Rapid and Efficient Preparation of Monoclonal Antibodies against 35 kDa Lipoprotein ofMycoplasma penetrans

Abstract

To develop a rapid and efficient method for preparing monoclonal antibodies (MAb) against 35 kDa lipoprotein of Mycoplasma penetrans, BALB/c mice were injected into the footpads for immunization, and the popliteal lymph nodes were isolated 19 days later for MAb-producing hybridomas, from which the mAbs against the 35 kDa lipoprotein were screened. The identification of the mAb against the 35 kDa lipoprotein was performed using indirect enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Using popliteal lymph node procedures, we generated several positive clones, one of which we characterized by ELISA and immunoblot. The clone 1D41B8 was identified as the immunoglobulin G1 (IgG1) isotype, kappa chain with affinity constants (Ka) of 2.95 x 10(9) M(-1). The MAbs did not cross-react with a number of control bacteria, which included Mycoplasma fermentans, Mycoplasma hominis, and Mycoplasma genitalium. This is the first report on the preparation of mAbs against M. penetrans that is specific to 35 kDa lipoprotein using popliteal lymph nodes. The high-specificity and high-affinity MAbs produced by two methodologies used of hybridomas provide a basis for further research on the pathogenesis and early diagnosis of M. penetrans. This simple approach may become a method of choice for the generation and production of MAbs in a short period of time.

The Design of New Adjuvants for Mucosal Immunity toNeisseria meningitidisB in Nasally Primed Neonatal Mice for Adult Immune Response

“…Murine anti-Stx1 and anti-Stx2 mAbs were secreted from hybridomas generated by a fusion between the lymph node cells of BALB/c mice immunized with Stx1 or Stx2 toxoids and P 3.653 Ag 8 myeloma cells, as described previously [22–25]. To test the action and specificity of these mAbs, the culture supernatant was assayed for the presence of secreted antibodies by capture ELISA using biotinylated isotypes mouse antibodies (Kirkegaard and Perry, Gaithersburg, MD).…”