CN102841203A - Competitive ELISA (enzyme-linked immuno sorbent assay) non-toxic detection method for zearalenone (ZEN) - Google Patents
Competitive ELISA (enzyme-linked immuno sorbent assay) non-toxic detection method for zearalenone (ZEN) Download PDFInfo
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Abstract
The invention discloses a competitive ELISA (enzyme-linked immuno sorbent assay) non-toxic detection method for zearalenone. The method disclosed by the invention is a competitive ELISA method built by ZEN-BSA (zearalenone-bovine serum albumin), a ZEN monoclonal antibody and a beta-anti-idiotype monoclonal antibody of ZEN. A ZEN standard substance is replaced by the beta-anti-idiotype monoclonal antibody of ZEN, so as to avoid the damage of toxin to an operator and the possibility of scattering toxin to the environment, and non-toxic detection is achieved. According to the method, the ZEN standard substance is not needed, so that the cost can be greatly reduced; and zearalenone polluted by foods, grains, feed and the like can be rapidly and sensitively detected at a large scale, therefore, the method has a good market prospect.
Description
Technical field
The present invention relates to the compound test field, the competitive ELISA that is specifically related to a kind of zearalenone does not have virus detection method.
Background technology
Zearalenone (Zenralenone; ZEN) be the mycotoxin of the kind estrogen-like that produces by multiple sickle-like bacteria; Pollution range is extensive; The cereals raw material in the field, results process, results backs storage period and feed and many links such as food products storage, use, all might receive the pollution of ZEN.ZEN has than Johnson & Johnson and grows toxicity and teratogenesis, can cause that animal is taken place by the hyperfunction disease of estrogen, causes the infertile or miscarriage of animal, pig, poultry, ruminant is all influenced bigger, brings very large economy loss to animal husbandry; ZEN also has cytotoxicity, immunotoxicity, hepatotoxicity wind agitation, potential carcinogenicity etc., and ZEN is difficult for removing the serious harm human health to high-temperature stable.
In view of zearalenone has stronger toxic action to human and other animals, JECFA in 2000 has announced that human interim maximum intake for ZEN is 40 μ g/ kg body weight.By 2004, limited the content of ZEN in food and feed existing 27 countries, and this number is also among continuation increases.It is 100,75 and 50 μ g/ kg that the ZEN maximum that EEC 253/ 2004 rules that on July 1st, 2006, European Union implemented have clearly been segmented the undressed cereal, flour, fast food and the breakfast food that do not comprise corn is limited the quantity of, and has also stipulated be that the infant food ZEN maximum of principal ingredient limits the quantity of with the treating grain to be 20 μ g/ kg.The ZEN maximum level is 500 μ g/kg in China's forage health standard regulation in 2006 corn class feed, and grain hygienic standard regulation ZEN is no more than 60 μ g/kg.
At present, immunologic detection method has become one of important method of food security fast detecting, in food safety detection, has brought into play vital role.ZEN is a kind of micromolecule toxin; The immunological method that is used for the residual detection by quantitative of ZEN at present need use the ZEN standard items; The use of ZEN standard items not only brings harm to operating personnel; Simultaneously also increased the discharging of poisonous and harmful substance in the environment, therefore, the nontoxic detection that the substitute of seeking ZEN is used for ZEN has important practical significance.β type anti-idiotype (β-Anti-idiotype antibody, β-AId or Ab2 β) has " interior image " effect with initial (partly) antigen same antigen determinant, can substitute small-molecule substance.
Summary of the invention
The objective of the invention is to according to the above-mentioned deficiency that exists in the prior art, utilize the β type antiidiotype monoclonal antibody of ZEN to substitute the ZEN standard items, provide a kind of zearalenone competitive ELISA not have virus detection method and application thereof.
