Background: A simple filter paper method was developed for, the transport and storage of monoclonal antibodies (Mabs) at room temperature or -20°C after spotting on filter paper, for subsequent serotyping of outer membrane antigens of N.meningitidis by dot-blot ELISA.

A new monoclonal antibody (5F81A4P1.9), which is specific for subtype 9 antigen of meningococci, was studied. The antibodies were raised against a previously non-typable (NT) serogroup B strain from Brazilian patients and were found to react with the subtype antigen of prototype reference strains for subtype 9 (M982), as well as with those of homologous strains. The subtype 9 epitope was found in 6.8% of serogroup B strains among 602 strains of Neisseria meningitidis case isolates, including representative isolates from Brazilian states. Subtype P1.9 was predominantly related to serogroup B in Brazil among the isolates collected during the N. meningitidis epidemic in 1992. No significant differences were observed in the occurrence of subtype P1.9 among strains isolated from several Brazilian states. Fluorescence-activated cell-sorter analysis showed that 5F81A4 MAb recognized a 46 kDa protein on the surface of a homologous strain of N. meningitidis (B:4:P1.9). These results, in association with a bactericidal activity assay for 5F81A4, and with experimental passive protection in mice, demonstrated the importance of subtype 9 class 1 proteins of N meningitidis in Brazil. Serotyping is essential for the development of vaccination strategies.

A rapid and efficient method for preparing monoclonal antibody (MAb) serotypes using Neisseria meningitidis outer membrane were used in BALB/c mouse footpads for the immunization. The popliteal lymph nodes were isolated 19 days later for MAb-producing hybridomas, from which the MAbs against the 37 kDa protein were screened. Variations in class 2/3 (PorB) proteins form the basis for meningococcal serotyping. This is the first report on the preparation of MAbs against N. meningitidis that is specific to PorB protein using popliteal lymph nodes. The new monoclonal antibodies were specific for PorB outer membrane protein FL24(PL)Br, a new serotype 24 class 3 antigens of non-typeable (NT:NST) serogroup B strain, and FL14(PL)Br specific for the serotype 14, and reacted with the S3446 reference strain analyzed. A total of 12% of the case isolates reacted with one or more of the monoclonal antibodies. The high-affinity MAbs produced by hybridoma methodology provide a basis for further research on the pathogenesis and early diagnosis of meningococcus.

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.