CN103232975B - Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody - Google Patents
Hybridoma cell strain 2D3, monoclonal antibody to zearalenone secreted by same and application of monoclonal antibody Download PDFInfo
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Abstract
The invention provides a hybridoma cell strain 2D3, a monoclonal antibody to zearalenone secreted by the hybridoma cell strain 2D3 and application of the monoclonal antibody. The hybridoma cell strain 2D3 is preserved in China Center for Type Culture Collection with an accession number of CCTCC No. C201328 and can be used for preparation of a high-titer monoclonal antibody to zearalenone. According to detection results of enzyme linked immunosorbent assay (ELISA), the titer of the monoclonal antibody to zearalenone prepared through purification of mouse ascites can reach 1.5 * 10<5>. The monoclonal antibody to zearalenone has high sensitivity, half maximal inhibitory concentration IC50 of 20 pg/mL to zearalenone and cross reactivity of 4.9%, 3.3% and 3.2% with beta-zearalanel, alpha-zearalanel and beta-zeranol, respectively. The monoclonal antibody to zearalenone can be used for determination of the content of zearalenone.
Description
Technical field
The present invention relates to anti-zearalenone monoclonal antibody and the application thereof of hybridoma cell strain 2D3, its generation.
Background technology
Zearalenone (ZEA) is mainly produced by Fusarium graminearum, and first from have the corn of head blight, separation obtains.Zearalenone has estrogen-like effects, can cause animal acute and chronic poisoning, causes that Reproduction is extremely even dead, can cause tremendous economic loss to stock-farms.The edible various wheaten food of making containing Gibberella saubinetii flour also can cause the toxicity symptom of central nervous system, as feel sick, feel cold, headache, mind depression and ataxia etc.Zearalenone mainly pollutes the cereal such as corn, wheat, rice, barley, millet and oat.Wherein the positive rate of corn is 45%, and the positive rate of wheat is 20%.Zearalenone has residual and is difficult for destroyed property, if the food of domestic animal, the pollution of poultry Long term Animal use zearalenone, zearalenone can constantly be put aside in vivo, and deposition in animal body.If people has eaten the livestock product of this quality, also can cause the generation of some diseases.In order to ensure human consumer's food safety consumption, should develop effective detection technique, the agricultural-food such as edible corn, wheat and feed are strictly supervised with the zearalenone content in agricultural-food, the pollution condition of zearalenone is carried out to supervision and prevention and control, ensure people's food consumption safety.
The existing detection method to zearalenone comprises thin layer chromatography, precision instrument analytical method and immune analysis method.When thin layer chromatography detects zearalenone, do 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 comprises spectrophotofluorimetry and high performance liquid chromatography, and it is highly sensitive, and accuracy is good, but instrument is expensive, require the degree of purification of zearalenone sample high, sample pretreatment process is loaded down with trivial details, length consuming time, high to experimental situation requirement, be difficult to realize rapid detection.The immuno analytical method growing up has in recent years 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.Immunoassay is to utilize biology, physics or the chemical amplification of the marker on the specific association reaction of antigen and antibody and antibody, antigen to carry out qualitative and quantitative analysis to ultramicron residue, wherein, antibody is as immunoreactive core reagent, in order to set up the immunology detection technology for zearalenone, must first make the antibody of anti-zearalenone.
Summary of the invention
Problem to be solved by this invention is to provide anti-zearalenone monoclonal antibody and the application thereof of hybridoma cell strain 2D3, its generation.
The invention provides hybridoma cell strain 2D3, this hybridoma cell strain was preserved in Chinese Typical Representative culture collection center (CCTCC) on March 7th, 2013, and preservation address is, China, and Wuhan, Wuhan University, deposit number is CCTCC NO.C201328.It has in sequence table the anti-zearalenone monoclonal antibody variable region of light chain coding gene sequence shown in SEQ ID No.2 in the anti-zearalenone monoclonal antibody variable region of heavy chain coding gene sequence shown in SEQ ID No.1 and sequence table.
Anti-zearalenone monoclonal antibody, the hybridoma cell strain 2D3 secretion that it is CCTCC NO.C201328 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 in sequence table.This anti-zearalenone monoclonal antibody can be identified zearalenone, the 50% inhibition concentration IC to zearalenone
50for 20pg/mL.
The application of anti-zearalenone monoclonal antibody in zearalenone assay.
Hybridoma cell strain 2D3 provided by the invention adopts two step screening method to obtain, its concrete steps are: by BALB/c mouse after zearalenone complete antigen ZEA-BSA immunity 4-6 time, with 2 times of zearalenone complete antigen ZEA-BSA to a front immunizing dose, make last booster immunization, 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, then adopt ELISA method to screen in two steps fused cell: the first step adopts indirect elisa method to filter out anti-zearalenone 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 zearalenone, 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 2D3.
