CN102964447A - Cry1Ab/Ac genetically modified ingredient paired monoclonal antibody preparation and detection method - Google Patents
Cry1Ab/Ac genetically modified ingredient paired monoclonal antibody preparation and detection method Download PDFInfo
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Abstract
The invention discloses a method for preparation of a Cry1Ab/Ac genetically modified ingredient paired monoclonal antibody and double-antibody sandwich ELISA detection. The preparation method comprises: using g transgenic protein to induce an immunized female BALB/c mouse of 6 weeks old, then fusing an immunized mouse spleen cell with a myeloma cell SP2/0, screening positive cloning holes, conducting cell line establishment and expansion culture, and cryopreserving hybridoma cell lines with liquid nitrogen, thus obtaining the desired product. Then, an HRP coupled antibody is employed to screen out paired antibodies about Bt Cry1Ab protein and Bt Cry1Ac protein by the double-antibody sandwich ELISA detection method. The found paired antibodies are utilized to detect an actual sample and a spiked sample so as to test the recovery rate.
Description
Technical field
The present invention relates to a kind of preparation of monoclonal antibody, especially a kind of Cry1Ab/Ac transgene component pairing monoclonal antibody preparation and double antibodies sandwich ELISA detection method.
Background technology
To be the specific b cells that produces with the myeloma cell with through certain antigen induction merge through the fusogen effect such as PEG or electric laser monoclonal antibody technique, form hybridoma then screening and separating go out single positive hybridoma cell.Again with the final separation of its amplification, the process of antibody purification.Hybridoma has dual nature can continue breeding as the myeloma cell, can secrete again the specific antibody for single epitope as the B cell.Monoclonal antibody and antiserum(antisera) claim again polyclonal antibody (polyclonal antibody PcAb), and the key distinction is that monoclonal antibody is a kind of immunoglobulin molecules of homogeneous, has the specificity for an antigenic determinant; Compare with polyclonal antibody, monoclonal antibody has unrivaled superiority:, good reproducibility good such as high specific, high purity, uniformity, the height of tiring, etc. advantage, be widely used at therapeutic treatment and diagnosis etc.
Summary of the invention
One of purpose of the present invention is achieved through the following technical solutions:
Select the female BALB/c mouse (4) in 6 ages in week as immune animal, adopt subcutaneous multi-point injection (100 μ g/ only) after transgene protein and the emulsification of equivalent Freund's complete adjuvant.Interval 4 all booster immunizations, multiple spot subcutaneous injection after transgene protein and the emulsification of equivalent Freund's incomplete adjuvant (100 μ g/ are only), booster immunization 2 times.Week docking blood sampling behind the last booster immunization, separation of serum is measured the mice serum antibody titer take transgene protein as detectable antigens.
Shift to an earlier date 3 days during cytogamy and carry out booster immunization with 30 μ g antigens to merging mouse.Selection mode good, to the SP2/0 cell strain of HAT sensitivity, immune mouse spleen cell mixes by 1: 1 with myeloma cell SP2/0, PEG-4000 is fusogen, spread into 96 porocyte culture plates merging good cell, cultivate with the HAT nutrient solution, change liquid after three days, use the HT nutrient solution instead.With ELISA method (transgene protein is detectable antigens) screening positive clone hole, limiting dilution assay clone 3 times reaches 100%, cell strain built, enlarged culturing, and liquid nitrogen cryopreservation hybridoma cell strain to positive rate.Get 7 the week age BALB/c mouse abdominal injection whiteruss, every mouse 0.2mL.The hybridoma of 10 days pneumoretroperitoneum inoculation some amounts.Mouse web portion obviously expands, and gathers ascites with connector bend dropping tube, and indirect ELISA is measured ascites antibody and tired.
