B-cell receptor-based multitargeting method for simultaneous production of novel multiple monoclonal antibodies.

Until now, various kinds of monoclonal antibodies have been raised against many antigens. Nevertheless, the production of these monoclonal antibodies was usually limited to only one antigen. If simultaneous generation of monoclonal antibodies against multiple antigens were available at one time, we could reduce not only laborious work, but also experimental animals. Here, we developed a multitargeting (MT) method that enables simultaneous production of monoclonal antibodies against multiple antigens on the basis of strict selection of sensitized B lymphocytes by the target antigens via B-cell receptors. After immunization using multiple antigens, monoclonal antibodies against four different antigens containing lower antigenic one were successfully generated only in one experiment. At maximum, more than 90 % of ELISA-positive wells to hybridoma-positive ones was obtained by this advanced technology. This must be attributed to strict selection of sensitized B lymphocytes by different antigens. In the MT method, sensitized B lymphocytes were selected by means of each desired antigen regardless of their antigenic differences. Selective fusion of B cell-myeloma cell complexes by electrical pulses was also of critical importance for efficient generation of hybridoma cells secreting desired monoclonal antibodies. This study strongly suggests that simultaneous production of novel monoclonal antibodies directed against multiple antigens of interest by the MT method can be feasible.

Evaluation of the Safety and Tolerability of Conjunctival Ring for Posterior Segment of the Eye.

PURPOSE: To evaluate the safety and tolerability of conjunctival rings (CRs), a novel device for drug delivery to the posterior segment of the eye. METHODS: In animal studies, CRs containing 5% dexamethasone sodium phosphate (DSP) or vehicle solution were placed on the right and left eyes of C57BL/6J mice, respectively. Contact lenses (CLs) containing vehicle solution were used as a control. Twenty-four hours after placement of the CRs, corneal fluorescein staining was graded based on the McDonald-Shadduck scoring system, ranging from 0 to 4. In humans, CRs containing vehicle solution were placed on the right eye of healthy volunteers for 9 hours. The corneal curvature, corneal thickness, intraocular pressure, visual acuity, tear production (Schirmer I test), tear film break-up time and fluorescein staining scores of the cornea (scores ranging from 0 to 3) and conjunctiva (scores ranging from 0 to 6) were assessed before and after wearing the CRs. The release characteristics of DSP from CRs were also evaluated. RESULTS: In animal experiments, corneal fluorescein staining scores were 1 or less in all the groups, and there was no significant difference between the CR group and the CL group. In the preclinical safety evaluation of CR for humans, ophthalmic examination revealed that CR caused no significant changes in all the parameters investigated including corneal curvature (p = 0.77), corneal thickness (p = 0.96), intraocular pressure (p = 0.59), visual acuity (p = 0.14), Schirmer I test results (p = 0.76), tear film break-up time (p = 0.68), corneal fluorescein staining scores (p = 0.64), and conjunctival fluorescein staining scores (p = 0.52). The DSP release from CRs occurs within a few hours, which is similar to the drug-release property of medicated CL, as reported previously. CONCLUSIONS: The current data showed the safety and tolerability of CR as a drug delivery device for the treatment of posterior segment diseases.