CN103215230B - Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit - Google Patents
Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit Download PDFInfo
- Publication number
- CN103215230B CN103215230B CN201310115872.8A CN201310115872A CN103215230B CN 103215230 B CN103215230 B CN 103215230B CN 201310115872 A CN201310115872 A CN 201310115872A CN 103215230 B CN103215230 B CN 103215230B
- Authority
- CN
- China
- Prior art keywords
- aflatoxin
- resolved fluorescence
- pad
- monoclonal antibody
- time resolved
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a hybridoma cell strain AFM1B7, a monoclonal antibody thereof and an aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit. The hybridoma cell strain AFM1B7 is collected in China Center for Type Culture Collection (CCTCC), and the collection number is CCTCC No.C201020. The monoclonal antibody secreted by the hybridoma cell strain AFM1B7 has high sensitivity and good specificity, the 50% inhibition concentration against aflatoxin M1 is 52pg/mL, and the cross reaction rate with aflatoxins B1, B2, G1 and G2, vomitoxin, zearalenone and fumonisin is less than 0.3%. The aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit prepared from the monoclonal antibody can be used for quantitatively measuring the content of aflatoxin M1, is simple and quick to operate, and has high accuracy.
Description
Technical field
The present invention relates to the mobile immune time resolved fluorescence quick testing reagent box that lags behind of hybridoma cell strain AFM1B7, its monoclonal antibody and aflatoxin M 1.
Background technology
Aflatoxin is mainly the secondary metabolite being produced by flavus and Aspergillus parasiticus secretion, is the natural toxic compounds that can cause the various infringements of people and animals.Aflatoxin has found that more than 20 plant at present, mainly comprises aflatoxin A1(AFA1), B2(AFB2), AFG and M1(AFM1) etc.Aflatoxin M 1(AFM1) be the hydroxylation metabolism product of AFB1, Mammals is taken in after the feed of AFB1 pollution, can secrete in the middle of milk in vivo through hydroxylation.Conventionally when animal, taken in after the food of AFB1 pollution, the output of AFM1 is 1%～3% of AFB1 intake.Toxicity and the carinogenicity of a large amount of investigators to AFM1 conducts in-depth research, and result of study also impels international cancer research institution that the carcinogenic grade of AFM1 is become to a class carcinogenic substance from two class carcinogenic substances.AFM1 stable in properties, even through pasteurize, also almost completely can not be destroyed.In many milk-product, all contain AFM1.Because milk-product are main sources of infant's food, therefore AFM1 pollution problem has caused the extensive concern of countries in the world, and AFM1 has been carried out to strict limitation.China belongs to the heavier area of aflatoxin contamination, therefore strengthens the detection of aflatoxin M 1 in milk and milk products, particularly speed and surveys, and understands and grasp in time the health information of milk and milk products, significant to ensureing China's food consumption safety.
The detection method of existing aflatoxin comprises thin layer chromatography, precision instrument analytical method and immune analysis method.Wherein thin layer chromatography is early for detection of the most frequently used detection method of aflatoxin, this method does not need special plant and instrument, common laboratory all can be carried out, but reagent dosage is large, complex operation, other component serious interference, poor accuracy, can not accurate quantitative analysis, and larger to experimenter and surrounding environment pollution hazard, be unsuitable for field quick detection.Precision instrument analytical method mainly comprises spectrophotofluorimetry and high performance liquid chromatography, these methods are highly sensitive, accuracy is good, but there is instrument costliness, require aflatoxin Sample Purification on Single degree high, sample pretreatment process is loaded down with trivial details, length consuming time, experimental situation is required to high deficiency, be difficult to realize rapid detection.Immuno analytical method has overcome the above two shortcoming, there is high specificity, highly sensitive, sample pre-treatments is simple, cost is low, little to the pollution hazard of experimenter and surrounding environment, be suitable for the advantages such as on-the-spot batch detection, be applied to multiple fields such as food, medical treatment.
Summary of the invention
Problem to be solved by this invention is to provide the mobile immune time resolved fluorescence quick testing reagent box that lags behind of hybridoma cell strain AFM1B7, its monoclonal antibody and aflatoxin M 1.
The invention provides hybridoma cell strain AFM1B7, this cell strain was preserved in Chinese Typical Representative culture collection center (CCTCC) on July 13rd, 2010, and preservation address is, China, and Wuhan, Wuhan University, deposit number is CCTCC NO.C201020.It has in sequence table the monoclonal antibody variable region of light chain coding gene sequence shown in SEQ ID No.2 in the monoclonal antibody variable region of heavy chain coding gene sequence shown in SEQ ID No.1 and sequence table.
Monoclonal antibody, the hybridoma cell strain AFM1B7 secretion that it is CCTCC NO.C201020 by deposit number produces.Its variable region of heavy chain has the aminoacid sequence shown in SEQ ID No.3 in sequence table; Variable region of light chain has the aminoacid sequence shown in SEQ ID No.4.This monoclonal antibody can be identified aflatoxin M 1, to 50% inhibition concentration IC of aflatoxin M 1
50for 52pg/mL.
The mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1, it is characterized in that: the example reaction bottle of the aspergillus flavus resisting toxin M1 monoclonal antibody dried frozen aquatic products that it comprises fluorescent test paper strip and contains europium mark, wherein: described fluorescent test paper strip comprises cardboard, the one side of cardboard is pasted absorbent pad from top to bottom successively, detecting pad and sample pad, the overlapping connection in junction of adjacent each pad, described detecting pad is take nitrocellulose filter as base wad, horizontal nature controlling line and detection line are set on nitrocellulose filter from top to bottom, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, coated aflatoxin M 1-bovine serum albumin conjugate (AFB1-BSA) on described detection line, the monoclonal antibody that the hybridoma cell strain AFM1B7 secretion that described aspergillus flavus resisting toxin M1 monoclonal antibody is is CCTCC NO.C201020 by deposit number produces.
Press such scheme, the aspergillus flavus resisting toxin M1 monoclonal antibody of described europium mark prepares in accordance with the following methods: monoclonal antibody (the aspergillus flavus resisting toxin M1 monoclonal antibody) carbonate buffer solution that the hybridoma cell strain AFM1B7 secretion that is CCTCC NO.C201020 by deposit number produces is dialysed afterwards and the ratio of europium labelled reagent take mass ratio as 0.5～2:1 fully mixes rear hold over night, then through the aspergillus flavus resisting toxin M1 monoclonal antibody of Sephadex G-50 chromatography column Separation Europium mark, wash-out, collects target product.
Press such scheme, the long 15～20mm of absorbent pad in described fluorescent test paper strip, wide 3～4mm; Long 25～the 30mm of detecting pad, wide 3～4mm; Long 12～the 18mm of sample pad, wide 3～4mm, the overlapping length of adjacent each pad is 1～3mm.
