Worm Breeder's Gazette 12(2): 20 (January 1, 1992)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Background. The extracellular matrix (ECM) plays an important role in maintaining the structural integrity and cellular functions of a multicellular organism. ECM components, including collagens, fibronectin, laminin, vitronectin, tenascin, entactin, and proteoglycans, have been identified and characterized in mammalian tissues. However, the structure and organization of these components in the intact matrix remain unclear. The inability to isolate mutants that are defective in a single ECM component in higher organisms impedes this type of analysis in vivo. With the advantage of the well-characterized genetics in C. elegans, we initiated a project to study the organization and structure of ECM using C. elegans as a model system. Our approach was to generate and characterize a panel of monoclonal antibodies against C. elegans ECM to use as markers for structural studies including immunofluorescent microscopy. In the future, we plan to use these monoclonal antibodies in affinity chromatography for the purification of ECM components and to use as probes for isolating cDNA.
Experimental Approach. The ECM components were extracted from a mixed population of adult and juvenile C. elegans using the procedure shown in Figure 1. [See Figure 1] BALB/c mice were immunized intraperitoneally with 100µg of ECM extract homogenized in complete Freund's adjuvant followed by three bi-weekly injections of 50µg of ECM extract in incomplete Freund's adjuvant. Spleen cells from immunized mice were fused with myeloma cells and plated at a concentration of 2.5 x10 +E5cells/well.
Results. Of the 1,200 wells screened.approximately 32% of the hybridoma supernatants tested positive in ELISA against C. elegans ECM extracts. Supernatants from positive wells were tested by immunoblotting against C. elegans ECM extracts and by immunofluorescent microscopy on whole or fragmented C. elegans Protein species ranging from 20,000-180,000 daltons were detected by the hybridoma supernatants in immunoblotting, and supernatant recognition ranged from complex patterns of multiple bands to a single band. Immunofluorescent studies also revealed diverse staining patterns, which included staining between muscle and hypodermis, around muscle bundles, in layers surrounding intestines and gonads, and around the cuticle. Seven of the hybridomas were purified by limiting dilution cloning and injected into mice for ascites tumor production. We would like to make these monoclonal antibodies available to interested investigators in the near future. (This project was part of a course, Biotechnology Laboratory: Molecular Recognition (BSC 352), which was composed of 70% graduate students and 30% undergraduate students, and was supported by the Department of Biological Sciences and the College of Arts and Sciences at Illinois State University. We also would like to thank Robert Barstead for providing worm fragments and helpful discussion.)