Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Taq enzyme 5'-3' exonuclease activity blocking monoclonal antibody and application thereof

A technology of monoclonal antibody and exonuclease, which is applied in the field of bioengineering to achieve the effect of improving stability, good stability and good sealing effect

Active Publication Date: 2021-02-26
YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
View PDF5 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no 5'-3' exonuclease blocking antibody of Taq enzyme in the domestic market, so it is necessary to fill the gap in this area and improve the quality of q-PCR products

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Taq enzyme 5'-3' exonuclease activity blocking monoclonal antibody and application thereof
  • Taq enzyme 5'-3' exonuclease activity blocking monoclonal antibody and application thereof
  • Taq enzyme 5'-3' exonuclease activity blocking monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Mouse immunity and serum titer determination

[0047] Three six-week-old Balb / c mice were immunized, and Taq enzyme was emulsified with Freund's complete adjuvant for the first immunization, and Taq enzyme was emulsified with Freund's incomplete adjuvant for subsequent immunizations, and the immunization dose was 100 μg per mouse Protein, immunized once a week, immunized for two months, by subcutaneous multi-point injection. Orbital blood was collected before each immunization after the third immunization, and the serum titer was detected by enzyme-linked immunosorbent assay (ELISA). The specific method is to coat the plate with 1 μg / mL Taq enzyme (dissolved in PBS), block with 5% skimmed milk powder, serially dilute the serum with PBS, use HRP-labeled goat anti-mouse IgG as the secondary antibody, and develop color with TMB. Three days before cell fusion, 100 μg Taq enzyme (dissolved in PBS) was injected into the peritoneal cavity of mice.

Embodiment 2

[0048] Example 2 Cell Fusion and Hybridoma Screening

[0049] Cultivate a sufficient amount of SP2 / 0 in advance, not less than 10 8 to ensure that the cells are in the logarithmic growth phase. Collect SP2 / 0 and wash three times with serum-free RPMI1640 medium. The spleens of the mice immunized with shock were taken, and the splenocytes were obtained by grinding, and the splenocytes were washed three times with RPMI1640 medium. Splenocytes and SP2 / 0 cells were mixed at a ratio of 2:1 to 5:1. 1000 rpm, 10 min, remove the medium, add 1 mL PEG1450 into the cells within 1 minute, and stir gently while adding. Within two minutes after adding PEG1450, add 2 mL of RPMI1640 culture medium at a uniform speed to terminate the effect of PEG1450, and then add RPMI1640 medium to a volume of 50 mL within 2 minutes. Gently shake the centrifuge tube to disperse the cells as much as possible and put them in the cell culture incubator for 10 min. Centrifuge at 1000 rpm for 10 min to remove...

Embodiment 3

[0051] Example 3 Hybridoma monoclonalization

[0052] Two days after the cells were cultured in the 24-well plate, the ELISA test was performed, the cells in the positive wells were counted, and 200 cells were taken and cultured in the 96-well plate. After culturing for about a week, pick wells with only 1 to 3 clones for ELISA detection, and transfer the cells in positive wells to 24-well culture. This is one monoclonalization, and so repeated, monoclonalization was performed four times, and 52 antibody strains were obtained. The medium containing HT was used for the initial monocloning, and then changed to a medium without HT.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a Taq enzyme 5'-3' exonuclease activity blocking monoclonal antibody. The Taq enzyme 5'-3' exonuclease activity blocking monoclonal antibody comprises a light chain variable region containing LCDR1, LCDR2 and LCDR3 sequences and a heavy chain variable region containing HCDR1, HCDR2 and HCDR3 sequences, wherein the sequence of the LCDR1 is shown as SEQ ID NO: 10, the sequence of the LCDR2 is shown as SEQ ID NO: 11, the sequence of the LCDR3 is shown as SEQ ID NO: 12, the sequence of the HCDR1 is shown as SEQ ID NO: 6, the sequence of the HCDR2 is shown as SEQ ID NO: 7, and the sequence of the HCDR3 is shown as SEQ ID NO: 8. The invention further discloses application of the monoclonal antibody. The blocking effect of the monoclonal antibody is good. The stability ofa prepared double-blocking hot-start enzyme is good.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a Taq enzyme 5'-3' exonuclease activity blocking monoclonal antibody and its application. Background technique [0002] People in the nucleic acid detection industry pay special attention to the stability of detection reagents. Whether it is long-term storage stability or short-term deviation from normal storage conditions, it directly affects the validity of test results. However, the stability of short-term deviation from normal storage conditions is particularly important in the actual use and transportation process. For example, in the case of large sample size, adaptation automation, waiting in line for the machine, etc., the reaction system needs to be placed at room temperature or 4°C. When the transportation time is long and the temperature is difficult to control, the detection system is exposed to risks. How to ensure stability and how to ensure that...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K16/40C12Q1/686C12N9/22
CPCC07K16/40C12Q1/686C12N9/22C07K2317/565C07K2317/56C12Q2521/101C12Q2527/127C12Q2549/101
Inventor 易红飞袁灿灿滕以刚王亚茹毛瑞祥沈晓波任晓律周其好
Owner YEASEN BIOTECHNOLOGY (SHANGHAI) CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com