With HRP coupling antibody, thereby carry out the antibody pair test:
(1) take by weighing 4mg HRP and be dissolved in the 1mL pure water, add 200 μ L 100mM sodium periodates (now with the current), 4 ℃ are stirred 20min, are blackish green to solution;
(2) mentioned solution is placed 1mM pH4.4 acetate buffer solution dialysed overnight;
(3) transferring above-mentioned HRP pH value of solution with 0.1M CB (carbonate buffer solution) is 9.0;
(4) an amount of antibody is added in the HRP solution, 4 ℃ are stirred 2h;
(5) continue to add 100 μ L 4mg/ml sodium borohydrides, 4 ℃ of lucifuges stir 2h;
(6) use pH 8.5 ammonium sulfate saturated solutions that above solution is carried out ammonium sulfate precipitation, the centrifugal end gained brown throw out of managing afterwards is experiment gained antibody connection enzyme.Filter out pairing antibody about Bt Cry1Ab albumen, Bt Cry1Ac albumen with the method for ELISA, the steps include: (1) with the mouse monoclonal antibody with the carbonate buffer solution of pH9.6 according to 1: 100 times of dilution;
The sealing of (2) 10% sheep blood serums, every hole 150 μ L, 37 ℃ of sealings of constant temperature 3 hours;
(3) adding concentration is transgenosis Cry1Ab, the Cry1Ac antigen standard substance of 50ppb/0ppb, 100 μ L/ holes, 37 ℃, incubation 60min;
(4) press 1: 100 dilution proportion enzyme labelled antibody D06-HRP, D07-HRP with diluent, abundant mixing, 37 ℃, incubation 60min;
(5) color reaction reads the OD value.
Detect actual sample and add sample, the test rate of recovery by the pairing antibody that has found.
Description of drawings
Fig. 1 is the transgene protein standard working curve.
Embodiment
Below will be described in detail the preferred embodiments of the present invention; Should be appreciated that preferred embodiment only for the present invention is described, rather than in order to limit protection scope of the present invention.
Select the female BALB/c mouse (4) in 6 ages in week as immune animal, adopt subcutaneous multi-point injection (100 μ g/ only) after transgene protein and the emulsification of equivalent Freund's complete adjuvant.Interval 4 all booster immunizations, multiple spot subcutaneous injection after transgene protein and the emulsification of equivalent Freund's incomplete adjuvant (100 μ g/ are only), booster immunization 2 times.Week docking blood sampling behind the last booster immunization, separation of serum is measured the mice serum antibody titer take transgene protein as detectable antigens.
Shift to an earlier date 3 days during cytogamy and carry out booster immunization with 30 μ g antigens to merging mouse.Selection mode good, to the SP2/0 cell strain of HAT sensitivity, immune mouse spleen cell mixes by 1: 1 with myeloma cell SP2/0, PEG-4000 is fusogen, spread into 96 porocyte culture plates merging good cell, cultivate with the HAT nutrient solution, change liquid after three days, use the HT nutrient solution instead.With ELISA method (transgene protein is detectable antigens) screening positive clone hole, limiting dilution assay clone 3 times reaches 100%, cell strain built, enlarged culturing, and liquid nitrogen cryopreservation hybridoma cell strain to positive rate.
Get 7 the week age BALB/c mouse abdominal injection whiteruss, every mouse 0.2mL.The hybridoma of 10 days pneumoretroperitoneum inoculation some amounts.Mouse web portion obviously expands, and gathers ascites with connector bend dropping tube, and indirect ELISA is measured ascites antibody and tired.
The centrifugal 15min of mouse ascites (2000rpm, room temperature) chooses the upper strata grease, dropwise slowly adds saturated ammonium sulphate to semi-saturation under 4 ℃ of stirrings, continues to stir 30min, and centrifugal 30min (13000rpm, 4 ℃) abandons supernatant; Precipitation is dissolved in an amount of PBS (0.01M pH7.4); Dropwise slowly add saturated ammonium sulphate to 33% under 4 ℃ of stirrings, continue to stir 30min, centrifugal 30min (13000rpm, 4 ℃) abandons supernatant; Precipitation is dissolved in an amount of PBS (0.01M, pH7.4), and 4 ℃ of dialysed overnight are measured protein content, and-20 ℃ frozen for subsequent use.