The present invention realizes above-mentioned purpose through following technical scheme:
A kind of zearalenone competitive ELISA of the present invention does not have virus detection method; It is to encapsulate 96 hole ELISA Plates to detect antigen ZEN-BSA; The β type antiidiotype monoclonal antibody (the standard items competition thing of Ab2 β-1D5) or testing sample (be suspected to be food that ZEN pollutes, cereal, feed etc. handle sample) that adds ZEN; Add ZEN monoclonal antibody (Ab1-1G4) again; Detect antigen ZEN-BSA and combine the ZEN monoclonal antibody with free Ab2 β-1D5 or free ZEN competition, Ab2 β-1D5 and Ab1-1G4-Ab2 β-1D5 compound or ZEN and Ab1-1G4-ZEN compound that flush away dissociates, the Ab1-1G4 that combines with envelope antigen ZEN-BSA close with two resistive connections of enzyme labeling again; Cessation reaction after the substrate colour developing is with the absorbance (OD) in each hole of ELIASA mensuration.The OD value is big more, and ZEN content is few more freely in the sample, the Regression Equations of reference standards gained, the content of ZEN in the calculation sample.The characteristic of this method is to have following testing process and step:
1. the preparation of ZEN monoclonal antibody (Ab1-1G4)
Adopt active ester method that ZEN and OVA coupling are prepared immunizing antigen; ZEN and BSA coupling preparation detect antigen, immune BALB/c mouse, Fusion of Cells; Obtain hybridoma through the screening back, prepare ascitic type ZEN monoclonal antibody in a large number through inducing method in the mouse body; The hybridoma 1G4G10D3 of secretion ZEN monoclonal antibody is preserved in Chinese typical culture collection center, deposit number CCTCC NO:C201237 on April 5th, 2012.
2. the ZEN antiidiotype monoclonal antibody (preparation of Ab2 β-1D5)
ZEN monoclonal antibody and KLH coupling, conjugate immunity BALB/c mouse, through serum tire with sensitivity detect qualified after; Carry out the conjugate lumbar injection again; Get mouse boosting cell after 3 days, merge, select the medium culture fused cell with HAT or HT with the myeloma cell; Adopt indirect ELISA and indirect competitive ELISA method screening positive clone simultaneously, obtain hybridoma cell strain 1D5; Prepare ascitic type ZEN antiidiotype monoclonal antibody in a large number through inducing method in the mouse body; Hybridoma cell strain 1D5 is preserved in Chinese typical culture collection center, deposit number CCTCC NO:C201238 on April 5th, 2012.
3. the purifying of ascitic type ZEN monoclonal antibody, ZEN antiidiotype monoclonal antibody
Ascites is carried out purifying with Protein G affinity column again after the saturated ammonium sulphate method is just pure.
(1) saturated ammonium sulfate is slightly pure
1. from-20 ℃ of refrigerators, get 6.5 mL ascites, 4 ℃ slowly melt, and the centrifugal 30min of 12000r/min gets supernatant;
2. under ice bath stirs, dropwise add 6.5 mL saturated ammonium sulfate solution, 4 ℃ of placements are spent the night;
3. the centrifugal 30min of 7500 r/min abandons supernatant, and deposition is with the dissolving of 6.5 mL 0.1M binding buffer liquid (20mM phosphate buffer, pH 7.0), mixing;
With the bag filter that above-mentioned antibody-solutions is packed into and handled, in the chromatography cabinet, more than the binding buffer liquid dialysis 6h, every at a distance from dislysate of 2h replacing.
(2) processing before the upper prop
Antibody is with 5 times of 20mM binding buffer liquid dilutions, with going up Protein G post again after the 0.45 μ m filter membrane ultrafiltration.
