CN102747043A - Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same - Google Patents
Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same Download PDFInfo
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Abstract
The present invention relates to a hybridoma cell line 3G1 and an anti-alfatoxin B1 monoclonal antibody produced by the hybridoma cell line 3G1. The hybridoma cell line 3G1 (CCTCC NO.C201014) can be used for preparation of a high titer anti-aflatoxin B1 monoclonal antibody, wherein an enzyme-linked immunosorbent assay (ELISA) method is adopted to determine a titer, and the titer is 6.40*10<6>. The anti-aflatoxin B1 monoclonal antibody of the present invention has characteristics of high sensitivity and good specificity, wherein 50% inhibiting concentration on aflatoxin B1 by the monoclonal antibody is 1.6 ng/mL, cross reaction rate with aflatoxin B2 is 6.4%, and cross reaction rates with aflatoxin G1 and G2 are less than 1%. In addition, the anti-aflatoxin B1 monoclonal antibody of the present invention can be used for determination of aflatoxin B1.
Description
Technical field
The present invention relates to the aspergillus flavus resisting toxin B1 monoclonal antibody of hybridoma cell strain 3G1 and generation thereof.
Background technology
Toxins, afla mainly is the secondary metabolite that is produced by flavus and Aspergillus parasiticus secretion, is the natural toxic compounds that can cause the various infringements of people and animals.Kind surplus Toxins, afla has found 20 at present, wherein the toxicity of AFB1 (AFB1) is the strongest, and its toxicity is 10 times of Potssium Cyanide, 68 times of arsenic.A lot of chain initiation human poisonings of AFB1 contaminated food took place in history.After milk cow has been taken in the polluted cereal of AFB1, behind internal metabolism, form another kind of carcinogenic substance aflatoxin M 1 (AFM1), AFM1 gets into then can the serious threat human health in the milk.In addition, AFB1 can also pollute plurality of raw materials, comprises fruit, dry fruit, vegetables, seasonings, oil crops, tobacco and herbal medicine etc., this shows that: AFB1 is extremely extensive to the pollution of food chain, almost can both find to exist in each link of food chain.Remove this, the recall rate of AFB1 is higher in the torrid zone and subtropical zone agricultural-food and food, pollutes more serious; Peanut, corn and peanut wet goods oil and foodstuffs pollute the most serious; Simultaneously, AFB1 exceeds standard and the International Agricultural Trade dispute incident that causes also takes place often, and 1999,2000, the peanut of calendar year 2001 China's outlet European Union are behind bank; Detected total aflatoxin content by European Union more than 40% and the AFB1 component exceeds standard; Returned goods or compelled entrepot trade, cause the agricultural products in China foreign trade to be sustained a great loss, influenced agricultural products in China world market reputation.Therefore, strengthen detection, the particularly speed of AFB1 in agricultural-food such as peanut and the goods and survey, the pollution condition of in time understanding and grasping AFB1 in the agricultural-food is significant to ensureing China's food consumption safety.
Existing Toxins, afla detection method mainly comprises thin layer chromatography, precision instrument analytical method and immune analysis method.Wherein thin layer chromatography is that Toxins, afla detects the most frequently used detection method; This method does not need special plant and instrument; Common laboratory all can be carried out, but have that reagent consumption is big, complex operation, result are subject to disturb, poor accuracy, can not be accurately quantitatively; And bigger etc. not enough to experimenter and surrounding environment pollution hazard, be inappropriate for field quick detection.The precision instrument analytical method comprises spectrophotofluorimetry and HPLC etc., and these a little methods are highly sensitive, and detected result is accurate; But required plant and instrument is expensive; Sample pretreatment process is loaded down with trivial details, requires Toxins, afla sample degree of purification high, and testing process is consuming time; Experimental situation is required height, be difficult to realize rapid detection.In recent years; The immuno analytical method that is used for the Toxins, afla detection grows up gradually; The above two some shortcomings have been overcome; Have high specificity, highly sensitive, sample pre-treatments simple, detect cost low, to advantages such as the pollution hazard of experimenter and surrounding environment are little, be suitable for on-the-spot batch detection etc.Immunoassay is that specific association reaction and the antibody that utilizes antigen and antibody, biology, physics or the chemical amplification of the affinity tag on the antigen come the ultramicron residue is carried out qualitative and quantitative analysis; So study any immunology detection technology of setting up to AFB1, all must obtain the antibody of aspergillus flavus resisting toxin B1 earlier.
