclonal antibody is the hybridoma cell strain 2C9 generation of CCTCC NO. C201018 by preserving number.
Described hybridoma cell strain 2C9 has been preserved in Chinese typical culture collection center (CCTCC) on July 13rd, 2010, the preservation address is, China, and Wuhan, Wuhan University, deposit number is CCTCC NO. C201018.It has in the sequence table aspergillus flavus resisting toxin M1 monoclonal antibody variable region of light chain coding gene sequence shown in the SEQ Gene No.2 in the aspergillus flavus resisting toxin M1 monoclonal antibody variable region of heavy chain coding gene sequence shown in the SEQ Gene No.1 and sequence table.
Aspergillus flavus resisting toxin M1 monoclonal antibody, it is the hybridoma cell strain 2C9 secretion generation of CCTCC NO. C201018 by deposit number.Its variable region of heavy chain has the amino acid sequence shown in the SEQ Protein No.1 in the sequence table; Variable region of light chain has the amino acid sequence shown in the SEQ Protein No.2 in the sequence table.This aspergillus flavus resisting toxin M1 monoclonal antibody can be discerned aflatoxin M 1, to 50% inhibition concentration IC of aflatoxin M 1
50Be 67 pg/mL.
The aspergillus flavus resisting toxin M1 MONOCLONAL ANTIBODIES SPECIFIC FOR method that the present invention adopts, step is as follows:
(1) adopt two step screening method to obtain hybridoma cell strain 2C9: with BALB/c mouse after aflatoxin complete A antigen FM1-BSA immunity 4-6 time, make last booster immunization with 2 times of aflatoxin complete A antigen FM1-BSA to a preceding immunizing dose, carry out Fusion of Cells after 3 days, adopt the ELISA method to screen fused cell in two steps: 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 nutrient solution that the first step filters out is detected, former with aflatoxin M 1 as competition, select light absorption value and all higher hole of sensitivity, adopt limiting dilution assay to clone, clone and adopted same two step screening method to detect in back about 10 days, behind the repeated cloning like this 2-3 time, final screening obtains hybridoma cell strain 2C9;
(2) the hybridoma cell strain 2C9 that obtains is injected the BALB/c mouse of handling with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, purifying promptly gets aspergillus flavus resisting toxin M1 monoclonal antibody.
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 M 1-bovine serum albumin(BSA) conjugate (AFM1-BSA) of every centimetre of detection line is 75～375ng 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 50～500ng.
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 M1 monoclonal antibody of the nano gold mark that the last every centimetre of spraying length of described gold mark pad is required is 200～600ng.
The preparation method of aforesaid aflatoxin M 1 immuno-chromatographic test paper strip 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 (AFM1-BSA) of aflatoxin M 1-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 (AFM1-BSA) of required aflatoxin M 1-bovine serum albumin(BSA) is 75～375ng on every centimetre of detection line, under 37～40 ℃ of conditions dry 5～20 minutes then;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is mixed with the coating buffer B of 0.1～0.5mg/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 50～500ng on every centimetre of nature controlling line, under 37～40 ℃ of conditions dry 5～20 minutes then;
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid A soak, take out, drying is 4～10 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 4～10 hours under 37～40 ℃ of conditions, on dry glass fibre membrane, laterally spray with the aspergillus flavus resisting toxin M1 monoclonal anti liquid solution of a spray mode nano gold mark, the aspergillus flavus resisting toxin M1 monoclonal antibody of every centimetre of required nano gold mark of spraying length is 200～600ng, and vacuum freeze drying 2～6h puts room temperature preservation in the exsiccator then;
Described aspergillus flavus resisting toxin M1 monoclonal antibody is the hybridoma cell strain 2C9 generation of CCTCC NO. C201018 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 aflatoxin M 1 immuno-chromatographic test paper strip.
