Scholarlink

Abstract <p>The development of a method to degrade amyloid via specificantibodies to haptens, being part of the amyloid composition, appearsrelevant for the introduction of immune therapy for amyloidosisinto practice. The experimental development of this approach iscomplicated by a possible nonspecific binding of immunoglobulins(IgG) to amyloid fibrils, which is considered here on the exampleof beta-2-microglobulin (β2M) fibrils. This study was aimed to identifyimmunoglobulins binding to amyloid and to detect antibodies specificto β2M fibrils. The purified IgG preparation Neocytotect and purified insulinActrapid were used for experiments. Insulin fibrils were incubatedin glycine-HCl for 7 days at 37°C; lysozyme fibrils were obtainedat 55°C and low pH. The monomer purity was tested via PAAG electrophoresisand mass spectrometry. Amyloid fibrils were detected using Congored staining, thioflavin T fluorescence, and microscopy. The synthesisof a recombinant yellow fluorescent immunoglobulin-binding protein(YFP-GB1) was induced by the IPTG reagent, the protein was isolatedby ultrasound, and purified by metal-chelate affinity chromatography.HRP-conjugated anti-human (IgG) secondary antibodies were used forimmunoblotting visualization and YFP-GB1 for fluorescence imaging.During medium acidification, β2M fibrils form spontaneously, andtheir stabilization was found to be attainable by the adsorptionon nitrocellulose filters (NCFs). When running dot immunoblottingon NCF, subsequent Congo red staining confirmed the presence offibrils on the membrane and their stability at physiological pH,whereas the dots of β2M monomer application were not stained. Therewas found the fact of nonspecific interaction of human IgG and secondaryantibodies with β2M fibrils. The revealed interaction of human andother species’ immunoglobulins with β2M fibrils indicates the necessityto consider this phenomenon when developing methods of immunotherapyfor amyloidosis and in immunodetecting anti-amyloid fibril antibodiesfor the diagnosis of β2M-related and other amyloidoses.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Nonspecific Interaction of Immunoglobulins (IgG) with Human Beta-2 Microglobulin Fibrils

  • 期刊论文

  • 发表日期：

    2025年1月1日

查看全文
  • D. S. Polyakov,
  • N. A. Grudinina,
  • O. I. Antimonova,
  • L. I. Klysbaeva,
  • R. G. Sakhabeev,
  • M. M. Shavlovsky

摘要

Abstract

The development of a method to degrade amyloid via specificantibodies to haptens, being part of the amyloid composition, appearsrelevant for the introduction of immune therapy for amyloidosisinto practice. The experimental development of this approach iscomplicated by a possible nonspecific binding of immunoglobulins(IgG) to amyloid fibrils, which is considered here on the exampleof beta-2-microglobulin (β2M) fibrils. This study was aimed to identifyimmunoglobulins binding to amyloid and to detect antibodies specificto β2M fibrils. The purified IgG preparation Neocytotect and purified insulinActrapid were used for experiments. Insulin fibrils were incubatedin glycine-HCl for 7 days at 37°C; lysozyme fibrils were obtainedat 55°C and low pH. The monomer purity was tested via PAAG electrophoresisand mass spectrometry. Amyloid fibrils were detected using Congored staining, thioflavin T fluorescence, and microscopy. The synthesisof a recombinant yellow fluorescent immunoglobulin-binding protein(YFP-GB1) was induced by the IPTG reagent, the protein was isolatedby ultrasound, and purified by metal-chelate affinity chromatography.HRP-conjugated anti-human (IgG) secondary antibodies were used forimmunoblotting visualization and YFP-GB1 for fluorescence imaging.During medium acidification, β2M fibrils form spontaneously, andtheir stabilization was found to be attainable by the adsorptionon nitrocellulose filters (NCFs). When running dot immunoblottingon NCF, subsequent Congo red staining confirmed the presence offibrils on the membrane and their stability at physiological pH,whereas the dots of β2M monomer application were not stained. Therewas found the fact of nonspecific interaction of human IgG and secondaryantibodies with β2M fibrils. The revealed interaction of human andother species’ immunoglobulins with β2M fibrils indicates the necessityto consider this phenomenon when developing methods of immunotherapyfor amyloidosis and in immunodetecting anti-amyloid fibril antibodiesfor the diagnosis of β2M-related and other amyloidoses.