Continuing behind the ammonium sulfate precipitation just has Protein G pillar to carry out purifying, and new pillar is crossed post with the 5ml ultrapure water first, uses 5mL0.4M PB damping fluid (pH 7.0) balance purifying pillar again; Antibody is crossed post, requires the slow post of crossing in the process, better is combined on the binding site in the hope of antibody protein; Continue 10mL0.4M PB damping fluid (pH 7.0) balance purifying pillar; 5mL0.1M the antibody on glycine-hydrochloride buffer (pH 7.0) elution of bound site, and add among the 1M Tris-HCI (pH 2.7) and glycine, pH is remained be fit to the neutrality that antibody is preserved; 10mL0.4M PB damping fluid (pH 7.0) balance purifying pillar; 5mL 20% ethanolic soln is crossed post, preserves the purifying pillar under 4 ℃ of conditions.
With HRP coupling antibody, carry out the antibody pair test:
(1) take by weighing 4mg HRP and be dissolved in the 1mL pure water, add 200 μ L 100mM sodium periodates (now with the current), 4 ℃ are stirred 20min, are blackish green to solution;
(2) mentioned solution is placed 1mM pH4.4 acetate buffer solution dialysed overnight;
(3) transferring above-mentioned HRP pH value of solution with 0.1M CB (carbonate buffer solution) is 9.0;
(4) an amount of antibody is added in the HRP solution, 4 ℃ are stirred 2h;
(5) continue to add 100 μ L 4mg/ml sodium borohydrides, 4 ℃ of lucifuges stir 2h;
(6) use pH 8.5 ammonium sulfate saturated solutions that above solution is carried out ammonium sulfate precipitation, the centrifugal end gained brown throw out of managing afterwards is experiment gained antibody connection enzyme.
Filter out pairing antibody about Bt Cry1Ab albumen, Bt Cry1Ac albumen with the method for ELISA:
1. configuration reagent
(1) configuration coating buffer: 0.3g NaCO3 and 0.58g NaHCO3 are added distilled water be settled to 100mL.
(2) configuration diluent, it is as follows to fill a prescription:
(3) configuration washings, it is as follows to fill a prescription:
2. coated
The mouse monoclonal antibody (ammonium sulfate precipitation) of (1) inciting somebody to action uses the carbonate buffer solution (coating buffer, lower same) of pH9.6 according to 1: 100 times of dilution, 100 μ L/ holes, and 4 ℃ are spent the night.
(2) washing lotion is cleaned coated plate 3 times, each washing lotion consumption 300uL, and the time is 1min.
3. sealing
(1) 10% sheep blood serum sealing (using the carbonate buffer solution of PH9.6 according to 1: 9 times of dilution), every hole 150 μ L, 37 ℃ of sealings of constant temperature 3 hours.
(2) washing lotion is cleaned coated plate 3 times, each washing lotion consumption 300uL, and the time is 1min.
4 application of samples
(1) adding concentration is transgenosis Cry1Ab, the Cry1Ac antigen standard substance of 50ppb/0ppb, 100 μ L/ holes, 37 ℃, incubation 60min.
(2) washing lotion is cleaned coated plate 3 times, each washing lotion consumption 300 μ L, and the time is 1min.
5 is enzyme-added
(1) presses 1: 100 dilution proportion enzyme labelled antibody D06-HRP, D07-HRP with diluent, abundant mixing, 37 ℃, incubation 60min.
(2) washing lotion is cleaned coated plate 3 times, each washing lotion consumption 300 μ L, and the time is 1min.
6 color reactions, detection
Every hole adds TMB 100 μ L, and 37 ℃ of reaction 15min add 2M H2SO4,100 μ L/ holes, and microplate reader is surveyed reading: 450nm and is read OD (absorbancy) value.