Press such scheme, described coating buffer A obtains according to following method preparation: with the commercially available aflatoxin M 1-of 10～50mg bovine serum albumin(BSA) conjugate (AFM1-BSA), 1～2g bovine serum albumin(BSA), 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 10～50mg 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 labeling method of the aspergillus flavus resisting toxin M1 monoclonal anti liquid solution of described nano gold mark 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 M1 monoclonal antibody aqueous solution that slowly adds 2.5mL 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 centrifugal 30 min of supernatant 12000r/min, abandoning supernatant adds the washing of 50.0mL mark and preserves liquid; Again with centrifugal 30 min 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 M1 monoclonal anti liquid solution of nano gold mark is 0.05mg/mL;
Described mark washing is preserved liquid and is obtained according to following method preparation: 2.0g Macrogol 2000 0, 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 aforesaid aflatoxin M 1 immuno-chromatographic test paper strip, method is as follows: get 1mL liquid cow's milk to be measured and goods thereof, add 0.25～0.5mL water, mixing, obtain sample solution, get 100 μ L and diluted good sample solution as detecting the sample pad that liquid dropwise is added drop-wise to an aflatoxin M 1 immuno-chromatographic test paper strip, with it as test strip, get the blank milk sample that 1mL does not contain aflatoxin M 1 simultaneously, add 0.25～0.5mL water, mixing, obtain negative control solution, get 100 μ L negative control solutions, dropwise be added drop-wise to the sample pad of another aflatoxin M 1 immuno-chromatographic test paper strip, with its test strips in contrast, read 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 the content of aflatoxin M 1 in the testing sample is greater than or equal to 0.5ng/mL; (2) feminine gender: when the nature controlling line and the detection line of test strip all demonstrates red lines, and detection line color and control stripes bar detection line color near the time, be judged to negative findings, show that the content of aflatoxin M 1 in the testing sample is lower than 0.5ng/mL; (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 this aflatoxin M 1 immuno-chromatographic test paper strip: 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 M1 monoclonal antibody of nano gold mark was dissolved.When containing aflatoxin M 1 in the sample, the aspergillus flavus resisting toxin M1 monoclonal antibody of the nano gold mark that free aflatoxin M 1 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 with limited antigen binding site on the aspergillus flavus resisting toxin M1 monoclonal antibody of aflatoxin M 1 competition combining nano gold mark, aflatoxin M 1 content is high more in the sample, antigen on the detection line can in conjunction with the aspergillus flavus resisting toxin M1 monoclonal antibody of nano gold mark will be few more, the colour developing band color of formation is shallow more.When the aspergillus flavus resisting toxin M1 monoclonal antibody of the nano gold mark of antigen institute combination is less than certain quantity, the detection line place will not have red lines and occur.No matter whether contain aflatoxin in the sample, the bond of the aspergillus flavus resisting toxin M1 monoclonal antibody of the nano gold mark that antigen on the not tested survey line is intercepted and captured or the aspergillus flavus resisting toxin M1 monoclonal antibody of nano gold mark and aflatoxin M 1 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 M 1 in the sample, be two red stripes when being negative, nature controlling line and detection line are redness; Contain a certain amount of aflatoxin M 1, 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:
(l) detect aflatoxin M 1.The antibody that aflatoxin M 1 immuno-chromatographic test paper strip provided by the invention uses is used for specificity fast detecting aflatoxin M 1 as aspergillus flavus resisting toxin M1 monoclonal antibody specific;
(2) simple to operate.Only need when detecting sample solution dropwise is added on the sample pad of test strips and get final product with this aflatoxin M 1 immuno-chromatographic test paper strip, for single step is operated, simple, convenient;
(3) testing process does not need aflatoxin M 1 standard solution as positive control.Do not need to use aflatoxin M 1 standard solution as positive control during aflatoxin M 1 immuno-chromatographic test paper strip test sample provided by the invention, and only need get final product as negative control with blank sample;
(4) detection sensitivity height.Aflatoxin M 1 immuno-chromatographic test paper strip provided by the invention is limited to 0.5ng/mL to the lowest detection of aflatoxin M in the sample 1.
