Aynur Başalp

Immune Response to 17β-Estradiol Involved in Polymer Gels: Antigen Specificity and Affinity of Hybridoma Clones

The immunogenic properties of 17beta-estradiol, immobilized in negatively charged polymer gels, were investigated, and the specificity of antibodies produced was analyzed. The polymer gels developed were composed of a hydrophobic estradiol core surrounded by hydrophilic polyanions as corona. As an immunogen, it was conceived to function via a dual mode, that is as a hapten-delivery system (prolongation effect) and as a polyelectrolyte adjuvant. Polymer gels containing estradiol appeared to possess a high estradiol-specific immunogenicity even without the addition of traditional adjuvants. A comparative study of estradiol trapped in polymer gels versus estradiol conjugated to bovine serum albumin (BSA.E) + Incomplete Freunds Adjuvant (IFA) mixtures revealed similar immunogenic properties in terms of induction of specific antibodies. Following a short immunization procedure based on the use of 17beta-estradiol immobilized in polymer gels, we developed 10 specific monoclonal antibodies with Kd values ranging between 1.2 X 10(-7) and 8 X 10(-8) M.

Generation of Anti-Idiotypic Antibodies that Mimic HBsAg and Vaccination against Hepatitis B Virus

The advent of hybridoma technology has opened up a new avenue in vaccine development, and antigen-mimicking properties of anti-idiotypic antibodies have provided promising alternatives in the generation of experimental anti-idiotypic vaccines. In the present study, mice were immunized with anti-hepatitis B virus (HBV) mouse monoclonal and anti-HBV goat polyclonal antibody to produce anti-idiotypic antibodies. Two mouse monoclonal antibodies (6C9, 6H9) were obtained from the fusions, and the immunogenic properties and specificity of antibodies were analyzed. BALB/c mice were immunized with varying concentrations of anti-idiotypic antibodies (25, 50, 75, and 100 micrograms of anti-Id), and it was shown that anti-idiotypic antibodies generated hepatitis B surface antigen (HBsAg), as well as a BSA-specific antibody response. A simple method for the purification of monoclonal antibodies by dialyzing antibody against water has also been reported.

Development of mouse hybridomas by fusion of myeloma cells with lymphocytes derived from spleen, lymph node, and bone marrow

Since its discovery by Kohler and Milstein in 1975, hybridoma technology has found a wide use in almost every field of biology and medicine. A general and simple approach for developing monoclonal antibodies is to use splenocytes from immunized mice. In the present study, 10 fusion experiments were carried out to analyze the hybridization efficiencies of mouse myeloma cells with lymphocytes derived from spleen, lymph node, and bone marrow and we found a higher yield of antigen specific antibody producing hybridoma lines when the lymph nodes were used.

Enhancement of the immune response to hepatitis B virus vaccine by antigen specific IgM

In the present study, modulation of antibody response induced by Hepatitis B virus vaccine-IgM complex was investigated. Purified IgM-type anti HBv monoclonal antibody (1B11) was complexed to commercially available HBv vaccine (GenHevac B Pasteur, France) at varying concentrations of HBsAg (0.5, 1, 1.5 microg of HBsAg) and used to immunize BALB/c mice. An enhanced humoral immune response was obtained with the HBv vaccine-IgM complex at all the doses compared with those immunized by vaccine alone and increased antibody levels were observed with increased concentrations of HBsAg in vaccine formulation. Immunization with HBv vaccine-IgM complex mostly generated IgG-type antibodies in the sera of mice, and also gave rise to the development of hybrid cells which predominantly produced IgG-type monoclonal antibodies. Hence, results from this study indicate that 1B11 can be effectively used to obtain a better immune response to HBv vaccine.

Monoclonal Antibodies Specific for Hepatitis B e Antigen and Hepatitis B Core Antigen

Despite effective vaccination programs in many countries, HBV infection is still a serious health problem throughout the world; more than 2 billion people have been infected with Hepatitis B virus (HBV). The serologic markers are crucial indicators in clinical diagnosis of HBV infection. The persistent presence of anti-HBc is associated with chronicity, and anti-HBe is an indicator for active viral replication. In the present study, two different hybridoma clones, 12E5 and 16F8, secreting anti-HBeAg and anti-HBc antibody were developed using hybridoma technology. BALB/c mice were immunized with HBe antigen (HBeAg), and monoclonal antibodies were generated from the spleen and lymph nodes of mice. Immunoglobulin types of antibodies were found to be IgG2a and IgG1, respectively. Monoclonal antibodies (MAbs) were produced in large scale, purified with affinity chromatography, and epitope analysis was performed. The results have shown that 12E5 and 16F8 monoclonal antibodies can be used for detection of HBcAg and HBeAg, indicating that they have the potential for use in clinical diagnosis.

Cloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISA

ABSTRACT Hepatitis B virus (HBV) infection is a worldwide health problem. More than 400 million people are chronic HBV carriers in the world. Infected individuals are at a high risk of developing liver cirrhosis and hepatocellular carcinoma as the main consequences of HBV. The discoveries of fast diagnostic systems and new therapeutic applications are crucial in the fight against viral hepatitis. In this paper we present the generation of a single-chain variable fragment (scFv) from a mouse monoclonal antibody specific to the HBV surface antigen (HBsAg) and demonstrate its expression as a bacterial alkaline phosphatase (AP) fusion protein. In this study, we constructed scFvs from hybridoma cells expressing HBsAg-specific antibody using phage display technology and expressed them in Escherichia coli. The anti-HBsAg scFvs were inserted into pQE-2 vector to produce scFv antibody genetically fused to bacterial AP. Reproducibility of the recombinant HBsAg-scFv fusion protein was tested using Enzyme-linked Immunosorbent Assay (ELISA). Present preliminary findings indicate that the anti-HBsAg-scFv AP conjugate could be further used for the development of one-step ELISA for the detection of HBV.