Press such scheme, on the detection line in described fluorescent test paper strip on detecting pad and nitrocellulose filter, the spacing on edge is 15～20mm, and the spacing of nature controlling line and detection line is 5～10mm; The bayonet socket bottle that described example reaction bottle is 1-5mL.
Press such scheme, in described fluorescent test paper strip, on detecting pad, the package amount of the required aflatoxin B1-bovine serum albumin conjugate of every centimetre of detection line is 50～100ng; The package amount of the required anti-mouse polyclonal antibody of rabbit of every centimetre of nature controlling line is 30～100ng; In described example reaction bottle, the content of the aspergillus flavus resisting toxin M1 monoclonal antibody dried frozen aquatic products of europium mark is 0.05-0.25 μ g.
Press such scheme, described fluorescent test paper strip is to adopt following methods to obtain:
(1) thieving paper is cut out to obtain to absorbent pad;
(2) preparation of detecting pad:
The conjugate AFM1-BSA of aflatoxin M-bovine serum albumin is mixed with to the coating buffer that concentration is 0.1～0.4mg/mL, in the position along 10～15mm on nitrocellulose filter, it is laterally coated on nitrocellulose filter by line spray mode, obtain detection line, the package amount of the conjugate AFM1-BSA of the every centimetre of required aflatoxin M 1-of detection line bovine serum albumin is 50～100ng, then under 37～40 ℃ of conditions, is dried 30～60 minutes;
Anti-rabbit mouse polyclonal antibody is made into the coating buffer that concentration is 0.1～0.4mg/mL, in the position apart from detection line 5～10mm, it is laterally coated on nitrocellulose filter by line spray mode, obtain nature controlling line, the package amount of the required anti-mouse polyclonal antibody of rabbit of every centimetre of nature controlling line is 30～100ng, then under 37～40 ℃ of conditions dry 30～60 minutes;
(3) preparation of sample pad:
Glass fibre membrane is put into confining liquid and soak, take out, under 37～40 ℃ of conditions, be dried 3～6 hours, obtain sample pad, then put room temperature preservation in moisture eliminator;
(4) assembling of fluorescent test paper strip:
One side at cardboard is pasted absorbent pad, detecting pad, sample pad from top to bottom successively, the overlapping connection in junction of adjacent each pad, and overlapping length is 1～3mm, obtains fluorescent test paper strip.
Press such scheme, in the preparation of described fluorescent test paper strip, preparing the coated damping fluid using in aflatoxin M 1-bovine serum albumin conjugate (AFM1-BSA) coating buffer is: in every 10mL, contain bovine serum albumin 0.1g, sodiumazide 0.002g, sodium-chlor 0.08g, disodium hydrogen phosphate 0.029g, Repone K 0.002g, potassium primary phosphate 0.002g;
The coated damping fluid using in the anti-mouse polyclonal antibody of preparation rabbit coating buffer is: in every 10mL, contain sodiumazide 0.002g, sodium-chlor 0.08g, disodium hydrogen phosphate 0.029g, Repone K 0.002g, potassium primary phosphate 0.002g;
The confining liquid using in the preparation of described fluorescent test paper strip is: in every 100mL, contain oralbumin 0.5-2g, sucrose 2g, sodiumazide 0.02g, sodium-chlor 0.8g, disodium hydrogen phosphate 0.29g, Repone K 0.02g, potassium primary phosphate 0.02g.
Press such scheme, the mobile immune time resolved fluorescence quick testing reagent box that lags behind of described aflatoxin M 1 also comprises sample diluting liquid and diluted sample liquid straw, and described sample diluting liquid is that volume fraction is 0.01～0.30% the tween 20 aqueous solution.
The mobile application of immune time resolved fluorescence quick testing reagent box in aflatoxin M 1 content detection that lag behind of above-mentioned aflatoxin M 1: analyte sample fluid is added in example reaction bottle, mix, insert fluorescent test paper strip, 37 ℃ of reactions are after 6 minutes, detect with temporal resolution fluorometric investigation instrument, obtain the ratio of detection line (T) time resolved fluorescence intensity level and nature controlling line (C) time resolved fluorescence intensity level on fluorescent test paper strip; Based on the fluorescent test paper strip detection line time resolved fluorescence intensity and the ratio (T/C) of nature controlling line time resolved fluorescence intensity and the relation curve of aflatoxin M 1 concentration that obtain in advance, obtain the content of aflatoxin M 1 in analyte sample fluid.
Press such scheme, described fluorescent test paper strip detection line time resolved fluorescence intensity and the ratio (T/C) of nature controlling line time resolved fluorescence intensity adopt following methods to obtain with the relation curve of aflatoxin M 1 concentration:
(1) preparation obtains aflatoxin M 1 standard solution of a series of concentration;
(2) aflatoxin M 1 standard solution of appropriate above-mentioned each concentration is joined respectively in example reaction bottle, mix, insert fluorescent test paper strip, 37 ℃ are reacted 6 minutes, with temporal resolution fluorescence immunity analyzer, detect the time resolved fluorescence intensity level that obtains detection line on each fluorescent test paper strip (T) and nature controlling line (C), obtain thus the ratio (T/C) of each fluorescent test paper strip detection line time resolved fluorescence intensity and nature controlling line time resolved fluorescence intensity;
(3) through matching, obtain the ratio (T/C) of fluorescent test paper strip detection line time resolved fluorescence intensity and nature controlling line time resolved fluorescence intensity and the relation curve of aflatoxin M 1 concentration.
Hybridoma cell strain AFM1B7 in the present invention adopts two step screening method to obtain, its concrete steps are: by BALB/c mouse after aflatoxin M 1 complete A antigen FM1-BSA immunity 4-6 time, make last booster immunization with 2 times of aflatoxin M 1 complete A antigen FM1-BSA to a front immunizing dose, after 3 days, carry out cytogamy, 2-3 week after cytogamy, with micropipet, individual cells colony is moved to 96 porocyte culture plates and adopt liquid amplification culture, clone for the first time, adopt ELISA method to screen in two steps fused cell: the first step adopts indirect elisa method to filter out aspergillus flavus resisting toxin M1 and the positive hole of not anti-carrier proteins BSA, the positive hole nutrient solution that second step adopts indirect competitive ELISA method to filter out the first step detects, former as competition with aflatoxin M 1, select all higher holes of light absorption value and sensitivity, adopt limiting dilution assay to carry out subclone, after subclone, adopt same two step screening method to detect, after repetition subclone like this 2-3 time, final screening obtains hybridoma cell strain AFM1B7.
The preparation method of monoclonal antibody in the present invention, step is as follows: the hybridoma cell strain AFM1B7 of acquisition is expelled to the belly of the BALB/c mouse of processing with freund 's incomplete adjuvant in advance, collects the ascites of this mouse, purifying and get final product.
