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Human AD7c-NTP monoclonal antibodies as well as preparation method and application thereof

A monoclonal antibody, ad7c-ntp technology, applied in the preparation method of peptides, chemical instruments and methods, anti-animal/human immunoglobulin, etc., can solve the problems of complex structure, insufficient sensitivity and specificity, and achieve high The effect of good sensitivity, specificity, and reliable clinical reference value

Pending Publication Date: 2020-07-24
BEIJING BIOSYNTHESIS BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most domestic AD7c-NTP in vitro diagnostic kits are developed based on AD7c-NTP polypeptide antigens, and their sensitivity and specificity are insufficient. Therefore, obtaining high-affinity and high-specificity anti-AD7c-NTP antibodies is the key to the successful development of AD7c-NTP in vitro diagnostic kits. The essential
AD7c-NTP is a membrane protein with a relatively complex structure. It has not been reported to obtain a large number of AD7c-NTP protein antigens through DNA recombination technology, prepare anti-AD7c-NTP monoclonal antibodies, and develop AD7c-NTP in vitro diagnostic kits.

Method used

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  • Human AD7c-NTP monoclonal antibodies as well as preparation method and application thereof
  • Human AD7c-NTP monoclonal antibodies as well as preparation method and application thereof
  • Human AD7c-NTP monoclonal antibodies as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Preparation of Human AD7c-NTP Recombinant Protein

[0032] (1), construction of human AD7c-NTP prokaryotic expression vector

[0033] Human AD7c-NTP gene sequence SEQ ID NO.2 was artificially synthesized, inserted into the prokaryotic expression plasmid pET30a digested with NdeI and XhoI, and transformed into competent Escherichia coli BL21(DE3). 0.1g / L kanamycin resistance plate screens positive clones, and induces them with a final concentration of 1mM IPTG at 16°C, 25°C, 30°C, and 37°C respectively, preferably at 25°C, shaking at 200rpm Cultured for 14h to induce the expression of human AD7c-NTP recombinant protein. Finally identified by SDS-PAGE, the results are as follows figure 1 .shown.

[0034] Note: M: pre-stained protein marker, lanes 1-3 are pET30a-AD7c-NTP bacteria solution before induction, supernatant of pET30a-AD7c-NTP bacteria solution after induction, pET30a-AD7c-NTP bacteria solution after induction and precipitation after ultrasound . ...

Embodiment 2

[0049] Example 2: Preparation of anti-human AD7c-NTP monoclonal antibody

[0050] (1), animal immunity

[0051] Using purified human AD7c-NTP recombinant protein as the immunogen, immunize 8-12 week old BALB / c female mice by conventional methods, emulsify with Freund's complete adjuvant and antigen for the first time, and immunize subcutaneously and intraperitoneally at multiple points, the dose is 100 μg / mouse; booster immunization once every two weeks, a total of three times, the adjuvant is Freund’s incomplete adjuvant, the dose is 100 μg / mouse / time; the titer of mouse tail blood was detected by indirect ELISA method>1:10^4, fusion In the first three days, intraperitoneal injection of 100 μg human AD7c-NTP antigen was carried out for booster immunization.

[0052] (2), cell fusion

[0053] Mix immune spleen cells and myeloma cells (SP2 / 0) at a ratio of 5:1, centrifuge at 1000rpm for 10min, discard the supernatant; resuspend the cells in RPMI-1640 medium, centrifuge at 100...

Embodiment 3

[0067] Example Three: Monoclonal Antibody Purification of Anti-human AD7c-NTP Recombinant Protein

[0068] (1) Purification of monoclonal antibodies

[0069] The monoclonal antibody ascites obtained in Example 2 was purified by Protein A affinity chromatography, and the specific steps were as follows:

[0070] The basic steps are: equilibration-loading-equilibrium-elution-equilibrium-preservation

[0071] Balance solution: 20mM PB (pH7.2) buffer

[0072] Eluent: 0.1M pH 3.0 glycine-HCl buffer

[0073] Neutralization buffer: 1M, pH9.0, Tris-HCl buffer

[0074] Preservation solution: 20% absolute ethanol

[0075]Take 3-5ml of ascitic fluid, precipitate with 50% saturated ammonium sulfate for more than 2 hours, centrifuge at 10,000rmp for 20 minutes, discard the supernatant; dissolve the precipitate with equilibrium solution, filter it with a 0.45μm filter membrane, and prepare for loading; Protein A affinity column Pre-equilibrated with 5 times the column volume of the bala...

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Abstract

The invention discloses human AD7c-NTP monoclonal antibodies as well as a preparation method and application thereof. The monoclonal antibodies are prepared by the following steps: taking AD7c-NTP recombinant protein as an immunogen, immunizing a BALB / c mouse, performing fusion and subcloning on mouse splenocyte and mouse myeloma cells, screening monoclonal antibodies with high specificity and titer, and finally performing ascites preparation and purification to obtain the monoclonal antibodies resisting human AD7c-NTP protein. Compared with the prior art, the method has the advantages that: the invention provides a hybridoma cell strain I1B10 and a hybridoma cell strain X1C11 capable of generating the anti-human-AD7c-NTP monoclonal antibodies, and the anti-human-AD7c-NTP monoclonal antibodies: I1B10 and X1C11. The antibody pair can be used for detecting the concentration of AD7c-NTP in human body fluid by virtue of a double-antibody sandwich method, and has an important application prospect.

Description

technical field [0001] The invention relates to the field of monoclonal antibody preparation, in particular to a human AD7c-NTP monoclonal antibody and its preparation method and application. Background technique [0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by a chronic progressive process of impaired intellectual function and memory loss. In 2001, 11 million people worldwide were living with Alzheimer's disease (Gadit, 2001). By 2005, there were 24.2 million people with dementia in the world [Reitz C,2011]; in 2010, this number increased to 36 million, to 66 million by 2030, and to 1.15-1.35 by 2050 billion [The Global Impact of Dementia 2013–2050]. Currently, Alzheimer's disease is the sixth leading cause of death (AA, 2012). Alzheimer's disease accounts for about 70 percent of all dementia patients. The latest diagnostic guidelines divide Alzheimer's disease into three stages: the dementia stage; the symptomatic, pre-dementia (also kn...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K1/22G01N33/68G01N33/577
CPCC07K16/18G01N33/68G01N33/577C07K2317/35G01N2333/47
Inventor 赵风强董兵杨春吴妤邬晓东
Owner BEIJING BIOSYNTHESIS BIOTECH
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