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Antibody pair for detecting content of RANKL in serum and application of antibody pair

A technology for detecting antibodies and antibody pairs, which is applied in the field of medicine and can solve problems such as paired antibody reports

Inactive Publication Date: 2020-10-20
廊坊天光生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, up to now, there is no paired antibody report for clinical detection of RANKL

Method used

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  • Antibody pair for detecting content of RANKL in serum and application of antibody pair
  • Antibody pair for detecting content of RANKL in serum and application of antibody pair
  • Antibody pair for detecting content of RANKL in serum and application of antibody pair

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Preparation of monoclonal antibodies AntiRANKL_N and AntiRANKL_C

[0033] 1. To analyze the antigenicity of RANKL (SEQ ID NO: 1) protein, divide the RANKL antigen into two sequences of N-terminal (SEQ ID NO: 2) and C-terminal (SEQ ID NO: 3), and synthesize the N-terminal expression of RANKL respectively (SEQ ID NO:2) and the C-terminal epitope of PF4 (SEQ ID NO:3), respectively coupled with KLH to obtain N-terminal immunogen and C-terminal immunogen; after immunization of mice, the mice were 8 weeks old female Balb / c mice.

[0034] 2. Animal immunization: 8-week-old female Balb / c mice were immunized with N-terminal immunogen and C-terminal immunogen respectively, and 100ug of antigen was used to immunize mice 4 times with an interval of 4 weeks between each time.

[0035] 3. Cell fusion: kill the mice by neck dislocation, place them in 75% ethanol solution for 5-10 minutes, take out the spleen under aseptic conditions, squeeze and gently grind the spleen cel...

Embodiment 2

[0044] Example 2: Verifying the monoclonal antibody obtained in Example 1

[0045] (1) Coat the whole RANKL antigen (SEQ ID NO:1), the N-terminal antigen of RANKL (SEQ ID NO:2) and the C-terminal antigen of RANKL (SEQ ID NO:3) respectively, and the coating concentration is 2ug / ml, at 4°C for 2 hours, wash the plate twice with 0.9% NaCl washing solution, pat dry, add 200 μl of blocking solution (0.5mol PBS+1%BSA+2.5% sucrose), block at 37°C for 2 hours, then pour out the wells Medium liquid, pat dry;

[0046] (2) Add different volumes of AntiRANKL_N and AntiRANKL_C obtained in Example 1 (as shown in Table 1), and react at 37°C for 1 hour;

[0047] (3) Wash the plate 3 times with 0.9% NaCl washing solution and pat dry;

[0048] (4) Add HRP-labeled goat anti-mouse IgG secondary antibody and react at 37°C for 1 hour;

[0049] (5) Wash the plate 5 times with 0.9% NaCl washing solution and pat dry;

[0050] (6) add substrate liquid luminol at last, measure its luminescence valu...

Embodiment 3

[0055] Example 3: Sequencing of monoclonal antibodies AntiRANKL_N and AntiRANKL_C and preparation of corresponding antibodies by hybridoma cells

[0056] 1. Extract the total RNA of the AntiRANKL_N hybridoma cells in Example 1, use the cDNA synthesis reverse transcription kit, synthesize the first strand cDNA with the RNA as a template, and then use the cDNA as a template to amplify the variable region gene of the monoclonal antibody AntiRANKL_N . The variable region PCR product sequence was cloned by T / A, and positive colonies were selected for sequencing, and the amino acid translation analysis was performed on the sequencing results.

[0057] The results showed that the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of the AntiRANKL_N monoclonal antibody are listed in SEQ ID NO: 4, 5 and 6, respectively. In addition, the light chain variable region gene and the encoded amino acid sequence comprising the above variable region are shown in ...

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Abstract

The invention provides an antibody pair for detecting the content of RANKL in serum and an application of the antibody pair. RANKL antigen is divided into an N-terminal (SEQ ID NO:2) sequence and a C-terminal (SEQ ID NO:3) sequence, and mice are immunized with the two sequences respectively; the high-specificity and high-sensitivity antibody pair AntiRANKL_N and AntiRANKL_C are screened out; and the content of the RANKL in the serum can be effectively determined through the antibody pair, so that diagnosis and treatment of osteoporosis are facilitated.

Description

technical field [0001] The present application relates to the field of medicine, in particular to an antibody pair for detecting the content of RANKL in serum and its application. Background technique [0002] Osteoporosis is an age-related chronic systemic metabolic disease, which is mainly characterized by decreased bone mass, destruction of bone microarchitecture, and increased bone fragility. Due to its increasing morbidity and mortality, the disease has become a global public health problem. In view of the high incidence of osteoporosis and its serious consequences, people have conducted in-depth research on its pathogenesis. [0003] RANKL protein is considered to be an essential biological molecule in the process of osteoclast activation and proliferation. It has three subtypes and all of them can promote osteoclast proliferation. RANKL can be expressed by a variety of cells. RANKL expressed in cartilage tissue is regulated by 1,25(OH)2D3 BMP2 and Wnt / b-catenin sign...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13G01N33/68
CPCC07K16/2875G01N33/68C07K2317/56G01N2333/70575
Inventor 刘静程勇刘继来王征
Owner 廊坊天光生物技术有限公司
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