Fluorescent microtiter screening assay for immunocytochemically reactive antibodies

Area of Science:

• Immunology
• Molecular Biology
• Biochemistry

Background:

• Hybridoma technology is crucial for producing monoclonal antibodies.
• Screening hybridomas for specific antibody reactivity is often time-consuming.
• Efficient screening methods are needed for antibody discovery.

Purpose of the Study:

• To develop a rapid in situ screening procedure for hybridomas.
• To enable high-throughput prescreening of hundreds of hybridomas in a 96-well format.
• To identify monoclonal antibodies specific to peroxisome proliferator-activated receptor gamma (PPAR gamma).

Main Methods:

• Utilizing fluorescence immunostaining of cells cultured in 96-well plates.
• Employing a fluorescence plate reader for initial detection of reactive antibodies.
• Confirming positive immunostaining via fluorescence microscopy.

Main Results:

• Successfully developed and validated a rapid in situ screening procedure.
• Identified monoclonal antibodies specific to the nuclear receptor PPAR gamma.
• Demonstrated the assay's applicability for cell surface and intracellular antigens.

Conclusions:

• The developed fluorescence-based assay is efficient for prescreening hybridomas.
• This method aids in the initial identification of antibodies reactive in immunocytochemical procedures.
• The assay is adaptable for various antigen targets, including PPAR gamma.