(3) dress post and post excessively
1. adorn post: the positioned vertical cylinder, connect the constant current pump line, and fill pillar with binding buffer liquid, carry out the balance pillar, approximately 5-10 times of column volume after balance finishes returns to zero the nucleic acid-protein detector;
2. go up appearance: use the constant flow pump sample introduction, sample is pumped into pillar, note not having bubble during sample introduction;
3. clean: clean pillar to nucleic acid-protein detector numerical value with about 10 times of column volume binding buffer liquid and no longer change;
4. wash-out: with 5 times of column volume elution buffers (0.1M Gly-HCl, pH 2.7) wash-out, the peak of this moment is the immune globulin white peak; Collect the purpose product, treat to collect when nucleic acid-protein detector numerical value begins to change, treat that numerical value is reduced to and stop to collect when minimum; And in collection process, ceaselessly add an amount of neutralizer (60-200uL; 1M Tris-HCl, pH 9.0), pH is transferred to 7.0;
5. balance: with about 10 times of column volume binding buffer liquid balance pillars;
6. clean pillar with about 5 times of column volume 20% ethanol, build pillar two lid, be stored in 4 ℃ of refrigerators;
7. concentrate the ultrafiltration pipe of the antibody of collection with 50kD, 4 ℃, the 5000g centrifugal ultrafiltration;
8. with the PBS about 1ml antibody is swept away from the ultrafiltration pipe, packing is stored in-20 ℃.The sample that takes a morsel carries out the mensuration of AC and purity.
(4) mensuration of antibody purification concentration
Application of B CA
TMThe concentration of protein quantification kit measurement antibody.
(5) evaluation of antibody purification purity
Using SDS-PAGE identifies the ascites purification effect.
4. the foundation of correlativity between ZEN toxin and ZEN antiidiotype monoclonal antibody
The standard that adopts indirect competitive ELISA to set up ZEN and Ab2 β-1D5 respectively suppresses curve
1. encapsulate damping fluid ZEN-BSA is diluted to 1 μ g/mL, 3 h are hatched for 37 ℃ in 100 μ L/ holes;
2. abandon reactant liquor, 300 μ L PBST wash plate 3 times, 3 min/ time;
3. add the sealing of 200 μ L, 5% skimmed milk power, hatch 1 h for 37 ℃;
4. abandon reactant liquor, 300 μ L PBST wash plate 3 times, 3 min/ time;
5. add the ZEN standard items (0-50ng) or the variable concentrations Ab2 β-1D5 (0-2.48 μ g) of 50 μ L variable concentrations, adding 50 μ L concentration simultaneously is the ZEN monoclonal antibody of 0.025 μ g/mL, hatches 1 h for 37 ℃;
6. abandon reactant liquor, 300 μ L PBST wash plate 3 times, 3 min/ time;
7. add the HRP mark sheep anti-mouse igg of 100 μ L 1:4000 dilution, hatch 40 min for 37 ℃;
8. abandon reactant liquor, 300 μ L PBST wash plate 5 times, 3 min/ time;
9. add 100 μ L tmb substrates, lucifuge 10 min that develop the color;
10. add 50 μ L 2M sulfuric acid cessation reactions, ELIASA is measured the OD450 value.
The standard of drawing ZEN and AId-1D5 according to experimental result suppresses curve and corresponding linear regression equation.While, between 20%-80%, under identical inhibiting rate condition, concentration corresponding relation between ZEN and anti-ZEN antiidiotype monoclonal antibody was drawn alternate standard curve and corresponding equation of linear regression, as quantitative reduction formula according to inhibiting rate.
5. encapsulate the preparation of bar in advance:
(1) encapsulate: with encapsulating damping fluid is that the 0.05M sodium carbonate buffer of pH9.6 will detect antigen ZEN-BSA and be diluted to 1 μ g/mL, and every hole 100 μ L join in the polystyrene micropore plate, hatch 3h for 37 ℃;
(2) washing: pour out encapsulate damping fluid in the micropore plate hole after, with wash bottle or hyperchannel pipettor 200 μ l cleansing solutions are added in the hand-hole; Pour out the liquid in the hole after 3～5 minutes once more, remove the liquid in the hole fully; Wash 3 ~ 5 times; Said cleansing solution is the 0.01M phosphate buffer (KH that PBS-T promptly contains 0.05% Tween-20
2PO
40.2g, Na
2PO
412H
2O 2.9g, NaCl 8g is settled to 1000ml, pH7.4 adds the 0.5ml Tween-20 again);