The preparation method of anti-zearalenone monoclonal antibody provided by the invention, step is as follows: the belly that the hybridoma cell strain 2D3 of above-mentioned acquisition is expelled to the BALB/c mouse of processing with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, purifying obtains anti-zearalenone monoclonal antibody.
Press such scheme, described purification process is caprylic acid-ammonium, concrete steps are: 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, with pure water, dialyse, the protein solution of fully having dialysed is put to-70 ℃ of refrigerator freezings, use afterwards freeze drier freeze-drying, collect lyophilized powder, obtain the anti-zearalenone 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 2D3 provided by the invention can be for the preparation of the anti-zearalenone monoclonal antibody of high-titer, and for antibody, enzyme linked immunosorbent assay analysis method (ELISA) records the purified anti-zearalenone making of mouse ascites fluid tires and can reach 1.5 * 10
5.
(2) anti-zearalenone monoclonal antibody provided by the invention is highly sensitive, the 50% inhibition concentration IC to zearalenone
50for 20pg/mL, with β-zearalenol, α-zearalenol, the cross reacting rate of β-ZER is respectively 84.9%, 3.3%, and 3.2%.
(3) anti-zearalenone monoclonal antibody provided by the invention can be applicable to the mensuration of zearalenone content.
Accompanying drawing explanation
Fig. 1 is the front view of the zearalenone immuno-chromatographic test paper strip prepared of application anti-zearalenone monoclonal antibody provided by the invention.
Fig. 2 is the side-view of the zearalenone immuno-chromatographic test paper strip prepared of application anti-zearalenone monoclonal antibody provided by the invention.
Fig. 3 applies the result process decision chart that zearalenone immuno-chromatographic test paper strip prepared by anti-zearalenone monoclonal antibody provided by the invention detects sample in embodiment 4.
In figure: 1 cardboard; 2 absorbent pad; 3 detecting pads; 4 gold medal mark pads; 5 sample pad; 6 nature controlling lines; 7 detection lines; 8 control stripes bars; 9 test strip.
Embodiment
Embodiment 1: the screening of hybridoma cell strain 2D3
1. animal immune
Buy 6 of BALB/c mouse in 6 week age, the zearalenone complete antigen ZEA-BSA that immunity is commercially available.Immunity is by after zearalenone 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.After being immune to for the second time 4 weeks, carry out, adopt freund 's incomplete adjuvant and the emulsification of isopyknic zearalenone complete antigen, 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 100 μ g.Latter 8 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.Zearalenone complete antigen ZEA-BSA is purchased from German aokin 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 5～8 ︰ 1 than mixing, with RPMI-1640 basic culture solution, wash cell mixing, with 50%PEG, merge, 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 (HFCS) 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-and L-glutaminate are purchased from Hyclone company; 1% xanthoglobulin-aminopterin-thymidine is that HAT and 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, with micropipet, clone is drawn from this substratum, 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 there being the culture hole of Growth of Hybridoma Cell to screen, screening is carried out in two steps, and the first step adopts indirect elisa method to filter out anti-zearalenone 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 zearalenone, 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 2D3.
Embodiment 2: anti-zearalenone monoclonal antibody hybridoma cell strain 2D3 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 obtain hybridoma cell strain 2D3;
(2) synthetic cDNA: total RNA that the step 1 of take obtains is template, oligo (dT)
15for primer, according to SuperScript
tM-2 II ThermoScript II specification sheetss carry out reverse transcription, synthetic cDNA the first chain; Primer oligo (dT)
15by Invitrogen, buied;
(3) PCR method clone variable region gene: according to the conservative site design primer of GENEBANK small mouse antibody gene sequence, take cDNA as masterplate amplification 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 separation 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 CTC ACC 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 315bp 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 105 amino acid, sequence is as shown in SEQ ID NO:3.The long 329bp 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 109 amino acid, and sequence is as shown in SEQ ID NO:4.
Embodiment 3: the preparation of anti-zearalenone monoclonal antibody, purifying, hypotype and CHARACTERISTICS IDENTIFICATION
The BALB/c mouse that the anti-zearalenone monoclonal antibody hybridoma cell strain 2D3 injection that embodiment 1 is obtained was processed with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, adopt caprylic acid-ammonium antibody purification, concrete operation step is: 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 buffer is resuspended, pack dialysis tubing into, pure water is dialysed, the protein solution of fully having dialysed is put to-70 ℃ of refrigerator freezings, use afterwards freeze drier freeze-drying, collect lyophilized powder, obtain the anti-zearalenone 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 0.1mol/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 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 hypotype of identifying the anti-zearalenone monoclonal antibody of hybridoma cell strain 2D3 secretion with commercially available hypotype identification kit is IgG2b.