7.ELISA detected result
(+represent signal values is strong and weak, + signal value 0.2-0.5, ++ 0.5-1.0, +++1.0-1.5, ++ ++ 1.5 above mark indistinction explanation blank values and the basic indistinction of positive value, the blank high explanation blank value of mark is higher, but still has any different with positive value, do not have mark indistinction explanation blank value and positive value that obvious difference is arranged, blank value is about 0.2)
| Coated antibody | D06-HRP | D07-HRP | Coated antibody | D06-HRP | D07-HRP |
| 006-9 | ++++ | 007-36 | + | ++++ | |
| 006-12 | ++ | 007-37 | + | ++++ | |
| 006-13 | ++ blank high | ++ blank high | 007-38 | + | ++++ |
| 006-14 | ++ | ++++ | 007-39 | + indistinction | ++ |
[0055]
| 006-16 | + | ++++ | 007-40 | + indistinction | |
| 006-19 | +++ | 007-41 | ++++ | ||
| 006-23 | ++ | 007-42 | + | +++ | |
| 007-6 | + | 007-43 | + | ||
| 007-9 | ++ | ++++ | 007-44 | + | |
| 007-10 | +++ | ++++ | 007-45 | + | + |
| 007-11 | + | 007-46 | + | ++ | |
| 007-12 | +++ | +++ | 007-48 | + | |
| 007-13 | ++++ | ++++ | 007-49 | + indistinction | |
| 007-14 | + | +++ | 007-50 | ++ | ++ |
| 007-15 | + | 007-51 | + | ++++ | |
| 007-16 | ++ | 007-52 | ++++ | ||
| 007-30 | + | +++ | 007-53 | + | |
| 007-31 | ++ | 007-54 | + | ++++ | |
| 007-32 | ++++ | 007-55 | + | ++++ | |
| 007-33 | ++ blank high | 007-56 | + | ++++ | |
| 007-34 | + | +++ | 007-58 | + blank high |
Select and the reasonable monoclonal antibody coupling of how anti-pairing effect HRP, again just pairing experiment
| 13-H | 32-H | 36-H | 37-H | 38-H | 41-H | 51-H | 52-H | 54-H | 55-H | 56- |
0 |
| 0.4 | 0.3 | 0.3 | 0.5 | 0.4 | 0.3 | 0.3 | 0.3 | 0.3 | 0.5 | 0.3 | 7-9 |
| 0.2 | 0.1 | 0.3 | 0.8 | 0.5 | 0.3 | 0.3 | 0.2 | 0.3 | 0.6 | 0.2 | 7-10 |
| 0.2 | 0.1 | 0.1 | 0.3 | 0.2 | 0.2 | 0.2 | 0.2 | 0.1 | 0.4 | 0.2 | 7-12 |
| 0.1 | 0.2 | 0.3 | 0.6 | 0.3 | 0.2 | 0.3 | 0.2 | 0.3 | 0.5 | 0.3 | 7-13 |
| 0.2 | 0.2 | 0.1 | 0.2 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.2 | 0.1 | 7-32 |
| 0.2 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.2 | 0.1 | 7-36 |
| 0.1 | 0.2 | 0.3 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.1 | 0.3 | 0.2 | 7-37 |
| 0.1 | 0.1 | 0.2 | 0.5 | 0.3 | 0.2 | 0.2 | 0.1 | 0.2 | 0.5 | 0.1 | 7-38 |
| 0.3 | 0.2 | 0.3 | 0.4 | 0.3 | 0.3 | 0.2 | 0.3 | 0.3 | 0.5 | 0.4 | 7-41 |
[0058]
| 0.1 | 0.1 | 0.2 | 0.2 | 0.1 | 0.2 | 0.1 | 0.1 | 0.1 | 0.4 | 0.1 | 7-51 |
| 0.2 | 0.1 | 0.1 | 0.3 | 0.2 | 0.2 | 0.1 | 0.3 | 0.2 | 0.4 | 0.2 | 7-52 |
| 0.3 | 0.1 | 0.3 | 0.4 | 0.2 | 0.3 | 0.4 | 0.3 | 0.3 | 0.5 | 0.1 | 7-54 |
| 0.3 | 0.1 | 0.3 | 0.9 | 0.6 | 0.2 | 0.2 | 0.1 | 0.3 | 0.8 | 0.2 | 7-55 |
| 0.2 | 0.1 | 0.1 | 0.2 | 0.1 | 0.1 | 0.1 | 0.1 | 0.2 | 0.4 | 0.3 | 7-56 |
| 0.9 | 0.1 | 0.3 | 0.6 | 0.6 | 0.2 | 0.2 | 0.2 | 0.3 | 1.8 | 1.2 | D06 |
