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United States Department of Agriculture

Agricultural Research Service

Title: Quantitative Detection of Serum Immunoglobulin in Channel Catfish by Competitive Inhibition Elisa

Authors
item Shelby, Richard
item Xu, Dehai
item Klesius, Phillip

Submitted to: Veterinary Immunology International Symposium
Publication Type: Abstract Only
Publication Acceptance Date: July 25, 2004
Publication Date: July 25, 2004
Citation: Shelby, R.A., Xu, D., Klesius, P.H. 2004. Quantitative detection of serum immunoglobulin in channel catfish by competitive inhibition elisa. Veterinary Immunology International Symposium.

Technical Abstract: Serum immunoglobulin (IG) levels in channel catfish (Ictalurus punctatus) are indicative of immune response to pathogens as well as general health of the fish. Precise quantitative measurement is possible using competitive inhibition ELISA (CI-ELISA), if pure IG can be produced in sufficient quantity and of sufficient purity for use a quantitative standard. We purified serum from channel catfish in a series of preparative scale steps involving precipitation with polyethylene glycol, anion exchange, and size exclusion chromatography. Purity was assessed by analytical size exclusion HPLC of native IG and denaturing SDS-PAGE. The quantitative standards and test sera are added to the wells along with anti-catfish IG monoclonal antibody, followed by anti-mouse peroxidase conjugate. Tetra methylbenzidine peroxidase substrate was added and absorbance values were analyzed by linear regression of standard curves generated by absorbance vs. µg/mL of purified IG. Regression curves were linear in the range of 1-100 µg/mL. Catfish sera, when diluted 1/100, generally fell within the linear portion of the standard curve. Serum IG of channel catfish ranged from 1 to 5 mg/mL in this study. Culture fluid from skin cultures of channel catfish generally fell within the range of 10-300 µg/mL. This method provides rapid and accurate quantitative measurement of channel catfish IG in serum and culture fluid.

Last Modified: 7/28/2014
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