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All-round nuclease Benzonase ELISA detection kit

A detection kit and nuclease technology, applied in the biological field, can solve the problems of low detection sensitivity and lack of compatibility of Benzonase, and achieve the effects of high detection accuracy, improved detection sensitivity, and high sensitivity

Pending Publication Date: 2021-12-10
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, major domestic manufacturers of biological products mainly use Merck’s Benzonase ELISA detection kit (Cat. No.: 101681), with a detection range of 1-10 ng / ml and a detection sensitivity of 0.2 ng / ml. There is no good kit in China, and domestic use The detection sensitivity of the kit prepared with polyclonal antibody as the raw material is not high, and monoclonal antibody is used as the raw material, which is not compatible with Benzonase from different manufacturers

Method used

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  • All-round nuclease Benzonase ELISA detection kit
  • All-round nuclease Benzonase ELISA detection kit
  • All-round nuclease Benzonase ELISA detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Preparation and Purification of Anti-Benzonase Polyclonal Antibody

[0062] 1.1 Preparation of rabbit immune serum

[0063] Mix equal volumes of Benzonase and Freund's complete adjuvant, and use a syringe to promote complete emulsification. The emulsified antigen was used for subcutaneous multi-point immunization on the back of New Zealand rabbits for 12 weeks, once every two weeks. Benzonase was emulsified with Freund's complete adjuvant for initial immunization, and Benzonase was emulsified with Freund's incomplete adjuvant for booster immunization. One group received 0.5 mg / rat for primary immunization, 0.25 mg / rat for booster immunization, and 0.5 mg / rat for booster immunization. After 3 times of immunization, take venous blood ELISA to test the serum titer, and then test the titer every 2 weeks. If the serum titer reaches 1:200,000 or more, stop the immunization. Take rabbit whole blood from the carotid artery. After standing at room temperature for 1 h...

Embodiment 2

[0096] Example 2 Biotin-labeled antibody

[0097] (1) Take the biotin reagent out of the refrigerator and equilibrate to room temperature before opening in step 3.

[0098] (2) Dilute the antibody with PBS, dissolve 1-10 mg antibody in 0.5-2.0 ml PBS.

[0099] (3) Prepare a 10 mM biotin reagent solution in DMSO. 2 mg NHS-Biotin was dissolved in 590 μl DMSO.

[0100] (4) Mix biotin reagent and antibody solution at a ratio of 20:1 molar ratio of biotin to antibody.

[0101] (5) Incubate on ice for 2 hours or at room temperature for 30 minutes.

[0102] (6) At this point, the antibody labeling is complete, and the excess biotin reagent is removed by dialysis.

[0103] The labeling effect of the antibody was detected by ELISA. 1 μg / ml of Benzonase was coated on the ELISA plate, blocked with 5% skimmed milk powder, and the labeled antibody was diluted 1000 times (antibody final concentration 1 μg / ml) into 7 gradients, incubated and washed. HRP-tagged avidin. TMB color develo...

Embodiment 3

[0106] Example 3 Sandwich ELISA detection Benzonase method debugging.

[0107]Sandwich ELISA steps are: rabbit polyclonal antibody coating, plate washing, ELISA plate blocking, adding samples, washing plate, adding biotinylated rabbit polyclonal antibody, washing plate, adding HRP-labeled avidin, washing plate, adding TMB Color development, add stop solution to stop, OD450 reading. At the beginning of the commissioning, it was found that the OD value of the negative sample was greater than 0.2, which was related to the simultaneous use of polyclonal antibodies as the coating antibody and the detection antibody. It is only meaningful to debug and optimize other conditions when the value drops below 0.2. In the present invention, the debugging sequence is as follows: optimize the concentration of the coating antibody and the concentration of the detection antibody, optimize the coating buffer, coating temperature and time, optimize the best blocking conditions, optimize the inc...

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Abstract

The invention provides an all-round nuclease Benzonase ELISA (Enzyme-Linked Immuno Sorbent Assay) detection kit. The Benzonase ELISA detection kit comprises: a rabbit-derived Benzonase polyclonal antibody; a rabbit-derived Benzonase polyclonal antibody marked by biotin; an HRP labeled by avidin; a standard substance Benzonase; a color developing solution, a stop solution, a confining solution and a washing solution. The lower detection limit of the detection kit is 24 pg / ml, the quantitative detection range is 0.047-3 ng / ml, Benzonase of different manufacturers can be detected, and an imported Benzonase detection kit can be replaced. Meanwhile, methodological system evaluation is carried out on a constructed ELISA detection system, and the kit is good in specificity and high in detection accuracy.

Description

technical field [0001] The patent of the invention relates to a kind of totipotent nuclease Benzonase ELISA detection kit, which belongs to the field of biotechnology. Background technique [0002] As more and more biological products (recombinant proteins, Vero cell vaccines, etc.) Good stability is easy to form residues in the production process, and when these biological products are applied to the prevention and treatment of human diseases, they will bring uncontrollable risk factors. The host nucleic acid residues in the final product must comply with the regulations of WHO, FDA, EMEA and my country's NMPA. [0003] Benzonase is a genetically engineered endonuclease from Serratia marcescens. Benzonase can degrade double-stranded, single-stranded, linear, and circular DNA and RNA, and completely degrade nucleic acids into 5'-monophosphate oligonucleotides with a length of 3 to 5 bases. Because it can efficiently degrade any form of DNA and RNA, it is also called "univ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/573G01N33/543
CPCG01N33/573G01N33/543G01N2333/922
Inventor 易红飞王亚如
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
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