| 2 | 0.2 | 0.7 | 1.3 | 1.5 | 0.2 | 0.2 | 0.2 | 0.2 | 2.4 | 0.2 | D07 |
| 100 | |||||||||||
| 1.9 | 1.5 | 1.7 | 1.9 | 1.8 | 1.8 | 1.5 | 1.7 | 1.7 | 1.7 | 1.4 | 7-9 |
| 1.2 | 1.4 | 1.5 | 1.7 | 1.2 | 1.2 | 1.4 | 1.4 | 1.1 | 1.8 | 0.9 | 7-10 |
| 0.9 | 1.8 | 2.1 | 1.9 | 1.1 | 0.9 | 1.4 | 1.8 | 1.4 | 2 | 1.7 | 7-12 |
| 0.9 | 1.5 | 2 | 1.8 | 1.5 | 0.8 | 1.4 | 1.1 | 1.3 | 2 | 1.3 | 7-13 |
| 0.4 | 0.5 | 1.6 | 1.3 | 1.7 | 1.2 | 0.8 | 1.4 | 1 | 1.7 | 1.9 | 7-32 |
| 0.7 | 0.8 | 0.3 | 0.8 | 1 | 0.9 | 0.8 | 1.3 | 0.6 | 1.5 | 0.7 | 7-36 |
| 0.7 | 0.5 | 0.5 | 0.6 | 1.1 | 0.8 | 0.7 | 1 | 0.7 | 1.3 | 0.7 | 7-37 |
| 0.7 | 1.1 | 1.6 | 1.6 | 0.9 | 1 | 1.1 | 1.3 | 1.4 | 2 | 1.6 | 7-38 |
| 1.3 | 1.8 | 1.9 | 2 | 1.4 | 1 | 1.4 | 1.8 | 1.4 | 2.1 | 1.6 | 7-41 |
| 1.6 | 1.1 | 1.8 | 1.7 | 1.3 | 1 | 0.7 | 0.9 | 0.9 | 1.9 | 0.8 | 7-51 |
| 1.2 | 1.9 | 1.8 | 2 | 1.3 | 0.9 | 0.7 | 0.7 | 1 | 1.9 | 0.9 | 7-52 |
| 0.7 | 1.6 | 1.7 | 1.8 | 1 | 0.8 | 1.3 | 1.2 | 1.3 | 2.2 | 1.7 | 7-54 |
| 1 | 0.4 | 1.4 | 1.5 | 1.5 | 1.2 | 0.8 | 1 | 1 | 1.2 | 0.8 | 7-55 |
| 1.8 | 1.4 | 2.1 | 2 | 2 | 1.9 | 1.1 | 1.3 | 1.4 | 2.1 | 1.1 | 7-56 |
| 2.1 | 1.5 | 2 | 1.8 | 1.9 | 1.5 | 1.4 | 1.5 | 1.4 | 2.6 | 1.4 | D06 |
| 2.4 | 2.2 | 2.2 | 2.2 | 2.1 | 1.5 | 1.3 | 1.9 | 1.8 | 2.1 | 1.8 | D07 |
1, most of antibody pair, positive value and 0 's value has significant difference, can think and can successfully match.
2, choose positive value and 0 point value and differ greatly, and the lower antibody of 0 point value pair, the antigen of low concentration is carried out lower detection
| 5 | 0.5 | 0 | |
| 7-9 7-41-H | 0.163 | 0.137 | 0.108 |
| 7-12 7-32-H | 0.126 | 0.082 | 0.069 |
| 7-12 7-36-H | 0.196 | 0.136 | 0.117 |
| 7-12 7-37-H | 0.262 | 0.23 | 0.208 |
| 7-12 7-52-H | 0.206 | 0.154 | 0.113 |
| 7-13 7-36-H | 0.242 | 0.186 | 0.136 |
| 7-13 7-38-H | 0.323 | 0.253 | 0.233 |
| 7-13 7-52-H | 0.134 | 0.111 | 0.099 |
| 7-13 7-55-H | 0.922 | 0.461 | 0.461 |
| 7-13 7-16-H | 0.109 | 0.093 | 0.1 |
| 7-36 7-52-H | 0.167 | 0.149 | 0.128 |
| 7-38 7-36-H | 0.364 | 0.251 | 0.237 |
| 7-38 7-56-H | 0.185 | 0.177 | 0.179 |
| 7-41 7-32-H | 0.157 | 0.158 | 0.161 |
| 7-41 7-36-H | 0.233 | 0.213 | 0.225 |
| 7-41 7-37-H | 0.168 | 0.152 | 0.152 |
| 7-51 7-13-H | 1.443 | 1.355 | 1.428 |
| 7-51 7-36-H | 0.479 | 0.183 | 0.143 |
[0064]
| 7-51 7-37-H | 0.203 | 0.18 | 0.121 |
| 7-41 7-52-H | 0.427 | 0.226 | 0.202 |
| 7-52 7-32-H | 0.24 | 0.163 | 0.107 |
| 7-52 7-36-H | 0.164 | 0.16 | 0.148 |
| 7-52 7-37-H | 0.228 | 0.244 | 0.29 |
| 7-54 7-56-H | 0.291 | 0.147 | 0.222 |
| 7-56 7-13-H | 0.369 | 0.305 | 0.326 |
| 7-56 7-32-H | 0.128 | 0.115 | 0.155 |
| 7-56 7-36-H | 0.193 | 0.129 | 0.168 |
| 7-56 7-37-H | 0.217 | 0.215 | 0.229 |
| 7-56 7-38-H | 0.278 | 0.207 | 0.273 |