Summary of the invention
Problem to be solved by this invention provides the aspergillus flavus resisting toxin B1 monoclonal antibody of hybridoma cell strain 3G1 and generation thereof.
The invention provides hybridoma cell strain 3G1, this cell strain is preserved in Chinese typical culture collection center (CCTCC) on July 13rd, 2010, and the preservation address is; China, Wuhan, Wuhan University; Deposit number is CCTCC NO. C201014, classification called after mouse hybridoma cell 3G1.It has in the sequence table aspergillus flavus resisting toxin B1 monoclonal antibody variable region of light chain coding gene sequence shown in the SEQ ID NO.2 in the aspergillus flavus resisting toxin B1 monoclonal antibody variable region of heavy chain coding gene sequence shown in the SEQ ID NO.1 and sequence table.
Aspergillus flavus resisting toxin B1 monoclonal antibody, it is the hybridoma cell strain 3G1 secretion generation of CCTCC NO. C201014 by deposit number.Its variable region of heavy chain has the aminoacid sequence shown in the SEQ ID NO.3 in the sequence table; Variable region of light chain has the aminoacid sequence shown in the SEQ ID NO.4 in the sequence table.This aspergillus flavus resisting toxin B1 monoclonal antibody can be discerned AFB1, to 50% inhibition concentration IC of AFB1
50Be 1.6ng/mL.
The application of aspergillus flavus resisting toxin B1 monoclonal antibody in AFB1 is measured.
Hybridoma cell strain 3G1 provided by the invention adopts two step screening method to obtain, and its concrete steps are: with AFB1 at H
2SO
4Be converted into its semi-acetal form AFB under the effect
2a, react with the amino aldimine condensation that takes place of BSA then, at NaBH
4C=N is reduced to C-N under the reductive action, generates AFB
2a-BSA mixture complete A antigen FB1-BSA uses its immune BALB/c mouse 4-6 time, and last booster immunization is with 2 times of AFB to a preceding immunizing dose
2a-BSA, immunity was carried out cytogamy after 3 days.Adopt the ELISA method to screen fused cell in two steps: the first step adopts indirect elisa method only to filter out can the aspergillus flavus resisting toxin and the positive colony of not anti-carrier proteins BSA; Second step adopted the indirect competitive ELISA method that the positive colony nutrient solution that the first step filters out is detected; Adopt AFB1 former as competition; The selection light absorption value is low, sensitivity high positive clone carries out limiting dilution assay and continues the clone; Clone and adopted same two step screening method to carry out antibody test in back about 10 days, so behind repeated cloning 2-3 time, final screening acquisition hybridoma cell strain 3G1.
Aspergillus flavus resisting toxin B1 MONOCLONAL ANTIBODIES SPECIFIC FOR method provided by the invention; Step is following: the hybridoma cell strain 1C8 that obtains is injected the BALB/c mouse of handling with freund 's incomplete adjuvant in advance; Collect the ascites of this mouse, obtain aspergillus flavus resisting toxin B1 monoclonal antibody after the purification process.
Press such scheme, described purification process is sad-ammonium sulfate method, and concrete operations are: with double-deck filter paper filtering mouse ascites, the ascites after the filtration is in 4 ℃; More than the centrifugal 15min of 12000r/min, draw supernatant, the acetate buffer of supernatant with 4 times of volumes mixed, slowly add n-caprylic acid while stirring; Every milliliter of required n-caprylic acid volume of ascites is 30～35 μ L, mixed at room temperature 30～60min, and 4 ℃ leave standstill more than the 2h, 4 ℃ then; More than the centrifugal 30min of 12000r/min, abandon deposition, with the supernatant that obtains with double-deck filter paper filtering after, the volumetric molar concentration that adds 1/10 filtrate volume is the phosphate buffered saline buffer of 0.1mol/L and pH7.4; Regulate the pH to 7.4 of this mixed solution with the sodium hydroxide solution of 2 mol/L, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL; 4 ℃ leave standstill more than the 2h, and 4 ℃ then, more than the centrifugal 30min of 12000r/min; Abandon supernatant, the gained deposition is resuspended with the 0.01mol/L phosphate buffered saline buffer of former ascites volume 1/10, the dialysis tubing of packing into; With the pure water dialysis, it is freezing that the protein solution that fully dialysis is good is put-70 ℃ of refrigerators, uses the frozen vacuum dryer freeze-drying then; Collect lyophilized powder, promptly get the good aspergillus flavus resisting toxin B1 monoclonal antibody of purifying, antibody is put in-20 ℃ of refrigerators subsequent use;
Acetate buffer: the 0.29g sodium-acetate adds 0.141mL acetic acid, and pure water is settled to 100mL;
0.1mol/L phosphate buffered saline buffer: 0.8g sodium-chlor, the 0.29g disodium hydrogen phosphate, 0.02g Repone K, potassium primary phosphate 0.02g adds water and is settled to 100mL.
