Autoantibodies to 1-adrenergic receptor may play a role in the development of certain cardiovascular system diseases.Synthetic peptide-based enzyme-linked immunosorbent assay (eliSA), traditionally used for the detection of such antibodies, does not allow to obtain adequate data, probably due to the poor similarity of peptide conformations to the spatial structure of the extracellular regions.Aim of the study: to improve the peptide-based eliSA method for the detection of autoantibodies to 1 adrenergic receptor by a separate determination of the igG1, igG2, igG3, igG4, igM and igA autoantibody isotypes.Material and methods: a modification of the eliSA method with peptides corresponding to the sequences of the 1-adrenergic receptor first and second extracellular loops, and secondary antibodies specific to the abovementioned human immunoglobulin isotypes.the method has been tested on a panel of samples obtained from patients with dilated cardiomyopathy (dcM), arrhythmia and coronary heart disease.ResultS: small excess of the signal mean values of individual antibody isotypes have been recorded for some groups of patients in comparison with the group of healthy volunteers.however, these differences were not statistically significant.the proportion of signals «above the 1 ооо «МонА», ул.кастанаевская, 38, стр. 1, Москва, 121108, российская федерация; 2 фГБу «национальный медицинский исследовательский центр кардиологии» Минздрава россии, ул.3-я черепковская, 15а, Москва, 121552, российская федерация Резюме Аутоантитела к 1-адренорецептору могут играть важную роль в развитии некоторых заболеваний сердечно-сосудистой системы.иммуноферментный анализ (ифА) с использованием синтетических пептидов, традиционно применяемый с целью детекции таких антител, не позволяет получать адекватные данные, вероятно, вследствие слабого сходства конформаций пептидов с пространственной структурой внеклеточных участков 1-адренорецептора.Цель исследования.усовершенствовать метод ифА для детекции аутоантител к 1-адренорецептору за счет раздельного определения аутоантител изотипов igG1, igG2, igG3, igG4, igM и igA.Материал и методы.предложена модификация метода ифА с использованием пептидов, соответствующих последовательностям первой и второй внеклеточных петель 1-адренорецептора, и вторых антител, специфических к вышеуказанным изотипам иммуноглобулинов человека.Метод опробован на панели образцов сыворотки крови, полученных от пациентов с дилатационной кардиомиопатией (дкМп), желудочковой аритмией и ишемической болезнью сердца.Результаты. для некоторых групп больных зафиксированы небольшие превышения средних значений сигналов отдельных изотипов антител по сравнению с группой здоровых добровольцев, однако эти отличия не являются статистически значимыми.доля сигналов «выше уровня нормы» в группе больных дкМп составила 21% для изотипа igG1 и от 6 до 16% для остальных изотипов, что весьма далеко от частоты встречаемости аутоантител к 1-адренорецептору (70-80%), наблюдаемой у больных дкМп при использовании функциональных методов анализа.в других группах доля таких сигналов не превысила 30% ни для одного изотипа антител.

OBJECTIVE:This study aimed to assess the level of anti-1-adrenergic receptor autoantibodies in patients with ventricular arrhythmias with no signs of organic heart disease and with presence of cardiovascular pathology in comparison with a group of healthy volunteers. MATERIAL AND METHODS:The study included 44 patients with ventricular arrhythmias with no signs of organic heart disease ("idiopathic"), 34 patients with diagnosed dilated cardiomyopathy (DCM) of inflammatory origin, 35 patients with coronary heart disease and ventricular arrhythmias, 12patients with coronary heart disease with no ventricular arrhythmias, and 19 healthy volunteers (control group). The level of autoantibodies against the 1-adrenergic receptor was determined by the developed competitive cell-based enzyme-linked immunosorbent assay (ELISA) and by the standard ELISA using peptides corresponding to the second extracellular loop of the 1-adrenergic receptor. RESULTS:Elevated level of autoantibodies detected by a competitive cell-based ELISA was observed in 62% of patients with DCM compared to 21% of healthy volunteers (p=0.0006). In patients with "idiopathic" ventricular arrhythmias, the level of 1-adrenergic receptor autoantibodies was lower than in healthy subjects (p=0.003). Coronary heart disease patients with or without ventricular arrhythmias exhibited no differences from the control group. The number of significantly positive signals in peptide-based ELISA did not exceed 10% in any of the groups. No correlation between the data from competitive cell-based ELISA and peptide-based ELISA was found. CONCLUSIONS:This study demonstrated that competitive cell-based ELISA technique can be applied for detection of 1-adrenergic receptor autoantibodies. The results in DCM patients generally correspond to the expected. Decreased level of autoantibodies in patients with "idiopathic" ventricular arrhythmias indicates that this disease is related to changes in the immune system. Such relation is not observed in the case of coronary heart disease patients.

Fragments corresponding to the 83–98 sequence of the first extracellular loop and to the 168–192 and 171–182 sequences of the second extracellular loop of the M2-muscarinic receptor (antibodies to this receptor could be markers of early symptoms of heart disorders) were synthesized by solid phase method using the Fmoc-SPPS strategy. A new conformational antigen with the natural location of the disulfide bridge was prepared by selective formation of disulfide bond between the corresponding cysteine residues in the synthe-sized peptides and characterized. The comparative analysis of reactivity of the synthesized peptides towards sera from patients which had no organic heart disease was performed. A new conformational antigen was effectively bound to the sera from patients with idiopathic arrhythmias, but without symptoms of organic heart disease.

Linear peptides corresponding to fragment 83-98 of the first loop and fragments 168-192 and 171-182 of the second extracellular loops of M2-muscarinic receptor (marker of early cardiac disorders and arrhythmias) were synthesized by Fmoc-SPPS method. A new conformational antigen was synthesized by method of selective ligation of linear peptides by disulfide bond with native localization. Peptides were studied in reaction with sera from patients with idiopathic arrhythmias. A new conformational antigen was recognized by sera from patients with idiopathic arrhythmias with high reactivity.