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Abstract
Our aim was to characterize anti-EPO antibodies in serum samples of the patients treated with erythropoietin. 106 serum samples from the patients treated with erythropoietin (EPO) were collected and assayed. 134 serum samples of patients who did not receive EPO were taken for comparative analysis. The anti-EPO antibody detection was performed in ELISA test with rhEPO, by passive capture on ELISA plates, using steptavidin-biotin immunochemical system. Mouse monoclonal antibodies to human IgG, IgG1, IgG2, IgG3 and IgG4 conjugated to horseradish peroxidase were used to detect anti-EPO antibodies, and protein-A peroxidase conjugate was used for quantitative assays. Rabbit anti-human EPO polyclonal antibodies at known concentrations were used as a calibration standard. Six calibration samples at the concentration range of 16-1000 ng/ml were used to plot calibration curves. The lower detection limit was 12 ng/mL, and the quantitative detection limit was 31 ng/ml. Immunochemical capturing led to increasing of total IgG antibody detection by 3.2 times, IgG1 – by 1.1 times IgG2 – by 1.25 times, IgG3 – by 1.5 times, IgG4 – by 1.7 times. Antibodies of mixed isotype were found in most patients. IgG1 or IgG4 antibodies to EPO were determined only in 3 samples. Specific IgM was not detectable among 106 sera samples, whereas total IgG antibodies were detected in 36.8 % of cases. In 34% of sera, their presence was confirmed by detection of at least one of the subclasses. IgG1 antibody was detected in 83.3%; IgG4, in 80.6% of the samples positive for total IgG antibodies. In all cases, IgG2 and/or IgG3 were detected in presence of IgG1 or IgG4 antibodies. The antibody concentration was 3.2 to 35.5 µg/mL in sera from 28 patients, in 8 cases the level of antibodies was > 50 µg/ml, however, being below the limit of quantitative detection in 3 patients. Only 6 samples contained antibodies with avidity index of > 50%. Immunochemical capturing of the antigen led to increased sensitivity for detecting all subclasses of specific antibodies. The specific IgG antibodies to EPO were found in more than 1/3 of serum samples from the patients treated with erythropoietin. Low-avidity antibodies of IgG1 and IgG4 subclasses were determined in most cases.
Highlights
Выявление АТ к ЭПО иммуноферментный анализ (ИФА) Erythropoietin antibodies detected by ELISA многоуровневый подход, включающий метод выявления суммарных антител к препарату, характеристику выявленных антител в отношении их изотипов, концентрации и авидности [3, 10]
Задачей настоящего исследования являлась разработка методов определения антител к эритропоэтину методом твердофазного иммуноферментного анализа и их использование для характеристики специфических антител у группы пациентов, проходящих терапию препаратами рекомбинантного эритропоэтина
The achieved significance level p is indicated for the Wilcoxon test in each group
Summary
Кудряшова А.М.1, Нестеренко Л.Н.1, Генералова Г.А.2, Абасеева Т.Ю.2, Михайлова Н.А.1, Борисова О.В.1. Целью исследования является характеристика антител к эритропоэтину в сыворотках крови пациентов, проходящих терапию препаратами эритропоэтина. Для сравнительного анализа были исследованы 134 сыворотки пациентов, не получавших препараты ЭПО. В выборке из 106 образцов IgМ-антитела не определялись, суммарные IgG-антитела были выявлены в 36,8% случаев, в 34% образцов их наличие было подтверждено выявлением антител хотя бы одного из подклассов. Во всех случаях антитела IgG2 и/или IgG3 подклассов были обнаружены в присутствии IgG1- или IgG4-антител. Только в 6 образцах были выявлены антитела с индексом авидности более 50%. Борисова «Характеристика антител к эритропоэтину у пациентов, проходящих терапию препаратами рекомбинантного человеческого эритропоэтина» // Медицинская иммунология, 2020. Медицинская Иммунология Medical Immunology (Russia)/Meditsinskaya Immunologiya сывороток крови пациентов, получавших препараты ЭПО, преимущественно определялись низкоавидные антитела IgG1- и IgG4-подклассов. Ключевые слова: эритропоэтин, твердофазный ИФА, антитела, иммуногенность, авидность, сыворотки крови человека
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