Described purification process is caprylic acid-ammonium, concrete operation step: with double-deck filter paper filtering mouse ascites, 4 ℃, more than the centrifugal 15min of 12000r/min, draw supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, under stirring, slowly add n-caprylic acid, every milliliter of required n-caprylic acid volume of ascites is 30～35 μ L, mixed at room temperature 30～60min, more than 4 ℃ of standing 2h, then 4 ℃, more than the centrifugal 30min of 12000r/min, abandon precipitation, by the supernatant liquor obtaining with after double-deck filter paper filtering, the volumetric molar concentration that adds 1/10 filtrate volume is 0.1mol/L, pH value is 7.4 phosphate buffered saline buffer, with the sodium hydroxide solution of 2mol/L, regulate the pH value to 7.4 of this mixed solution, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL, more than 4 ℃ of standing 2h, then 4 ℃, more than the centrifugal 30min of 12000r/min, abandon supernatant, 0.01mol/L by gained precipitation with former ascites volume 1/10, pH value is that 7.4 phosphate buffered saline buffer is resuspended, pack dialysis tubing into, dialyse with pure water, the protein solution of fully dialysing is put to-70 ℃ of refrigerator freezings, use afterwards freeze drier freeze-drying, collect lyophilized powder, obtain the monoclonal antibody that purifying is good, antibody is put in-20 ℃ of refrigerators standby,
Described acetate buffer is 0.29g sodium-acetate, and 0.141mL acetic acid adds water constant volume to 100mL gained; The phosphate buffered saline buffer of described 0.01mol/L is 0.8g sodium-chlor, 0.29g disodium hydrogen phosphate, and 0.02g Repone K, potassium primary phosphate 0.02g, adds water constant volume to 100mL gained; The phosphate buffered saline buffer of described 0.1mol/L is 8g sodium-chlor, 2.9g disodium hydrogen phosphate, and 0.2g Repone K, potassium primary phosphate 0.2g, adds water constant volume to 100mL gained.
Beneficial effect of the present invention:
(1) hybridoma cell strain AFM1B7 provided by the invention can be for the preparation of high-titer monoclonal antibody, and tiring that mouse hydroperitoneum antibody of RGDV Enzyme Linked Immunoadsorbent Assay (ELISA) method records can reach 2.2 × 10
5.
(2) monoclonal antibody provided by the invention is highly sensitive, specificity good, to 50% inhibition concentration IC of aflatoxin M 1
50for 52pg/mL, with aflatoxin B1, B2, G1, G2, vomitoxin, zearalenone, the cross reacting rate of fumonisin is all less than 0.3%.
(4) the mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1 that prepared by monoclonal antibody provided by the invention can be used for the quantitative assay of aflatoxin M 1 content, and simple to operate, and fast, accuracy is high.
Accompanying drawing explanation
Fig. 1 is the structural representation of fluorescent test paper strip in the mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1 provided by the invention.In figure: 1 sample pad, 2 detecting pads, 3 detection lines, 4 nature controlling lines, 5 absorbent pad.
Embodiment
Embodiment 1: the screening of hybridoma cell strain AFM1B7
1. animal immune
Buy 6 of BALB/c mouse in 6 week age, the aflatoxin M 1 complete A antigen FM1-BSA that immunity is commercially available.Immunity is by after aflatoxin M 1 complete antigen and isopyknic Freund's complete adjuvant emulsification, in the subcutaneous multi-point injection in mouse carotid back for the first time.After being immune to for the second time 4 weeks, carry out, adopt freund 's incomplete adjuvant and the 1 complete antigen emulsification of isopyknic aflatoxin M, in mouse peritoneal, inject.Immunity for the third time and immune interval for the second time 4 weeks, immunization ways is identical with it, carries out after being immune to immune 3 weeks for the third time for the 4th time, and immunization ways, with immune identical for the second time, is similarly abdominal injection.4 times immunizing dose is identical, is every mouse 80 μ g.Latter 8～10 days of 3 times each immunity, tail vein blood, separation of serum, adopts indirect elisa method monitoring mice serum to tire.Latter 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 by indirect competitive ELISA method, selection is tired, sensitivity all relatively high mouse corresponding to serum carry out last booster immunization, immunizing dose is above 2 times.
Aflatoxin M 1 complete A antigen FM1-BSA is purchased from Sigma-Aldrich company.
2. cytogamy
In last booster immunization after 3 days, adopt 50%(weight percentage) polyoxyethylene glycol be that PEG(molecular weight is 1450) make fusogen, carry out according to a conventional method cytogamy, concrete steps: kill immune mouse under aseptic condition, separating Morr. cell, with mouse source myeloma cell SP2/0 with the number of 5 ︰ 1 than mixing, wash cell mixing with RPMI-1640 basic culture solution, merge with 50%PEG, merge 1 minute, then slowly add RPMI-1640 basic culture solution, centrifugal, remove supernatant, the fused cell that mouse boosting cell and mouse source myeloma cell SP2/0 form is resuspended containing the cell perfect medium of 1%HAT with 20mL, the cell having hanged is joined in 80mL semisolid medium, after mixing, be added on 6 porocyte culture plates, 1.5mL/ hole, being placed in 37 ℃ of CO2gas incubator cultivates.
The described cell perfect medium containing 1%HAT contains 20%(percent by volume) foetal calf serum, 75%(percent by volume) RPMI-1640 basic culture solution, 1%(weight percentage) L-glutaminate, 1%(percent by volume) HEPES, 1%(percent by volume) dual anti-(the 10000 every ml penicillins of unit and every milliliter of Streptomycin sulphate of 10000 micrograms), 2%(weight percentage) somatomedin and 1%(weight percentage) xanthoglobulin-aminopterin-thymidine is HAT; Semisolid medium is for containing 1%(mass percent) the cell perfect medium of methylcellulose gum;
RPMI-1640 basic culture solution, HEPES, dual anti-, L-glutaminate are purchased from Hyclone company; 1% xanthoglobulin-aminopterin-thymidine is that HAT, methylcellulose gum are purchased from Sigma-Aldrich company.
3. the screening of cell strain and clone
2-3 week after cytogamy, cell colony grows to people's naked eyes when visible, clone is drawn from this substratum with micropipet, move to 96 porocyte culture plates and adopt liquid amplification culture, every hole moves into 1 clone, at the bottom of cell grows to full hole 1/2～2/3 o'clock, draw culture supernatant and carry out positive detection, carry out antibody test.Adopt ELISA method to screen the culture hole that has Growth of Hybridoma Cell, 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 proteins BSA; The positive hole that second step adopts indirect competitive ELISA method to filter out the first step is detected, former as competition with aflatoxin M 1, all (the higher finger competition of light absorption value was that 0 hole is that the final tested volume in positive control hole is higher originally, competition original content that is IC when the higher finger inhibiting rate of sensitivity is 50% higher hole to select light absorption value and sensitivity
50be worth less), adopt limiting dilution assay to carry out subclone, after subclone, adopt same two-step approach to detect, so repeat after subclone 2-3 time acquisition hybridoma cell strain AFM1B7.
