Получение поликлональных антител к AL-каппа легкой цепи амилоидного белка

Abstract

Introduction. Amyloidoses are classified based on the precursor protein that forms amyloid fibrils, and either systemic or localized distribution of amyloid deposition. Light chain (AL) amyloidosis, the most common form of systemic amyloidosis, comprises the full length of immunoglobulin fragments of light chains of kappa or lambda isotype. In contrast to other amyloid deposits, AL amyloidosis is considered as the most difficult one for classification by immunohistochemistry analysis, since every patient seems to have individual amino acid sites in the variable region. Thus, the use of standard antibody kits will not enable to obtain accurate results in all cases. The aim of this study was to obtain polyclonal antibodies to peptide epitopes of the variable and constant regions of kappa light chains of the amyloid protein. Material and methods. Using the Database of the National Center for Biotechnology Information (NCBI) and the data from scientific publications, we conducted a search and analysis of five classes of amyloidogenic kappa light chains.The Tcoffe Regular program was used for processing all amino acid chains. Protein secondary structure was determined according to the method described by Parker (1986), using the Peptide Companion software (CoshiSoft/PeptiSearch, USA). After the selection of amino acid sequences, two polypeptides were synthesized and isolated by high performance liquid chromatography (HPLC). To obtain polyclonal peptide antibodies, New Zealand rabbits were immunized with synthetic peptides according to a standardized protocol (Pineda Antibody-Service, Germany). Blood serum for the study was taken on day 60 and day 100 after the start of immunization. Immunization success was tested by dot-blotting method. The IgG fraction was isolated by affinity chromatography on a Hi Trap Protein G HP column (Amersham Biosciences AB, Germany). Results. Analysis of the 112 amino acid sequences of kappa light chains showed the subclass I to be the most common finding (87 cases). Therefore, it was selected for composing a hypothetical amino acid chain. The first antigenic epitope was selected from the most common amino acid residues of the variable region in the hypothetical chain and it corresponded to amino acid positions 3–17 (QMTQSPSSLSASVGD). The constant region in all five subclasses was the same. As the second epitope, a section of the constant region was selected which corresponded to amino acid positions 116–130 (FIFPPSDEQLKSGTA). The synthesized peptides NH2-CQMTQSPSSLSASVGD-CONH2 and NH2-CFIFPPSDEQLKSGTA-CONH2 were purified by high-performance liquid chromatography (HPLC) technique with the achieved purity of 86% and 89%, respectively and used for immunization. Its success was tested by dot-blotting method using the synthesized peptides as a positive control. IgG antibodies isolated from blood serum showed positive amyloid reaction in all cases of AL-kappa amyloidosis and negative, in AL-lambda, AA- and transtyretin amyloidosis. Conclusion. As a result, we obtained four new polyclonal antibodies directed against NH2-CQMTQSPSSLSASVGD-CONH2 (AK1 and AK2) and NH2-CFIFPPSDEQLKSGTA-CONH2 (AK3 and AK4) polypeptides of the variable and constant regions of the immunoglobulin kappa light chain. These antibodies can be recommended for immunohistochemical typing of amyloidosis in different organs and tissues in order to clarify the diagnosis of AL-kappa amyloidosis. Keywords: amyloidosis, AL-kappa amyloidosis, antibodies, dot-blotting, immunohistochemistry.