(3) sealing: add in the every hole of microwell plate 150～200 μ L contain 0.5%～3%BSA (bovine serum albumin(BSA)) 0.01M PBS (phosphate buffer, pH7.4), 37 ℃ of sealing 0.5h～1.5h;
(4) set by step (2) washing micropore is 3～5 times;
(5) add 20% sucrose phosphate buffer room temperature protection 3h;
(6) drying: after the microwell plate drying, preserve for use in the packaging bag that contains drying agent of packing into;
6. the detection of ZEN:
(1) add respectively in each plate hole the ZEN of 50 μ l series concentration β type antiidiotype monoclonal antibody (sample extracting solution such as standard items liquid of Ab2 β-1D5) or the food of handling well, cereal, feed in micropore, mixing;
(2) add 50 μ L concentration be the ZEN monoclonal antibody (Ab1-1G4) of 0.025 μ g/mL to micropore, mix, hatch 1～2h for 37 ℃;
(3) the washing micropore is 3～5 times;
(4) goat anti-mouse igg antibody of the HRP mark of adding 100 μ L 1:4000 dilution is hatched 40 min for 37 ℃;
(5) the washing micropore is 3～5 times;
(6) each micropore add 100 μ L colour developing liquid TMB (3,3 ', 5,5 '-tetramethyl benzidine) the substrate working fluid, reaction 10～20min;
(7) each micropore adds 50 μ L 2M H
2SO
4(stop buffer) cessation reaction, and the mixing that vibrates gently;
(8) in 15min, join readout instrument and be determined at OD (absorbance) value under the 450nm wavelength with enzyme;
(9) gained titer and sample absorbance multiply by 100% again divided by the absorbance of 0 standard (titer of 0 concentration); With this titer calculated value is ordinate; The semilog of ZEN antiidiotype monoclonal antibody concentration (μ g/kg) is a horizontal ordinate drawing standard curve, reads the concentration of corresponding ZEN antiidiotype monoclonal antibody from typical curve according to the B/B0 value of each sample; [y=5901.1x-4.5101 (r=0.9862), y are ZEN concentration (ng/mL) according to the quality conversion formula again; X is anti-idiotype concentration (ng/mL)] calculate corresponding ZEN concentration, multiply by corresponding extension rate again, calculate the concentration of ZEN actual in the sample.
7. the separating of zearalenone in food, cereal and the feed
Food, cereal and feed make it 90% through 20 order mesh screens after crushed.Take by weighing 5g and be added in the 100mL tool plug conical flask, and adding 25mL 70% methanol/water solution (v:v, 70:30).Oscillator extracts 10min, and extract is through quick filter paper filtering.Get 1mL filtrating, add the 4mL distilled water diluting, shake up, be test liquid.
Preservation information:
Hybridoma cell strain 1D5, preservation date: on April 5th, 2012, depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University, deposit number: CCTCC NO:C201238.
Hybridoma 1G4G10D3, preservation date: on April 5th, 2012, depositary institution: Chinese typical culture collection center, address: China. Wuhan. Wuhan University, deposit number CCTCC NO:C201237.
Description of drawings
Fig. 1. the standard of ZEN suppresses curve.
Fig. 2. the standard of β-AId-1D5 suppresses curve.
Fig. 3. concentration transformation curve between ZEN toxin and anti-idiotype.
Embodiment
Embodiment 1 preparation zearalenone monoclonal antibody
1. the preparation of immunizing antigen ZEN-OVA, detection antigen ZEN-BSA
Adopt active ester method that ZEN and OVA coupling are prepared immunizing antigen (ZEN-OVA), ZEN and BSA coupling preparation detect antigen (ZEN-BSA).
ZEN derives and is ZEN-CMO
(1) take by weighing 6.5mg ZEN, add the 1ml pyridine, add 13mg O-ethyloic azanol then, stirring at room reaction 24h, last vacuum is drained.
(2) 4m dissolved in distilled water dissolution residual substance transfers to 8.0 with 1M NaOH with pH.
(3) with 4ml benzene extraction 2 times, remove unreacted ZEN, with 1M HCl pH is transferred to 3.0 then.
(4) with 8ml ethyl acetate extraction 4 times, collect extract, vacuum is drained, and residue is a zearalenone ethyloic oxime (ZEN-CMO)
ZEN-BSA is synthetic:
(1) preparation Acibenzolar: take by weighing 3.0mg ZEN-CMO, dissolve in 1mlDMF, and then add 1.8mg NHS, 1.8mg EDC, the stirring at room reaction is spent the night, and the centrifugal 10min of 5000rpm gets supernatant and obtains A liquid.