| 7-56 7-41-H | 0.197 | 0.139 | 0.184 |
| 7-56 7-52-H | 0.266 | 0.201 | 0.24 |
| 7-56 7-55-H | 0.759 | 0.62 | 0.639 |
Wherein 7-51 is coated, and the effect of 7-36-HRP is best antibody pair.Detection sensitivity can reach 5ppb.
Sample detection detects actual sample and adds sample, the test rate of recovery by the pairing antibody that has found.
1. experimental raw
| Title | Lot number | Originate in | Supply of material company |
| NaCO 3 | 060623 | Upper marine rainbow photoinitiator chemical factory | Chemical reagents corporation of traditional Chinese medicines group |
| NaHCO 3 | 060517 | Upper marine rainbow photoinitiator chemical factory | Chemical reagents corporation of traditional Chinese medicines group |
| NaCl | F20061225 | Chemical reagents corporation of traditional Chinese medicines group | Chemical reagents corporation of traditional Chinese medicines group |
| Na 2HPO 4*12H 2O | F20060912 | Chemical reagents corporation of traditional Chinese medicines group | Chemical reagents corporation of traditional Chinese medicines group |
| NaH 2PO 4*2H 2O | F20060622 | Chemical reagents corporation of traditional Chinese medicines group | Chemical reagents corporation of traditional Chinese medicines group |
| KCl | F20100713 | Chemical reagents corporation of traditional Chinese medicines group | Chemical reagents corporation of traditional Chinese medicines group |
| Tween-20 | F20061031 | Chemical reagents corporation of traditional Chinese medicines group | Chemical reagents corporation of traditional Chinese medicines group |
[0069]
| Casein | C8200 | Sigma | Beijing Suo Laibao |
| BSA | 11/2009 | Amresco | Jiangyin Tylenol |
| Sheep blood serum | Zhengzhou Yi Kang | ||
| TMB | 52-00-04 | KPL | West Beijing is U.S. outstanding |
Bt Cry1Ab albumen, Bt Cry1Ac albumen are by U.S.'s imported with original packaging;
Antibody 007-51 is the mouse immune preparation; 007-36-HRP is autonomous coupling preparation.
Corn sample 3 is that sample 1 extracting solution adds Bt Cry1Ab protein 10 ppb;
Corn sample 4 is that sample 2 extracting solutions add Bt Cry1Ab albumen 30ppb;
Corn sample 5 is that sample 1 extracting solution adds Bt Cry1Ac protein 10 ppb;
Corn sample 6 is that sample 2 extracting solutions add Bt Cry1Ac albumen 30ppb
2. experimental implementation
2.1 configuration reagent
(1) configuration coating buffer: 0.3g NaCO3 and 0.58g NaHCO3 are added distilled water be settled to 100mL.
(2) configuration diluent, it is as follows to fill a prescription:
(3) configuration washings, it is as follows to fill a prescription:
2.2 coated
(1) with 007-51 antibody with the carbonate buffer solution of pH9.6 (coating buffer, lower with) according to 1: 100 times of dilution, 100 μ L/ holes, 4 ℃ are spent the night.
