title: Purification and development of ELISAs for two forms of vitellogenin in Indian walking catfish, Clarias batrachus (L.).
abstract: Two forms of vitellogenin (Vg: Vg1 and Vg2) were purified from the plasma of estradiol-17β (E2)-treated Indian walking catfish, Clarias batrachus, by gel filtration and adsorption chromatography. Native Vg1 and Vg2 had apparent molecular masses of 375 and 450 kDa, respectively, and both Vgs resolved into two similar major bands (95 and 67 kDa) in SDS-PAGE under reducing condition. Polyclonal antisera raised against each form of Vg were absorbed with a combination of hypophysectomized male catfish serum proteins and alternate Vg to ensure specificity. Immunological analyses verified the presence of Vg1 and Vg2 in the plasma of female catfish. Homologous ELISAs were developed for Vg1 and Vg2 using their respective harvested antisera, which exhibited the detection limit of 100 ng ml-1for Vg1 and 40 ng ml-1for Vg2, and low level of cross-reactivity (not parallel to the standard) was found with alternate Vg in each assay. Treatment of male catfish with E2induced both Vgs showing a proportionate ratio of Vg1 to Vg2 at 5.6:1. Plasma concentrations of both Vgs measured by ELISAs at different reproductive phases of field collected female catfish increased in accordance with the ovarian development, keeping the proportionate ratio of Vg1 to Vg2 at about 2:1 in fish undergoing vitellogenesis during prespawning period and 1:20 during spawning period, suggesting that Vg1 may be the major Vg to contribute in yolk formation, whereas Vg2, besides its role in yolk formation, may facilitate other physiological functions. The present study, thus, demonstrates the occurrence of two unequally synthesized Vgs in the catfish.
authors.affiliation: Department of Zoology, Visva-Bharati University, Santiniketan,, Kolkata, West Bengal, 731235, India.
authors.forename: S
authors.initials: S
authors.lastname: Mahapatra
authors.affiliation: Department of Zoology, Visva-Bharati University, Santiniketan,, Kolkata, West Bengal, 731235, India.
authors.forename: Sk
authors.initials: S
authors.lastname: Kabita
authors.affiliation: Department of Zoology, Visva-Bharati University, Santiniketan,, Kolkata, West Bengal, 731235, India.
authors.forename: D
authors.initials: D
authors.lastname: Bhattacharya
authors.affiliation: Department of Zoology, Visva-Bharati University, Santiniketan,, Kolkata, West Bengal, 731235, India.
authors.forename: S
authors.initials: S
authors.lastname: Sarkar
authors.affiliation: Department of Zoology, Visva-Bharati University, Santiniketan,, Kolkata, West Bengal, 731235, India.
authors.forename: S K
authors.initials: SK
authors.lastname: Juin
authors.affiliation: Department of Zoology, Visva-Bharati University, Santiniketan,, Kolkata, West Bengal, 731235, India.
authors.forename: S
authors.initials: S
authors.lastname: Maitra
authors.affiliation: Department of Zoology, Visva-Bharati University, Santiniketan,, Kolkata, West Bengal, 731235, India. pnath1614@rediffmail.com.
authors.forename: P
authors.initials: P
authors.lastname: Nath
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002397:Catfishes / Q000097:blood*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004958:Estradiol / Q000494:pharmacology; D005260:Female; D029941:Fish Proteins / Q000097:blood / Q000276:immunology / Q000302:isolation & purification; D007106:Immune Sera / Q000276:immunology; D008297:Male; D014819:Vitellogenins / Q000097:blood / Q000276:immunology / Q000302:isolation & purification
chemical_list: D029941:Fish Proteins; D007106:Immune Sera; D014819:Vitellogenins; D004958:Estradiol
keywords: Catfish; Clarias batrachus; ELISA; Vitellogenin