Isolation, Purification, and Molecular Weight Determination of Serum Immunoglobulin from Gulf Menhaden: Development of an Enzyme-Linked Immunosorbent Assay to Assess Serum Immunoglobulin Concentrations from Atlantic Menhaden.

Abstract

An immunoglobulin M (IgM)-like immunoglobulin was isolated by polyethylene glycol precipitation from pooled serum collected from healthy gulf menhaden Brevoortia patronus. The immunoglobulin (Ig) was purified by Sephacryl-400 gel filtration chromatography. The molecular weight of unreduced, purified Ig was determined to be 850 kilodaltons (kD) by high-performance liquid chromatography. A goat antiserum against the purified Ig was produced and determined to react with the serum Ig of both gulf and Atlantic menhaden B. tyrannus by double gel diffusion. When reacted with sera from taxonomically unrelated species of fish, sheepshead minnow Cyprinodon variegatus, striped mullet Mugil cephalus, gulf flounder Paralichthys albigutta, and hybrid striped bass (white bass Morone chrysops × striped bass M. saxatilis), no precipitation bands developed. Furthermore, the specificity of the goat antiserum was shown by Western blot analysis to be for the 77,000-molecular-weight heavy chain of reduced and alkylated gulf and Atlantic menhaden Ig. An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the assessment of Ig concentrations in Atlantic menhaden serum. To illustrate the applicability of the ELISA, we assessed the Ig concentration in the serum of 542 healthy Atlantic menhaden collected from inland bays of Delaware and Maryland in 2000 and 2001. The amount of Ig was estimated to be in the range 0.26-23.50 mg/mL, with a mean of 7.37 and a standard deviation of 5.12 mg/mL. The ELISA was reproducible, as determined by the inter- and intra-assay coefficients of variation (100·SD/mean) of 11.2% and 6.8%, respectively, and used very small amounts (1-2 μL) of serum to assess the Ig concentrations from menhaden.