Embodiment 2: hybridoma cell strain AFM1B7 antibody variable region sequencing
(1) extract total RNA: adopt the total RNA extraction reagent box of Tian Gen company and extract to specifications total RNA that can produce hybridoma cell strain AFM1B7;
(2) synthetic cDNA: the total RNA obtaining take step 1 is template, oligo (dT)
15for primer, according to SuperScriptTM-2 II ThermoScript II specification sheets, carry out reverse transcription, synthetic cDNA the first chain; Primer oligo (dT) 15 is buied by Invitrogen;
(3) PCR method clone variable region gene: according to the conservative site design primer of GENEBANK small mouse antibody gene sequence, increasing take cDNA as masterplate, antibody is light, heavy chain variable region gene.PCR program is: 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 1min, and 30 circulations of increasing, last 72 ℃ are extended 10min.PCR product is through 1%(weight percentage) agarose gel electrophoresis separate after, with test kit, purify and reclaim DNA fragmentation, be connected in carrier pMD18-T, transform bacillus coli DH 5 alpha competent cell, picking positive colony, delivers to Sani bio tech ltd, Shanghai and checks order.Wherein the sequence of primer is respectively: variable region of heavy chain primer is 5,-AGG TSM ARC TGC AGS AGT CWG G-3, (22mer) He 5,-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC-3, (32mer) wherein S, M, R and W are merger base, M=A/C, R=A/G, S=C/G, W=A/T, variable region of light chain primer is 5,-GAC ATT GAG CTCACC CAG CTT GGT GCC-3, (24mer) with 5 ,-CCG TTT CAG CTC CAG CTT GGT CCC-3, (24mer).
The gene order result obtaining: the long 339bp of variable region of heavy chain coding gene sequence, sequence is as shown in SEQ ID NO:1, according to obtained gene order, derive the coded variable region of heavy chain of this gene order and be comprised of 113 amino acid, sequence is as shown in SEQ ID NO:3.The long 318bp of variable region of light chain coding gene sequence, sequence, as shown in SEQ ID NO:2, is derived the coded variable region of light chain of this gene order according to obtained gene order and is comprised of 106 amino acid, and sequence is as shown in SEQ IDNO:4.
Embodiment 3: monoclonal antibody prepare purifying, hypotype and CHARACTERISTICS IDENTIFICATION
The hybridoma strain AFM1B7 that embodiment 1 is obtained injects the BALB/c mouse of processing with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt caprylic acid-ammonium 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, under stirring, slowly add n-caprylic acid, every milliliter of required n-caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min, 4 ℃ of standing 2h, then 4 ℃, the centrifugal 30min of 12000r/min, abandon precipitation, by the supernatant liquor obtaining with after double-deck filter paper filtering, the volumetric molar concentration that adds 1/10 filtrate volume is the phosphate buffered saline buffer that 0.1mol/L and pH value are 7.4, with the sodium hydroxide solution of 2mol/L, regulate the pH value to 7.4 of this mixed solution, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL, 4 ℃ of standing 2h, then 4 ℃, the centrifugal 30min of 12000r/min, abandon supernatant, 0.01mol/L by gained precipitation with former ascites volume 1/10, pH value is that 7.4 phosphate buffered saline buffers are resuspended, pack dialysis tubing into, pure water is dialysed, the protein solution of fully dialysing is put to-70 ℃ of refrigerator freezings, use afterwards freeze drier freeze-drying, collect lyophilized powder, obtain the monoclonal antibody that purifying is good, antibody is placed in to-20 ℃ of refrigerators standby,
Described acetate buffer is 0.29g sodium-acetate, and 0.141mL acetic acid adds water and is settled to 100mL gained; The phosphate buffered saline buffer of described 0.01mol/L is 0.8g sodium-chlor, 0.29g disodium hydrogen phosphate, and 0.02g Repone K, potassium primary phosphate 0.02g, adds water and is settled to 100mL gained; The phosphate buffered saline buffer of described 0.1mol/L is 8g sodium-chlor, 2.9g disodium hydrogen phosphate, and 0.2g Repone K, potassium primary phosphate 0.2g, adds water constant volume to 100mL gained.
The hypotype of identifying the monoclonal antibody of hybridoma cell strain AFM1B7 secretion with commercially available hypotype identification kit is IgG1.
The tiring of BALB/c mouse ascites antibody that records injection AFM1B7 hybridoma cell strain by the non-competing Enzyme Linked Immunoadsorbent Assay of routine (ELISA) method can reach 2.2 × 10
5, i.e. mouse ascites antibody dilution 2.2 × 10
5times time measured in solution result positive.Identify its 50% inhibition concentration IC to aflatoxin M 1 by conventional indirect competitive ELISA method
50for 52pg/mL, with aflatoxin B1, B2, G1, G2, vomitoxin, zearalenone, the cross reacting rate of fumonisin is all less than 0.3%.
Embodiment 4: aflatoxin M 1 mobile lag behind immune time resolved fluorescence quick testing reagent box and application thereof
The mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1,
Example reaction bottle, sample diluting liquid and the diluted sample liquid straw of the aspergillus flavus resisting toxin M1 monoclonal antibody dried frozen aquatic products that it comprises fluorescent test paper strip, contain europium mark, described fluorescent test paper strip comprises cardboard, the one side of cardboard is pasted absorbent pad, detecting pad and sample pad from top to bottom successively, the overlapping connection in junction of adjacent each pad, overlapping length is 1mm, wherein: the long 15mm of absorbent pad, wide 4mm; The long 25mm of detecting pad, wide 4mm; The long 13mm of sample pad, wide 4mm.Described detecting pad is take nitrocellulose filter as base wad, horizontal nature controlling line and detection line are set on nitrocellulose filter from top to bottom, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, its package amount is 40ng/cm nature controlling line, coated aflatoxin B1-bovine serum albumin conjugate (AFB1-BSA) on described detection line, its package amount is 65ng/cm detection line, and on detection line and nitrocellulose filter, the spacing on edge is 15mm, and the spacing of nature controlling line and detection line is 5mm.
