Abstract
The aim of the work was development of a monoclonal antibody-based latex agglutination test to identify the causative agent of melioidosis, and the approbation of a freeze-dried experimental preparation for screening of environmental bacterial isolates in Vietnam.Materials and methods. The carriers of specific antibodies were polyacrolein latex particles with active aldehyde groups on the surface. Typical strains of the causative agents of melioidosis and glanders with a full-fledged antigenic structure, as well as the strains Burkholderia thailandensis, Burkholderia cepacia, Pseudomonas aeruginosa, and Pseudomonas putida were used to control the test specificity. The latex agglutination reaction was carried out on plastic Petri dishes with daily bacterial cultures, from which suspensions were prepared at a concentration of 1–2·109 m.c./ml. The results of the reaction were registered visually for 5–8 min using a 4-cross system against a dark background under lighting. The reaction to 3–4 crosses was recorded as positive. Colonies suspected of belonging to pathogenic Burkholderia from primary inoculations were transferred to L-agar with polymyxin B and grown for 36 hours at (37±1) °C. The species of the selected suspicious colonies was determined by multiplex PCR.Results and discussion. With collection strains, latex test demonstrated high sensitivity agglutinating 97.7 % of B. pseudomallei and all B. mallei strains. At the same time, it was negative with B. thailandensis, B. cepacia, P. aeruginos and P. putida. In microbiological screening of bacterial cultures isolated from environmental objects, the latex test had a diagnostic sensitivity of 89.4 %. Using the latex test at the stage of primary screening, it is possible to significantly reduce the time when processing a lot of samples received for analysis, as well as to reduce the consumption of reagents used at the subsequent stages of identification.
Highlights
The aim of the work was development of a monoclonal antibody-based latex agglutination test to identify the causative agent of melioidosis, and the approbation of a freeze-dried experimental preparation for screening of environmental bacterial isolates in Vietnam
The latex agglutination reaction was carried out on plastic Petri dishes with daily bacterial cultures, from which suspensions were prepared at a concentration of 1–2·109 m.c./ml
Colonies suspected of belonging to pathogenic Burkholderia from primary inoculations were transferred to L-agar with polymyxin B and grown for 36 hours at (37±1) °C
Summary
Цель работы – разработка латекс-теста для выявления возбудителя мелиоидоза, где в качестве сенситина использовались моноклональные антитела, а также апробация лиофильно высушенного экспериментального препарата на бактериальных изолятах из объектов внешней среды, собранных на территории Социалистической Республики Вьетнам. Для контроля специфичности диагностикума использовали типичные штаммы возбудителей мелиоидоза и сапа с полноценной антигенной структурой, а также штаммы Burkholderia thailandensis, Burkholderia cepacia, Pseudomonas aeruginosa и Pseudomonas putida. Реакцию латекс-агглютинации выполняли с бактериальными взвесями 1–2·109 м.к./мл на пластиковых чашках Петри. Результаты реакции оценивали визуально на темном фоне по 4-крестовой системе в течение 5–8 мин. Изолированных из объектов внешней среды, чувствительность диагностикума составила 89,4 %. Использование латекс-теста на этапе первичного скрининга большого количества образцов существенно сокращает время выделения чистых культур возбудителей мелиоидоза и сапа и их идентификацию. Ключевые слова: моноклональные антитела, латексные микрочастицы, патогенные буркхольдерии, реакция латекс-агглютинации, чувствительность, специфичность.
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