Development of a new sensitive immunoassay for the detection of cardiac troponins in sera of patients with cardiovascular diseases

摘要

Troponin complex plays an important role in regulating skeletal and cardiac muscle contraction.It consists of three different subunits(I,C,T)each of which is responsible for different functions.Cardiac troponins are released in the bloodstream during irreversible cardiac muscle damage and they are being detected in patients' sera 4-6 hours after the cardiovascular incident.The development of biological assays for the detection of cardiac troponins is based on the application of specific antibodies against the whole complex or individual subunits.The high homology between cardiac and skeletal muscle troponins,establish the problem of cross-reactions.This fact combined with the need of signifying improvement of the method's sensitivity enforces the search of more sensitive and specific reagents.In order to contribute in this field,our first theoretical and experimental approach was based on developing a pair of antibodies,which will recognize different regions of the troponin complex,with high specificity and affinity.According to this consideration,we performed:i)a systematic mapping of the antigenic regions of cardiac troponin I,using an antigenicity predicting software,ii)Selection of the cardiac troponin I regions,which represent the minimum homology compared to the skeletal isoforms,utilizing homology software.From the entire regions that have been determined,we have chosen the 19-31(RRRSSNVRAYATE)and 118-131(TKNITEIADLTQKI)regions of cardiac troponin I isoform for production of antibodies.In order to increase the immunogenicity of the selected sequences,especially of the low immunogenic region 118-131,we conjugated them to the synthetic peptide carrier,formed by the repetitive moiety Lys-Aib-Gly,namely Sequential Oligopeptide Carrier,SOCn,Ac-(Lys-Aib-Gly)_n-OH,(n=4),which has been proved to increase the immunogenicity of the conjugated epitopes.The constructs are denoted as Ac-SOC_4-[TRIC(19-31)]_4-OH(M.W:7588.4)and Ac-SOC_4[TRIC(118-113)]_4-OH(M.W:7420.7).The analogues have been synthesized by solid phase peptide synthesis method using the Boc/Bzl strategy,purified by semi-preparative HPLC and identified by mass spectrometry(ESI-MS).Rabbits immunizations with the above analogues gave high immune response.