Description of drawings
Fig. 1 is the front elevation of aflatoxin M 1 immuno-chromatographic test paper strip 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 aflatoxin M 1 immuno-chromatographic test paper strip 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: aspergillus flavus resisting toxin M1 MONOCLONAL ANTIBODIES SPECIFIC FOR method
Aspergillus flavus resisting toxin M1 monoclonal antibody is the hybridoma cell strain 2C9 generation of CCTCC NO. C201018 by preserving number, and its preparation method may further comprise the steps:
1. adopt two step screening method to obtain hybridoma cell strain 2C9
(1) animal immune
6 of purchase BALB/c mouse in 6 age in week, the aflatoxin complete A antigen FM1-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 FM1-BSA purchases the company in Sigma-Aldrich;
(2) Fusion of Cells
In last booster immunization after 3 days, adopt the 50%(percent by weight) polyglycol be that the PEG(molecular weight is 1450) make 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 the number of 5 ﹕ 1 than mixing, wash cell mixing with the RPMI-1640 basic culture solution, merge with 50%PEG, merged 1 minute, fill it up with the RPMI-1640 basic culture solution then, 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, and resuspended cell is added drop-wise in the 96 porocyte culture plates, 2/hole, put 37 ℃ of CO2gas incubator and cultivate, described RPMI-1640 basic culture solution is for containing the 20%(percent by volume) hyclone, the 2%(percent by weight) growth factor and 1%(percent by weight) 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) 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 aspergillus flavus resisting toxin M1 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, former with aflatoxin M 1 as competition, (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 2C9.
Adopt following method to measure hybridoma cell strain 2C9 antibody variable region sequence:
(1) measures the total RNA of extraction: adopt day total RNA extraction reagent box of root company and extract total RNA that can produce hybridoma cell strain 2C9 to specifications;
(2) synthetic cDNA: the total RNA that obtains with step 1 is a template, oligo (dT)
15Be primer, according to SuperScript
TM-2 II reverse transcriptase instructionss carry out reverse transcription, synthetic cDNA first chain; Primer oligo (dT)
15Buy by Invitrogen;
(3) PCR method clone variable region gene:, be light, the heavy chain variable region gene of masterplate amplification antibody with cDNA according to the conservative site design primer of the medium and small mouse antibody genes sequence of GENEBANK.The PCR program is: 94 ℃ of 30s, 55 ℃ of 1min, 72 ℃ of 1min, and 30 circulations of increasing, last 72 ℃ are extended 10min.PCR product process 1%(percent by weight) after agarose gel electrophoresis separates, reclaim dna fragmentation with the kit purifying, be connected among the carrier pMD18-T transformed into escherichia coli DH5 α competent cell, the picking positive colony is delivered to Shanghai Sani's bio tech ltd and is checked order.Wherein the sequence of primer is respectively: the variable region of heavy chain primer is 5
,-AGG TSM ARC TGC AGS AGT CWG G-3
,(22mer) with 5
,-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC-3
,(32mer) wherein S, M, R and W are the merger base, M=A/C, and R=A/G, S=C/G, W=A/T, variable region of light chain primer are 5
,-GAC ATT GAG CTC ACC CAG CTT GGT GCC-3
,(24mer) with 5
,-CCG TTT CAG CTC CAG CTT GGT CCC-3
,(24mer);
The gene order result who obtains: the long 354bp of variable region of heavy chain coding gene sequence, sequence is shown in SEQ ID NO:1, derive the coded variable region of heavy chain of this gene order according to the gene order that is obtained and be made up of 117 amino acid, sequence is shown in SEQ ID NO:3.The long 332bp of variable region of light chain coding gene sequence, sequence is derived the coded variable region of light chain of this gene order according to the gene order that is obtained and is made up of 110 amino acid shown in SEQ ID NO:2, and sequence is shown in SEQ ID NO:4.
2. aspergillus flavus resisting toxin M1 Purification of Monoclonal Antibodies
The hybridoma cell strain 2C9 that step 1 is obtained injects the BALB/c mouse of handling 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 2 mol/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 M1 monoclonal 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.
The hypotype of identifying the aspergillus flavus resisting toxin M1 monoclonal antibody of hybridoma cell strain 2C9 secretion with commercially available hypotype identification kit is IgG2a.The tiring of mouse ascites fluid antibody that records 2C9 with the non-competing Enzyme Linked Immunoadsorbent Assay of routine (ELISA) method can reach 4.26 * 10
6, promptly the mouse ascites fluid antibody dilution 4.26 * 10
6Times the time measured in solution result positive.Identify that with conventional indirect competitive ELISA method its sensitivity to aflatoxin M 1 is 67 pg/mL, with aflatoxin B1, B2, G1, the G2 cross reacting rate is all less than 0.1%.
