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Title: A MODIFIED ENZYME-LINKED IMMUNOSORBENT ASSAY FOR DETECTION OF CUTANEOUS ANTIBODY AGAINST ICHTHYOPHTHIRIUS MULTIFILIIS

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

Submitted to: European Association of Fish Pathologists
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/1/2003
Publication Date: 12/1/2003
Citation: Xu, D., Klesius, P.H., Shelby, R.A. 2003. A modified enzyme-linked immunosorbent assay for detection of cutaneous antibody against Ichthyophthirius multifiliis. European Association of Fish Pathologists. 23(5):228-234.

Interpretive Summary: Ichthyophthirius multifiliis (Ich) is a protozoan parasite that infects species of cultured and wild fish. The parasite damages gills and skin of fish, leads to high mortality, and causes heavy economic loss in the tens of million dollars annually. The fish recovered from Ich infection develop resistance. Cutaneous antibody was found in mucus or skin culture fluid of fish resistant to Ich. The assays currently used to measure the cutaneous anti-Ich antibody are not sensitive enough for low levels of antibody. The purpose of this study was to modify and optimize an enzyme-linked immunosorbent assay (ELISA) for detection of low levels of cutaneous antibody against Ich in channel catfish and in water. The following factors were determined for the modified ELISA: 1) concentration of coating protein and coating time, 2) suitable substrate, 3) conjugate concentration and 4) incubation time of substrates. The modified ELISA required less theront coating protein, shorter coating time for the plate, and low concentration of conjugate. The sensitivity of modified ELISA was approximately 4-fold higher than the original assay when measuring anti-Ich antibody in the immune skin culture fluid. The modified ELISA was able to detect the antibody in water samples collected from tanks with Ich resistant fish. The original assay failed to detect the antibody even though the water samples were concentrated 40-fold. This study showed that the modified ELISA was a sensitive, quantitative and time saving assay to measure low concentrations of anti-Ich cutaneous antibody excreted from skin of channel catfish resistant to Ich.

Technical Abstract: Fish surviving Ichthyophthirius multifiliis (Ich) infection develop an immune response against this parasite. Enzyme-linked immunosorbent assays (ELISA) currently used to determine the cutaneous anti-Ich antibody lack sensitivity. This study modified an ELISA assay for detection of low levels of cutaneous antibody against Ich in channel catfish and in water. The modified ELISA used tetramethylbenzidine (TMB) as substrate for horseradish peroxidase (HRP). The modified ELISA required less theront coating protein, short coating time for the plate, and low concentration of conjugate. The optical density readings using modified ELISA were approximately 4-fold higher than the ELISA using o-phenylenediamine (OPD) when measuring anti-Ich antibody in the immune skin culture fluid. The modified ELISA was able to detect the antibody in water samples collected from tanks with Ich immune fish. The ELISA by using OPD failed to detect the antibody even though the water samples were concentrated 40-fold. The results of this study showed that the modified ELISA by using TMB as a HRP substrate was a sensitive, quantitative and time saving assay to measure low concentrations of anti-Ich cutaneous antibody excreted from skin of channel catfish immune to Ich.