title: Specific antibody responses to subtilisin Carlsberg (Alcalase) in mice: development of an intranasal exposure model.
abstract: An intranasal (i.n.) dosing model was developed in mice as a potential alternative to more difficult, time-consuming, and costly guinea pig intratracheal (GPIT) or mouse intratracheal models for assessment of the respiratory immunogenicity of detergent enzymes. Using a benchmark enzyme, Alcalase (protease subtilisin Carlsberg), studies were conducted to standardize the model in terms of mouse strain, dosing and serum harvest regimen, and the primary immunoglobulin endpoint to use. The primary assay endpoint selected was the enzyme-specific IgG1 titer determined by an Alcalase-specific ELISA. This is not the primary allergenic antibody in mice (IgE is); however, IgG1 is coregulated with IgE via the IL-4/TH2 pathway and may have a role in mediating allergic-type responses. BDF1 mice (C57B1/6 x DBA/2) were selected as representative of high responder strains, with high response associated with the H-2b (C57B1/6) parent. The dosing regimen used for most studies incorporated three i.n. exposures (Days 1, 3, and 10) and bleeding of the animals on Day 15. The animals were anesthetized and then immunized by allowing them to inhale 5-microliters aliquots of dosing solution into each nostril at each immunization. Positioning of the animals with their heads down (vs up) may have allowed more of the dosing solution to remain in the nasal region for a slightly longer period of time, but did not change the eventual GI tract migration and excretion of each dose. The presence of a detergent matrix in the enzyme dosing solution enhanced the IgG1 response. Immunizing with enzyme plus detergent gave highly consistent dose-response curves for Alcalase when evaluated over many studies. An enzyme-specific allergic antibody (IgE) response was weak and inconsistent under the dosing regimen used to generate the IgG1 response, but was stronger with longer-term dosing, consistent with the delay in IgE vs IgG1 responses seen in some other studies. Using IgG1 as a surrogate for allergic sensitization, we have preliminary data showing similar differential potencies between Alcalase and other test enzymes as detected in previous GPIT tests. On the basis of these data, we believe the i.n. immunization/IgG1 response model is a robust technique that may be useful in determining the relative immunogenicities of detergent enzymes and other proteins.
authors.affiliation: Procter & Gamble Company, Cincinnati, Ohio 45253, USA.
authors.forename: M K
authors.initials: MK
authors.lastname: Robinson
authors.forename: L S
authors.initials: LS
authors.lastname: Babcock
authors.forename: P A
authors.initials: PA
authors.lastname: Horn
authors.forename: T T
authors.initials: TT
authors.lastname: Kawabata
mesh_terms: D000281:Administration, Intranasal; D000485:Allergens / Q000008:administration & dosage; D000818:Animals; D003902:Detergents; D004306:Dose-Response Relationship, Immunologic; D005260:Female; D007074:Immunoglobulin G / Q000096:biosynthesis*; D007700:Kinetics; D051379:Mice; D008954:Models, Biological; D009297:Nasal Mucosa / Q000187:drug effects / Q000473:pathology; D013045:Species Specificity; D013381:Subtilisins / Q000008:administration & dosage / Q000276:immunology* / Q000494:pharmacology
chemical_list: D000485:Allergens; D003902:Detergents; D007074:Immunoglobulin G; D013381:Subtilisins