Validation of a Protein A-based ELISA for quantifying Immunoglobulin G in a non-traditional wildlife species, the Steller sea lion (Eumatopias jubatus)

Abstract

Abstract Immunoglobulins are key proteins that function to preserve immune homeostasis and are quantified to infer changes to the acquired humoral immune response in mammals. Measuring immunoglobulins in non-traditional wildlife for immune surveillance often requires ingenuity, and rigorous standardization of methodologies to provide consistent and reliable results especially for species lacking species-specific reagents. We modified and optimized existing ELISA methodology that utilizes the binding properties of Staphylococcus derived Protein A (Prt A) with immunoglobulin G (IgG). We validated the assay for accurately quantifying IgG in Steller sea lion (SSL) serum using critical measures of validation including parallelism, spike and percent recoveries, and internal controls. Of the modifications made, heat inactivation of SSL serum proved pivotal to enhance accuracy and precision of IgG detection by improving linearity and percent recovery in parallelism dilutions and serum spikes. Purified canine IgG standard was not affected by the heat inactivation protocol. These results support that confounding serum proteins likely interfere with the binding of Prt A with IgG, and demonstrate the need for heat inactivation of serum to ensure optimal IgG quantification using the PrtA-ELISA. Further, incorporation of spike and recovery are essential validation measures to ensure proper standardization of this assay. Consequently, the improved and validated PrtA-ELISA guarantees a species-independent IgG detection with validation criteria to enhance accuracy, and precision for addressing future immunological questions concerning SSLs and other non-traditional wildlife in a clinical, ecological, and conservation context.