The acquisition of described fluorescent test paper strip:
(1) preparation of absorbent pad
Thieving paper is cut out to growth 15mm, and the specification of wide 4mm, obtains absorbent pad;
(2) preparation of detecting pad
Being coated with of detection line:
The conjugate AFM1-BSA of aflatoxin M 1-bovine serum albumin is mixed with to the coating buffer that concentration is 0.15mg/mL with coated damping fluid; In the position along 15mm on nitrocellulose filter, it is laterally coated on nitrocellulose filter by line spray mode, obtain detection line, the package amount of every centimetre of required AFM1-BSA of detection line is 65ng, then under 37 ℃ of conditions dry 30 minutes;
Described coated damping fluid is: 0.1g bovine serum albumin, and 0.002g sodiumazide, 0.08g sodium-chlor, 0.029g disodium hydrogen phosphate, 0.002g Repone K, 0.002g potassium primary phosphate, adds water constant volume to 10mL gained;
Being coated with of nature controlling line:
Anti-rabbit mouse polyclonal antibody is made into the coating buffer that concentration is 0.1mg/mL with coated damping fluid; In the position apart from detection line 6mm, it is laterally coated on nitrocellulose filter by line spray mode, obtain nature controlling line, the package amount of the required anti-mouse polyclonal antibody of rabbit of every centimetre of nature controlling line is 40ng, then under 37 ℃ of conditions dry 1 hour;
Described coated damping fluid is 0.002g sodiumazide, 0.08g sodium-chlor, and 0.029g disodium hydrogen phosphate, 0.002g Repone K, 0.002g potassium primary phosphate, adds water and is settled to 10mL gained;
(3) preparation of sample pad:
Glass fibre membrane is cut out to growth 13mm, and the specification of wide 4mm, puts into confining liquid and soaks, and takes out, and under 37 ℃ of conditions, is dried 6 hours, obtains sample pad, then puts room temperature preservation in moisture eliminator;
Described confining liquid is 1g oralbumin, 2g sucrose, and 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 100mL gained;
(4) assembling of fluorescent test paper strip:
One side at cardboard is pasted absorbent pad, detecting pad, fluorescent-labeled antibody reacting pad and sample pad from top to bottom successively, the overlapping connection in junction of adjacent each pad, and overlapping length is 2mm, obtains fluorescent test paper strip (see figure 1).
The acquisition of the aspergillus flavus resisting toxin M1 monoclonal antibody of described europium mark:
Get the monoclonal antibody in 1mg above-described embodiment 3, with after the carbonate buffer solution repetitive scrubbing of 100mmol/L pH9.3 6 times, itself and 0.5mg europium labelled reagent are fully mixed, in 4 ℃, spend the night.Then joined in the Sephadex G-50 chromatography column of 1.9cm × 60cm, with the 50mmol/L Tris-HCl elutriant wash-out containing 0.9%NaCl, collect effluent liquid (1ml/ pipe), by pipe, measure light absorption value (A280nm), merge peak pipe, obtain the aspergillus flavus resisting toxin M1 monoclonal antibody of target product europium mark.Above-mentioned europium labelled reagent is purchased from Shanghai Uni Bio-Tech. Co., Ltd., but is not limited to this.
The acquisition of the example reaction bottle of the described aspergillus flavus resisting toxin M1 monoclonal antibody lyophilized powder that contains europium mark: the aspergillus flavus resisting toxin M1 monoclonal antibody 0.05 μ g that gets above-mentioned europium mark is put in 3mL bayonet socket bottle, after adopting conventional freezing vacuum drying method to drain, both obtained the aspergillus flavus resisting toxin M1 monoclonal antibody lyophilized powder of europium mark, 4 ℃ of preservations, standby.
Described sample diluting liquid is: the tween 20 aqueous solution that volume fraction is 0.01%.
The application of above-mentioned mobile hysteresis immunity time resolved fluorescence quick testing reagent box in milk sample aflatoxin M 1 detects:
The foundation of the ratio (T/C) of I fluorescent test paper strip detection line time resolved fluorescence intensity and nature controlling line time resolved fluorescence intensity and the relation curve of aflatoxin M 1 concentration:
(1), to detecting as the milk sample of aflatoxin M 1 feminine gender carries out aflatoxin M 1 mark-on through high performance liquid chromatography (HPLC), join to obtain aflatoxin M 1 standard solution of 4ng/mL, 2ng/mL, 1ng/mL, 0.5ng/mL, 0.25ng/mL, 0.1ng/mL, 0.05ng/mL, 0.02ng/mL and 0ng/mL;
(2) get the each 200 μ L of aflatoxin M 1 standard solution of above-mentioned each concentration, join respectively in example reaction bottle, mix, insert fluorescent test paper strip, 37 ℃ are reacted 6 minutes, blot sample pad residual liquid with thieving paper, at once with temporal resolution fluorescence immunity analyzer, detect (excitation wavelength: 365nm, measure wavelength: 615nm), obtain the time resolved fluorescence intensity level that on each fluorescent test paper strip, detection line place (T) and nature controlling line (C) locate, obtain thus the ratio (T/C) of each fluorescent test paper strip detection line time resolved fluorescence intensity and nature controlling line time resolved fluorescence intensity,
(3) take AFM1 concentration as X-coordinate, the corresponding detection line time resolved fluorescence intensity/nature controlling line of the aflatoxin M 1 standard solution time resolved fluorescence intensity of each concentration is that T/C value is ordinate zou, and matching obtains the ratio (T/C) of fluorescent test paper strip detection line time resolved fluorescence intensity and nature controlling line time resolved fluorescence intensity and the relation curve of aflatoxin M 1 concentration.The valid analysing range of the method is 0.05～2ng/mL.
The detection of aflatoxin M 1 content in II milk sample:
Get 5 parts of milk samples to be measured, each 200 μ L add in example reaction bottle, mix, insert fluorescent test paper strip, 37 ℃ of reactions are after 6 minutes, blot sample pad residual liquid with thieving paper, with temporal resolution fluorescence immunity analyzer, detect (excitation wavelength: 365nm immediately, measure wavelength: 615nm), obtain the ratio (T/C) of each fluorescent test paper strip detection line time resolved fluorescence intensity and nature controlling line time resolved fluorescence intensity, then the relation curve with aflatoxin M 1 concentration by the ratio (T/C) of fluorescent test paper strip detection line time resolved fluorescence intensity obtained above its substitution and nature controlling line time resolved fluorescence intensity, : the detected result of 5 milk samples is followed successively by: <0.05ng/mL, 0.12ng/mL, <0.05ng/mL, 1.2ng/mL and 0.83ng/mL.
The method detected result and ELISA detected result are compared: the method detected result is consistent with national standard efficient liquid-phase chromatography method detected result height, and coincidence rate is up to 97.5%; Separately, the method single sample detects required time and is also only about 1/10th of high performance liquid chromatography standard method, has significantly improved detection speed.
Embodiment 5: aflatoxin M 1 mobile lag behind immune time resolved fluorescence quick testing reagent box and application thereof
Long 15～the 20mm of absorbent pad in described fluorescent test paper strip, wide 3～4mm; Long 25～the 30mm of detecting pad, wide 3～4mm; Long 12～the 18mm of sample pad, wide 3～4mm, the overlapping length of adjacent each pad is 1～3mm.
The mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1, example reaction bottle, sample diluting liquid and the diluted sample liquid straw of the aspergillus flavus resisting toxin M1 monoclonal antibody dried frozen aquatic products that it comprises fluorescent test paper strip, contain europium mark, described fluorescent test paper strip comprises cardboard, the one side of cardboard is pasted absorbent pad, detecting pad and sample pad from top to bottom successively, the overlapping connection in junction of adjacent each pad, overlapping length is 1mm, wherein: the long 18mm of absorbent pad, wide 3mm; The long 28mm of detecting pad, wide 3mm; The long 15mm of sample pad, wide 3mm.Described detecting pad is take nitrocellulose filter as base wad, horizontal nature controlling line and detection line are set on nitrocellulose filter from top to bottom, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, its package amount is 100ng/cm nature controlling line, coated aflatoxin B1-bovine serum albumin conjugate (AFB1-BSA) on described detection line, its package amount is 100ng/cm detection line, and on detection line and nitrocellulose filter, the spacing on edge is 20mm, and the spacing of nature controlling line and detection line is 10mm.
The acquisition of described fluorescent test paper strip:
(1) preparation of absorbent pad
Thieving paper is cut out to growth 18mm, and the specification of wide 3mm, obtains absorbent pad;
(2) preparation of detecting pad
Being coated with of detection line:
The conjugate AFM1-BSA of aflatoxin M 1-bovine serum albumin is mixed with to the coating buffer that concentration is 0.4mg/mL with coated damping fluid; In the position along 20mm on nitrocellulose filter, it is laterally coated on nitrocellulose filter by line spray mode, obtain detection line, the package amount of every centimetre of required AFM1-BSA of detection line is 100ng, then under 40 ℃ of conditions dry 30 minutes;
Described coated damping fluid is: 0.1g bovine serum albumin, and 0.002g sodiumazide, 0.08g sodium-chlor, 0.029g disodium hydrogen phosphate, 0.002g Repone K, 0.002g potassium primary phosphate, adds water constant volume to 10mL gained;
Being coated with of nature controlling line:
Anti-rabbit mouse polyclonal antibody is made into the coating buffer that concentration is 0.4mg/mL with coated damping fluid; In the position apart from detection line 6mm, it is laterally coated on nitrocellulose filter by line spray mode, obtain nature controlling line, the package amount of the required anti-mouse polyclonal antibody of rabbit of every centimetre of nature controlling line is 40ng, then dry 30min under 40 ℃ of conditions;
Described coated damping fluid is 0.002g sodiumazide, 0.08g sodium-chlor, and 0.029g disodium hydrogen phosphate, 0.002g Repone K, 0.002g potassium primary phosphate, adds water and is settled to 10mL gained;
The long 28mm of described nitrocellulose filter, wide 3mm.
(3) preparation of sample pad:
Glass fibre membrane is cut out to growth 15mm, and the specification of wide 3mm, puts into confining liquid and soaks, and takes out, and under 37 ℃ of conditions, is dried 6 hours, obtains sample pad, then puts room temperature preservation in moisture eliminator;
Described confining liquid is 1g oralbumin, 2g sucrose, and 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 100mL gained;
(4) assembling of fluorescent test paper strip:
One side at cardboard is pasted absorbent pad, detecting pad, fluorescent-labeled antibody reacting pad and sample pad from top to bottom successively, the overlapping connection in junction of adjacent each pad, and overlapping length is 2mm, obtains fluorescent test paper strip (see figure 1).
The acquisition of the aspergillus flavus resisting toxin M1 monoclonal antibody of described europium mark:
Get the monoclonal antibody in 1mg above-described embodiment 3, with after the carbonate buffer solution repetitive scrubbing of 100mmol/L pH9.3 6 times, itself and 2mg europium labelled reagent are fully mixed, in 4 ℃, spend the night.Then joined in the Sephadex G-50 chromatography column of 1.9cm × 60cm, with the 50mmol/L Tris-HCl elutriant wash-out containing 0.9%NaCl, collect effluent liquid (1ml/ pipe), by pipe, measure light absorption value (A280nm), merge peak pipe, obtain the aspergillus flavus resisting toxin M1 monoclonal antibody of target product europium mark.Above-mentioned europium labelled reagent is purchased from Shanghai Uni Bio-Tech. Co., Ltd., but is not limited to this.
The acquisition of the example reaction bottle of the described aspergillus flavus resisting toxin M1 monoclonal antibody lyophilized powder that contains europium mark: the aspergillus flavus resisting toxin M1 monoclonal antibody 0.25 μ g that gets above-mentioned europium mark is put in 3mL bayonet socket bottle, after adopting conventional freezing vacuum drying method to drain, both obtained the aspergillus flavus resisting toxin M1 monoclonal antibody lyophilized powder of europium mark, 4 ℃ of preservations, standby.
Described sample diluting liquid is: the tween 20 aqueous solution that volume fraction is 0.30%.
The application of above-mentioned mobile hysteresis immunity time resolved fluorescence quick testing reagent box in milk sample aflatoxin M 1 detects:
Getting milk sample 200 μ L to be measured adds in example reaction bottle, mix, insert fluorescent test paper strip, 37 ℃ of reactions are after 6 minutes, blot sample pad residual liquid with thieving paper, with temporal resolution fluorescence immunity analyzer, detect (excitation wavelength: 365nm immediately, measure wavelength: 615nm), obtain the ratio (T/C) of each fluorescent test paper strip detection line time resolved fluorescence intensity and nature controlling line time resolved fluorescence intensity, then the relation curve with aflatoxin M 1 concentration by the ratio (T/C) of fluorescent test paper strip detection line time resolved fluorescence intensity obtained above its substitution and nature controlling line time resolved fluorescence intensity, : in this milk sample, the content of aflatoxin M 1 is 1ng/mL.
Claims (10)
1. hybridoma cell strain AFM1B7, is characterized in that: it is preserved in Chinese Typical Representative culture collection center (CCTCC), and deposit number is CCTCC NO. C201020.
2. monoclonal antibody, is characterized in that: the hybridoma cell strain AFM1B7 secretion that it is CCTCC NO. C201020 by deposit number produces.
3. the mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1, it is characterized in that: the example reaction bottle of the aspergillus flavus resisting toxin M1 monoclonal antibody dried frozen aquatic products that it comprises fluorescent test paper strip and contains europium mark, wherein: described fluorescent test paper strip comprises cardboard, the one side of cardboard is pasted absorbent pad from top to bottom successively, detecting pad and sample pad, the overlapping connection in junction of adjacent each pad, described detecting pad is take nitrocellulose filter as base wad, horizontal nature controlling line and detection line are set on nitrocellulose filter from top to bottom, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, coated aflatoxin M 1-bovine serum albumin conjugate (AFB1-BSA) on described detection line, described aspergillus flavus resisting toxin M1 monoclonal antibody is that deposit number claimed in claim 1 is the monoclonal antibody of the hybridoma cell strain AFM1B7 secretion generation of CCTCC NO. C201020.