Beneficial effect of the present invention is:
(1) hybridoma cell strain 3G1 provided by the invention can be used to prepare the height aspergillus flavus resisting toxin B1 monoclonal antibody of tiring, and tiring that aspergillus flavus resisting toxin B1 mouse ascites antibody ELISA immuning adsorpting analysis (ELISA) method records can reach 6.40 * 10
6
(2) aspergillus flavus resisting toxin B1 monoclonal antibody provided by the invention is highly sensitive, specificity good, to 50% inhibition concentration IC of AFB1
50Be 1.6ng/mL, with AFB 2 cross reacting rates be 6.4%, with the cross reacting rate of aflatoxin G 1 and AFG 2 all less than 1%.
(3) aspergillus flavus resisting toxin B1 monoclonal antibody provided by the invention can be applicable to measure AFB1 content.
Description of drawings
Fig. 1 is the front view of the AFB1 immuno-chromatographic test paper strip of the embodiment of the invention 4 preparations.Among the figure: 1 cardboard, 2 absorbent pad; 3 detecting pads; 4 gold medals mark pad; 5 sample pad; 6 nature controlling lines; 7 detection lines.
Fig. 2 is the left view of the AFB1 immuno-chromatographic test paper strip of the embodiment of the invention 4 preparations.Among the figure: 1 cardboard; 2 absorbent pad; 3 detecting pads; 4 gold medals mark pad; 5 sample pad.
Fig. 3 is the process decision chart as a result of the AFB1 immuno-chromatographic test paper strip test sample using the embodiment of the invention 4 and provide.Among the figure: 8 control stripes bars; 9 test strip; 6 nature controlling lines; 7 detection lines.
Embodiment
Embodiment 1: the screening of hybridoma cell strain 3G1
1. antigen synthesizes and animal immune
Buying commercially available AFB1 standard substance, to carry out complete antigen synthetic, and concrete synthesis step is following: 4 mg AFB1 are dissolved in 2 mL acetone, add 40 μ L, 10% H
2SO
4, mixture is at 56 ℃ of stirring reaction 4 h; Behind the product evaporate to dryness, add the H of 5 mL
2O with 25 mL chloroform extraction twice, uses 20 mL H then
2O washs organic layer, keeps organic layer; Boil off organic solvent, get yellow solid product.Get 1.0 mg products, to wherein adding 2 mL, 0.5% BSA solution (37 ℃ of reaction 30 min of 4 mL PBS (phosphate buffered saline buffer, pH7.4) dissolving 20 mg BSA); Add 100 μ L, 6.5 mM NaBH
4, 4 ℃ of reaction 30 min; Add 50 μ L 0.1mol/L HCl, remove excessive N aBH
4Under 4 ℃ of conditions, (phosphate buffered saline buffer, pH7.4) dialysis 3d removes AFB1 and AFB to PBS solution
2a, carrying out conventional UV scanning method at last and identify, qualification result shows and has prepared AFB1 complete A antigen FB
2a-BSA.
6 of purchase female BALB/c mouses in 6 ages in week, immunity is synthetic AFB1 complete A antigen FB voluntarily
2a-BSA.Immunity is with complete A antigen FB for the first time
2a-BSA and equivalent Fu Shi Freund's complete adjuvant mixing and emulsifying, the subcutaneous multi-point injection in mouse carotid back then.For the second time be immune to head and exempt to carry out after 4 weeks, adopt freund 's incomplete adjuvant and equal-volume complete A antigen FB
2a-BSA emulsification, the mouse peritoneal injection.Immunity for the third time and immunity 4 weeks of pitch time for the second time, immunization ways and dosage be with for the second time identical, carries out after being immune to immune for the third time 3 weeks the 4th time, and immunization ways and immunizing dose are immune identical with for the second time, and immunizing dose is every mouse 50 μ g at every turn.3 times each immunity one week of back, tail vein blood, separation of serum adopts indirect elisa method monitoring mice serum antibody titer.A week after the 4th immunity; Tail vein blood; Separation of serum adopts indirect elisa method monitoring mice serum antibody titer, and measures mice serum sensitivity with the indirect competitive ELISA method; The mouse that the serum that selection is tired, sensitivity is all higher relatively is corresponding carries out last booster immunization, and immunizing dose is before 2 times.AFB1 purchases the company in Sigma-Aldrich.