By the BALB/c mouse ascites that the non-competing Enzyme Linked Immunoadsorbent Assay of routine (ELISA) method records injection hybridoma cell strain 2D3, purify the tiring of antibody obtaining and can reach 1.5 * 10
5, i.e. anti-zearalenone monoclonal antibody dilution 1.5 * 10
5times time measured in solution result positive.By conventional indirect competitive ELISA method, identify that it is 20pg/mL to the sensitivity of zearalenone, with β-zearalenol, α-zearalenol, the cross reaction of β-ZER is respectively 84.9%, 3.3%, and 3.2%.
Embodiment 4: antibody application
The anti-zearalenone monoclonal antibody of hybridoma cell strain 2D3 secretion is prepared to zearalenone immuno-chromatographic test paper strip, and for the detection of zearalenone content, its concrete preparation method comprises the following steps:
(1) preparation of absorbent pad
Thieving paper is cut out to the 15～20mm that grows up, and the specification of wide 3.4mm, obtains absorbent pad;
(2) preparation of detecting pad
Being coated with of detection line:
The conjugate ZEA-BSA of zearalenone-bovine serum albumin is mixed with to the coating buffer that concentration is 0.4mg/mL with coated damping fluid; In on nitrocellulose filter along the position of 15mm, by a spray mode, it is laterally coated on nitrocellulose filter, obtain detection line, the package amount of the conjugate of every centimetre of required zearalenone-bovine serum albumin of detection line is 200ng, then under 37 ℃ of conditions, is dried 30 minutes;
Being coated with of nature controlling line:
The anti-mouse polyclonal antibody of rabbit is made into the coating buffer that concentration is 0.25mg/mL with coated damping fluid; In the position apart from detection line 6mm, by a some spray mode, it is laterally coated on nitrocellulose filter, obtain nature controlling line, the package amount of the anti-mouse polyclonal antibody of rabbit that every centimetre of nature controlling line is required is 100ng, then under 37 ℃ of conditions dry 1 hour;
In the described every 10mL of coated damping fluid, contain bovine serum albumin 0.1g, sodium-chlor 0.08g, disodium hydrogen phosphate 0.029g, Repone K 0.002g, potassium primary phosphate 0.002g;
The long 25mm of described nitrocellulose filter, wide 3.4mm;
(3) preparation of sample pad:
Glass fibre membrane is cut out to the specification of the wide 3.4mm of growth 13mm, put into confining liquid A and soak, take out, under 37 ℃ of conditions, be dried 6 hours, obtain sample pad, then put room temperature preservation in moisture eliminator;
Described confining liquid A is 2g bovine serum albumin, 2.5g 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) preparation of gold mark pad:
Glass fibre membrane is cut out to the specification of the wide 3.4mm of growth 10mm, putting into confining liquid B soaks, take out, under 37 ℃ of conditions, be dried 6 hours, on dried glass fibre membrane, by some spray mode, the anti-zearalenone monoclonal antibody solution of nano gold mark is laterally sprayed, the anti-zearalenone monoclonal antibody of every centimetre of required nano gold mark of spraying length is 350ng, then vacuum lyophilization 2.5h, puts room temperature preservation in moisture eliminator;
Described confining liquid B is: 2g bovine serum albumin, 2.5g sucrose, 1.6775g sodium-chlor, 0.05g tween 20,0.3g polyvinylpyrrolidone, 0.02g sodiumazide, 0.29g disodium hydrogen phosphate, 0.02g Repone K, 0.02g potassium primary phosphate, adds water and is settled to 100mL gained;
The concrete marking method of the anti-zearalenone monoclonal antibody solution of described nano gold mark is: measure 50.0mL mass concentration and be 0.01% nano-Au solution, by 425 μ L0.1mol/L wet chemical regulator solution pH values; The anti-zearalenone monoclonal antibody aqueous solution that slowly adds 2.5mL0.1mg/mL under the state stirring, continues stirring reaction 30min; To add mass concentration be 10% Bovine Serum Albumin in Aqueous Solution to the whole mass concentration of bovine serum albumin be 1%, continue to stir 30min; After 4 ℃ of placement 2h, the centrifugal 15min of 1500r/min, gets supernatant liquor, abandons precipitation; By the centrifugal 30min of supernatant liquor 12000r/min, abandoning supernatant, adds the washing of 30.0mL mark to preserve liquid; Again with the centrifugal 30min of 12000r/min, abandoning supernatant, will precipitate that with mark washing, to preserve liquid resuspended, obtain 5.0mL enriched material, put 4 ℃ of refrigerators standby, and wherein the mass concentration of the anti-zearalenone monoclonal antibody solution of nano gold mark is 0.05mg/mL;
In described nano-Au solution, the particle diameter of nanometer gold is 15nm;
Described 0.1mol/L wet chemical is: 13.8g salt of wormwood is dissolved in pure water and is settled to 1000mL, 0.22 μ m membrane filtration gained; The anti-zearalenone monoclonal antibody of the described 0.1mg/mL aqueous solution is that the anti-zearalenone monoclonal antibody of 1mg is dissolved in 10mL pure water and makes; Described 10% Bovine Serum Albumin in Aqueous Solution is that 10g bovine serum albumin is dissolved in 100mL pure water, 0.22 μ m membrane filtration gained; Described mark washing is preserved liquid and is: 2.0g PEG-400, and 0.2g sodium azide, 0.1235g boric acid, pure water is settled to 1000mL, 0.22 μ m membrane filtration gained;
(5) assembling of test strip:
In the one side of cardboard, paste successively from top to bottom absorbent pad, detecting pad, gold mark pad and sample pad, adjacent each pad overlapping connection in junction, overlapping length is 1～2mm, obtains zearalenone immuno-chromatographic test paper strip, sees Fig. 1 and Fig. 2.
