Indirect Enzyme-Linked Immunosorbent Assay Based on Immunoglobulin Y Antibodies for the Evaluation of Humoral Immunity Against Flavobacterium oreochromis in Colossoma macropomum: A Preliminary Study

Abstract

Infections with Flavobacterium oreochromis pose risks to the production of Colossoma macropomum. Since no therapeutic treatment exists for this disease in this aquatic host, prophylaxis through vaccination is one method for disease control. The enzyme-linked immunosorbent assay (ELISA) is widely used in aquaculture to assess animal immunity and, mainly, to validate vaccination protocols. The method based on the use of immunoglobulin Y (IgY) is an alternative to mammalian antibodies that should be considered due to its advantages over antibodies produced by small mammals. This study aimed to standardize an indirect ELISA (iELISA) protocol to validate the immunization of C. macropomum against F. oreochromis. For this, a hen and fish were immunized to obtain anti-C. macropomum and anti-F. oreochromis antibodies, respectively, which were used to standardize the test. Fish were inoculated with bacterin + adjuvant, bacterin alone, adjuvant alone, or phosphate-buffered saline, subjected to blood sampling, and monitored for up to 60 days. The iELISA was composed of the inactivated bacteria in the coating step, C. macropomum anti-Flavobacterium oreochromis as the primary antibody, and anti-C. macropomum IgY as the secondary antibody. The results demonstrate that the highest antibody titers were found in the organisms immunized with bacterin + adjuvant and bacterin. The iELISA enabled the detection of antibodies produced by C. macropomum, which were mediated by IgY specificity.

Keywords: IgY; immunoassay; plasma; tambaqui; vaccine.

Figure 1

Dynamic changes in antibody production of immunized animals in each group over time at 0, 21, 30, 40, 50, and 60 days. Similar results were observed between G1 and G2, with differences compared to G3 and G4.

Figure 2

Representation of the model’s prediction. Cut-off determined at 0.088 (3× standard deviation, upper dashed line) and 0.077 (2× standard deviation, lower dashed line) OD₄₅₀.