Neonatal Foreskin Substrate Has Limitations for the Immunofluorescent Screening of Monoclonal Antibodies

Abstract

Two monoclonal antibodies to type IV collagen showed a marked decrease in the labeling of the dermal-epidermal junction of neonatal foreskin while the basement membrane around dermal blood vessels was brightly stained. In contrast, these antibodies labeled the junction and dermal blood vessels with approximately equal intensity when adult skin of nonforeskin site was used as substrate. Other antibodies to matrix molecules (bullous pemphigoid antigen, epidermolysis bullosa acquisita antigen, and laminin) showed excellent staining of both the dermal-epidermal junction and dermal blood vessels in both neonatal foreskin and adult skin. Further, the ultrastructural appearance of the substrates appeared identical. The implication is that neonatal foreskin is not a good substrate to use for the routine screening of monoclonal antibodies to matrix components by indirect immunofluorescence since a "false negative" evaluation may occur.

Laminin inhibits human keratinocyte migration

“…Pepsinized human placental type IV collagen was obtained from Sigma Chemical, St. Louis, MO. Human placental type I and type V collagen were prepared by pepsinization, acid extraction, and sequential salt precipitation, as previously described (Burgeson et al, 1976;Woodley et al, 1987b). Fibronectin was prepared from human plasma by gelatin-Sepharose and heparin-Sepharose chromatography, as previously described (Hayashi and Yamada, 1982;O'Keefe et al, 1984).…”

“…In order t o determine whether the gold salts influenced keratinocyte migration, we also assessed migration in the absence of gold salts in a second type of assay. We added matrix molecules directly to Petri dishes containing coverslips not previously coated with gold salts and then visualized migration tracks by indirect immunofluorescence, using antibodies to type IV collagen (Takiya et al, 1983), bovine serum albumin (Cappel Laboratories, Malvern, PA), type V collagen (Woodley et al, 1987b), fibronectin (O'Keefe et al, 1985 and laminin (BRL Laboratories, Bethesda, MD) diluted 1:50, and fluorescein-conjugated second antibodies, as previously described (Woodley et al, 1987a). Normal rabbit and sheep serum served as a control.…”