4. the mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1 according to claim 3, it is characterized in that: the aspergillus flavus resisting toxin M1 monoclonal antibody of described europium mark prepares in accordance with the following methods: the monoclonal antibody carbonate buffer solution that the hybridoma cell strain AFM1B7 secretion that is CCTCC NO. C201020 by deposit number claimed in claim 1 produces is dialysed afterwards and the ratio of europium labelled reagent take mass ratio as 0.5～2:1 fully mixes rear hold over night, then through the aspergillus flavus resisting toxin M1 monoclonal antibody of Sephadex G-50 chromatography column Separation Europium mark, wash-out, collect target product.
5. the mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1 according to claim 3, is characterized in that: the long 15～20mm of absorbent pad in described fluorescent test paper strip, wide 3～4mm; Long 25～the 30mm of detecting pad, wide 3～4mm; Long 12～the 18mm of sample pad, wide 3～4mm, the overlapping length of adjacent each pad is 1～3mm; On described detection line and nitrocellulose filter, the spacing on edge is 15～20mm, and the spacing of nature controlling line and detection line is 5～10mm; The bayonet socket bottle that described example reaction bottle is 1-5mL.
6. the aflatoxin M according to claim 31 immune time resolved fluorescence quick testing reagent box that lags behind that flows, is characterized in that: in described fluorescent test paper strip, on detecting pad, the package amount of the required aflatoxin B1-bovine serum albumin conjugate of every centimetre of detection line is 50～100ng; The package amount of the required anti-mouse polyclonal antibody of rabbit of every centimetre of nature controlling line is 30～100ng; In described example reaction bottle, the content of the aspergillus flavus resisting toxin M1 monoclonal antibody dried frozen aquatic products of europium mark is 0.05-0.25 μ g.
7. the mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1 according to claim 3, is characterized in that: described fluorescent test paper strip is to adopt following methods to obtain:
(1) thieving paper is cut out to obtain to absorbent pad;
(2) preparation of detecting pad:
The conjugate AFM1-BSA of aflatoxin M-bovine serum albumin is mixed with to the coating buffer that concentration is 0.1 ~ 0.4mg/mL, in the position along 10 ~ 15mm on nitrocellulose filter, it is laterally coated on nitrocellulose filter by line spray mode, obtain detection line, the package amount of the conjugate AFM1-BSA of the every centimetre of required aflatoxin M 1-of detection line bovine serum albumin is 50 ~ 100ng, then under 37 ~ 40 ℃ of conditions, is dried 30 ~ 60 minutes;
Anti-rabbit mouse polyclonal antibody is made into the coating buffer that concentration is 0.1 ~ 0.4mg/mL, in the position apart from detection line 5 ~ 10mm, it is laterally coated on nitrocellulose filter by line spray mode, obtain nature controlling line, the package amount of the required anti-mouse polyclonal antibody of rabbit of every centimetre of nature controlling line is 30 ~ 100ng, then under 37 ~ 40 ℃ of conditions dry 30 ~ 60 minutes;
(3) preparation of sample pad:
Glass fibre membrane is put into confining liquid and soak, take out, under 37 ~ 40 ℃ of conditions, be dried 3 ~ 6 hours, obtain sample pad, then put room temperature preservation in moisture eliminator;
(4) assembling of fluorescent test paper strip:
One side at cardboard is pasted absorbent pad, detecting pad, sample pad from top to bottom successively, the overlapping connection in junction of adjacent each pad, and overlapping length is 1 ~ 3 mm, obtains fluorescent test paper strip.
8. the mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1 according to claim 6, it is characterized in that: in the preparation of described fluorescent test paper strip, preparing the coated damping fluid using in aflatoxin M 1-bovine serum albumin conjugate (AFM1-BSA) coating buffer is: in every 10mL, contain bovine serum albumin 0.1g, sodiumazide 0.002g, sodium-chlor 0.08g, disodium hydrogen phosphate 0.029g, Repone K 0.002g, potassium primary phosphate 0.002g;
The coated damping fluid using in the anti-mouse polyclonal antibody of preparation rabbit coating buffer is: in every 10mL, contain sodiumazide 0.002g, sodium-chlor 0.08g, disodium hydrogen phosphate 0.029g, Repone K 0.002g, potassium primary phosphate 0.002g;
The confining liquid using in the preparation of described fluorescent test paper strip is: in every 100mL, contain oralbumin 0.5-2g, sucrose 2g, sodiumazide 0.02g, sodium-chlor 0.8g, disodium hydrogen phosphate 0.29g, Repone K 0.02g, potassium primary phosphate 0.02g.
9. the mobile immune time resolved fluorescence quick testing reagent box that lags behind of aflatoxin M 1 according to claim 3, it is characterized in that: it also comprises sample diluting liquid and diluted sample liquid straw, described sample diluting liquid is that volume fraction is 0.01 ~ 0.30% the tween 20 aqueous solution.