The application of above-mentioned zearalenone immuno-chromatographic test paper strip:
The processing of corn sample: get 20g1# and 2# corn sample to be measured and grind with shredder, after grinding, add 80mL70%(volume fraction) methanol-water, stirring reaction 2 minutes, make sample mix liquid, and this mixed solution is manually rocked 3 minutes, then use double-deck filter paper filtering, collect filtrate 2mL, add 4mL pure water dilution filtrate, mix, obtain 1# and 2# testing sample and detect liquid.Separately get the blank corn sample of known zearalenone and process through same, the white maize sample detection of having leisure liquid.
Zearalenone ELISA test strip corn sample: draw 1# and 2# testing sample and detect each 100 μ L of liquid, dropwise add respectively the sample pad of zearalenone immuno-chromatographic test paper strip, using it as test strip; Separately get 100 μ L and containing the blank corn sample of zearalenone, do not detect the sample pad that liquid dropwise adds another zearalenone immuno-chromatographic test paper strip, by its test strip in contrast, reading result after 15 minutes.
Detected result: the nature controlling line of control stripes bar and detection line all demonstrate red stripes; The nature controlling line of 1# sample detection test strip demonstrates red lines, and detection line does not develop the color, judge thus: its positive result, and the content that testing sample detects zearalenone in liquid is equal to or higher than 2ng/mL, see Fig. 3-1, then through convert in 1# corn sample the content of zearalenone be equal to or higher than 24ng/g.
The nature controlling line of 2# sample detection test strip demonstrates red lines, and detection line color is more shallow than contrast ELISA test strip line color, judge thus: its positive result, and the content that testing sample detects zearalenone in liquid is equal to or higher than 0.5ng/mL, and be less than 2ng/mL, see Fig. 3-2, then through convert in 2# corn sample to be measured zearalenone content be equal to or higher than 6ng/g, and be less than 24ng/g.
Claims (5)
1. hybridoma cell strain 2D3, is characterized in that: it is preserved in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO.C201328.
2. anti-zearalenone monoclonal antibody, is characterized in that: the hybridoma cell strain 2D3 secretion that it is CCTCC NO.C201328 by deposit number produces.
3. the application of anti-zearalenone monoclonal antibody claimed in claim 2 in zearalenone assay.
4. the preparation method of anti-zearalenone monoclonal antibody according to claim 2, it is characterized in that: the BALB/c mouse that hybridoma cell strain 2D3 injection claimed in claim 1 was processed with freund 's incomplete adjuvant in advance, collect the ascites of this mouse, purifying obtains anti-zearalenone monoclonal antibody.
5. the preparation method of anti-zearalenone monoclonal antibody according to claim 4, it is characterized in that: described purification process is caprylic acid-ammonium, concrete steps are: 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, with pure water, dialyse, the protein solution of fully having dialysed is put to-70 ℃ of refrigerator freezings, use afterwards freeze drier freeze-drying, collect lyophilized powder, obtain the anti-zearalenone 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.
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| CN106950375B (en) * | 2017-03-03 | 2018-08-07 | 重庆市畜牧科学院 | A kind of zearalenone monoclonal antibody and its application |
| CN106970223B (en) * | 2017-03-07 | 2018-12-11 | 中国农业科学院油料作物研究所 | Purify five kinds of mycotoxin immunosorbent and the compound affinity columns such as fumonisin B1, aflatoxin B1 |
| CN110396126A (en) * | 2019-07-23 | 2019-11-01 | 山东绿都生物科技有限公司 | A kind of monoclonal antibody and its application identifying zearalenone |
| CN114214288B (en) * | 2021-12-24 | 2023-09-01 | 江南大学 | Zearalenone monoclonal antibody hybridoma cell strain and application thereof |
| CN114958774B (en) * | 2022-05-08 | 2023-10-27 | 中国医学科学院医学生物学研究所 | Anti-rabies virus monoclonal antibody, hybridoma cell strain secreting antibody and application |
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