(2) washing lotion is cleaned coated plate 3 times, each washing lotion consumption 300 μ L, and the time is 1min.4.3.3 sealing
(1) 10% sheep blood serum sealing (using the carbonate buffer solution of pH9.6 according to 1: 9 times of dilution), every hole 150 μ L, 37 ℃ of sealings of constant temperature 3 hours.
(2) washing lotion is cleaned coated plate 3 times, each washing lotion consumption 300 μ L, and the time is 1min.
2.3. sample pre-treatments
(1) gets corn 15g, put into homogenizer and grind;
(2) Semen Maydis powder after taking by weighing 5g and grinding adds the 25mL diluent, vibration 10min;
(3) the centrifugal 10min of 4000g gets supernatant liquor and detects.
2.4 application of sample
(1) adds transgenosis Cry1Ab, Cry1Ac antigen standard substance, corn sample and the corn that concentration gradient is 45ppb, 15ppb, 5ppb, 0ppb (being diluent) and add sample, 100 μ L/ holes, 37 ℃, incubation 60min.
(2) washing lotion is cleaned coated plate 3 times, each washing lotion consumption 300 μ L, and the time is 1min.
2.5 enzyme-added
(1) presses 1: 100 dilution proportion enzyme labelled antibody 007-36-HRP with diluent, fully mixing; 37 ℃, incubation 60min.
(2) washing lotion is cleaned coated plate 3 times, each washing lotion consumption 300 μ L, and the time is 1min.
2.6 color reaction, detection
Every hole adds TMB 100 μ L, and 37 ℃ of reaction 15min add 2M H2SO4,100 μ L/ holes, and microplate reader is surveyed reading: 450nm and is read OD (absorbancy) value.
The ELISA detected result
Deduct OD value after the blank well as ordinate zou, Bt Cry1Ac protein concentration is mapped as X-coordinate, as shown in Figure 1.
Claims (7)
1. Cry1Ab/Ac transgene component pairing method for preparing monoclonal antibody, it is characterized in that, induce the immune female BALB/c mouse in 6 ages in week with transgene protein, merge with immune mouse spleen cell and myeloma cell SP2/0 again, the screening positive clone hole, cell strain built, enlarged culturing, the liquid nitrogen cryopreservation hybridoma cell strain obtains required product.
2. the preparation method of a kind of Cry1Ab/Ac transgene component pairing monoclonal antibody as claimed in claim 1, it is characterized in that, select the female BALB/c mouse in 6 ages in week as immune animal, adopt subcutaneous multi-point injection after transgene protein and the emulsification of equivalent Freund's complete adjuvant, 100 μ g/ only, interval 4 all booster immunizations, multiple spot subcutaneous injection after transgene protein and the emulsification of equivalent Freund's incomplete adjuvant, 100 μ g/ only, booster immunization 2 times, week docking blood sampling behind the last booster immunization, separation of serum is measured the mice serum antibody titer take transgene protein as detectable antigens.
3. the preparation method of a kind of Cry1Ab/Ac transgene component pairing monoclonal antibody as claimed in claim 1, it is characterized in that, the establishment step of hybridoma cell strain is: shift to an earlier date 3 days during cytogamy with 30 μ g antigens and carry out booster immunization to merging mouse, selection mode is good, SP2/0 cell strain to the HAT sensitivity, immune mouse spleen cell mixes by 1: 1 with myeloma cell SP2/0, PEG-4000 is fusogen, spread into 96 porocyte culture plates merging good cell, cultivate with the HAT nutrient solution, change liquid after three days, use the HT nutrient solution instead, with ELISA method screening positive clone hole, limiting dilution assay clone 3 times reaches 100%, cell strain built to positive rate, enlarged culturing obtains hybridoma cell strain.
4. the application of a kind of Cry1Ab/Ac transgene component pairing monoclonal antibody as claimed in claim 1 is characterized in that, utilizes this monoclonal antibody to detect actual sample and add sample, the test rate of recovery by double antibodies sandwich ELISA detection technique.