2. cytogamy
Last booster immunization is after 3 days, and adopting the polyoxyethylene glycol of 50% (weight percentage) is that PEG (molecular weight is 1450) makes fusogen, carries out cytogamy by ordinary method, and concrete steps are following:
(1) the cervical vertebra dislocation method is put to death immune mouse, under aseptic condition, gets spleen, 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 then; Time of fusion 1 minute is filled it up with the RPMI-1640 basic culture solution then, and is centrifugal; Remove supernatant, the fused cell that mouse boosting cell and mouse source myeloma cell SP2/0 form is resuspended with 72mL RPMI-1640 basic culture solution, and resuspended cell is added drop-wise in the 96 porocyte culture plates; 2/hole; Put 37 ℃ of CO2gas incubators and cultivate, described RPMI-1640 basic culture solution is for containing 20% (percent by volume) foetal calf serum, 2% (weight percentage) growth factor and 1% (weight percentage) xanthoglobulin-aminopterin-thymidine.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% xanthoglobulin-aminopterin-thymidine (HAT) is purchased the company in Sigma-Aldrich.
3. the screening of cell strain and clone
Treated after the cytogamy about the 12nd day, cell colony long at the bottom of account for the hole 1/2 area big or small, 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 B1 and the positive hole of not anti-carrier proteins BSA; Second step adopted the indirect competitive ELISA method that the positive hole that the first step filters out is detected; Former with AFB1 as competition; (it was that 0 hole is that the final measured value of negative control hole is higher originally that the higher finger of light absorption value is competed, 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 3G1.
Embodiment 2: the strain of aspergillus flavus resisting toxin B1 monoclonal antibody hybridoma cell is a 3G1 antibody variable region sequencing
(1) extracts total RNA: adopt day total RNA extraction reagent box of root company and extract to specifications to produce total RNA that hybridoma cell strain is 3G1;
(2) synthetic cDNA: the total RNA that obtains with step 1 is a template, oligo (dT)
15Be primer, according to SuperScript
TM-2 II ThermoScript II specification sheetss 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.After the agarose gel electrophoresis separation of PCR product through 1% (weight percentage); With test kit purifying and recovering dna fragmentation, 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 347bp 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 115 amino acid, sequence is shown in SEQ ID NO:3.The long 338bp 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.
Embodiment 3: aspergillus flavus resisting toxin B1 MONOCLONAL ANTIBODIES SPECIFIC FOR purifying, hypotype and CHARACTERISTICS IDENTIFICATION
The aspergillus flavus resisting toxin B1 monoclonal antibody hybridoma cell strain that embodiment 2 is obtained is the BALB/c mouse that the 3G1 injection was handled with freund 's incomplete adjuvant in advance, collects the ascites of this mouse, adopts sad-ammonium sulfate method antibody purification, and concrete operations are: with double-deck filter paper filtering mouse ascites; 4 ℃, the centrifugal 15min of 12000r/min draws supernatant, and gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes; Stir the slow down n-caprylic acid that adds, every milliliter of required n-caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min; 4 ℃ leave standstill 2h, and 4 ℃ then, the centrifugal 30min of 12000r/min; Abandon deposition, 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 buffered saline buffer; With the pH value to 7.4 that the sodium hydroxide solution of 2 mol/L is regulated this mixed solution, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL; 4 ℃ leave standstill 2h, and 4 ℃ then, the centrifugal 30min of 12000r/min; Abandon supernatant, the gained deposition is resuspended with the 0.01mol/L phosphate buffered saline buffer of former ascites volume 1/10, the dialysis tubing 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, uses the freeze drier freeze-drying afterwards; Collect lyophilized powder, promptly get the good aspergillus flavus resisting toxin B1 monoclonal antibody of purifying, antibody is put in-20 ℃ of refrigerators subsequent use;
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 buffered saline buffer of described 0.1mol/L is a 0.8g sodium-chlor, the 0.29g disodium hydrogen phosphate, and 0.02g Repone K, potassium primary phosphate 0.02g adds water and is settled to the 100mL gained.
