After the last washing step, substrate solution was added and absorption values at 405nm were measured with a microtiter plate reader and compared with the wells without supernatants (20). hydrocarbons (PAHs) are an important class of environmental pollutants that are a concern because several of its members K-7174 have been known to cause cancer (3, K-7174 4). Therefore, antibody-based immunoassays have also been widely employed in the detection of environmental PAHs, including bioelectrochemical immunoassay (5), enzyme-linked K-7174 immunosorbent assays (ELISAs) (6) and biosensors (7). All of these antibody-based immunoassay methods require the use of sensitive anti-PAH mAbs. The traditional protocol for mAb production is immunizing mice with the appropriate immunogen to induce specific B cells that are obtained from the spleen, and then fusing these with suitable myeloma Id1 cells. The resulting hybridomas are evaluated by immunological screening procedures, such as competitive ELISAs for the desired affinities and specificities (8) and are able to produce a uniform population of antibodies with the preferred characteristics. This whole process is time-consuming (1) and it usually requires 3 to 6 months to produce suitable mAbs (9). Typically during the PAH mAb development procedure, half of the time is used to monitor the sera for a satisfactory titer prior to fusion. A number of mAbs against PAHs have been developed in the recent past (10-12). The immunization strategies from those reports are various. However, the immunization periods utilized for antiserum production were all at least 12 weeks. Antibody persistence is definitely thought to be determined by long-lived plasma and memory space B cells (13-15). It has been reported that most Ab-secreting cells are generated during secondary (memory space) immune response (16), which are present one month after immunization and later on (17). Thus, it may be possible to use shorter immunization periods and obtain successful mAbs by optimizing the immunization strategy. To this purpose, PBA-KLH (pyrene butyric acid-keyhole limpet hemocyanin) immunogen was given to mice and the immunological response in sera and fusion success were evaluated at different week intervals post-immunization. The goal of this study was to appraise numerous post-immunization periods in the production of anti-PBA antibodies and to optimize a time effective immunization strategy to create sensitive anti-PAH antibodies. Experimental Chemicals and reagents Bovine serum albumin (BSA) was from Fisher Scientific (Pittsburgh, PA, USA). Anhydrous N,N-dimethylformamide (DMF) was from Acros Organics (Morris Plains, NJ, USA). The Bicinchoninic acid (BCA) protein assay Kit, N-hydroxysuccinimide (NHS) and K-7174 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) were from Pierce (Rockford, IL, USA). Keyhole limpet hemocyanin (KLH), HAT medium product, Freund’s adjuvant total (FAC) and Freund’s adjuvant incomplete (FAI) were purchased from SigmaCAldrich (St. Louis, MO, USA). RPMI-1640 medium with L-glutamine and fetal bovine serum (FBS) were from Hyclone (Logan, UT, USA). Goat anti-mouse immunoglobulin G secondary antibody conjugated with horseradish peroxidase (GAM IgG-HRPO) and cells tradition plates (Costar, Corning) were purchased from Fisher Scientific (Pittsburgh, PA, USA). The 1-pyrene butyric acid (PBA) was from Acros (NJ, USA). All other reagents were of analytical grade and purchased from Fisher Scientific (Pittsburgh, PA, USA). Balb/c mice were bred at our own facility with the original mice purchased from your Jackson Laboratory, (Pub Harbor, ME, USA) All animal protocols were examined and authorized by the Institutional Animal Care and Use Committee of the College of William and Mary. Preparation of PBA-protein conjugates Two conjugations were prepared with this study. The immunogen PBA-KLH and the covering antigen PBA-BSA which was used in titer and affinity analyses.