CN103215230A - 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
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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 hybridoma cell strain AFM1B7, its monoclonal antibody and the aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind.
Background technology
Aflatoxin 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 aflatoxin has found 20 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 can secrete in the middle of milk through hydroxylation after taking in the feed of AFB1 pollution in vivo.Usually after animal had been taken in the food of AFB1 pollution, the output of AFM1 was 1%～3% of an AFB1 intake.A large amount of investigators has carried out deep research to toxicity and the carinogenicity of AFM1, and result of study also impels international cancer research institution that the carcinogenic grade of AFM1 is become a class carcinogenic substance by two class carcinogenic substances.The 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, so the AFM1 pollution problem caused the extensive concern of countries in the world, and AFM1 has been carried out strict limiting the quantity of.China belongs to the heavier area of aflatoxin contamination, therefore strengthens the detection of aflatoxin M 1 in the milk and milk products, particularly speed and surveys, and in time understands and grasp the health information of milk and milk products, and is significant to ensureing China's food consumption safety.
Existing examination of aflatoxin method comprises thin layer chromatography, precision instrument analytical method and immune analysis method.Wherein thin layer chromatography is early to be used to detect 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 big, complex operation, other component serious interference, poor accuracy, can not be accurately quantitative, and bigger to experimenter and surrounding environment pollution hazard, be unsuitable for field quick detection.The precision instrument analytical method mainly comprises spectrophotofluorimetry and high performance liquid chromatography, these methods are highly sensitive, accuracy is good, but the instrument costliness is arranged, require aflatoxin sample degree of purification height, sample pretreatment process is loaded down with trivial details, length consuming time, experimental situation is required high deficiency, be difficult to realize rapid detection.Immuno analytical method has overcome the above two shortcoming, have 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 advantages such as on-the-spot batch detection, be applied to a plurality of fields such as food, medical treatment.
Summary of the invention
Problem to be solved by this invention provides hybridoma cell strain AFM1B7, its monoclonal antibody and the aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind.
The invention provides hybridoma cell strain AFM1B7, this cell strain was preserved in Chinese typical culture collection center (CCTCC) on July 13rd, 2010, and the preservation address is, China, and Wuhan, Wuhan University, deposit number is CCTCC NO.C201020.It has in the sequence table monoclonal antibody variable region of light chain coding gene sequence shown in the SEQ ID No.2 in the monoclonal antibody variable region of heavy chain coding gene sequence shown in the SEQ ID No.1 and sequence table.
Monoclonal antibody, it is the hybridoma cell strain AFM1B7 secretion generation of CCTCC NO.C201020 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.This monoclonal antibody can be discerned aflatoxin M 1, to 50% inhibition concentration IC of aflatoxin M 1
50Be 52pg/mL.
The aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind, it is characterized in that: it comprises fluorescent test paper strip and contains the example reaction bottle of the aspergillus flavus resisting toxin M1 monoclonal antibody dried frozen aquatic products of 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, adjacent each pad overlaps in the junction and connects, described detecting pad is base wad with the nitrocellulose filter, horizontal nature controlling line and detection line are set on the nitrocellulose filter from top to bottom, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, and bag is by aflatoxin M 1-bovine serum albumin conjugate (AFB1-BSA) on the described detection line; Described aspergillus flavus resisting toxin M1 monoclonal antibody is for by deposit number being the monoclonal antibody of the hybridoma cell strain AFM1B7 secretion generation of CCTCC NO.C201020.
Press such scheme, the aspergillus flavus resisting toxin M1 monoclonal antibody of described europium mark prepares in accordance with the following methods: with deposit number is that monoclonal antibody (aspergillus flavus resisting toxin M1 monoclonal antibody) that the hybridoma cell strain AFM1B7 secretion of CCTCC NO.C201020 produces is standing over night behind the abundant mixing of ratio of 0.5～2:1 with mass ratio with carbonate buffer solution dialysis back and europium labelled reagent, the aspergillus flavus resisting toxin M1 monoclonal antibody of separating the europium mark then through Sephadex G-50 chromatography column, wash-out is collected target product.
Press such scheme, the long 15～20mm of the absorbent pad in the 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, the spacing of detection line in the described fluorescent test paper strip on the detecting pad and nitrocellulose filter upper 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 the described fluorescent test paper strip on the detecting pad package amount of the required aflatoxin B1-bovine serum albumin conjugate of every centimetre of detection line be 50～100ng; The package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 30～100g; 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 in the described example reaction bottle.
