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Combined Biomarker Measurements of Fibrosis

A technology for fibrotic diseases and binding capacity, which can be used in biological testing, measuring devices, biomaterial analysis, etc., and can solve problems such as undetermined binding epitopes

Active Publication Date: 2022-04-05
NORDIC BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0017] Bayer (33) disclosed a sandwich ELISA using 2 Detector monoclonal antibody to N-GSPGPPGICQSCPTGPQNYSP-COOH (SEQ ID NO: 3), but binding epitope undetermined

Method used

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  • Combined Biomarker Measurements of Fibrosis
  • Combined Biomarker Measurements of Fibrosis
  • Combined Biomarker Measurements of Fibrosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1 - Monoclonal Antibody NB61-N62

[0067] Production of monoclonal antibodies

[0068] The N-terminal propeptide sequences of type III collagen were aligned between human, rat, and mouse species, and homology between species and uniqueness among other ECM proteins were selected by protein blasting. The amino acid sequence 145'-CPTGPQNYSP-'153 (SEQ ID NO: 6) in the PIIINPα1 chain is 100% homologous between human and rat ( figure 1 ). Monoclonal initiation was initiated by subcutaneously immunizing 4-5 week-old Balb / C mice with 200 μL of emulsified antigen and 50 μg of the PIIINP neoepitope C-terminal sequence (OVA-CGG-CPTGPQNYSP (SEQ ID NO: 10)) using Freund's incomplete adjuvant. Antibody production. Immunizations were repeated every 2 weeks until a stable serum titer level was reached. The mouse with the highest serum titer was selected for fusion. Mice were rested for one month and then boosted intravenously with 50 μg of the PIIINP neoepitope C-terminal...

Embodiment 2

[0076] Example 2 - PRO-C3 ELISA using NB61N-62

[0077] Supernatants from antibody-producing hybridomas were collected and monoclonal antibodies were purified using HiTrap affinity columns (GE Healthcare LifeScience, Little Chalfont, Buckinghamshire, UK) and Lightning-Link according to the manufacturer's instructions. TM The HRP Conjugation Kit (Innova Biosciences, Babraham, Cambridge, UK) was labeled with HRP.

[0078] The process of PRO-C3 competitive ELISA was as follows: the biotinylated peptide biotin-CGG-CPTGPQNYSP (SEQ ID NO: 11) dissolved in coating buffer (50 mM PBS-BTE+10% sorbitol, pH7.4) 96-well streptavidin-coated ELISA plates from Roche, cat. 11940279 were coated, incubated at 20° C. in the dark for 30 minutes, and then washed in wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Thereafter, 20 μL of peptide calibrators or samples were added to appropriate wells, followed by 100 μL of HRP-conjugated monoclonal antibody dissolved in incubation buffer (50 mM PBS-BTB +...

Embodiment 3

[0097] Example 3 - Determining the ratio of binding affinities

[0098] To determine the ratio of the binding affinity of the mAb to the target sequence versus the binding affinity of the mAb to the extended or shortened sequence, each sequence was synthesized and used as a calibrator peptide in the PRO-C3 ELISA as described in Example 2. The resulting calibration curve was used to determine the IC for each sequence / antibody combination 50 value. IC 50 [Target] / IC 50 The ratio of [lengthening or shortening] defines the ratio of binding affinities.

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Abstract

Provided herein is a sandwich immunoassay for the detection of cross-linked PIIINPs having at least two PIIINP chains linked together by interchain crosslinks, each chain having N protease composed of intact type III procollagen C-terminal neo-epitopes of PIIINP generated by cleavage. A biological sample with cross-linked PIIINP is contacted with a first surface-bound monoclonal antibody, followed by a second monoclonal antibody, both of which are specifically reactive with the neo-epitope in the C-terminal sequence of PIIINP, and then Binding of the second monoclonal antibody was determined. Also provided is a method of assessing the efficacy of an antagonist drug targeting lysyl oxidase by an immunoassay and a kit comprising a solid support bound to a first monoclonal antibody and comprising a second monoclonal antibody.

Description

technical field [0001] The present invention relates to a sandwich immunoassay for the detection of cross-linked PIIINP in biological samples and its use in assessing the efficacy of drugs targeting lysyl oxidase (LOX). The invention also relates to kits for performing sandwich immunoassays. Background technique [0002] Fibrotic diseases, including those listed in Table 1, are major causes of morbidity and mortality, for example, cirrhosis of the liver accounts for 800,000 deaths per year globally (1). [0003] Table 1. Different fibrotic disorders (2) [0004] [0005] [0006] A "fibrotic disease" is any disease that causes fibrosis, whether as a primary or secondary symptom. Fibrosis is the end result of a chronic inflammatory response to a variety of stimuli, including persistent infection, autoimmune responses, allergic reactions, chemical insults, radiation, and tissue damage. Fibrosis is characterized by the accumulation and reorganization of the extracellul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577G01N33/68
CPCG01N33/577G01N33/6887G01N2333/78G01N2800/085G01N2800/52G01N2800/7052Y02A50/30G01N33/581G01N2500/00
Inventor 费德里卡·杰诺韦塞梅特·尤尔·尼尔森莉萨·拉森黛安娜·朱莉·奥尔内斯-利明莫滕·卡斯达尔
Owner NORDIC BIOSCI
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