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Bt crystallin CrylAc radio-immunity test reagent box, preparing and detecting method thereof

A technology for detecting kits and crystal proteins, applied in biochemical equipment and methods, biological testing, measuring devices, etc., can solve the problems of long reaction time, cumbersome operation, and many operation steps, so as to achieve simple operation and reduce sample testing time. , the effect of increasing the reaction rate

Inactive Publication Date: 2004-06-02
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There are following defects in the ELISA method used in the prior art for detecting Bt crystal protein: 1, the detection result is unstable, because the enzymatic reaction is easily affected by the reaction conditions; 2, the reaction time is long, and the sampling time is as long as 24 hours Above (including covering time); 3. There are many operation steps, which are relatively cumbersome
[0004] Radioimmunoassay detection of Bt crystal protein, although the results are stable, but the traditional radioimmunoassay requires centrifugation to separate the antigen-antibody complex, the operation is cumbersome, and the shortest detection time is 3 hours
The existing relatively new technology radioimmunoassay antigen-antibody reaction is carried out at the solid phase interface without centrifugation, but the reaction time is longer than the traditional radioimmunoassay
[0005] Therefore, none of the methods for detecting Bt crystal protein in the prior art can meet the requirements of quickness and simplicity.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1. Extraction and purification medium of Bt crystal protein CrylAc:

[0031] Liquid LB (see translation by Jin Dongyan et al., original work J. Sambrook, 1992. Molecular Cloning Experiment Guide 2nd Edition, Science Press): Tryptone1%, Yeast extract 0.5%, NaCl1%, pH7.0, 15 lbs, 20min.

[0032] Liquid Z's (see Zuo Yahui et al., 1999, a preliminary study on the screening of several Bt medium and crystal protein purification methods, Plant Protection, 25(3), 31-31): peptone 1%, yeast powder 0.2%, soluble starch 0.3%, Glucose 0.2%, K 2 HPO 4 0.1%, KH 2 PO 4 1%, CaCO 3 0.2%, pH7.0, 15 pounds, 20min. Activated strain:

[0033] Pick a single colony (HD-73) in LB liquid medium, 37°C, 200rpm, and shake the flask for 20-24 hours. Extraction steps:

[0034] Transfer the activated bacterium solution to Z's liquid medium according to the inoculation amount of 1%, and culture in shake flask at 37°C for 36-48 hours. Centrifuge, 5000rpm, 5min, collect all bacteria, Na 2 CO 3...

Embodiment 2

[0065] Embodiment 2 kit composition

[0066] 1. Bt crystal protein CrylAc standard solution (0.05M phosphate buffer pH7.4) series, the concentrations are 1000ng / ml, 500ng / ml, 100ng / ml, 50ng / ml, 10ng / ml, 1ng / ml and 0ng / ml , a total of 7 bottles, each 1ml.

[0067] 2. A bottle of Bt crystal protein CrylAc antibody solution (Tris-HCl buffer) linked to magnetic particles, 10ml. blue color.

[0068] For the preparation of the Bt crystal protein CrylAc antibody, refer to the literature (Edited by Li Zhenjia, Han Chunsheng and Wang Jianxun, Practical Radioimmunology. Science and Technology Press pp21-40).

[0069] Preparation of magnetic particle-linked Bt crystal protein CrylAc antibody: Aerosol 604 in 1% glutaraldehyde solution with Fe 3 o 4 The aqueous solution was mixed and adjusted to a certain pH, deoxygenated by nitrogen, shaken for 24 hours (to maintain the pH), dialyzed against water, and centrifuged to obtain 100nm particles. The microparticles were centrifuged after b...

Embodiment 3

[0077] Embodiment 3 sampling process

[0078] 1. Sample collection and crude protein extraction

[0079] Take 1g of grains or fresh leaves, add 2ml of protein extract (2.66g of anhydrous sodium carbonate, 2.92g of sodium chloride and Vclg in 1L of distilled water), grind it into a homogenate, transfer it to a small test tube, shake it slightly, and let it rest for 5 minute. Aspirate the supernatant for testing.

[0080] 2. Operating procedures

[0081] 1) Numbering on plastic test tubes, including non-specific tubes (NSB), zero-binding tubes (S 0 ), standard pipe (S 1 -S 6 ), quality control tubes (Qcl, 2, 3), sample tubes (U), and the above test tubes must be labeled with double tubes.

[0082] 2) Add 200 μl zero standard to NSB tube, S 0 Add 100 μl of zero standard to the tube and add 100 μl of the corresponding standard, quality control or sample to the other tube.

[0083] 3) Add 100 [mu]l microparticle-linked antiserum to all tubes except NSB tubes.

[0084] 4) A...

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PUM

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Abstract

The present invention provides one kind of Bt crystallin Cry lAc radioimmunoassay kit and its preparation process and detection method. Magnetic particle is prepared with glutaraldehyde, Aerosol 604 and ferroferric oxide, and connected to Bt crystallin Cry lAc antibody. Under the action of magnetic field, Bt crystallin Cry lAc and Bt crystallin Cry lAc antibody react specifically in fast speed and the sample testing period is reduced to 40 min. The present invention also connect magnetic particle to IgG to prepare immunological separating agent, and under the action of magnetic field, the antigen antibody composition deposits automatically for separation without needing centrifugation.

Description

Technical field: [0001] The invention relates to a Bt crystal protein CrylAc radioimmunoassay kit and a preparation method thereof, and also relates to a Bt crystal protein radioimmunoassay method. Background technique: [0002] Transgenic Bt plants have good insect resistance, so Bt genes have been widely transferred into crops such as cotton and corn. Bt crystal protein is the Bt gene expression product of Bt gene-transfer organisms, and is an important indicator for identifying insect resistance. Its rapid and accurate determination provides technical support for breeding, pest management and gene identification of transgenic plants. [0003] There are following defects in the ELISA method used in the prior art for detecting Bt crystal protein: 1, the detection result is unstable, because the enzymatic reaction is easily affected by the reaction conditions; 2, the reaction time is long, and the sampling time is as long as 24 hours Above (including coating time); 3. There...

Claims

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Application Information

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IPC IPC(8): C12Q1/00G01N21/00G01N33/53G01N33/534G01N33/58G01N33/68
Inventor 潘家荣张维张杰乔艳红宋平李锦波林敏黄大昉
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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