Press such scheme, described fluorescent test paper strip is to adopt following method to obtain:
(1) thieving paper is cut out absorbent pad;
(2) preparation of detecting pad:
The conjugate AFM1-BSA of aflatoxin M-bovine serum albumin is mixed with the coating buffer that concentration is 0.1～0.4mg/mL, position in distance nitrocellulose filter upper edge 10～15mm, with line spray mode it is laterally wrapped by on nitrocellulose filter, 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, under 37～40 ℃ of conditions dry 30～60 minutes then;
The anti-mouse polyclonal antibody of rabbit is made into the coating buffer that concentration is 0.1～0.4mg/mL, position in distance detection line 5～10mm, with line spray mode it is laterally wrapped by on nitrocellulose filter, get nature controlling line, the package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 30～100ng, under 37～40 ℃ of conditions dry 30～60 minutes then;
(3) preparation of sample pad:
Glass fibre membrane is put into confining liquid soak, take out, drying is 3～6 hours under 37～40 ℃ of conditions, gets sample pad, puts room temperature preservation in the moisture eliminator then;
(4) assembling of fluorescent test paper strip:
One side at cardboard is pasted absorbent pad, detecting pad, sample pad from top to bottom successively, and adjacent each pad overlaps in the junction and connects, and overlapping length is 1～3mm, promptly gets fluorescent test paper strip.
Press such scheme, in the preparation of described fluorescent test paper strip in preparation aflatoxin M 1-bovine serum albumin conjugate (AFM1-BSA) coating buffer employed bag be cushioned liquid and be: contain bovine serum albumin 0.1g among every 10mL, sodiumazide 0.002g, sodium-chlor 0.08g, disodium hydrogen phosphate 0.029g, Repone K 0.002g, potassium primary phosphate 0.002g;
Employed bag is cushioned liquid and is in the preparation rabbit anti-mouse polyclonal antibody coating buffer: contain sodiumazide 0.002g among every 10mL, sodium-chlor 0.08g, disodium hydrogen phosphate 0.029g, Repone K 0.002g, potassium primary phosphate 0.002g;
The confining liquid that uses in the preparation of described fluorescent test paper strip is: contain oralbumin 0.5-2g among every 100mL, 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 described aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind 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 above-mentioned aflatoxin M 1 mobile application of immune time resolved fluorescence quick testing reagent box in aflatoxin M 1 content detection that lag behind: analyte sample fluid is added in the example reaction bottle, mixing, insert fluorescent test paper strip, 37 ℃ of reactions are after 6 minutes, differentiate the fluorometric investigation instrument with the time and detect, the ratio of detection line (T) time resolved fluorescence intensity level and nature controlling line (C) time resolved fluorescence intensity level on the acquisition fluorescent test paper strip; Based on the ratio (T/C) of fluorescent test paper strip detection line time resolved fluorescence intensity that obtains in advance and nature controlling line time resolved fluorescence intensity and the relation curve of aflatoxin M 1 concentration, obtain the content of aflatoxin M 1 in the analyte sample fluid.
Press such scheme, the ratio (T/C) of described fluorescent test paper strip detection line time resolved fluorescence intensity and nature controlling line time resolved fluorescence intensity adopts following method 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 above-mentioned each concentration joins respectively in the example reaction bottle in right amount, mixing, insert fluorescent test paper strip, 37 ℃ were reacted 6 minutes, differentiate fluorescence immunity analyzer with the time and detect the time resolved fluorescence intensity level that obtains detection line on each fluorescent test paper strip (T) and nature controlling line (C), 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 thus;
(3) 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 through match.
Hybridoma cell strain AFM1B7 among the present invention adopts two step screening method to obtain, its concrete steps are: with 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 preceding immunizing dose, carry out cytogamy after 3 days, treat 2-3 week after the cytogamy, with micropipet the individual cells colony is moved to 96 porocyte culture plates and adopt the liquid amplification culture, carry out clone's first time, adopt the ELISA method to screen fused cell in two steps: 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; Second step adopted the indirect competitive ELISA method that the positive hole nutrient solution that the first step filters out is detected, former with aflatoxin M 1 as competition, select light absorption value and all higher hole of sensitivity, adopt limiting dilution assay to carry out subclone, adopt same two step screening method to detect behind the subclone, behind the repetition subclone like this 2-3 time, final screening obtains hybridoma cell strain AFM1B7.