Use commercially available hypotype identification kit to identify that the hypotype of hybridoma cell strain 3G1 excretory aspergillus flavus resisting toxin B1 monoclonal antibody is IgG
2a
The tiring of mouse ascites fluid antibody that records 3G1 with conventional non-competing Enzyme Linked Immunoadsorbent Assay (ELISA) method can reach 6.4 * 10
6, promptly the mouse ascites fluid antibody dilution 6.4 * 10
6Times the time measured in solution result positive.Use conventional indirect competitive ELISA method to identify that its sensitivity to AFB1 is 1.6ng/mL, with AFB 2 cross reacting rates be 6.4%, the cross reacting rate of aflatoxin G 1 and G2 is all less than 1%.
Embodiment 4: antibody is used
Hybridoma cell strain 3G1 excretory aspergillus flavus resisting toxin B1 monoclonal antibody is used to prepare the AFB1 immuno-chromatographic test paper strip, and the preparation method may further comprise the steps:
(1) preparation of absorbent pad
Specification with thieving paper is cut out the wide 3mm of growth 16mm promptly gets absorbent pad;
(2) preparation of detecting pad
Encapsulating of detection line:
Toxins, afla synthetic complete A antigen FB2a-BSA is mixed with the coating buffer A of 0.1mg/mL; In position apart from nitrocellulose filter upper edge 15mm; With some spray mode coating buffer A is laterally encapsulated on nitrocellulose filter; Obtain detection line, the package amount of every centimetre of required complete A antigen FB2a-BSA of detection line is 75ng, under 37 ℃ of conditions dry 15 minutes then;
Described coating buffer A is: 10mg Toxins, afla synthetic complete A antigen FB2a-BSA, 1g bovine serum albumin, 0.02g sodiumazide; 0.8g sodium-chlor, 0.29g disodium hydrogen phosphate, 0.02g Repone K; 0.02g potassium primary phosphate adds water and is settled to the 100mL gained;
Encapsulating of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is made into the coating buffer B of 0.2mg/mL; In position apart from detection line 5mm, with a some spray mode coating buffer B is laterally encapsulated 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 sodiumazide, and 0.8g sodium-chlor, the 0.29g disodium hydrogen phosphate, 0.02g Repone K, the 0.02g potassium primary 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 moisture eliminator then;
Described confining liquid A is the 2g bovine serum albumin, 2.5g sucrose, and the 0.02g sodiumazide, 0.8g sodium-chlor, the 0.29g disodium hydrogen phosphate, 0.02g Repone K, the 0.02g potassium primary phosphate adds water and is settled to the 100mL gained;
(4) preparation of gold mark pad:
With the specification that glass fibre membrane is cut out the wide 3mm of growth 9mm, put into confining liquid B and soak, 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 B1 monoclonal anti liquid solution transverse jet of some spray mode nano gold mark, the aspergillus flavus resisting toxin B1 monoclonal antibody of every centimetre of required nano gold mark of spraying length is 600ng; Vacuum lyophilization 6h puts room temperature preservation in the moisture eliminator then;
Described confining liquid B is the 2g bovine serum albumin, 0.1mL triton x-100,0.3g Vinylpyrrolidone polymer, 2.5g sucrose; 0.02g sodiumazide, 0.8g sodium-chlor, 0.29g disodium hydrogen phosphate; 0.02g Repone K, the 0.02g potassium primary phosphate adds water and is settled to the 100mL gained;
The concrete marking method of the aspergillus flavus resisting toxin B1 monoclonal anti liquid solution of described nano gold mark is: measure the 50.0mL mass concentration and be 0.01% nano-Au solution, using 0.1 mol/L wet chemical regulator solution pH value is 5.5; Under the state that stirs, slowly add the aspergillus flavus resisting toxin B1 monoclonal antibody aqueous solution of 2.5mL 0.1mg/mL, continue to stir 30min; The adding mass concentration is that the whole mass concentration of 10% Bovine Serum Albumin in Aqueous Solution to bovine serum albumin is 1%, continues to stir 30min; Behind 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min gets supernatant, abandons deposition; 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 enriched material; It is subsequent use to put 4 ℃ of refrigerators, and wherein the mass concentration of the aspergillus flavus resisting toxin B1 monoclonal anti liquid solution of nano gold mark is 0.06mg/mL;
The particle diameter of nanometer gold is 15nm in the said nano-Au solution;
Described 0.1 mol/L wet chemical is: 13.8g salt of wormwood is dissolved in pure water and is settled to 1000mL, 0.22 μ m membrane filtration gained; The described 0.1mg/mL aspergillus flavus resisting toxin B1 monoclonal antibody aqueous solution is that 1mg aspergillus flavus resisting toxin B1 monoclonal antibody is dissolved in the 10 mL pure water and processes; Described 10% Bovine Serum Albumin in Aqueous Solution is dissolved in the 100mL pure water for the 10g bovine serum albumin, 0.22 μ m membrane filtration gained; Described mark washing is preserved liquid and is: 2.0g polyoxyethylene glycol-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 strip: paste absorbent pad, 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 the AFB1 immuno-chromatographic test paper strip, sees Fig. 1 and Fig. 2.
