Evaluation of barcoded magnetic bead-based immunoassays for the simultaneous detection of feline leukemia virus antigen and antibody against feline immunodeficiency virus

Abstract

Testing platforms that leverage automation, require minimal sample volume, and enable various tests to be performed simultaneously on a single sample have the potential to improve workflow and efficiency in veterinary diagnostic laboratories. We evaluated a barcoded magnetic bead (BMB) technology using established immunoassays for detection of feline leukemia virus (FeLV) p27 antigen and antibody against feline immunodeficiency virus (FIV). Analytical sensitivity, limit of blank, and limit of detection were used to establish a functional sensitivity of 1.00 ng/mL of inactivated FeLV antigen and 35.7 ng/mL of anti-FIV monoclonal antibody. Common interferents, such as hemoglobin, lipid, and bilirubin, were not found to interfere with the performance of the assay. Intra- and inter-assay CVs were <13% for both assays using manufactured samples. Using a set of 116 feline samples, the diagnostic accuracy of our multiplex assay was 100% compared to reference assays. Performance in a convenience set of 1,000 feline samples submitted to a commercial diagnostic laboratory revealed a proportion of positive results of 1.3% for FeLV and 3.7% for FIV. BMB technology should enable rapid screening of samples for various markers in a single immunoassay well.

Keywords: antibody; antigen; feline immunodeficiency virus; feline leukemia virus; immunoassay; multiplex.

Figure 1.

Performance of the feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) barcoded magnetic bead (BMB) assay with manufactured and native feline samples. A. Detection of a serial dilution of inactivated FeLV in the FeLV BMB assay. B. Detection of a serial dilution of anti-FIV monoclonal antibody in the FIV BMB assay. C. FeLV/FIV BMB assay results for a characterized sample set of 116 feline sera. Median fluorescence intensity (MFI) cutoffs were 1.00 ng/mL (1,270 MFI) for FeLV (black dashed line) and 35.7 ng/mL (738 MFI) for FIV (red dashed line). D. Distribution of the FeLV/FIV BMB assay MFI results from a set of 1,000 feline samples obtained from a commercial diagnostic laboratory.