MONOCLONAL ANTIBODIES SPECIFIC FOR method among the present invention, step is as follows: the hybridoma cell strain AFM1B7 that obtains is expelled in advance the belly of the BALB/c mouse of handling with freund 's incomplete adjuvant, collects the ascites of this mouse, purifying promptly.
Described purification process is sad-ammonium sulfate method, concrete operations 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, stir and slowly add n-caprylic acid down, every milliliter of required n-caprylic acid volume of ascites is 30～35 μ L, mixed at room temperature 30～60min, 4 ℃ leave standstill more than the 2h, 4 ℃ then, more than the centrifugal 30min of 12000r/min, abandon precipitation, with the supernatant liquor that obtains with double-deck filter paper filtering after, the volumetric molar concentration that adds 1/10 filtrate volume is 0.1mol/L, the pH value is 7.4 phosphate buffered saline buffer, regulate the pH value to 7.4 of this mixed solution with the sodium hydroxide solution of 2mol/L, 4 ℃ of precoolings, slowly adding ammonium sulfate to ammonium sulfate final concentration is 0.277g/mL, 4 ℃ leave standstill more than the 2h, 4 ℃ then, more than the centrifugal 30min of 12000r/min, abandon supernatant, with the 0.01mol/L of gained precipitation with former ascites volume 1/10, the pH value is that 7.4 phosphate buffered saline buffer is resuspended, 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, use the freeze drier freeze-drying afterwards, collect lyophilized powder, promptly get the good monoclonal antibody of purifying, antibody is put in-20 ℃ of refrigerators standby;
Described acetate buffer is the 0.29g sodium-acetate, and 0.141mL acetic acid adds the water constant volume to the 100mL gained; The phosphate buffered saline buffer of described 0.01mol/L is a 0.8g sodium-chlor, the 0.29g disodium hydrogen phosphate, and 0.02g Repone K, potassium primary phosphate 0.02g adds the water constant volume to the 100mL gained; The phosphate buffered saline buffer of described 0.1mol/L is a 8g sodium-chlor, the 2.9g disodium hydrogen phosphate, and 0.2g Repone K, potassium primary phosphate 0.2g adds the water constant volume to the 100mL gained.
Beneficial effect of the present invention:
(1) hybridoma cell strain AFM1B7 provided by the invention can be used to prepare the high monoclonal antibody of tiring, and tiring that mouse ascites fluid antibody ELISA immuning adsorpting analysis (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
50Be 52pg/mL, with aflatoxin B1, B2, G1, G2, vomitoxin, zearalenone, the cross reacting rate of fumonisin is all less than 0.3%.
(4) the aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind of Monoclonal Antibody provided by the invention can be used for the quantitative assay of aflatoxin M 1 content, and simple to operate, fast, and the accuracy height.
Description of drawings
Fig. 1 is the structural representation of fluorescent test paper strip in the aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind provided by the invention.Among the 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
6 of purchase BALB/c mouse in 6 age in week, the aflatoxin M 1 complete A antigen FM1-BSA that immunity is commercially available.Immunity is with after aflatoxin M 1 complete antigen and the emulsification of isopyknic Fu Shi Freund's complete adjuvant, in the subcutaneous multi-point injection in mouse carotid back for the first time.Carry out after for the second time being immune to for 4 weeks, adopt freund 's incomplete adjuvant and the 1 complete antigen emulsification of isopyknic aflatoxin M, inject in mouse peritoneal.Immunity for the third time and immunity for the second time be 4 weeks at interval, and immunization ways is identical with it, carry out after being immune to immune for the third time 3 weeks the 4th time, and immunization ways is immune identical with the second time, is similarly abdominal injection.4 times immunizing dose is identical, is every mouse 80 μ g.3 times each immunity back 8～10 days, tail vein blood, separation of serum adopts indirect elisa method monitoring mice serum to tire.Back 8 days of the 4th immunity, tail vein blood, separation of serum, adopt indirect elisa method monitoring mice serum to tire, and measure mice serum sensitivity with the indirect competitive ELISA method, the mouse of the serum correspondence that selection is tired, sensitivity is all higher relatively carries out last booster immunization, and immunizing dose is 2 times of front.
Aflatoxin M 1 complete A antigen FM1-BSA purchases the company in Sigma-Aldrich.
