Specific binding of fibronectin--antifibronectin immune complexes to procollagen: a new pitfall in immunostaining.

Abstract

We observed intense intracellular immunofluorescence of rat lung fibroblasts stained with hybridoma culture supernatant containing monoclonal antibodies to human plasma fibronectin, but no pericellular matrix staining. Immunoprecipitation and absorption experiments revealed that this intracellular staining by hybridoma-conditioned medium was due to binding of fibronectin-antifibronectin immune complexes via the fibronectin to intracellular procollagen . The anomalous staining patterns we encountered were not revealed by the usual controls for immunohistochemical specificity, and also occurred in rat tissue sections . This general phenomena-binding of serum antigens present in hybridoma medium to cellular components-could in principle result in artifactual staining with monoclonal antibodies to other serum components, so investigators using monoclonal antibodies should be aware of this new artifact. Our results also demonstrate that fibronectin binds specifically to native procollagen . Monoclonal antibodies may be useful for studying fibronectin-procollagen and other macromolecular interactions .Monoclonal antibodies (7) are powerful tools for cell biologists, but their use may be accompanied by unusual pitfalls. For example, it is well documented that monoclonals may bind genetically distinct polypeptides (8) . Using hybridoma culture medium containing monoclonal antibody to human fibronectin (FN)' to stain rat lung fibroblasts (RLF), we encountered a previously unreported artifact . Monoclonal antifibronectin-fibronectin complexes bound to intracellular collagenase-sensitive ligands, presumably procollagen, resulting in intense intracellular immunofluorescence. As monoclonals are often used in the form of conditioned medium from serum-containing hybridoma cultures, this general phenomena is important to recognize as a potential artifact of monoclonal antibody staining. Our findings also have implications for fibronectin interactions with procollagen, which are briefly discussed. MATERIALS AND METHODS 1042Immunoreagents and FN: Monoclonal antibodies to human 'Abbreviations used in thispaper: FITC, fluorescein isothiocyanate; FN, fibr6nectin; RLF, rat lung fibroblasts. plasma FN were obtained by fusing spleen cells from immunized BALB/c mice with SP2/O-Ag 14 myeloma cells (19). Positive fusions were cloned in soft agar and antibody-secreting clones detected by enzyme-linked immunosorbent assay (4). Hybridomas were expanded in cell culture using DME plus 10% agammaglobulinemic horse serum, 5% calf serum, and nonessential amino acids ("hybridoma medium") . Ascites fluid containing 2-5 mg/ml of IgG was obtained by intraperitoneal injection of 106 myeloma cells into pristane primed BALB/c mice followed by paracentesis after 1-2 wk. Ascites was clotted at 25°C for 1 h, centrifuged (1,000 g, 10 min), protease inhibitors (I mM phenylmethylsulfonylfluoride, 1 mM N-ethylmaleimide, 10 mM EDTA) and 0.02% NaN3 were added, and the supernate ("ascites") stored at -20°C. In some experiments IgG monoclonals were purifi...

A monoclonal antibody to the carboxyterminal domain of procollagen type I visualizes collagen-synthesizing fibroblasts. Detection of an altered fibroblast phenotype in lungs of patients with pulmonary fibrosis.

“…These results suggest that Anti-pC selectively detects fibroblasts synthesizing type I procollagen and therefore represents a useful tool for the study of fibroproliferative disorders and wound healing in general. 2 (9). In some experiments, fibronectin was removed from labeled medium by gelatin-Sepharose absorption before immunoprecipitation to prevent its nonspecific precipitation.…”

“…Polyclonal rabbit anti-human plasma fibronectin and preimmune rabbit IgG were purified as described (1 1 Mapping ofthe Anti-pC binding domain using procollagen digestion products. Procollagen was subjected to digestion with pepsin, skin collagenase, and bacterial collagenase according to published methods (9,(13)(14)(15). Crystalline pepsin (Sigma P 6887) and highly purified bacterial collagenase (type VII; see reference 9) were purchased from Sigma Chemical Co., St. Louis, MO.…”

Fibronectin mediates adherence of rat alveolar type II epithelial cells via the fibroblastic cell-attachment domain.

“…In addition, immunoblots with anti-FN against reduced human plasma and human epidermal cell extracts after 5% SDS-PAGE showed only one band at 220,000 and 250,000, respectively, the molecular weights for monomeric plasma and cellular FN. Monoclonal antibodies were prepared to various FN domains as previously described (28). Polyclonal antibodies to human FG were purchased from Cappel Laboratories (Cochranville, PA).…”