Preparation of polyclonal and monoclonal anti-idiotypic antibodies against morphine-specific immunoglobulins

Abstract

The preparation and study of anti-idiotypic (secondary) antibodies (Ab2) against monoclonal primary antibodies (Ab1) specific to biologically active molecules with a known structure is of great scientific and practical importance. Due to partial antigenic similarity of Ab2 and the initial antigen structures, these antibodies can be the basis of the vaccine, if the antigen usage is not possible, or is limited by law. In particular, one may create Ab2-based preparations, designed for immunization, in order to prevent and treat the drug addiction. The value of Ab2 properties increases even more if Ab1, used to obtain them, recognize different parts of the antigen molecule, which makes it possible to obtain second-generation antibodies with a wide range of specificity. In this work, the morphine-like polyclonal and monoclonal Ab2 were obtained. In each case, as the first-generation immunoglobulins for immunization, we used two murine monoclonal antibodies (mAbs) specific to different morphine derivatives: 3K11 antibodies to 3-0-carboxymethyl (CMM) and 2-p-carboxyphenylazomethyl (FAM) derivatives, as well as 6G1 antibodies to 6-hemisuccinyl derivative (GSM). After immunization of the horse with Ab1 and development of immune response, three pools of specific polyclonal antibodies were isolated from the animal blood serum: horse anti-species antibodies to the total mouse immunoglobulins (HAM); horse anti-idiotypic antibodies against 3K11 antibodies (HAM-K11), and against 6G1 antibodies (HAM-G1). In parallel, immunization of mice with 3K11 and 6G1 antibodies and fusion of obtained lymphocytes with Sp2/0 mouse myeloma cells by the Milstein-Köhler method resulted in three producers of anti-idiotypic antibodies: a clone producing mouse monoclonal Ab2 specific for mAb-6G1 (AIG1), as well as clones producing anti-mAb-3K11 antibodies (AI-K11A and AI-K11B). The physico-chemical and antigenic properties of all the Ab2 obtained were characterized. It was shown that the horse anti-idiotypic immunoglobulins not only belong to different classes, but are also polyvalent, while all monoclonal Ab2 obtained are represented by IgM immunoglobulins, being also strictly specific to the corresponding first-generation antibodies. Subsequently, the morphine-like properties of the first domestic polyclonal and monoclonal Ab2 obtained in the work will be investigated in a cellular model. Likewise, we shall study their ability to induce Ab3 as well as morphine-specific Ab1.

Keywords

• anti-idiotypic antibodies
• antibodies
• competitive analysis
• elisa
• monoclonal antibodies
• morphine derivatives
• polyclonal antibodies