2. cytogamy
In last booster immunization after 3 days, adopt the 50%(weight percentage) polyoxyethylene glycol be that the PEG(molecular weight is 1450) make fusogen, carry out cytogamy according to a conventional method, concrete steps: kill immune mouse under the aseptic condition, separating Morr. cell, with mouse source myeloma cell SP2/0 with the number of 5 ︰ 1 than mixing, wash cell mixing with the RPMI-1640 basic culture solution, merge with 50%PEG, merged 1 minute, and slowly added the RPMI-1640 basic culture solution then, centrifugal, remove supernatant, the fused cell that mouse boosting cell and mouse source myeloma cell SP2/0 form is resuspended with the cell perfect medium that 20mL contains 1%HAT, and the cell that has hanged is joined in the 80mL semisolid medium, is added to behind the mixing on the 6 porocyte culture plates, 1.5mL/ the hole places 37 ℃ of CO2gas incubator to cultivate.
The cell perfect medium of the described 1%HAT of containing contains the 20%(percent by volume) foetal calf serum, the 75%(percent by volume) RPMI-1640 basic culture solution, the 1%(weight percentage) L-glutaminate, the 1%(percent by volume) HEPES, the 1%(percent by volume) two anti-(the every ml penicillin of 10000 units 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 the 1%(mass percent) the cell perfect medium of methylcellulose gum;
RPMI-1640 basic culture solution, HEPES, two anti-, L-glutaminate are purchased the company in Hyclone; 1% xanthoglobulin-aminopterin-thymidine is that HAT, methylcellulose gum are purchased the company in Sigma-Aldrich.
3. the screening of cell strain and clone
Treat 2-3 week after the cytogamy, cell colony is long to people's naked eyes when visible, to clone from this substratum with micropipet and to draw, move to 96 porocyte culture plates and adopt the liquid amplification culture, every hole moves into 1 clone, treat that cell grows at the bottom of the full hole at 1/2～2/3 o'clock, draw culture supernatant and carry out positive detection, promptly carry out antibody test.Adopt the ELISA method that the culture hole that the hybridoma growth is arranged is screened, screening is carried out in two steps, and the first step adopts indirect elisa method to filter out aspergillus flavus resisting toxin M1 and the positive hole of not anti-carrier proteins BSA; Second step adopted the indirect competitive ELISA method that the positive hole that the first step filters out is detected, former with aflatoxin M 1 as competition, (the higher finger competition of light absorption value was that 0 hole is that the final measured value in positive control hole is higher originally, and the higher finger inhibiting rate of sensitivity is 50% o'clock competition original content that is IC to select all higher hole of light absorption value and sensitivity
50Be worth less), adopt limiting dilution assay to carry out subclone, adopt same two-step approach to detect behind the subclone, so repeat subclone 2-3 time after, acquisition hybridoma cell strain AFM1B7.
Embodiment 2: hybridoma cell strain AFM1B7 antibody variable region sequencing
(1) extracts total RNA: adopt day total RNA extraction reagent box of root company and extract total RNA that can produce hybridoma cell strain AFM1B7 to specifications;
(2) synthetic cDNA: the total RNA that obtains with step 1 is a template, oligo (dT)
15Be primer, carry out reverse transcription, synthetic cDNA first chain according to SuperScriptTM-2 II ThermoScript II specification sheets; Primer oligo (dT) 15 is buied 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, 58 ℃ of 1min, 72 ℃ of 1min, and 30 circulations of increasing, last 72 ℃ are extended 10min.PCR product process 1%(weight percentage) after agarose gel electrophoresis separates, reclaim dna fragmentation with the test kit purifying, be connected among the carrier pMD18-T transformed into escherichia coli DH5 α competent cell, the picking positive colony is delivered to Shanghai Sani's bio tech ltd and is checked order.Wherein the sequence of primer is respectively: the variable region of heavy chain primer is 5,-AGG TSM ARC TGC AGS AGT CWG G-3, (22mer) with 5,-TGA GGA GAC GGT GAC CGT GGT CCC TTG GCC CC-3, (32mer) wherein S, M, R and W are the merger base, M=A/C, R=A/G, S=C/G, W=A/T, variable region of light chain primer are 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 who obtains: the long 339bp 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 113 amino acid, sequence is shown in SEQ ID NO:3.The long 318bp 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 106 amino acid shown in SEQ ID NO:2, and sequence is shown in SEQ IDNO:4.
