Биологические науки / 9.Биохимия и биофизика

Galkin O.Yu., Nikitina К.І., Savchenko А.А.

National Technical University of Ukraine “Kyiv Polytechnic Institute”, Ukraine

NEW monoclonal antibodIES to HUMAN IgЕ

One of the main tasks of the immune system is to protect the body against various infections. There are several factors of natural immunity, cellular and humoral responsible for combating infectious agents. Immunoglobulins that are in biological fluids, are important effectors of the humoral immune response. Immunoglobulin E is synthesized mainly in the lymphoid tissue of the skin, respiratory tract, intestines and lymph nodes that drain them. The main function of IgE is associated with protection of the organism from worms and protozoa parasites. IgE plays an important role in hypersensitivity type I, which is manifested by various allergic diseases such as allergic asthma, allergic rhinitis, food allergy, atopic dermatitis, anaphylaxis, etc.

Among the arsenal of reagents used for the determination of IgE, a leading place belongs to the respective specific antibodies. Polyclonal antibodies to human serum have a number of deficiencies (non-specific binding, cross-reactivity, lack of reproducibility studies, etc.), which significantly limits their use. More successful reagents in this case are an anti-IgE monoclonal antibodies. The advantages of the latter include exclusive specificity, homogeneity and the possibility of obtaining virtually unlimited quantities. Having armed panel MAbs to human immunoglobulins of particular class may select high affinity and specific clones, those that give the best results in a given test system. It is known that the development of any ELISA test kits should have a wide range of relevant MAbs, because usually a whole panel of antibodies only detect one or more satisfactory results.

The aim of our study was to select immunization schemes and obtaining monoclonal antibodies to human IgE, study their properties and selection of diagnostically significant MAbs.

Monoclonal antibodies were received on short period immunization scheme (7-8 days): antigen was injected into mice hindfoot pads in total dosage 50 μg ІgЕ per animal, first two injections were done with full Freund’s adjuvant (“Sіgma”, USA), the third injection was done without adjuvant. Hybridization was carried out by means of lymphocytes from regional mice lymph notes.

Table 1. Characteristics of monoclonal antibodies to human IgE

MAbs	OD in ELISA		Isotype	Titer in culture fluid	Affinity constant, 10^9 mol/l	Epitope
	IgE	Fcε-fragments
161A7	2.842	2.742	IgG1	1:800	10.0	А3
161H5	2.872	2.740	IgG1	1:800	10.0	А2
163C10	3.220	3.166	IgG1	1:500	8.0	А2
163D12	3.015	3.097	IgG2a	1:800	10.0	В
164F5	2.925	2.920	IgG1	1:200	10.0	А3
164H3	3.005	2.977	IgG2b	1:800	10.0	А2
164H10	3.017	3.029	IgG1	1:1000	14.0	В
165C12	2.097	2.111	IgG1	1:1000	10.0	А1
166A10	2.950	2.901	IgG1	1:500	8.0	А1
166B7	3.001	3.101	IgG2b	1:500	10.0	А3
166F3	2.895	2.861	IgG1	1:800	10.0	А2
167B4	2.906	2.972	IgG2b	1:1000	8.0	А1

The original set from 12 clones of hybridomas, producers of monoclonal antibodies against human IgЕ has been obtained. The study of biological properties of antibodies is conducted: their specificity, constant of affinity and titer in a cultural medium. Obtained MAbs are directed to the two epitop regions on IgE molecule: the epitop region A has three epitops (closest epitops A2 and A3, and relatively remote from them epitop A1); epitop region B has represented by only one epitop.