Embodiment 2-4: the preparation and the application of aflatoxin M 1 immuno-chromatographic test paper strip
The aspergillus flavus resisting toxin M1 monoclonal antibody that the aspergillus flavus resisting toxin M1 monoclonal antibody that following embodiment 2-4 uses prepares as embodiment 1.
The preparation method of aflatoxin M 1 immuno-chromatographic test paper strip 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 AFM1-BSA of aflatoxin M 1-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 the every centimetre of required aflatoxin M 1-of detection line bovine serum albumin(BSA) is 75ng, under 37 ℃ of conditions dry 15 minutes then;
Described coating buffer A is: the commercially available aflatoxin M 1-of 10mg bovine serum albumin(BSA) conjugate AFM1-BSA, 1g bovine serum albumin(BSA), 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 0.2mg/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 100ng, under 37 ℃ of conditions dry 15 minutes then;
Described coating buffer B is with the anti-mouse polyclonal antibody of 20mg 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 6 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, be coated with the aspergillus flavus resisting toxin M1 monoclonal anti liquid solution transverse jet of some spray mode nano gold mark, the aspergillus flavus resisting toxin M1 monoclonal antibody of every centimetre of required nano gold mark of spraying length is 600ng, 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 concrete labeling method of the aspergillus flavus resisting toxin M1 monoclonal anti liquid solution of described nano gold mark is: measuring the 50.0mL mass concentration and be 0.01% nano-Au solution, is 5.5 with 0.1 mol/L wet chemical regulator solution pH value; The aspergillus flavus resisting toxin M1 monoclonal antibody aqueous solution that slowly adds 2.5mL 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 centrifugal 30 min of supernatant 12000r/min, abandoning supernatant adds the washing of 50.0mL mark and preserves liquid; Again with centrifugal 30 min 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 M1 monoclonal anti liquid solution of nano gold mark is 0.05mg/mL;
The particle diameter of nm of gold is 15nm in the described nano-Au solution;
Described 0.1 mol/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 M1 monoclonal antibody aqueous solution is that 1mg aspergillus flavus resisting toxin M1 monoclonal antibody is dissolved in the 10 mL 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, and overlapping length is 1mm, promptly gets aflatoxin M 1 immuno-chromatographic test paper strip, sees Fig. 1 and Fig. 2.
The application of above-mentioned aflatoxin M 1 immuno-chromatographic test paper strip:
Testing result: the nature controlling line of 1# test strip demonstrates red lines, and detection line does not develop the color, and then is judged to positive findings, shows that the content of the aflatoxin M 1 in the testing sample is higher than 0.5ng/mL, 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 is judged to positive findings, shows that the content of the aflatoxin M 1 in the testing sample is greater than or equal to 0.5ng/mL, sees Fig. 3-2.
The preparation method of aflatoxin M 1 immuno-chromatographic test paper strip 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 AFM1-BSA of aflatoxin M 1-bovine serum albumin(BSA) is mixed with the coating buffer A of 0.2mg/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 the aflatoxin M 1-bovine serum albumin(BSA) that every centimetre of detection line is required is 150ng, under 38 ℃ of conditions dry 10 minutes then;
Described coating buffer A is: the commercially available aflatoxin M 1-of 20mg bovine serum albumin(BSA) conjugate AFM1-BSA, 1.5g bovine serum albumin(BSA), 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 0.2mg/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 200ng, under 38 ℃ of conditions dry 10 minutes then;
Described coating buffer B is with the anti-mouse polyclonal antibody of 20mg 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 5 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 5 hours under 38 ℃ of conditions, on dry glass fibre membrane, be coated with the aspergillus flavus resisting toxin M1 monoclonal anti liquid solution transverse jet of some spray mode nano gold mark, the aspergillus flavus resisting toxin M1 monoclonal antibody of every centimetre of required nano gold mark of spraying length is 450ng, 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 concrete labeling method of the aspergillus flavus resisting toxin M1 monoclonal anti liquid solution of described nano gold mark is: measuring the 50.0mL mass concentration and be 0.01% nano-Au solution, is 5.5 with 0.1 mol/L wet chemical regulator solution pH value; The aspergillus flavus resisting toxin M1 monoclonal antibody aqueous solution that slowly adds 2.5mL 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 centrifugal 30 min of supernatant 12000r/min, abandoning supernatant adds the washing of 50.0mL mark and preserves liquid; Again with centrifugal 30 min 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 M1 monoclonal anti liquid solution of nano gold mark is 0.05mg/mL;
The particle diameter of nm of gold is 17nm in the described nano-Au solution;
Described 0.1 mol/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 M1 monoclonal anti liquid solution is that 1mg aspergillus flavus resisting toxin M1 monoclonal antibody is dissolved in the 10 mL 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 gets aflatoxin M 1 immuno-chromatographic test paper strip, sees Fig. 1 and Fig. 2.