Embodiment 3: MONOCLONAL ANTIBODIES SPECIFIC FOR purifying, hypotype and CHARACTERISTICS IDENTIFICATION
The hybridoma cell strain that embodiment 1 is obtained is the BALB/c mouse that the AFM1B7 injection was handled with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt sad-ammonium sulfate method antibody purification, concrete operations are: with double-deck filter paper filtering mouse ascites, 4 ℃, the centrifugal 15min of 12000r/min, draw supernatant, gained ascites supernatant is mixed with the acetate buffer of 4 times of volumes, stir and slowly add n-caprylic acid down, every milliliter of required n-caprylic acid volume of ascites is 33 μ L, mixed at room temperature 30min, 4 ℃ leave standstill 2h, 4 ℃ then, the centrifugal 30min of 12000r/min, abandon precipitation, with the supernatant liquor 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 2mol/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, 4 ℃ then, the centrifugal 30min of 12000r/min abandons supernatant, with the 0.01mol/L of gained precipitation with former ascites volume 1/10, the pH value is that 7.4 phosphate buffered saline buffers are resuspended, 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, use the freeze drier freeze-drying afterwards, collect lyophilized powder, promptly get the good monoclonal antibody of purifying, antibody is placed-20 ℃ of refrigerators standby;
Described acetate buffer is the 0.29g sodium-acetate, and 0.141mL acetic acid adds water and is settled to the 100mL gained; The phosphate buffered saline buffer of described 0.01mol/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; The phosphate buffered saline buffer of described 0.1mol/L is a 8g sodium-chlor, the 2.9g disodium hydrogen phosphate, and 0.2g Repone K, potassium primary phosphate 0.2g adds the water constant volume to the 100mL gained.
The hypotype of identifying hybridoma cell strain AFM1B7 excretory monoclonal antibody with commercially available hypotype identification kit is IgG1.
The tiring of BALB/c mouse ascites antibody that records injection AFM1B7 hybridoma cell strain with the non-competing Enzyme Linked Immunoadsorbent Assay of routine (ELISA) method can reach 2.2 * 10
5, promptly the mouse ascites antibody dilution 2.2 * 10
5Times the time measured in solution result positive.Identify its 50% inhibition concentration IC with conventional indirect competitive ELISA method to aflatoxin M 1
50Be 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 aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind,
It comprises fluorescent test paper strip, contains 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 of 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, adjacent each pad overlaps in the junction and connects, 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 base wad with the nitrocellulose filter, horizontal nature controlling line and detection line are set on the nitrocellulose filter from top to bottom, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, its package amount is the 40ng/cm nature controlling line, bag is by aflatoxin B1-bovine serum albumin conjugate (AFB1-BSA) on the described detection line, its package amount is the 65ng/cm detection line, and the spacing of detection line and nitrocellulose filter upper 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 growth 15mm, and the specification of wide 4mm promptly gets absorbent pad;
(2) preparation of detecting pad
The bag quilt of detection line:
The conjugate AFM1-BSA of aflatoxin M 1-bovine serum albumin is cushioned liquid with bag is mixed with the coating buffer that concentration is 0.15mg/mL; In the position of distance nitrocellulose filter upper edge 15mm, with line spray mode with its laterally bag obtained detection line by on nitrocellulose filter, the package amount of every centimetre of required AFM1-BSA of detection line is 65ng, under 37 ℃ of conditions dry 30 minutes then;
Described bag is cushioned liquid: the 0.1g bovine serum albumin, and the 0.002g sodiumazide, 0.08g sodium-chlor, the 0.029g disodium hydrogen phosphate, 0.002g Repone K, the 0.002g potassium primary phosphate adds the water constant volume to the 10mL gained;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is cushioned liquid with bag is made into the coating buffer that concentration is 0.1mg/mL; In the position of distance detection line 6mm, with line spray mode with its laterally bag by on nitrocellulose filter, nature controlling line, the package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 40ng, under 37 ℃ of conditions dry 1 hour then;
It is the 0.002g sodiumazide that described bag is cushioned liquid, 0.08g sodium-chlor, and the 0.029g disodium hydrogen phosphate, 0.002g Repone K, the 0.002g potassium primary phosphate adds water and is settled to the 10mL gained;
(3) preparation of sample pad:
Glass fibre membrane is cut out growth 13mm, and the specification of wide 4mm is put into confining liquid and is soaked, and takes out, and drying is 6 hours under 37 ℃ of conditions, gets sample pad, puts room temperature preservation in the moisture eliminator then;
Described confining liquid is the 1g oralbumin, 2g 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) 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, and adjacent each pad overlaps in the junction and connects, and overlapping length is 2mm, promptly gets the 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 the foregoing description 3, behind the carbonate buffer solution repetitive scrubbing of 100mmol/L pH9.3 6 times,, spend the night in 4 ℃ with itself and the abundant mixing of 0.5mg europium labelled reagent.Then it is joined in the Sephadex G-50 chromatography column of 1.9cm * 60cm, with the 50mmol/L Tris-HCl elutriant wash-out that contains 0.9%NaCl, collect effluent liquid (1ml/ pipe), measure light absorption value (A280nm) by pipe, merge the peak pipe, obtain the aspergillus flavus resisting toxin M1 monoclonal antibody of target product europium mark.Above-mentioned europium labelled reagent is purchased in Shanghai Uni Bio-Tech. Co., Ltd., but is not limited thereto.