10. according to right, to require the mobile application of immune time resolved fluorescence quick testing reagent box in aflatoxin M 1 content detection that lag behind of aflatoxin M 1 described in 3, it is characterized in that: it is that analyte sample fluid is added in example reaction bottle, mix, insert fluorescent test paper strip, 37 ℃ of reactions are after 6 minutes, detect with temporal resolution fluorometric investigation instrument, obtain the ratio of detection line time resolved fluorescence intensity level and nature controlling line time resolved fluorescence intensity level on fluorescent test paper strip; Based on the time resolved fluorescence ELISA test strip line time resolved fluorescence intensity and the ratio of nature controlling line time resolved fluorescence intensity and the relation curve of aflatoxin M 1 concentration that obtain in advance, obtain the content of aflatoxin M 1 in analyte sample fluid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310115872.8A CN103215230B (en) | 2013-04-03 | 2013-04-03 | Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310115872.8A CN103215230B (en) | 2013-04-03 | 2013-04-03 | Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103215230A CN103215230A (en) | 2013-07-24 |
| CN103215230B true CN103215230B (en) | 2014-04-30 |
Family
ID=48813392
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310115872.8A Active CN103215230B (en) | 2013-04-03 | 2013-04-03 | Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103215230B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104251904B (en) * | 2014-10-09 | 2016-05-11 | 福建农林大学 | A kind of test paper of Quantitative detection pyrethrin pesticide |
| CN107083368B (en) * | 2017-04-10 | 2020-01-31 | 北京勤邦生物技术有限公司 | hybridoma cell strains secreting monoclonal antibodies against total aflatoxin and application thereof |
| CN107188961A (en) * | 2017-07-17 | 2017-09-22 | 齐齐哈尔大学 | A kind of aspergillus flavus resisting toxin M1 monoclonal antibodies and immunosorbent, immune affinity column and preparation method thereof |
| CN107561273A (en) * | 2017-08-29 | 2018-01-09 | 联合益康（北京）生物科技有限公司 | A kind of time-resolved fluorescence test strips for detecting Aflatoxins M1 and its application |
| CN111007245B (en) * | 2019-11-15 | 2021-09-21 | 中国农业科学院油料作物研究所 | Quick detection kit for flow lag immune time resolution fluorescence of ribes diacetylenium sickle enol |
| CN111007246B (en) * | 2019-11-15 | 2021-09-21 | 中国农业科学院油料作物研究所 | Time resolution fluorescence kit for synchronously detecting ribes diacetate sickle enol, deoxynivalenol and T-2 toxin |
| CN110988339A (en) * | 2019-12-30 | 2020-04-10 | 江南大学 | Time-resolved immune quantitative test strip for detecting aflatoxin M1 in milk |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220286A (en) * | 2010-12-21 | 2011-10-19 | 中国农业科学院油料作物研究所 | Hybridoma cell strain 2C9, anti-aflatoxin M1 monoclonal antibody produced by hybridoma cell strain 2C9 and application thereof |
| CN102253209A (en) * | 2010-12-21 | 2011-11-23 | 中国农业科学院油料作物研究所 | Aflatoxin M1 (AFM1) immunity chromatography test paper strip and preparation method thereof |
-
2013
- 2013-04-03 CN CN201310115872.8A patent/CN103215230B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102220286A (en) * | 2010-12-21 | 2011-10-19 | 中国农业科学院油料作物研究所 | Hybridoma cell strain 2C9, anti-aflatoxin M1 monoclonal antibody produced by hybridoma cell strain 2C9 and application thereof |
| CN102253209A (en) * | 2010-12-21 | 2011-11-23 | 中国农业科学院油料作物研究所 | Aflatoxin M1 (AFM1) immunity chromatography test paper strip and preparation method thereof |
Non-Patent Citations (8)
| Title |
|---|
| Imai，Y等.登录号DJ605028：Antibodies that specifically bind to a beta oligomers and use thereof.《EMBL数据库》.2012,氨基酸序列. * |
| Liu,P.C.等人.登录号AFR11387:anti-Staphylococcal enterotoxin K immunoglobulin light chain variable region.《GenBank数据库》.2012,氨基酸序列. * |
| 应用HTS-ELISA筛选方法制备抗黄曲霉毒素M1单抗;裴世春等;《微生物学报》;20101231;第50卷(第10期);全文 * |
| 张园园等.抗黄曲霉毒素M1单克隆抗体的制备及定量ELISA方法的建立.《中国预防兽医学报》.2008,第30卷(第10期),全文. |
| 张道宏.黄曲霉毒素杂交瘤细胞株的选育及免疫层析检测技术研究.《中国博士学位论文全文数据库》.2012,摘要，38页最后1段-第39页表2.7. |
| 抗黄曲霉毒素M1单克隆抗体的制备及定量ELISA方法的建立;张园园等;《中国预防兽医学报》;20081031;第30卷(第10期);全文 * |
| 裴世春等.应用HTS-ELISA筛选方法制备抗黄曲霉毒素M1单抗.《微生物学报》.2010,第50卷(第10期),全文. |
| 黄曲霉毒素杂交瘤细胞株的选育及免疫层析检测技术研究;张道宏;《中国博士学位论文全文数据库》;20120715;摘要，第28页最后段-29页表2.7 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103215230A (en) | 2013-07-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103278631B (en) | Aflatoxin B1 flow lag immunization time distinguishing fluorescence rapid-detection kit and application thereof | |
| CN103215230B (en) | Hybridoma cell strain AFM1B7, monoclonal antibody thereof and aflatoxin M1 flow lag immunization time-resolved fluorescence quick test kit | |
| CN101993855B (en) | Hybridoma cell line 1C11 and anti-aflatoxin general monoclonal antibody generated by same as well as applications thereof | |
| CN102747042B (en) | Hybridoma cell line 10G4 and anti-alfatoxin total (B1, B2, G1 and G2) monoclonal antibody produced by the same | |
| CN102220286B (en) | Hybridoma cell strain 2C9, anti-aflatoxin M1 monoclonal antibody produced by hybridoma cell strain 2C9 and application thereof | |
| CN106918704B (en) | Synchronous detection aflatoxin and the time-resolved fluoroimmunoassay of carbaryl composite pollution chromatograph kit, preparation method and application | |
| CN103217531B (en) | Immunochromatography test strip for synchronously detecting mixed pollution of aflatoxin, ochratoxin A and zearalenone, and preparation method | |
| CN106932370A (en) | The immunochromatography time-resolved fluorescence kit of five kinds of mycotoxin composite pollutions such as synchronous detection AFB1 and application | |
| CN103257230B (en) | Immunochromatographic test strip for synchronously detecting mixed pollution of aflatoxin and zearalenone, preparation method and application thereof | |
| CN102747043B (en) | Hybridoma cell line 3G1 and anti-alfatoxin B1 monoclonal antibody produced by the same | |
| CN103215229B (en) | Hybridoma cell strain AFB3G1, monoclonal antibody thereof and multi-detection line immunochromatography test strip for semi-quantitatively detecting aflatoxin B1 | |
| CN101865924B (en) | Method for detecting fumonisins based on immuno-magnetic bead combined enzyme-linked immunosorbent assay | |
| CN102719405B (en) | Hybridoma cell strain 1C8 and anti-aflatoxin G1 monoclonal antibody produced by same | |
| CN103215231B (en) | Hybridoma cell strain 1H2, anti-ochratoxin A monoclonal antibody produced from same and application thereof | |
| CN103278630B (en) | Immunity chromatography test strip for synchronously detecting aflatoxin and ochratoxin A mixed pollution, and preparation method and application thereof | |
| CN105652005B (en) | Quantitatively detect time-resolved fluoroimmunoassay chromatograph test strip, the preparation method and applications of Capsaicinoids | |
| CN109254155A (en) | A kind of detection African swine fever virus antigen colloidal gold immune chromatography test paper and preparation method and application | |
| CN101930006A (en) | High-sensitivity immunochromatographic test strip for detecting total aflatoxin content quickly and preparation method thereof | |
| CN102253209B (en) | Aflatoxin M1 (AFM1) immunity chromatography test paper strip and preparation method thereof | |
| CN101949937A (en) | Oophoroma tumor marker HE4 time-resolved fluoroimmunoassay detection kit | |
| CN101863981A (en) | Preparation method of anti-bisphenol A monoclonal antibody | |
| CN105541655A (en) | Universal artificial hapten and artificial complete antigen for capsaicins and application thereof | |
| CN103232975B (en) | Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody | |
| CN103849604B (en) | Hybridoma cell strain ST03, anti-aflatoxin biosynthesis precursor ST monoclonal antibody and application thereof | |
| CN102103144A (en) | Method for detecting ovarian tumor marker human epididymis (HE4) protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |