FORSE-1: A Positionally Regulated Epitope in the Developing Rat Central Nervous System

We designed a protocol to identify cell surface molecules expressed in restricted spatial patterns in the developing central nervous system (CNS) that might be regulated by regionally restricted transcription factors. The immunogen was a membrane fraction from NT2/D1 embryocarcinoma cells that were induced to differentiate into neurons and upregulate Hox gene expression in response to retinoic acid. One monoclonal antibody (mAb), FORSE-1, specifically labels the rostral rat CNS from the earliest stages. Staining is observed in the rostral but not caudal neural folds of the embryo prior to neural tube closure. Staining is enriched in the forebrain as compared to the rest of the CNS, until E18. Between E11.5 and E13.5, only certain areas of the telencephalon and diencephalon are labeled. Later, up to E17.5, FORSE-1 labeling is specifically restricted to the telencephalon, where a correlation with mitotic activity is apparent: the ventricular zone labels with FORSE-1, while the cortical plate is negative. The staining of the neuroepithelium is intensified by acetone fixation, which also reveals, between E11.5 and E13.5, a dorsoventrally restricted, FORSE-1- positive region of the spinal cord. After E18, the entire CNS is labeled, through adulthood. The mAb labels the surfaces of dissociated, living cells. Other, non-CNS areas of FORSE-1 labeling are nasal and otic placodes, nasal epithelium, nasal glands, and early (E9.5–10.5) endoderm. mAb FORSE-1 recognizes an epitope present on both a high- molecular-weight (> 200 kDa) proteoglycan from embryonic and early postnatal brain, and on a 80 kDa doublet that is restricted to the CNS in the adult. These findings suggest the FORSE-1 antigen as a candidate cell surface molecule for mediating regional specification from the earliest stages of CNS development

A more efficient and economical approach for monoclonal antibody production

We describe here an efficient, economical way to produce monoclonal antibodies in tissue culture flasks. Hybridoma cells are cultured in horizontal tissue culture flasks until 90% confluent, and then the flasks are filled with culture medium and placed in a standing position and left in the incubator for 3–4 weeks until most of the cells have died. At this time, the culture fluid is harvested for antibodies. With this method, which involves only a single cell plating and one change of medium, the yield of antibodies from one tissue culture flask is 6- to 9-fold higher than is obtained by the usual method of sequential replating and harvesting of cells and media. This approach therefore saves labor as well as costly materials, and minimizes risk of contamination

Comparison of two techniques for targeting the production of monoclonal antibodies against particular antigens

We have compared the efficacy of two methods for enhancing the probability of producing monoclonal antibodies against particular target antigens in a complex mixture. These methods use two tissue extracts, one extract that contains (A) and one extract that does not contain (B) the target antigen(s) of interest. In the chemical immunosuppression approach, cyclophosphamide is used to suppress the mouse's response to common antigens in extract B before injection of extract A containing the target antigens. In the tolerization approach, neonatal mice are tolerized against the common antigens in extract B before injection of extract A containing the target antigens. Although small numbers of animals were used in this initial comparison, immunodot assays clearly indicate that the cyclophosphamide immunosuppression method yields significantly more monoclonal antibodies specific for the target antigen-containing extract A than does the tolerization method

A modified method for obtaining large amounts of high titer polyclonal ascites fluid

When limited amounts of antigens are available, it is often difficult to obtain large quantities of polyclonal antisera. Although antisera can be raised in small rodents, yields are usually small. To circumvent these problems, we have designed a modified method for generating polyclonal ascites fluid (AF). Using the appropriate strain of mice and adjuvant, we generated high serum titers by injection of 1–100 μg of protein. Following i.p. injection of compatible sarcoma cells, 13–19 ml of high titer (1:1000–1:20,000), polyclonal ascites fluid were obtained from each mouse. Similar results were obtained using nine different antigens