The acquisition of the example reaction bottle of the described aspergillus flavus resisting toxin M1 monoclonal antibody lyophilized powder that contains the europium mark: the aspergillus flavus resisting toxin M1 monoclonal antibody 0.05 μ g that gets above-mentioned europium mark is put in the 3mL bayonet socket bottle, after adopting the conventional freezing vacuum drying method to drain, both got the aspergillus flavus resisting toxin M1 monoclonal antibody lyophilized powder of europium mark, 4 ℃ of preservations, standby.
Described sample diluting liquid is: volume fraction is 0.01% the tween 20 aqueous solution.
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 through high performance liquid chromatography (HPLC) to the milk sample of aflatoxin M 1 feminine gender carries out aflatoxin M 1 mark-on, join 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 each 200 μ L of aflatoxin M 1 standard solution of above-mentioned each concentration, join respectively in the example reaction bottle, mixing, insert fluorescent test paper strip, 37 ℃ were reacted 6 minutes, blot the sample pad residual liquid with thieving paper, at once differentiate fluorescence immunity analyzer with the time and detect (excitation wavelength: 365nm, measure wavelength: 615nm), obtain the time resolved fluorescence intensity level that detection line place (T) and nature controlling line (C) locate on each fluorescent test paper strip, 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 thus;
(3) be X-coordinate with AFM1 concentration, the aflatoxin M 1 standard solution relevant detection line time resolved fluorescence intensity/nature controlling line time resolved fluorescence intensity of each concentration is that the T/C value is ordinate zou, and match 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 this method is 0.05～2ng/mL.
The detection of aflatoxin M 1 content in the II milk sample:
Get 5 parts of milk samples to be measured, each 200 μ L adds in the example reaction bottle, mixing, insert fluorescent test paper strip, 37 ℃ of reactions are after 6 minutes, blot the sample pad residual liquid with thieving paper, differentiate fluorescence immunity analyzer with the time immediately and detect (excitation wavelength: 365nm, 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, with the ratio (T/C) of above-mentioned fluorescent test paper strip detection line time resolved fluorescence intensity that obtains of its substitution and nature controlling line time resolved fluorescence intensity and the relation curve of aflatoxin M 1 concentration, get: the detected result of 5 milk samples is followed successively by:＜0.05ng/mL then, 0.12ng/mL,＜0.05ng/mL, 1.2ng/mL and 0.83ng/mL.
