Efficacy of the Valita Titer™ Assay to Rapidly Quantify Serum IgG in Sheep

Abstract Evaluation of serum antibody responses is a powerful tool in animal agriculture research and research focus has shifted to evaluation of health metrics. The standard methodology for measuring immunoglobulin-G (IgG), the most abundant antibody in circulation, relies on enzyme-linked immunosorbent assays (ELISA). While these assays have demonstrated reliability, they are labor-intensive and require up to 5 hrs to complete. If IgG is to be used as biomarker for developing selection metrics, a different assay must be employed with the same reliability, specificity and sensitivity but in a high-throughput format. Recently a new assay to measure IgG has been developed based on the inherent affinity that protein-G has for IgG molecules across multiple species reducing the need for specie-specific reagents. The Valita Titer™ assay was designed to utilize fluorescently labeled protein-G molecules and their affinity for IgG in a 15-minute assay that quantifies IgG in a variety of media by measuring fluorescence and polarization. When the labeled-protein-G molecule is bound to IgG and illuminated with polarized light the result is slow rotation of the IgG-protein-G complex which retain polarization. Free, unbound labeled-protein-G has rapid rotation and loses polarization. Therefore, this assay uses both fluorescence and polarization to quantify IgG. To determine the efficacy of this assay in detecting sheep IgG, a sheep IgG standard (Millipore-Sigma) was diluted to 2,000, 1,600, 1,200, 800, 400, 300, 100 mg/L in 1X MOPS buffer and run in triplicate on a Valita Titer™ assay plate. The resulting polarization values revealed a CV between triplicate samples of less than 3% and a R2 value of 0.999. The results observed using purified sheep IgG were consistent with data generated in other species and confirm that this assay was able to detect sheep IgG at varying concentrations. To evaluate the efficacy of this assay in detecting IgG in serum, we used serum from hyperimmunized lambs and performed a serial dilution of serum. Serum from hyperimmunized lambs revealed that the most effective concentration present within the standard curve was 1:20 dilution. This assay is sensitive, specific, and rapid in the detection of sheep IgG and based on this work can be used to measure IgG in serum, thus, providing a high throughput means to generate data for biomarker analysis.