Validation of a cytometric bead-based immunoassay for tear interleukin 8 and interferon gamma inducible protein 10

Background Interleukin 8 (IL-8) and Interferon gamma inducible protein 10 (IP-10) are pro-inflammatory chemokines that may play key antagonistic roles in regulating corneal immune privilege and angiogenesis prevention. Some groups have reported that tear levels of IL-8 and IP-10 measured by multiplexed antibody-based assay are artifact prone and therefore unreliable, leading to their under-utilization as ocular surface disease and dry eye biomarkers. The current study compares tear levels of IL-8 and IP-10 measured by multiplexed cytometric bead-based assay (CBA) with single analyte ELISA assay. Mass spectrometry, a non-antibody based method, was also used to obtain confirmation of tear levels. Methods Matching pairs of micropipette-collected non-stimulated and stimulated tears from 20 non-dry eye subjects were assayed by sandwich ELISA and by BioRad 27-Plex cytokine assay on a Luminex 200 system. Subsequently, non-stimulated tear samples of 4 subjects from a group of 49 non-dry eye and aqueous deficient dry eye subjects, which were assayed by another investigator using CBA, were processed for IL-8 and IP-10 isolation and subjected to ABSciex 5600 Triple-TOF mass spectrometry for semi-quantitative analysis. Results Comparison of ELISA and CBA results showed strong correlation for IL-8 in non-stimulated tears and IP-10 in stimulated tears. There was a weaker relationship for non-stimulated IP-10 and stimulated IL-8. Bland Altman plots showed non-stimulated tear IL-8 to be valid by CBA, but non-stimulated IP-10 levels appeared to be subject to systematic interference by a matrix component and were therefore questionable. In both assays, IP-10 was consistently higher than IL-8. Tear IP-10 and IL-8 excised from electrophoresis gels were identified by mass spectrometry using strongly ionizing reference peptides. By both CBA and mass spectrometry, non-stimulated levels of tear IP-10 were higher than tear IL-8. Conclusion In this study, CBA was determined to be a valid assay for quantifying non-stimulated tear IL-8 and capable of replacing ELISA. This was not the case for tear IP-10, interference casting doubt on its potential to replace ELISA. Despite this limitation, mass spectrometry confirms that human tears contain much higher IP-10 levels than IL-8, supporting ELISA and CBA findings and further confirming the biomarker potential of IP-10.