This method detected result and ELISA detected result are compared: this method detected result is consistent with national standard efficient liquid-phase chromatography method detected result height, and coincidence rate is up to 97.5%; In addition, this method single sample detects required time and also only is 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 the 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 aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind, it comprises fluorescent test paper strip, contains 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 of 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, adjacent each pad overlaps in the junction and connects, 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 base wad with the nitrocellulose filter, horizontal nature controlling line and detection line are set on the nitrocellulose filter from top to bottom, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, its package amount is the 100ng/cm nature controlling line, bag is by aflatoxin B1-bovine serum albumin conjugate (AFB1-BSA) on the described detection line, its package amount is the 100ng/cm detection line, and the spacing of detection line and nitrocellulose filter upper 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 growth 18mm, and the specification of wide 3mm promptly gets absorbent pad;
(2) preparation of detecting pad
The bag quilt of detection line:
The conjugate AFM1-BSA of aflatoxin M 1-bovine serum albumin is cushioned liquid with bag is mixed with the coating buffer that concentration is 0.4mg/mL; In the position of distance nitrocellulose filter upper edge 20mm, with line spray mode with its laterally bag obtained detection line by on nitrocellulose filter, the package amount of every centimetre of required AFM1-BSA of detection line is 100ng, under 40 ℃ of conditions dry 30 minutes then;
Described bag is cushioned liquid: the 0.1g bovine serum albumin, and the 0.002g sodiumazide, 0.08g sodium-chlor, the 0.029g disodium hydrogen phosphate, 0.002g Repone K, the 0.002g potassium primary phosphate adds the water constant volume to the 10mL gained;
The bag quilt of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is cushioned liquid with bag is made into the coating buffer that concentration is 0.4mg/mL; In the position of distance detection line 6mm, with line spray mode with its laterally bag by on nitrocellulose filter, nature controlling line, the package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 40ng, then dry 30min under 40 ℃ of conditions;
It is the 0.002g sodiumazide that described bag is cushioned liquid, 0.08g sodium-chlor, and the 0.029g disodium hydrogen phosphate, 0.002g Repone K, the 0.002g potassium primary phosphate adds water and is settled to the 10mL gained;
The long 28mm of described nitrocellulose filter, wide 3mm.
(3) preparation of sample pad:
Glass fibre membrane is cut out growth 15mm, and the specification of wide 3mm is put into confining liquid and is soaked, and takes out, and drying is 6 hours under 37 ℃ of conditions, gets sample pad, puts room temperature preservation in the moisture eliminator then;
Described confining liquid is the 1g oralbumin, 2g 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) 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, and adjacent each pad overlaps in the junction and connects, and overlapping length is 2mm, promptly gets the 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 the foregoing description 3, behind the carbonate buffer solution repetitive scrubbing of 100mmol/L pH9.3 6 times,, spend the night in 4 ℃ with itself and the abundant mixing of 2mg europium labelled reagent.Then it is joined in the Sephadex G-50 chromatography column of 1.9cm * 60cm, with the 50mmol/L Tris-HCl elutriant wash-out that contains 0.9%NaCl, collect effluent liquid (1ml/ pipe), measure light absorption value (A280nm) by pipe, merge the peak pipe, obtain the aspergillus flavus resisting toxin M1 monoclonal antibody of target product europium mark.Above-mentioned europium labelled reagent is purchased in Shanghai Uni Bio-Tech. Co., Ltd., but is not limited thereto.
The acquisition of the example reaction bottle of the described aspergillus flavus resisting toxin M1 monoclonal antibody lyophilized powder that contains the europium mark: the aspergillus flavus resisting toxin M1 monoclonal antibody 0.25 μ g that gets above-mentioned europium mark is put in the 3mL bayonet socket bottle, after adopting the conventional freezing vacuum drying method to drain, both got the aspergillus flavus resisting toxin M1 monoclonal antibody lyophilized powder of europium mark, 4 ℃ of preservations, standby.
Described sample diluting liquid is: volume fraction is 0.30% the tween 20 aqueous solution.
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 the example reaction bottle, mixing, insert fluorescent test paper strip, 37 ℃ of reactions are after 6 minutes, blot the sample pad residual liquid with thieving paper, differentiate fluorescence immunity analyzer with the time immediately and detect (excitation wavelength: 365nm, 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, with the ratio (T/C) of above-mentioned fluorescent test paper strip detection line time resolved fluorescence intensity that obtains of its substitution and nature controlling line time resolved fluorescence intensity and the relation curve of aflatoxin M 1 concentration, get: the content of aflatoxin M 1 is 1ng/mL in this milk sample then.
Claims (10)
1. hybridoma cell strain AFM1B7, it is characterized in that: it is preserved in Chinese typical culture collection center (CCTCC), and deposit number is CCTCC NO.C201020.
2. monoclonal antibody is characterized in that: it is that the hybridoma cell strain AFM1B7 secretion of CCTCC NO.C201020 produces by deposit number.
