Production of a Monoclonal Antibody by In Vitro Immunization That Recognizes a Native Chondroitin Sulfate Epitope in the Embryonic Chick Limb and Heart

Abstract

SUMMARYWe report the production of a monoclonal antibody (d1C4) by in vitro immunization that has immunoreactivity with a native chondroitin sulfate epitope in embryonic chick limb and heart. Murine lymphocytes were stimulated by direct exposure to unfixed, unsolubilized precartilage mesenchymal aggregates in high-density micromass culture derived from Stage 22-23 chick limb buds. Specificity of d1C4 reactivity was demonstrated by sensitivity of immunohistochemical staining to pretreatment with chondroitinase ABC or AC, preferential immunoreactivity with chondroitin-6-sulfate glycosaminoglycan (CS-C GAG) in ELISA, and competition of immunohistochemical staining with CS-C GAG. Immunohistochemical analysis of the expression of the d1C4 epitope revealed a striking localization of immunoreactivity in the extracellular matrix (ECM) of precartilage aggregates of chick limb mesenchyme in high-density micromass culture by 16 hr and the prechondrogenic limb core at Stage 23 in vivo. Immunoreactivity in both cultured limb mesenchyme and the embryonic limb continued through differentiation of prechondrogenic condensations into cartilage tissue. In the developing chick heart, d1C4 staining was found throughout the ECM of atrioventricular cushion tissue by Stage 25, but was localized to mesenchyme adjacent to the myocardium in the outflow tract cushions. There was an abrupt demarcation between d1C4-reactive intracardiac mesenchyme and unreactive extracardiac mesenchyme of the dorsal mesocardium in the Stage 22 embryo. This study demonstrates the efficacy of in vitro immunization of lymphocytes for the production of MAbs to native ECM constituents, such as CS-GAGs. Immunohistochemical data utilizing d1C4 suggest that CS-GAGs bearing this epitope may be important in early morphogenetic events leading to cartilage differentiation in the limb and valvuloseptal morphogenesis in the heart.

Heterogeneity of chondroitin sulfate glycosaminoglycan localization during early development of the striped bass (Morone saxatilis)

“…Because of possible structural modifications in an individual CS-GAG chain, antigenic determinants are generated that may be quite different from the repetitive backbone disaccharide structure. While the precise epitopes recognized by the d1C4 and TC2 antibodies utilized in this study are not known at this time, it is clear from previous studies (Capehart et al, 1997(Capehart et al, , 1999) that they reside on native CS-GAG chains enriched in either chondroitin-4-(TC2) or chondroitin-6-sulfate chains (d1C4). The CS-56 epitope may be found on both chondroitin-4-and -6-sulfate chains (Avnur and Geiger, 1984), suggesting strongly that the determinant recognized by this antibody is a structural variation of the usual backbone of CS-GAG chains.…”

“…It recognizes a native epitope on GAGs enriched in chondroitin-4-sulfate (bovine trachea; Sigma Chemical Co., St. Louis, MO). MAb d1C4 was produced by in vitro immunization by direct exposure of naïve mouse splenocytes to unfixed, air-dried prechondrogenic micromass cultures of chick limb mesenchyme (Capehart et al, 1997). MAb d1C4 is reactive with a native epitope on chondroitin sulfate GAG chains enriched in chondroitin-6-sulfate (shark cartilage; Sigma).…”

“…In the present study three antibodies were utilized that recognize native epitopes on chondroitin sulfate GAG chains: 1) TC2 (Capehart et al, 1999) and 2) d1C4 (Capehart et al, 1997), which recognize, respectively, epitopes on GAGs enriched in chondroitin-4-and chondroitin-6-sulfate; and 3) CS-56 (Avnur and Geiger, 1984), which binds a different epitope on both chondroitin-4-and chondroitin-6-sulfate chains. Our results outline major sites of immunoreactivity with these three antibodies and demonstrate codistribution of chondroitin sulfate GAG epitopes in the ECM at some sites, but a striking heterogeneity of chondroitin sulfate GAG localization in several developing organs, including the eye and heart.…”

Heterogeneous Expression of Chondroitin Sulfate Glycosaminoglycans and Versican Proteoglycan during Early Development of Rathke’s Pouch

“…The d1C4 monoclonal IgM was produced by in vitro immunization through direct exposure of naïve mouse splenocytes to unfixed micromass cultures of precartilage chick limb mesenchyme [16] and recognizes a native chondroitinase-sensitive epitope on GAG chains enriched in chondroitin-6-sulfate from shark cartilage (Sigma). Initial experiments utilized CS-56 mouse IgM (Sigma) which also recognizes a native CS-GAG epitope and staining patterns showing marked overlap with d1C4 localization in neighboring sections (not shown).…”

“…Lectican family chondroitin sulfate proteoglycans (CSPGs) are important constituents of the ECM that are also selectively expressed in various embryonic basement membranes [16,17] and function in numerous roles including matrix organization, growth, migration, and cell differentiation. One lectican CSPG, versican, has widespread distribution in the early embryonic ECM and its misexpression has been shown to disrupt cardiac [18], limb [19,20], and joint development [20,21].…”