Generation of mouse monoclonal antibodies specific to tilapia immunoglobulin using fish immunoglobulin/BSA complex for monitoring of the immune response in Nile tilapia Oreochromis niloticus

Abstract

The simple immunoprecipitation method was used to isolate tilapia immunoglobulin (Ig) for immunization in order to produce monoclonal antibodies (MAbs) specific to tilapia Ig. First, the tilapia antiserum against bovine serum albumin (BSA) was prepared by peritoneal injection of BSA into tilapia, and the tilapia anti-BSA antiserum was used to precipitate BSA to form the Ig/BSA immune complex. The Ig/ BSA immune complex was then injected into Swiss mice for hybridoma production. After fusion, three hybridoma clones producing MAbs specific to the tilapia antibody were selected by dot blot and Western blot. All MAbs (101A, 59G, and 11A) were bound specifically to the heavy chain of immunoglobulin M (IgM). The MAbs 101A and 59G demonstrated twofold higher affinity than MAb 11A and the commercialized antibody. However, MAbs 11A could also bind to the heavy chain of IgM in Asian seabass, Lates calcarifer, as well. These MAbs can be used to monitor the immune responses of individual fish by indirect ELISA upon exposure to various antigens.

A multi‐epitope chimeric protein elicited a strong antibody response and partial protection against Edwardsiella ictaluri in Nile tilapia

“…Two experiments were carried out, Experiment A was incubated with infected striped catfish and tilapia serum, and Experiment B was incubated with non‐infected striped catfish and tilapia serum. The membranes were consecutively incubated with 1:100 serum dilution of the respective serum, followed by incubation with 1:50 mouse anti‐tilapia or mouse anti‐catfish IgM monoclonal antibodies (Soonthonsrima et al., 2019) (kindly provided by Prof. Dr. Paisarn Sithigorngul, Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand) and 1:500 Goat anti‐mouse IgG‐Horseradish peroxidase conjugate GAM‐HRP antibody (SeraCare) at 4°C for 2 hr with gently shaking. The membranes were washed 3 times with PBST at room temperature with gentle shaking between each consecutive incubation.…”

“…The coated wells were washed and blocked overnight at 4°C with 200 µl/well of 5% BSA in PBS. Then, the wells were washed and further incubated with test sera from all 5 groups diluted 1:500 (100 µl/well), added in triplicate and incubated at room temperature for 2 h. After that, the plates were washed and 100 µl/well of mouse anti‐tilapia IgM monoclonal antibodies (1:100) (Soonthonsrima et al., 2019) (kindly provided by Prof. Dr. Paisarn Sithigorngul, Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok, Thailand) was added to the wells and incubated at room temperature for 2 h followed by washing and subsequent addition of 100 µl of GAM‐HRP antibody (1:5000) with incubation for 1 h at room temperature. After washing, 100 μl/well TMB (3,3′,5,5′‐tetramethylbenzidine) was added, and the microplates were incubated in the dark at room temperature for 5 min.…”

Ozone nanobubble treatments improve survivability of Nile tilapia (Oreochromis niloticus) challenged with a pathogenic multidrug-resistantAeromonas hydrophila

“…In brief, 96 well EIA/RIA plates (Costar ® , Corning Inc., USA) were coated with formalin-killed A. hydrophila whole-cell antigen (OD 600nm = 1.0). Fish sera (dilution 1:256), anti-Tilapia IgM secondary antibody (1:200) (Soonthonsrima et al, 2019), and commercial goat anti mouse antibody horseradish peroxidase (HRP) conjugate (1:3000) were used for the ELISA assay in this study and samples were read at an absorbance of 450 nm using a SpectraMax ® iD5 Multi-Mode Microplate Reader (Molecular Devices, USA).…”

Immunization of Nile tilapia (Oreochromis niloticus) Broodstock With Tilapia Lake Virus (TiLV) Inactivated Vaccines Elicits Protective Antibody and Passive Maternal Antibody Transfer

“…The samples were then centrifuged at 5,000 g for 10 min at were prepared. The blocking reagent was removed from the wells and the diluted samples added to the ELISA plate, which was subsequently incubated overnight at 4 o C. The plates were rinsed 5 times with high salt wash buffer (HSWB, 2 mM Tris; 50 mM NaCl; 0.01% Tween 20, pH 7.7) before incubating them with an anti-tilapia IgM monoclonal antibody [32] (diluted 1:200 in 1X PBS + 1% BSA) for 2 h at 28 o C. The plates were then rinsed 5 times with HSWB, followed by incubation with goat anti-mouse antibody conjugated with horseradish peroxidase (diluted 1:3000 in LSWB + 1% BSA) for 1 h at 28 o C. Finally, the plates were rinsed 5 times with HSWB, each well was filled with 100 µL of 3,3′,5,5′-Tetramethylbenzidine (TMB), and the reaction was allowed to develop in the dark for 5-10 min before adding 50 µL of stop solution (2 M H2SO4).…”