Abstract

Complement system is an important component of innate immunity, providing primary protection against pathogens invading the body. In addition, it was shown that the complement system is associated with many diseases, not only autoimmune and infectious, but also mental disorders. In this regard, it is necessary to develop affordable and fast method of measuring activity of the complement system in real-time mode. We present a new semi-automated method for assessment of serum complement activity. The assay is based on cytolytic action of complement system upon the ciliate organism Tetrahymena pyriformis. This method consists in repeated counting of live Tetrahymena motile cells by means of specially developed Biolat device, which consists of two video cameras, light sources, and movable round plate. The plate has two rows of holes. The device also includes microprocessor control unit based on AutoCiliata software, intended for control of operation module and counting the surviving cell. The calculations are based on fixation of two sequential video-frames, with subsequent software image processing. Cell death events were observed upon incubation in triethanolamine (TEA) buffer containing 5% of blood serum. We have also compared complement activity in different buffers, i.e., standard medium for culturing of ciliates, Veronal-Medinalum buffer, and the TEA buffer. TEA buffer was found superior to the Veronal buffer when applied in the test system. The time of cell death in the TEA-buffered medium containing 5% serum was < 15 minutes for all the sera studied. The parameters denoting serum complement activity were as follows: a half-life time for the moving cells (TLD50), and a similar value for 100% cell inactivation (1/TLD50, functional activity of the complement system, ACS). The sensitivity of this assay was calculated from dependencies between TLD50 and ACS, and actual serum concentrations. We have suggested an opportunity for evaluation of an integral complement activity, and interrelations between the intensity of synthesis and consumption of its major effector proteins. In the course of this study, we have tested different concentrations of Ca++ and Mg++ ions in the incubation buffer, with optimal physiological concentrations of2.5 mMand1.5 mM, respectively. We have also estimated statistical precision characteristics for pre-analytical and analytical steps of the method. The average coefficients of variation (CV) were 3.9% and 2.7%, respectively, thus satisfying the reliability criteria in research. A short performance time of the study suggests its potential application in clinical practice, including online examination regimens. A method for semi-automatic measurement of serum complement activity could be applicable in daily clinical practice, including the online performance.

Highlights

  • Система комплемента (СК), самая древняя составляющая иммунитета, имеет важнейшее значение в защите организма человека и животных, но эта система часто бывает одним из значимых факторов неблагоприятного прогноза при многих заболеваниях, не только аутоиммунных или инфекционных

  • We present a new semi-automated method for assessment of serum complement activity

  • Поступила 05.03.2015 Отправлена на доработку 11.03.2015 Принята к печати 06.04.2015

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Summary

Immunological methods

Черемных Е.Г.1, Иванов П.А.1, Фактор М.И.1, Карпова Н.С.1, Васильева Е.Ф.1, Гусев К.В.2, Брусов О.С.1. В связи с этим необходима разработка доступного и быстрого метода определения активности системы комплемента в режиме реального времени. Представлен новый автоматизированный метод оценки функциональной активности системы комплемента (СК), основанный на цитолитическом действии этой системы для инфузорий Tetrahymena pyriformis. Время гибели всех клеток в буфере на основе триэтаноламина с концентрацией сыворотки 5% не превышает 15 минут для всех исследованных сывороток. Брусов, «Новый метод оценки функциональной активности системы комплемента» // Медицинская иммунология, 2015. Медицинская Иммунология Medical Immunology (Russia)/Meditsinskaya Immunologiya ваниям надежности результатов исследования, а короткое время исследования доказывает возможность применения метода в клинической практике в онлайн-режиме. Разработан метод полуавтоматического определения функциональной активности сывороточного комплемента, который может быть использован в повседневной клинической практике, том числе в онлайн-режиме. Ключевые слова: система комплемента, активность комплемента, Tetrahymena pyriformis, прибор БиоЛаТ, обработка изображения, подсчет клеток

A NEW METHOD TO ASSESS FUNCTIONAL ACTIVITY OF
Визуальное наблюдение гибели Tetrahymena pyriformis
Чувствительность метода

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