Galactomannan ELISA in bronchoalveolar lavage fluid with surface-enhanced raman scattering readout

BACKGROUND: Invasive aspergillosis is a threatening fungal infection with high mortality rates. One of the key factors for timely diagnostics of the disease is an approach based on detection of galactomannan, a polysaccharide from the pathogen’s cell wall. The existing galactomannan enzyme-linked immunosorbent assays (ELISA) employ the colorimetric detection. The emergence of the new highly sensitive techniques for the measurement of peroxidase label in ELISA, e.g., based on the effect of surface-enhanced Raman scattering (SERS), facilitates the development of a next generation assays with improved sensitivity and with potentially broader range of biological fluids applicable for analysis. An important step of developing the new test-system is the demonstration of its applicability to real clinical samples and comparability of the results with reference method. AIM: To evaluate the applicability of the previously developed highly sensitive and selective SERS-based ELISA protocol for the measurement of Aspergillus galactomannan in bronchoalveolar lavage fluid samples. METHODS: Ten bronchoalveolar lavage fluid samples were analyzed using both the GalMAg-EIA ELISA kit (XEMA LLC, Russia) and the previously developed SERS-based ELISA, which employs o-phenylenediamine substrate and silver nanoparticles colloid. RESULTS: Using standard samples prepared with in vitro cultivated galactomannan, the limit of detection for the SERS-based ELISA was estimated as 53 pg/mL with the working range spanning up to 10.8 ng/mL and the mean coefficient of variation within this range being 4%. Using the GalMAg-EIA kit, 6 out of 10 bronchoalveolar lavage fluid samples were identified as positive and 4 — as negative. The comparison of the results for these two methods demonstrates the non-linear dependence with the Spearman coefficient of correlation being 0.93–0.95 (p=2.3×10-5–1.1×10-4). CONCLUSION: For the bronchoalveolar lavage fluid samples, the comparability of the results was demonstrated when detecting the galactomannan using the commercially available GalMAg-EIA kit and the previously developed SERS-based ELISA. The results confirm the high sensitivity of the SERS-based Aspergillus galactomannan ELISA and its prospectiveness for practical laboratory applications. Compared to chromogenic detection, the advantages of surface-enhanced Raman scattering readout include a lower limit of detection for galactomannan and increased sensitivity at the lower end of the galactomannan-positivity index (<2). These findings could enable more accurate determination of the threshold for discriminating the positive and the negative samples. Thus, the research provides the basis for future larger scale trials of the SERS-based galactomannan ELISA in order to evaluate its applicability for clinical-laboratory diagnostics.