The application of aforesaid aflatoxin M 1 immuno-chromatographic test paper strip:
Draw milk sample 1mL to be measured, add 0.3mL water, mixing, obtain sample solution, get 100 μ L and dilute good sample solution as detecting the sample pad that liquid dropwise adds an aflatoxin M 1 immuno-chromatographic test paper strip, with it as test strip, get the blank milk sample of 1mL simultaneously, add 0.3mL water, mixing obtains negative control solution, get 100 μ L negative control solutions, the sample pad that dropwise adds another aflatoxin M 1 immuno-chromatographic test paper strip with its test strips in contrast, reads the result after 15 minutes;
Testing result: the nature controlling line of test strip and detection line all demonstrate red lines, and detection line color and control stripes bar detection line color are approaching, be judged to negative findings, show that the content of the aflatoxin M 1 in the testing sample is lower than 0.5ng/mL, see Fig. 4.
The preparation method of aflatoxin M 1 immuno-chromatographic test paper strip 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 AFM1-BSA of aflatoxin M 1-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 the aflatoxin M 1-bovine serum albumin(BSA) conjugate that every centimetre of detection line is required is 375ng, under 39 ℃ of conditions dry 8 minutes then;
Described coating buffer A is: the commercially available aflatoxin M 1-of 50mg bovine serum albumin(BSA) conjugate AFM1-BSA, 2g bovine serum albumin(BSA), 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, be coated with the aspergillus flavus resisting toxin M1 monoclonal anti liquid solution transverse jet of some spray mode nano gold mark, the aspergillus flavus resisting toxin M1 monoclonal antibody of every centimetre of required nano gold mark of spraying length is 200ng, 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 concrete labeling method of the aspergillus flavus resisting toxin M1 monoclonal anti liquid solution of described nano gold mark is: measuring the nano-Au solution that the 50.0mL mass concentration is 0.0.1%, is 5.5 with 0.1 mol/L wet chemical regulator solution pH value; The aspergillus flavus resisting toxin M1 monoclonal antibody aqueous solution that slowly adds 2.5mL 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 centrifugal 30 min of supernatant 12000r/min, abandoning supernatant adds the washing of 50.0mL mark and preserves liquid; Again with centrifugal 30 min 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 M1 monoclonal anti liquid solution of nano gold mark is 0.05mg/mL;
The particle diameter of nm of gold is 20nm in the described nano-Au solution;
Described 0.1 mol/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 M1 monoclonal antibody aqueous solution is that 1mg aspergillus flavus resisting toxin M1 monoclonal antibody is dissolved in the 10 mL 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, and overlapping length is 3mm, promptly gets aflatoxin M 1 immuno-chromatographic test paper strip, sees Fig. 1 and Fig. 2.
The application of aforesaid aflatoxin M 1 immuno-chromatographic test paper strip:
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. aflatoxin M 1 immuno-chromatographic test paper strip, it is characterized in that: 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 M 1-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 M1 monoclonal antibody of nano gold mark, and described aspergillus flavus resisting toxin M1 monoclonal antibody is the hybridoma cell strain 2C9 generation of CCTCC NO. C201018 by preserving number.
