title: Monoclonal antibodies for the measurement of class-specific antibody responses in the green turtle, Chelonia mydas.
abstract: Monoclonal antibodies (Mabs) were developed against the known immunoglobulin classes of the green turtle, Chelonia mydas. Plasma protein fractions enriched for 5.7S IgY, 7S IgY, and IgM turtle immunoglobulins were used to immunize Balb/c mice for hybridoma production and for hybridoma screening. Fifteen hybridomas produced Mabs with specificity for turtle immunoglobulins and for affinity purified dinitrophenol (DNP) specific turtle antibodies. Three Mabs specific for either turtle 5.7S IgY heavy chain (HL814), 7S IgY heavy chain (HL857), or IgM heavy chain (HL846) were purified and used in an enzyme-linked immunosorbent assay (ELISA) to measure antibody responses in two turtles immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) over a 10 month period. In both turtles the 7S IgY antibody response developed within 5 weeks of the first inoculation and remained high over the following 9 months. The 5.7S IgY antibody response was detected in one turtle at 3-4 months and in the other at 8 months, and reached high levels in both individuals by 10 months. The IgM responses were difficult to interpret. One turtle had pre-inoculation anti-DNP IgM antibody in its plasma and the other developed only a weak, transient response at about 4 months. The class-specific antibody activity in immune turtle plasma could be strongly inhibited by soluble DNP or by rabbit anti-DNP specific antiserum, showing that these antibody responses were directed predominantly to the DNP hapten on the DNP-BSA antigen. Antibody responses to the BSA carrier could not be detected in either turtle over the course of the immunization. Mab HL814, specific for an epitope on the 5.7S green turtle immunoglobulin heavy chain, will be useful for characterizing the molecular relationships of 5.7S, 7S and IgM heavy chains and the role of 5.7S antibody in humoral immunity in this species. All anti-turtle Ig Mabs were screened against the plasma globulins of Loggerhead (Caretta caretta), Olive Ridley (Lepidochelys olivacea), Kemp's Ridley (Lepidochelys kempi), Hawksbill (Eretmochelys imbricata), and Leatherback (Dermochelys coriacea). While the Mabs specific for IgM and 5.7S IgY reacted only with the green turtle, two Mabs specific for light chain reacted with all species except the leatherback, and nine mabs specific for 7S IgY heavy chain reacted with all five species. Thus, these Mabs may be useful for immunodiagnostic applications in these endangered species as well.
authors.affiliation: Department of Comparative and Experimental Pathology, College of Veterinary Medicine, University of Florida, Gainesville 32610, USA.
authors.forename: L H
authors.initials: LH
authors.lastname: Herbst
authors.forename: P A
authors.initials: PA
authors.lastname: Klein
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000918:Antibody Specificity / Q000276:immunology*; D002846:Chromatography, Affinity / Q000662:veterinary; D003429:Cross Reactions / Q000276:immunology; D004140:Dinitrophenols / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000662:veterinary; D005260:Female; D006241:Haptens / Q000276:immunology; D006825:Hybridomas; D007114:Immunization / Q000662:veterinary; D007132:Immunoglobulin Isotypes / Q000032:analysis* / Q000302:isolation & purification; D007136:Immunoglobulins / Q000145:classification / Q000276:immunology* / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D012710:Serum Albumin, Bovine / Q000276:immunology; D014426:Turtles / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D004140:Dinitrophenols; D006241:Haptens; D007132:Immunoglobulin Isotypes; D007136:Immunoglobulins; C034638:dinitrophenyl-bovine serum albumin; D012710:Serum Albumin, Bovine