Біотехнологія одержання та використання моноклональних антитіл до IgM людини

Доказана возможность использования конъюгатов 2-х МКАТ в составе ИФА тест-систем для диагностики цитомегалии и Эпштейн-Барр вирусной инфекции, также 3-х МКАТ в составе иммуносорбента наборов для диагностики токсоплазмоза, краснухи и урогенитального хламидиоза и простого герпеса.

Разработан высокочувствительный ИФА набор для количественного определения IgM-антител с аналитической чувствительностью 11 нг/мл. Предложена методика специфического выделения IgM человека с использованием иммуноаффинного сорбента на основе полученных анти-IgM МКАТ.

Ключевые слова: гибридомы, моноклональные антитела, иммуноглобулины, эпитопы, иммуноферментный анализ, иммуноаффинные сорбенты.

Summary

Galkin O.Yu. Biotechnology of obtaining and use of monoclonal antibodies to human IgM. - Manuscript.

Thesis for a PhD degree, specialty 03.00.20 - biotechnology. - I.I. Mechnikov Odesa National University of Ministry of Education and Science of Ukraine, Odesa, 2010.

The thesis presents the results of studies aimed for elaboration of biotechnological fundamentals of human anti-IgM MAb production and investigation. An original set of 21 novel clone hybridomas - producers of monoclonal antibodies to human IgM - has been obtained. Advanced study of biotechnological characteristics of the antibodies has been carried out: their specificity, affinity constant, and titer in culture medium have been determined. The epitope structure of human IgM molecule has been found to contain three basic epitope regions (ER): А, В, and С. ER А includes 1 predominant epitope А1, represented by the highest number of high affinity MAbs, and 2 adjacent epitopes А2 and А3 of medium affinity. ER В contains 2 adjacent epitopes В1 and В2 with medium affinity MAbs. ER С includes only 1 epitope with medium affinity MAbs. Epitope specificity of anti-IgM MAbs has been proven to depend on immunization scheme. The short-term immunization scheme results in mono-specificity of anti-ER-A antibodies, and the prolonged scheme causes the produced MAbs to be directed against antigenic determinants ER-B and ER-C. A comparative study of the possibility of using both immunization schemes for production of MAbs against human IgM has been conducted. Considering high immunochemical characteristics of MAbs, rapid results, and the absence of allergic reactions in Balb/c mice, the short-term immunization scheme appears more convenient and adequate.

Diagnostic characteristics of produced MAbs used as a complex with immunoenzyme conjugate and immunosorbent have been studied. Information capacity of one MAb conjugate exceeded that of a well-known analogue СН2 (Sorbent, Russia), and another three MAbs within the sorbent complex ensured better analytical properties versus МВ11 MAbs (Sigma, USA). A possibility of using 2 MAb-based conjugates in test systems to diagnose cytomegalic inclusion disease and Epstein-Barr viral infection, as well as 3 MABs within a test kit immunosorbent for diagnostics of toxoplasmosis, rubella, urogenital chlamidiosis, and simplex herpes has been proven.

A high sensitivity immunoenzyme kit for total IgM assay of analytical sensitivity 11 ng/ml has been elaborated. A well-reproducible and easy method of specific human IgM isolation using the produced anti-IgM MAb-based immunoaffine sorbent has been proposed.

Our MAb-based immunosorbents and peroxidase conjugates served as a base for elaboration and production of kits intended for detection of IgM antibodies against Toxoplasma gondii, Chlamydia trachomatis and viruses of rubella, herpes simplex, cytomegalic inclusion disease, and Epstain-Barr virus. Test kits for detection of IgM antibodies against other infectious agents, such as varicella-zoster virus or pneumonia agents can be developed using the obtained MAbs or MAb-based conjugates. The study results have also been implemented in lecture courses of the department of industrial biotechnology of National Technical University of Ukraine “KPI”.

Keywords: hybridomas, monoclonal antibodies, immunoglobulins, epitopes, enzyme immunoassay, immunoaffine sorbents.

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