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Preparation method of rabbit monoclonal antibody for resisting beta-glucuronidase

An aldolase and cloning antibody technology, applied in the direction of anti-animal/human immunoglobulin, cells modified by introducing foreign genetic material, fusion cells, etc., which can solve the problems of error and interference.

Inactive Publication Date: 2011-07-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Only preliminary judgment can be made by tissue staining gus Whether the gene has been transferred to the gene in the plant tissue, so this method is gus There will be large errors in the judgment of genes, and other interferences are relatively large

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] 1) Rabbit immunity

[0016] 400 μg of immune antigen β -Glucuronidase was mixed with an equal amount of Freund's incomplete adjuvant, and after complete emulsification, two New Zealand white rabbits were subcutaneously injected at 3 points on the back, and three, five, seven, and nine weeks after the start of immunization, respectively. 150~250mg of immune antigen protein is mixed with Freund's incomplete adjuvant and then immunized in the same way;

[0017] 2) cell fusion

[0018] After the fifth immunization, rabbits with high serum titer were selected for a booster immunization. After 8 days, a sterile spleen was taken to prepare a single cell suspension. 48% by mass of polyethylene glycol as a fusion agent, 18% by volume of HAT as a selective medium, spread in 40 96-well plates, where the concentration of H is 0.8 ×10 -6 mol / L of hypoxanthine, A is a concentration of 3.5×10 -9 mol / L of aminopterin, T is a concentration of 1.4×10 -7 mol / L thymidine;

[0019] 3...

Embodiment 2

[0023] 1) Rabbit immunity

[0024] 600 μg of immune antigen β -Glucuronidase was mixed with the same amount of Freund's incomplete adjuvant, and after complete emulsification, 3 New Zealand white rabbits were subcutaneously injected at 6 o'clock on the back. Immunize in the same way after mixing 250mg of immune antigen protein with Freund's incomplete adjuvant;

[0025] 2) cell fusion

[0026] After the fifth immunization, rabbits with high serum titer were selected for a booster immunization. After 10 days, a sterile spleen was taken to prepare a single cell suspension. 52% by mass percentage of polyethylene glycol as a fusion agent, 22% by volume of HAT as a selective medium, spread in 40 96-well plates, where the concentration of H is 1.2 ×10 -6 mol / L hypoxanthine, A is a concentration of 4.5×10 -9 mol / L of aminopterin, T is the concentration of 1.8×10 -7 mol / L thymidine;

[0027] 3) Screening of hybridoma cells

[0028] On the 12th day after cell fusion, 2 plates o...

Embodiment 3

[0031] 1) Rabbit immunity

[0032] 500 μg of immune antigen β -Glucuronidase was mixed with an equal amount of Freund's incomplete adjuvant, and after complete emulsification, 3 New Zealand white rabbits were subcutaneously injected at 5 points on the back, and three, five, seven, and nine weeks after the start of immunization, respectively. Immunize in the same way after mixing 200mg of immune antigen protein with Freund's incomplete adjuvant;

[0033] 2) cell fusion

[0034] After the fifth immunization, rabbits with high serum titer were selected for a booster immunization. After 10 days, a sterile spleen was taken to prepare a single cell suspension. 50% polyethylene glycol as a fusion agent, 20% HAT as a selective medium, spread in 40 96-well plates, where the concentration of H is 1.0 ×10 -6 mol / L of hypoxanthine, A is a concentration of 4.0×10 -9 mol / L of aminopterin, T is the concentration of 1.6×10 -7 mol / L thymidine;

[0035] 3) Screening of hybridoma cells

[...

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PUM

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Abstract

The invention discloses a preparation method of a rabbit monoclonal antibody for resisting beta-glucuronidase. The method comprises the following steps: high purity beta-glucuronidase protein is used as immunogen immune molecule, the titer of antiserum is measured, the rabbit B lymphocyte and the myeloma cell are fused, selective medium is used to perform selective cultivation, pre-screening, preliminary screening and secondary screening are performed, positive clone is selected to extensively proliferate, recombinant vector is constructed to perform in vitro culture, and the monoclonal antibody is obtained through separation and purification. The preparation method can be used in the immunologic methods such as enzyme-linked immuno sorbent assay (ELISA) and WesternBlotting to perform the qualitative or quantitative detection of beta-glucuronidase.

Description

technical field [0001] The present invention relates to anti- β - glucuronidase antibody, especially relates to an anti β -The preparation method of glucuronidase rabbit monoclonal antibody. Background technique [0002] gus The gene is a type of reporter gene obtained from bacteria, which can encode glucuronidase, which hydrolyzes the substrate 5-bromo-4chloro-3 indole-glucuronide in histochemical staining, and its hydrolyzate is blue. There is no endogenous gus activity in the plant tissue. Use the gus histochemical detection method to detect the transformed callus or regenerated plant sections. If it turns blue, it can be judged initially gus The gene has been transferred into plant cells and expresses glucuronidase. gus gene is β - glucuronidase gene, ie β-gus . Only preliminary judgment can be made by tissue staining gus Whether the gene has been transferred to the gene in the plant tissue, so this method is gus There will be large errors in the judgment of g...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N5/16
Inventor 郑晓冬倪庚沈金儿刘娜陆蕾冯劲松
Owner ZHEJIANG UNIV
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