3. the aflatoxin M 1 immune time resolved fluorescence quick testing reagent box that lags behind that flows, it is characterized in that: it comprises fluorescent test paper strip and contains the example reaction bottle of the aspergillus flavus resisting toxin M1 monoclonal antibody dried frozen aquatic products of 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, adjacent each pad overlaps in the junction and connects, described detecting pad is base wad with the nitrocellulose filter, horizontal nature controlling line and detection line are set on the nitrocellulose filter from top to bottom, described nature controlling line is coated with the anti-mouse polyclonal antibody of rabbit, and bag is by aflatoxin M 1-bovine serum albumin conjugate (AFB1-BSA) on the described detection line; Described aspergillus flavus resisting toxin M1 monoclonal antibody is that the described deposit number of claim 1 is the monoclonal antibody of the hybridoma cell strain AFM1B7 secretion generation of CCTCC NO.C201020.
4. the aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind 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: standing over night behind the abundant mixing of ratio that monoclonal anti body and function carbonate buffer solution dialysis back that the hybridoma cell strain AFM1B7 secretion that is CCTCC NO.C201020 with the described deposit number of claim 1 produces and europium labelled reagent with mass ratio are 0.5～2:1, the aspergillus flavus resisting toxin M1 monoclonal antibody of separating the europium mark then through Sephadex G-50 chromatography column, wash-out is collected target product.
5. the aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind according to claim 3 is characterized in that: the long 15～20mm of the absorbent pad in the 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 spacing of described detection line and nitrocellulose filter upper 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 the described fluorescent test paper strip on the detecting pad package amount of the required aflatoxin B1-bovine serum albumin conjugate of every centimetre of detection line be 50～100ng; The package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 30～100g; 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 in the described example reaction bottle.
7. the aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind according to claim 1, it is characterized in that: described fluorescent test paper strip is to adopt following method to obtain:
(1) thieving paper is cut out absorbent pad;
(2) preparation of detecting pad:
The conjugate AFM1-BSA of aflatoxin M-bovine serum albumin is mixed with the coating buffer that concentration is 0.1～0.4mg/mL, position in distance nitrocellulose filter upper edge 10～15mm, with line spray mode it is laterally wrapped by on nitrocellulose filter, 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, under 37～40 ℃ of conditions dry 30～60 minutes then;
The anti-mouse polyclonal antibody of rabbit is made into the coating buffer that concentration is 0.1～0.4mg/mL, position in distance detection line 5～10mm, with line spray mode it is laterally wrapped by on nitrocellulose filter, get nature controlling line, the package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 30～100ng, under 37～40 ℃ of conditions dry 30～60 minutes then;
(3) preparation of sample pad:
Glass fibre membrane is put into confining liquid soak, take out, drying is 3～6 hours under 37～40 ℃ of conditions, gets sample pad, puts room temperature preservation in the moisture eliminator then;
(4) assembling of fluorescent test paper strip:
One side at cardboard is pasted absorbent pad, detecting pad, sample pad from top to bottom successively, and adjacent each pad overlaps in the junction and connects, and overlapping length is 1～3mm, promptly gets fluorescent test paper strip.
8. the aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind according to claim 6, it is characterized in that: in the preparation of described fluorescent test paper strip in preparation aflatoxin M 1-bovine serum albumin conjugate (AFM1-BSA) coating buffer employed bag be cushioned liquid and be: contain bovine serum albumin 0.1g among every 10mL, sodiumazide 0.002g, sodium-chlor 0.08g, disodium hydrogen phosphate 0.029g, Repone K 0.002g, potassium primary phosphate 0.002g;
Employed bag is cushioned liquid and is in the preparation rabbit anti-mouse polyclonal antibody coating buffer: contain sodiumazide 0.002g among every 10mL, sodium-chlor 0.08g, disodium hydrogen phosphate 0.029g, Repone K 0.002g, potassium primary phosphate 0.002g;
The confining liquid that uses in the preparation of described fluorescent test paper strip is: contain oralbumin 0.5-2g among every 100mL, 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 aflatoxin M 1 mobile immune time resolved fluorescence quick testing reagent box that lags behind according to claim 1, it is characterized in that: it 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.
10. to require the 3 described aflatoxin Ms 1 mobile application of immune time resolved fluorescence quick testing reagent box in aflatoxin M 1 content detection that lag behind according to right, it is characterized in that: it is that analyte sample fluid is added in the example reaction bottle, mixing, insert fluorescent test paper strip, 37 ℃ of reactions are after 6 minutes, differentiate the fluorometric investigation instrument with the time and detect, the ratio of detection line time resolved fluorescence intensity level and nature controlling line time resolved fluorescence intensity level on the acquisition fluorescent test paper strip; Based on the time resolved fluorescence test strip detection 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 the analyte sample fluid.
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