(2) coupling:, A liquid is slowly added OVA solution, stirred overnight at room temperature with 1.2ml PBS dissolving 8.6mg OVA.
(3) dialysis: use 0.015M, the pH7.4 PBS conjugate of dialysing is removed to the haptens of the coupling 3d that dialyses altogether, changes dislysate every day 3 times.The centrifugal 10min of dialysis back 5000rpm gets supernatant, and packing is stored in-20 ℃.
ZEN-OVA is synthetic:
(1) preparation Acibenzolar: take by weighing 2.7mg ZEN-CMO, dissolve in 1ml DMF, and then add 1.6mg NHS, 1.6mg EDC, the stirring at room reaction is spent the night, and the centrifugal 10min of 5000rpm gets supernatant and obtains A liquid.
(2) coupling:, A liquid is slowly added OVA solution, stirred overnight at room temperature with 1.2ml PBS dissolving 6.3mg OVA.
(3) dialysis: use 0.015M, the pH7.4 PBS conjugate of dialysing is removed to the haptens of the coupling 3d that dialyses altogether, changes dislysate every day 3 times.The centrifugal 10min of dialysis back 5000rpm gets supernatant, and packing is stored in-20 ℃.
2. the preparation of ZEN monoclonal antibody.
Get 3 6～8 age in week healthy BALB/c mouse (available from animal institute of Nanfang Medical Univ), numbering respectively, every each immunizing dose of mouse is 100 μ g; Immunogene ZEN-OVA and equal-volume Freund's complete adjuvant (just exempting from) or Freund (reinforcement) emulsification (0.2 mL), the subcutaneous multi-point injection in back, the immunity cycle is 14 d; 7 d behind each booster immunization; Blood is got in docking, obtains serum, be stored in-20 ℃ subsequent use.ZEN-BSA is as coating antigen, and indirect ELISA is measured antiserum titre, and indirect competitive ELISA is measured sero-fast sensitivity.
Through 3 booster immunizations; Testing result shows that the sensitivity of No. 1 mouse is the highest, and the IC50 value is 39.8 ng/mL, selects No. 1 mouse spleen as the splenocyte donor; The preceding 3 d lumbar injections of Fusion of Cells 100 μ g ZEN-OVA (0.1 mL) get mouse boosting cell (3.75 * 10 behind 3 d
8), 50% PEG 4000 effect down with myeloma cell SP2/0 (purchase is from Chinese typical culture collection center) (3.75 * 10
7) merge; Select the medium culture fused cell with HAT or HT, adopt indirect ELISA and indirect competitive ELISA method screening positive clone simultaneously, through three time cloningizations; Obtain the hybridoma of the anti-ZEN monoclonal antibody of 1 strain stably excreting high sensitivity; Called after 1G4G10D3, the anti-ZEN monoclonal antibody called after monoclonal antibody 1G4G10D3 of its secretion is called for short Ab1-1G4.Be equipped with the ascitic type monoclonal antibody through inducing legal system in the mouse body, specifically referring to document (Liu Xiu is of ancient India, the application of monoclonal antibody on agricultural, Anhui science tech publishing house, in November, 1994).
HAT selects nutrient culture media to contain hypoxanthine (hypoxantin), aminopterin (aminopterin) and thymidine; HT selects nutrient culture media to contain hypoxanthine and thymidine.
Embodiment 2 preparation zearalenone antiidiotype monoclonal antibodies
1. the monoclonal antibody with the hybridoma 1G4G10D3 of the anti-ZEN monoclonal antibody of stably excreting high sensitivity secretion is an immunogene, immune mouse, Fusion of Cells, preparation zearalenone antiidiotype monoclonal antibody.
