Submitted to: Journal of Aquatic Animal Health
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 30, 2002
Publication Date: December 1, 2002
Citation: SHELBY, R.A., EVANS, J.J., KLESIUS, P.H. 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. JOURNAL OF AQUATIC ANIMAL HEALTH. 2002. 15(4):254-262. Interpretive Summary: The immunoglobin which is found in the blood of all animals is a defense mechanism which develops in response to pathogens or antigens. Its function is mainly to help destroy these foreign antigens. The presence of antibodies can tell us if the animal is responding normally to disease organisms, and with a specific assay, we can also determine to which disease or diseases the animal has been exposed. We developed this assay to measure immunoglobulin in menhaden, a commercially important marine fish species. This species is also important since it spends part of its life cycle in estuaries and bays of the Atlantic and Gulf coastal waters of the United States, and may be an indicator species of water quality in these areas. We purified the immunoglobulin from Gulf menhaden, and produced an antibody in goats which could, in turn, be used to measure immunoglobulin of 542 Atlantic menhaden from bays in Delaware and Maryland. Having established a baseline for levels of immunoglobulin in healthy menhaden, we can use the assay to examine the immunoglobulin levels of fish exposed to disease organisms or environmental stress.
Technical Abstract: An IgM-like immunoglobulin was isolated from pooled serum collected from healthy Gulf menhaden, Brevootia patronus by polyethylene glycol precipitation. The immunoglobulin (Ig) was purified by Sephacryl-400 gel filtration chromatography. The molecular weight of unreduced purified Ig was produced and determined to be 850kD 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 (B. patronus) and Atlantic (B. tryannus) menhaden by double gel diffusion. When reacted with serum from taxonomically unrelated species of fish, sheepshead minnow Cryprinodon variegatus, striped mullet Mugil cephalus, Gulf flounder Paralichthys albigutta, and hybrid striped bass Morone chrysops x M. saxatilis, no precipitation bands developed. Furthermore, the specificity of the goat antiserum was shown to be for the 77,000 molecular weight heavy chain of reduced and alkylated B. patronus and B. tryannus Ig by western blot analysis. An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the assessment of Ig concentrations in B. tryannus serum. To illustrate the applicability of the ELISA, we assessed the Ig concentration in the serum of 542 healthy B. tryannus collected from inland bays of Delaware and Maryland in 2000 and 2001. The amount of Ig was estimated to be in the range from 0.26 to 23.50mg/mL with a mean of 7.37 and standard deviation of 5.12mg/mL. The ELISA was reproducible, as determined by the inter- and intra-assay variability coefficients (11.2% and 6.8% respectively), and used very small amounts (1 to 2uL) of serum to assess the Ig concentrations from menhaden.