The application of above-mentioned AFB1 immuno-chromatographic test paper strip:
Getting known 1# and the 2# peanut sample that does not contain AFB1 respectively pulverizes; Adopt 10% methanol-water (V methyl alcohol/V water=1: 9) stirring and evenly mixing then; Draw 1# and each 1mL of 2# peanut sample to be measured; The AFB1 standard substance that the good sample solution of getting 100 μ L dilution adds different concns respectively are as the mark-on sample, and each 100 μ L adds the sample pad of AFB1 immuno-chromatographic test paper strip then, as test set; Get 100 μ L simultaneously and do not contain the sample pad of the blank peanut sample of AFB1, it as the control stripes bar, is read the result after 15 minutes as another AFB1 immuno-chromatographic test paper strip of negative control adding; Detected 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, and the content that shows the AFB1 in the testing sample is seen Fig. 3-1 more than or equal to 10ng/mL; 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 strip detection line, then is judged to positive findings, shows that the content of AFB1 is greater than or equal to 1ng/mL in the testing sample; And, see Fig. 3-2 less than 10 ng/mL.
Sequence table
＜110＞Inst. of Oil Crops, Chinese Academy of Agriculture
The aspergillus flavus resisting toxin B1 monoclonal antibody of＜120＞hybridoma cell strain 3G1 and generation thereof
＜160＞ 4
＜210＞ 1
＜211＞ 347bp
＜212＞ DNA
＜213＞mouse
＜400＞ 1
tgaggagacg gtgaccgtgg tcccttggcc ccagtagtcc atagcccagt 50
aggccgatct tgcacagtaa tatgtggctg tgtcctcagt agtcacagaa 100
tttaactgca ggtagtactg gttcttggat gtgtctcgag tgatggagat 150
tcgactcttg agagatggat tgtagtaagg gttaccactg tagctcatga 200
accccatgta ctcaagttta ttcccaggga atttccggat ccagtgccag 250
taatcactgg tgatggagtc gccagtgaca gaacaggtga gggacagagt 300
ctgagaaggt ttcacgaggc taggtcctga ctcctgcagc tgcacct 347
＜210＞ 2
＜211＞ 338bp
＜212＞ DNA
＜213＞mouse
＜400＞ 2
gacattgagc tcacccagtc tccaaaattc atgtccacat cagtaggaga 50
cagggtcagc atctcctgca aggccagtca ggatgtgggt actgatgtag 100
cctggtacgg agtccctgat cgcctcacag gcagtggatc tgggacagat 150
ttcactctca ccattagcaa tgtgcagtct gaagacttgg cagattattt 200
ctgtcaacaa tatagcagct atattcacgt tcggctcggg gacaaagttg 250
gaaataaaac gtttttgtgg agaggggcat gtcatagtcc tcactgtgtc 300
tcacgttcgg tgctgggacc aagctggagc tgaaacgg 338
＜210＞ 3
＜211＞ 115
＜212＞ PRT
＜213＞mouse
＜400＞ 3
Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr
1 5 10 15 20
Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Asp Tyr Trp His Trp Ile Arg Lys Phe Pro
25 30 35 40
Gly Asn Lys Leu Glu Tyr Met Gly Phe Met Ser Tyr Ser Gly Asn Pro Tyr Tyr Asn Pro
45 50 55 60
Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu Gln
65 70 75 80
Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Ala Tyr Trp
85 90 95 100
Ala Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
105 110 115
＜210＞ 4
＜211＞ 110
＜212＞ PRT
＜213＞mouse
＜400＞ 4
Asp Ile Glu Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser
1 5 10 15 20
Ile Ser Cys Lys Ala Ser Gln Asp Val Gly Thr Asp Val Ala Trp Tyr Gly Val Pro Asp
25 30 35 40
Arg Leu Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser
45 50 55 60
Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Ile His Val Arg Leu Gly
65 70 75 80
Asp Lys Val Gly Asn Lys Thr Phe Leu Trp Arg Gly Ala Cys His Ser Pro His Cys Val
85 90 95 100
Ser Arg Ser Val Leu Gly Pro Ser Trp Ser
105 110
Claims (4)
1. hybridoma cell strain 3G1, it is characterized in that: it is preserved in Chinese typical culture collection center, and deposit number is CCTCC NO. C201014.