(1) immunogene 1G4G10D3-KLH conjugate preparation
1. 25.6 mg EDC are added in the 4 mg/mL IgG solution (PBS, 500 μ L), stirring at room is reacted 30 min;
2. the KLH solution (PBS, 1 mL) with 2 mg/mL joins in the above-mentioned solution 4 ℃ of stirring reaction 12 h ~ 16 h;
3. last reactant liquor promptly obtains immunogene mAb-KLH conjugate (being the 1G4G10D3-KLH conjugate), packing, frozen subsequent use in-20 ℃ with 0.01 M PBS dialysis two days.
(2) preparation of the Fab antibody fragment of monoclonal antibody 1G4G10D3 and evaluation
1. saturated ammonium sulphate preliminary purification ZEN monoclonal antibody 1G4G10D3 is further purified through Protein G immune affinity chromatographic column then;
2. in 20 mM acetate buffers (pH4.5),, shake reaction 1 h in 37 ℃ of water-baths with the ZEN monoclonal antibody 1G4G10D3 of 250 μ L immobilization pepsin digestions, 10 mg purifying;
3. under 4 ℃ of conditions, the centrifugal 3min of digestion product 1000g gets supernatant, adds 1.5 mL, 10 mM Tris-HCl damping fluids (pH7.5) then, and the centrifuging and taking supernatant merges supernatant twice;
4. the supernatant that is obtained is collected effluent and concentrated through Protein G immune affinity chromatographic column, promptly obtains anti-ZEN monoclonal antibody 1G4G10D3 Fab fragment;
5. adopt BCA kit measurement Fab protein concentration; Identify that with indirect ELISA the Fab fragment is active, can find out that from table 1 the more complete IgG of IC50 of Fab has reduced more than 3 times, show that the sensitivity of antibody fragment is higher, active fine; Identify the Fab antibody fragment with non-reduced SDS-PAGE simultaneously, the result shows that the endonuclease bamhi molecular weight is 50 kD, coincide with the antibody passage Fab size.
Table 1 ZEN monoclonal antibody and the contrast of antibody passage Fab characteristic
(3) mouse immune and monoclonal antibody preparation
It is experimental subjects that the 6-7 Balb/c female mice in age in week of 10 health is selected in experiment for use; Immunogene (1G4G10D3-KLH conjugate) immunizing dose is 40 μ g/; With the abundant mixing of equal-volume novel adjuvant Qickadjuvant, through shank (back leg) intramuscular injection immune mouse; Pressed the same manner booster immunization one pin on the 21st day; Taked micro-tail blood on the 35th day; Separation of serum adopts indirect ELISA and indirect competitive ELISA to tire and sensitivity determination respectively, and the result shows that tiring of No. 1 mouse is the highest with sensitivity; Tire and be 1:1600; The IC50 value of competition thing ZEN is 33.8 ng/mL, detects and is limited to 0.06 ng/mL, the donor of splenocyte when therefore selecting No. 1 mouse as Fusion of Cells.The preceding 3 d lumbar injections of Fusion of Cells 40 μ g 1G4G10D3-KLH (0.1 mL); Get booster immunization mouse boosting cell (3.75 * 10 behind 3 d
8), in 50%PEG 4000 effect down and murine myeloma cell (3.75 * 10
7) merge; Select the medium culture fused cell with HAT or HT, when treating that hybridoma grows into the 1/4-1/3 of plate hole, adopt indirect ELISA and indirect competitive ELISA to detect cell conditioned medium, screening positive clone; Screen the hybridoma cell strain of the β type antiidiotype monoclonal antibody of 6 strains ability stably excreting ZEN, called after 1D5,2C11,2D9,3H8,3F8,6D8 in time preserve cell line respectively.Choose 8 age in week the Balb/c mouse prepare ascites, 1 week injection, 0.5 a mL/ incomplete Freund in advance, 1 all pneumoretroperitoneum inoculations 1 * 10
6Individual hybridoma, general 7-8 d extracts ascites, and centrifugal 10 min of 1000 r/min get supernatant and are stored in-20 ℃.
Wherein the IC50 of the antibody of hybridoma 1D5 generation is 10 ng/mL, and is minimum in this 6 strain of hybridoma, therefore selects 1D5 to set up the nontoxic ELISA detection technique of zearalenone.
