New Monoclonal Antibodies to Human IgA: Obtaining and Study of Biological Properties

Abstract

Aim: Preparation of monoclonal antibodies to human IgA, investigation of their properties and selection of the most appropriate McAbs for highly sensitive and specific immunoassay tests. Methodology: Balb/c mice were used for monoclonal antibodies (McAbs) production. Animals were immunized subcutaneously with purified preparation of human IgA. Immunization duration - 7 days, B cells source - regional lymph nodes. Hybridization of immunocompetent cells and myeloma cells was performed with polyethylene glycol as a fusogen. Screening and subsequent hybridoma clones selection was performed by ELISA-test using human IgA and IgA Fc-fragments, IgG and human IgM. To determine the isotype of McAbs, titer, affinity constants, and to identify its comparative epitopic specificity appropriate modifications of ELISA-test were used.  Results: In our experiments new hybrid clones selection scheme to define the most appropriate McAbs for highly sensitive and specific immunoassay tests was developed. It was established that the most prospective were hybridoma clones producing antibodies against Fc-region of IgA molecule. It was proposed to have a comprehensive description of antibodies, which included the establishment of its isotype, titer, and affinity constants. In view of the further use of obtained McAbs for development of highly informative immunoassay methods, only high titer and affinity antibodies were selected. Since IgM-antibodies are characterized by low specificity and affinity, McAbs of such isotype have not been chosen for further study. Focusing on more efficient McAbs purification on protein A based sorbents, antibodies with IgG2a, IgG2b, and IgG3 isotypes were selected. It was established that there is a correlation between the antibody titer in the culture fluid and its affinity constant: antibodies with titer more than 1:500 characterized by an affinity constant not less than 109 М-1. Conclusion: A set of 14 new monoclonal antibodies to human IgA has been obtained. Following biological properties of obtained McAbs has been studied: Activity in the indirect ELISA, specificity within IgA molecule (Fab- or Fc- fragment), cross-reactivity with other classes of serum immunoglobulins (human IgG, and IgM), titer in the culture fluid, affinity constant, and relative epitope specificity. Criteria of McAbs selection for their further use in highly sensitive and specific immunoassay methods have been developed. McAbs with following characteristics are the most promising for these purposes: they should  be directed to the Fc-fragment of IgA, have a high signal in the indirect ELISA, absence of cross-reactivity with other classes of immunoglobulins, titer in the culture fluid not less then 1:500, and affinity constant not less then 8.0×109 M-1. McAbs of IgM isotype  are not suitable for effective biotechnological approaches.