2. aflatoxin M 1 immuno-chromatographic test paper strip according to claim 1 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. aflatoxin M 1 immuno-chromatographic test paper strip according to claim 1 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. aflatoxin M 1 immuno-chromatographic test paper strip according to claim 1 is characterized in that: the package amount of the required aflatoxin M 1-bovine serum albumin(BSA) conjugate of every centimetre of detection line is 75～375ng 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 50～500ng.
5. aflatoxin M 1 immuno-chromatographic test paper strip according to claim 1 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 M1 monoclonal antibody of the nano gold mark that the last every centimetre of spraying length of described gold mark pad is required is 200～600ng.
6. the preparation method of aflatoxin M 1 immuno-chromatographic test paper strip according to claim 1 and 2 is characterized in that: 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 M 1-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 (AFM1-BSA) of required aflatoxin M 1-bovine serum albumin(BSA) is 75～375ng on every centimetre of detection line, under 37～40 ℃ of conditions dry 5～20 minutes then;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is mixed with the coating buffer B of 0.1～0.5mg/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 50～500ng on every centimetre of nature controlling line, under 37～40 ℃ of conditions dry 5～20 minutes then;
(3) preparation of sample pad
Glass fibre membrane is put into confining liquid A soak, take out, drying is 4～10 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 4～10 hours under 37～40 ℃ of conditions, on dry glass fibre membrane, laterally spray with the aspergillus flavus resisting toxin M1 monoclonal anti liquid solution of a spray mode nano gold mark, the aspergillus flavus resisting toxin M1 monoclonal antibody of every centimetre of required nano gold mark of spraying length is 200～600ng, and vacuum freeze drying 2～6h puts room temperature preservation in the exsiccator then;
Described aspergillus flavus resisting toxin M1 monoclonal antibody is the hybridoma cell strain 2C9 generation of CCTCC NO. C201018 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 aflatoxin M 1 immuno-chromatographic test paper strip.
7. the preparation method of aflatoxin M 1 immuno-chromatographic test paper strip according to claim 6, it is characterized in that: described coating buffer A obtains according to following method preparation: with the commercially available aflatoxin M 1-of 10～50mg bovine serum albumin(BSA) conjugate (AFM1-BSA), 1～2g bovine serum albumin(BSA), 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 10～50mg 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 aflatoxin M 1 immuno-chromatographic test paper strip 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 aflatoxin M 1 immuno-chromatographic test paper strip according to claim 6, it is characterized in that: the labeling method of the aspergillus flavus resisting toxin M1 monoclonal anti liquid solution of described nano gold mark 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 M1 monoclonal antibody aqueous solution that slowly adds 2.5mL 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 centrifugal 30 min of supernatant 12000r/min, abandoning supernatant adds the washing of 50.0mL mark and preserves liquid; Again with centrifugal 30 min 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 M1 monoclonal anti liquid solution of nano gold mark is 0.05mg/mL;
Described mark washing is preserved liquid and is obtained according to following method preparation: 2.0g Macrogol 2000 0, 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 claim 1 or 2 described aflatoxin M 1 immuno-chromatographic test paper strips, it is characterized in that: get 1mL liquid cow's milk to be measured and goods thereof, add 0.25～0.5mL water, mixing, obtain sample solution, get 100 μ L and diluted good sample solution as detecting the sample pad that liquid dropwise is added drop-wise to an aflatoxin M 1 immuno-chromatographic test paper strip, with it as test strip, get the blank milk sample that 1mL does not contain aflatoxin M 1 simultaneously, add 0.25～0.5mL water, mixing, obtain negative control solution, get 100 μ L negative control solutions, dropwise be added drop-wise to the sample pad of another aflatoxin M 1 immuno-chromatographic test paper strip, with its test strips in contrast, read 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 the content of aflatoxin M 1 in the testing sample is greater than or equal to 0.5ng/mL; (2) feminine gender: when the nature controlling line and the detection line of test strip all demonstrates red lines, and detection line color and control stripes bar detection line color near the time, be judged to negative findings, show that the content of aflatoxin M 1 in the testing sample is lower than 0.5ng/mL; (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.
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