A multiplexed assay to detect antimicrobial peptides in biological fluids and cell secretions

Abstract

Mucosal tissues represent the front line in defense against potential pathogens, and one means by which mucosa provide protection is via the secretion of antimicrobials which can interfere with potential pathogens as well as recruit and modify the responses of immune cells. Here we describe adaptation of ELISA assays to microsphere format, facilitating simultaneous quantification of antimicrobial peptides including elafin, MIP3α, HBD2, HBD3, SLPI, RANTES, SDF1, lactoferrin, LL-37, and HNP1-3. The multiplexed assay exhibits excellent reproducibility, shows linearity over a two order of magnitude concentration range for most analytes, is compatible with biological fluids such as cervicovaginal lavage fluid, and presents significant cost and sample savings relative to traditional ELISA assays.

Keywords: Antimicrobials; Chemokines; Cytokines; Female reproductive tract; Mucosal secretions; Multiplex assay.

Figure 1. Adaptation of antimicrobial factor assays to microsphere format

A. Dose-response curves for antimicrobial factors for ELISA assays (black, OD scale at right y-axis) and microsphere assays (blue, MFI scale at left y-axis). B. Intra-assay CVs. C. Inter-assay CVs. D. Comparison of CVs between microsphere and ELISA assays for the same sample run in duplicate (Wilcoxon matched pairs signed rank test).

Figure 2. Multiplexed assay performance

A. 9-plex assay standard curve using LL-37 dilution buffer for standards, PBS-B as detection buffer, and LL-37 wash buffer. B. 9-plex assay standard curve using PBS-T to dilute standards, HNP1-3 buffer for detection, and PBS-T for washes. C. Scatterplot comparing results of single and multiplexed assays for analyte detection. D. Signal observed on the RANTES bead set in the presence of RANTES analyte (+, green bar), or the presence of all off target analytes in the absence of RANTES analyte (−, black bar). E. Cross-reactivity profile of the RANTES analyte across bead sets. F. Scatterplot comparing MFI values observed for samples suspended in either PBS-B or tissue culture media (DMEM). G. Scatterplot comparing observed and expected MFI values for cervicovaginal lavage (CVL) samples.