Embodiment 3 sets up the zearalenone competitive ELISA does not have virus detection method
1. encapsulate the preparation of bar in advance:
(1) encapsulate: with encapsulating damping fluid is that the 0.05M sodium carbonate buffer of pH9.6 will detect antigen ZEN-BSA and be diluted to 1 μ g/mL, and every hole 100 μ L join in the polystyrene micropore plate, hatch 3h for 37 ℃;
(2) washing: pour out encapsulate damping fluid in the micropore plate hole after, with wash bottle or hyperchannel pipettor 200 μ l cleansing solutions are added in the hand-hole; Pour out the liquid in the hole after 3～5 minutes once more, remove the liquid in the hole fully; Wash 3 ~ 5 times; Said cleansing solution is the 0.01M phosphate buffer (KH that PBS-T promptly contains 0.05% Tween-20
2PO
40.2g, Na
2PO
412H
2O 2.9g, NaCl 8g is settled to 1000ml, pH7.4 adds the 0.5ml Tween-20 again);
(3) sealing: add in the every hole of microwell plate 150～200 μ L contain 0.5%～3%BSA (bovine serum albumin(BSA)) 0.01M PBS (phosphate buffer, pH7.4), 37 ℃ of sealing 0.5h～1.5h;
(4) set by step (2) washing micropore is 3～5 times;
(5) add 20% sucrose phosphate buffer room temperature protection 3h;
(6) drying: after the microwell plate drying, preserve for use in the packaging bag that contains drying agent of packing into.
2. the preparation of the β type antiidiotype monoclonal antibody standard items of ZEN
(1) saturated ammonium sulphate preliminary purification ZEN antiidiotype monoclonal antibody 1D5 is further purified through Protein G immune affinity chromatographic column then;
(2) antibody of purifying collection is with the ultrafiltration pipe of 50kD, 4 ℃, 5000g centrifugal ultrafiltration; 0.01M (phosphate buffer pH7.4) sweeps away antibody come from the ultrafiltration pipe PBS, and packing is stored in-20 ℃.The sample that takes a morsel carries out the mensuration of AC and purity.
(3) ZEN antiidiotype monoclonal antibody standard items concentration is 100ng/mL.
3. the detection of ZEN:
(1) add respectively in each plate hole the ZEN of 50 μ l series concentration β type antiidiotype monoclonal antibody (sample extracting solution such as standard items liquid of Ab2 β-1D5) or the food of handling well, cereal, feed in micropore, mixing;
(2) add 50 μ L concentration be the ZEN monoclonal antibody (Ab1-1G4) of 0.025 μ g/mL to micropore, mix, hatch 1～2h for 37 ℃;
(3) the washing micropore is 3～5 times;
(4) goat anti-mouse igg antibody of the HRP mark of adding 100 μ L 1:4000 dilution is hatched 40 min for 37 ℃;
(5) the washing micropore is 3～5 times;
(6) each micropore add 100 μ L colour developing liquid TMB (3,3 ', 5,5 '-tetramethyl benzidine) the substrate working fluid, reaction 10～20min;
(7) each micropore adds 50 μ L 2M H
2SO
4(stop buffer) cessation reaction, and the mixing that vibrates gently;
(8) in 15min, join readout instrument and be determined at OD (absorbance) value under the 450nm wavelength with enzyme;
(9) gained titer and sample absorbance multiply by 100% again divided by the absorbance of 0 standard (titer of 0 concentration); With this titer calculated value is ordinate; The semilog of ZEN antiidiotype monoclonal antibody concentration (μ g/kg) is a horizontal ordinate drawing standard curve, reads the concentration of corresponding ZEN antiidiotype monoclonal antibody from typical curve according to the B/B0 value of each sample; [y=5901.1x-4.5101 (r=0.9862), y are ZEN concentration (ng/mL) according to the quality conversion formula again; X is anti-idiotype concentration (ng/mL)] calculate corresponding ZEN concentration, multiply by corresponding extension rate again, calculate the concentration of ZEN actual in the sample.