2. aspergillus flavus resisting toxin B1 monoclonal antibody, it is that the hybridoma cell strain 3G1 secretion of CCTCC NO. C201014 produces by deposit number.
3. the application of aspergillus flavus resisting toxin B1 monoclonal antibody according to claim 2 in AFB1 is measured.
4. aspergillus flavus resisting toxin B1 monoclonal antibody according to claim 2 the preparation method; It is characterized in that: the hybridoma cell strain 1C8 that obtains is injected the BALB/c mouse of handling with freund 's incomplete adjuvant in advance; Collect the ascites of this mouse, obtain aspergillus flavus resisting toxin B1 monoclonal antibody after the purification process.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210117614 CN102747043B (en) | 2012-04-20 | 2012-04-20 | Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same |
| PCT/CN2013/070614 WO2013155883A1 (en) | 2012-04-20 | 2013-01-17 | Hybridoma cell line 3g1 and monoclonal antibody produced thereby against aflatoxin b1 |
| NZ613444A NZ613444A (en) | 2012-04-20 | 2013-01-17 | A hybridoma cell line 3g1 and a monoclonal antibody against aflatoxin b1 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210117614 CN102747043B (en) | 2012-04-20 | 2012-04-20 | Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same |
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| CN102747043A true CN102747043A (en) | 2012-10-24 |
| CN102747043B CN102747043B (en) | 2013-07-17 |
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| CN 201210117614 Active CN102747043B (en) | 2012-04-20 | 2012-04-20 | Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same |
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| Country | Link |
|---|---|
| CN (1) | CN102747043B (en) |
| NZ (1) | NZ613444A (en) |
| WO (1) | WO2013155883A1 (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103116024A (en) * | 2012-11-30 | 2013-05-22 | 中国农业科学院油料作物研究所 | Application and method of anti-aflatoxin universal monoclonal antibody 1C11 in aflatoxin B1 fluorescence quenching |
| CN103130892A (en) * | 2013-01-25 | 2013-06-05 | 中国农业科学院油料作物研究所 | Aflatoxin recombination single-chain antibody 2G7, encoding gene and application thereof |
| CN103215652A (en) * | 2013-01-25 | 2013-07-24 | 中国农业科学院油料作物研究所 | Aflatoxin positive antibody gene bank, establishing method and application thereof, and aflatoxin recombinant single-chain antibody 1A7 |
| CN103215229A (en) * | 2013-04-03 | 2013-07-24 | 中国农业科学院油料作物研究所 | Hybridoma cell strain AFB3G1, monoclonal antibody thereof and multi-detection line immunochromatography test strip for semi-quantitatively detecting aflatoxin B1 |
| WO2013155883A1 (en) * | 2012-04-20 | 2013-10-24 | 中国农业科学院油料作物研究所 | Hybridoma cell line 3g1 and monoclonal antibody produced thereby against aflatoxin b1 |
| CN104762267A (en) * | 2015-01-15 | 2015-07-08 | 北京科技大学 | Hybridoma cell AFB1-2A4 and aflatoxin B1 monoclonal antibody produced by hybridoma cell AFB1-2A4 |
| WO2015143833A1 (en) * | 2014-03-28 | 2015-10-01 | 中国农业科学院油料作物研究所 | Aflatoxin b1 nanobody 2014afb-g15 |
| CN105586317A (en) * | 2016-01-19 | 2016-05-18 | 北京龙科方舟生物工程技术有限公司 | Monoclonal antibody against aflatoxin B1 and application of monoclonal antibody |
| CN106970223A (en) * | 2017-03-07 | 2017-07-21 | 中国农业科学院油料作物研究所 | Purify five kinds of mycotoxin immunosorbent and the compound affinity columns such as fumonisin B1, aflatoxin B1 |
