Development of a multiplex assay for simultaneous quantification of endocrine analytes

Abstract Text: Since the advent of the radioimmunoassay in the 1950s, numerous immunologically-based methods have evolved for sample analysis. Although each immunological method possesses unique assets and liabilities, all share limited abilities in range of detection and the number of analytes resolvable simultaneously; most procedures are limited to one analyte determined per replicate per sample. The objective of this study was to adapt technology evolving from the genomics revolution for multiplexed hormonal analysis in livestock. We sought to complete this objective using sequencing technologies and tools: Illumina BeadXpress, Luminex xMAP, and quantitative immuno-PCR. The Illumina BeadXpress and Luminex xMAP both share similar characteristics; each platform consists of a laser spectrum analyzer and a bead-set. Each bead-set contains microscopic beads; each set with unique identifying signatures. As a test of proof of concept, the surface of a bead set was conjugated to an LH antibody. Using these technologies, we were able to establish an assay for LH on the Luminex platform, but not on the Illumina platform. The proprietary nature of both the Luminex and Illumina platforms however, greatly limited assay flexibility. Therefore, we chose to establish an assay for LH using quantitative immuno-PCR. Quantitative immuno-PCR exploits PCR amplification with antibody detection. Briefly, a sandwich immunoassay is performed with capture antibody immobilized to a PCR plate. A second detection antibody is conjugated to an oligonucleotide and after a series of washes, the plate is subjected to quantitative PCR (qPCR). Detection of LH was achieved, but background binding was a problem. Subsequently, to simplify the design and demonstrate proof of concept, a biotinylated oligonucleotide was incubated with streptavidin coated PCR plates and subjected to qPCR. The results of this latter experiment suggested that the assay performed well with over six orders of magnitude linearity. In conclusion, background binding was found to be a major problem with quantitative immuno-PCR but one that is believed to be resolvable. Moreover, our observations suggest that immuno-PCR has the potential to improve detection capabilities of hormonal assays with six orders of magnitude sensitivity, reproducibility, and ultimately in a multiplex capacity with the oligonucleotide serving as both a label and as a barcode for identifying the analyte. The technological leap in capabilities provided by successful multiplexing can be used for understanding the complex interaction of endocrine and metabolic signals in the dynamically changing animal.

Keywords: Multiplexing, endocrine profiling, immuno-PCR