Pollute separating of ZEN in embodiment 4 food, cereal and the feed
Food, cereal and feed make it 90% through 20 order mesh screens after crushed.Take by weighing 5g and be added in the 100mL tool plug conical flask, and adding 25mL 70% methanol/water solution (v:v, 70:30).Oscillator extracts 10min, and extract is through quick filter paper filtering.Get 1mL filtrating, add the 4mL distilled water diluting, shake up, be test liquid.
Claims (3)
1. the competitive ELISA of a zearalenone does not have virus detection method; Said method is the competitive ELISA detection method of setting up with the β type antiidiotype monoclonal antibody of ZEN-BSA, ZEN monoclonal antibody and ZEN; Wherein, Said ZEN monoclonal antibody is Ab1-1G4, and by hybridoma 1G4G10D3 secretion, hybridoma 1G4G10D3 was preserved in Chinese typical culture collection center on April 5th, 2012; The preservation place is a China. Wuhan. and Wuhan University, deposit number is CCTCC NO:C201237; The β type antiidiotype monoclonal antibody of said ZEN is Ab2 β-1D5; Secrete by hybridoma cell strain 1D5; Hybridoma cell strain 1D5 is preserved in Chinese typical culture collection center on April 5th, 2012, the preservation place is a China. Wuhan. and Wuhan University, deposit number is CCTCC NO:C201238;
Concrete detection method comprises the steps:
(A) encapsulate the preparation of bar in advance:
(1) encapsulate: with encapsulating damping fluid is that the 0.05M sodium carbonate buffer of pH9.6 will detect antigen ZEN-BSA and be diluted to 1 μ g/mL, and every hole 100 μ L join in the polystyrene micropore plate, hatch 3h for 37 ℃;
(2) washing: pour out encapsulate damping fluid in the micropore plate hole after, with wash bottle or hyperchannel pipettor 200 μ l cleansing solutions are added in the hand-hole, pour out the liquid in the hole behind 3～5min once more, remove the liquid in the hole fully, wash 3 ~ 5 times; Said cleansing solution is PBS-T;
(3) sealing: add the 0.01M PBS that 150～200 μ L contain 0.5%～3%BSA in the every hole of microwell plate, 37 ℃ of sealing 0.5h～1.5h;
(4) set by step (2) washing micropore is 3～5 times;
(5) add 20% sucrose phosphate buffer room temperature protection 3h;
(6) drying: after the microwell plate drying, preserve for use in the packaging bag that contains drying agent of packing into;
(B) detection of ZEN:
(7) β type antiidiotype monoclonal antibody titer or the sample extracting solution handled well of ZEN that adds 50 μ l series concentration in each plate hole respectively in micropore, mixing;
(8) add 50 μ L concentration be the ZEN monoclonal antibody of 0.025 μ g/mL to micropore, mixing is hatched 1～2h for 37 ℃;
(9) the washing micropore is 3～5 times;
(10) goat anti-mouse igg antibody of the HRP mark of adding 100 μ L1:4000 dilution is hatched 40 min for 37 ℃;
(11) the washing micropore is 3～5 times;
(12) each micropore adds 100 μ L colour developing liquid tmb substrate working fluid, reaction 10～20min;
(13) each micropore adds 50 μ L 2M H
2SO
4Cessation reaction, and the mixing that vibrates gently;
(14) in 15min, join readout instrument and be determined at the OD value under the 450nm wavelength with enzyme;
(15) calculate in the sample concentration of actual ZEN.
2. the competitive ELISA of zearalenone according to claim 1 does not have virus detection method, it is characterized in that said detection method is used for the detection that food, cereal or feed zearalenone pollute.
3. the competitive ELISA of zearalenone according to claim 2 does not have the separation method that virus detection method is characterized in that zearalenone in food, cereal or the feed and is: food, cereal or feed make it 90% through 20 order mesh screens after crushed; Take by weighing 5g and be added in the 100mL tool plug conical flask, and adding 25mL 70% methanol/water solution (v:v, 70:30); Oscillator extracts 10min; Extract is got 1mL filtrating through quick filter paper filtering, adds the 4mL distilled water diluting; Shake up, be test liquid.
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