| CN107012128A (en) * | 2017-04-10 | 2017-08-04 | 北京勤邦生物技术有限公司 | A kind of hybridoma cell strain for secreting aspergillus flavus resisting toxin B1 monoclonal antibodies and its application |
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| CN102747043B (en) * | 2012-04-20 | 2013-07-17 | 中国农业科学院油料作物研究所 | Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same |
| CN102746403A (en) * | 2012-04-20 | 2012-10-24 | 中国农业科学院油料作物研究所 | Standard substance universal alternate for aflatoxin detection by using ELISA, preparation method thereof, and ELISA detection method for aflatoxin |
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- 2012-04-20 CN CN 201210117614 patent/CN102747043B/en active Active
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- 2013-01-17 NZ NZ613444A patent/NZ613444A/en not_active IP Right Cessation
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| WO2013155883A1 (en) * | 2012-04-20 | 2013-10-24 | 中国农业科学院油料作物研究所 | Hybridoma cell line 3g1 and monoclonal antibody produced thereby against aflatoxin b1 |
| CN103116024A (en) * | 2012-11-30 | 2013-05-22 | 中国农业科学院油料作物研究所 | Application and method of anti-aflatoxin universal monoclonal antibody 1C11 in aflatoxin B1 fluorescence quenching |
| CN103116024B (en) * | 2012-11-30 | 2014-04-09 | 中国农业科学院油料作物研究所 | Application and method of anti-aflatoxin universal monoclonal antibody 1C11 in aflatoxin B1 fluorescence quenching |
| CN103215652B (en) * | 2013-01-25 | 2014-04-30 | 中国农业科学院油料作物研究所 | Aflatoxin positive antibody gene bank, establishing method and application thereof, and aflatoxin recombinant single-chain antibody 1A7 |
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| CN103215652A (en) * | 2013-01-25 | 2013-07-24 | 中国农业科学院油料作物研究所 | Aflatoxin positive antibody gene bank, establishing method and application thereof, and aflatoxin recombinant single-chain antibody 1A7 |
| CN103215229A (en) * | 2013-04-03 | 2013-07-24 | 中国农业科学院油料作物研究所 | Hybridoma cell strain AFB3G1, monoclonal antibody thereof and multi-detection line immunochromatography test strip for semi-quantitatively detecting aflatoxin B1 |
| WO2015143833A1 (en) * | 2014-03-28 | 2015-10-01 | 中国农业科学院油料作物研究所 | Aflatoxin b1 nanobody 2014afb-g15 |
| CN104762267A (en) * | 2015-01-15 | 2015-07-08 | 北京科技大学 | Hybridoma cell AFB1-2A4 and aflatoxin B1 monoclonal antibody produced by hybridoma cell AFB1-2A4 |
| CN105586317A (en) * | 2016-01-19 | 2016-05-18 | 北京龙科方舟生物工程技术有限公司 | Monoclonal antibody against aflatoxin B1 and application of monoclonal antibody |
| CN106970223A (en) * | 2017-03-07 | 2017-07-21 | 中国农业科学院油料作物研究所 | Purify five kinds of mycotoxin immunosorbent and the compound affinity columns such as fumonisin B1, aflatoxin B1 |
| CN106970223B (en) * | 2017-03-07 | 2018-12-11 | 中国农业科学院油料作物研究所 | Purify five kinds of mycotoxin immunosorbent and the compound affinity columns such as fumonisin B1, aflatoxin B1 |
| CN107012128A (en) * | 2017-04-10 | 2017-08-04 | 北京勤邦生物技术有限公司 | A kind of hybridoma cell strain for secreting aspergillus flavus resisting toxin B1 monoclonal antibodies and its application |
| CN107012128B (en) * | 2017-04-10 | 2020-04-28 | 北京勤邦生物技术有限公司 | Hybridoma cell strain secreting monoclonal antibody against aflatoxin B1 and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| NZ613444A (en) | 2016-09-30 |
| CN102747043B (en) | 2013-07-17 |
| WO2013155883A1 (en) | 2013-10-24 |
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