5. the application of a kind of Cry1Ab/Ac transgene component pairing monoclonal antibody as claimed in claim 4, it is characterized in that, with HRP coupling antibody, filter out pairing antibody about Bt Cry1Ab albumen, Bt Cry1Ac albumen with double antibodies sandwich ELISA detection method.
6. the application of a kind of Cry1Ab/Ac transgene component pairing monoclonal antibody as claimed in claim 5 is characterized in that double antibodies sandwich ELISA detection method step is:
(1) use the carbonate buffer solution of pH9.6 according to 1: 100 times of dilution the mouse monoclonal antibody;
The sealing of (2) 10% sheep blood serums, every hole 150 μ L, 37 ℃ of sealings of constant temperature 3 hours;
(3) adding concentration is transgenosis Cry1Ab, the Cry1Ac antigen standard substance of 50ppb/0ppb, 100 μ L/ holes, 37 ℃, incubation 60min;
(4) press 1: 100 dilution proportion enzyme labelled antibody D06-HRP, D07-HRP with diluent, abundant mixing, 37 ℃, incubation 60min;
(5) color reaction reads the OD value.
7. the application of a kind of Cry1Ab/Ac transgene component pairing monoclonal antibody as claimed in claim 4 is characterized in that, detects actual sample and adds sample, the test rate of recovery by the pairing antibody that has found.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN103267865A (en) * | 2013-05-22 | 2013-08-28 | 中国农业科学院棉花研究所 | Method for detecting Bt protein resistance of cotton bollworm |
| WO2015109951A1 (en) * | 2014-01-26 | 2015-07-30 | 江苏省农业科学院 | Anthropogenic pest-resistant gene, anti-cry1ab toxin idiotypic single-chain antibody encoded by same, and application |
| CN105777899A (en) * | 2016-04-25 | 2016-07-20 | 江苏省农业科学院 | Monoclonal antibody capable of resisting Bt Cry1Ab toxins, cell strain generating monoclonal antibody, and preparation method and application of monoclonal antibody |
| CN113325182A (en) * | 2021-05-26 | 2021-08-31 | 武汉华美生物工程有限公司 | Efficient screening method of paired antibodies |
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| CN1525173A (en) * | 2003-02-27 | 2004-09-01 | 重庆金标生物技术有限公司 | Insecticidal crystallin BT-Cry1Ab/1Ac rapid detection test bar |
| CN101914156A (en) * | 2010-08-20 | 2010-12-15 | 中华人民共和国北京出入境检验检疫局 | Protein chip kit and method for detecting transgenic crops |
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| CN1525173A (en) * | 2003-02-27 | 2004-09-01 | 重庆金标生物技术有限公司 | Insecticidal crystallin BT-Cry1Ab/1Ac rapid detection test bar |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103267865A (en) * | 2013-05-22 | 2013-08-28 | 中国农业科学院棉花研究所 | Method for detecting Bt protein resistance of cotton bollworm |
| CN103267865B (en) * | 2013-05-22 | 2014-12-03 | 中国农业科学院棉花研究所 | Method for detecting Bt protein resistance of cotton bollworm |
| WO2015109951A1 (en) * | 2014-01-26 | 2015-07-30 | 江苏省农业科学院 | Anthropogenic pest-resistant gene, anti-cry1ab toxin idiotypic single-chain antibody encoded by same, and application |
| EP3098315A4 (en) * | 2014-01-26 | 2017-06-21 | Jiangsu Academy of Agricultural Sciences | Anthropogenic pest-resistant gene, anti-cry1ab toxin idiotypic single-chain antibody encoded by same, and application |
| US9751952B2 (en) | 2014-01-26 | 2017-09-05 | Jiangsu Academy Of Agricultural Sciences Institute Of Food Safety And Monitoring Technology | Human-derived insect-resistant gene and anti-Cry1B toxin idiotype single-chain antibody encoded thereby and application thereof |
| CN105777899A (en) * | 2016-04-25 | 2016-07-20 | 江苏省农业科学院 | Monoclonal antibody capable of resisting Bt Cry1Ab toxins, cell strain generating monoclonal antibody, and preparation method and application of monoclonal antibody |
| CN113325182A (en) * | 2021-05-26 | 2021-08-31 | 武汉华美生物工程有限公司 | Efficient screening method of paired antibodies |
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