Background: Comprehensive analysis of bronchoalveolar lavage fluid (BALF) with commercially available ELISA and Meso Scale Discovery systems is limited because manufacturer?s validation is restricted to serum or plasma. Application to BALF is challenging due to variable analyte concentrations and matrix effects. We aimed to test suitability and validity of assays measuring 22 biomarkers in human BALF.

Methods: Assays were validated in accordance with ICH Guideline Q2(R1) including accuracy, precision, linearity, assay range, and sample stability. Biobanked BALF from healthy or asthmatic volunteers were used and measured before or after endotoxin or allergen challenge. Recombinant proteins were spiked in different concentrations into BALF over the full assay range.

Results: All analytes were measured with high intra-assay precision (CV for IL-8 = 2.9-8.1%, GM-CSF = 4.0-9.6%, ICAM-1 = 0.3-4.3%, IL-1? = 1.9-6.4%) with reliable repeatability on different days and by different operators. BALF storage at -80°C up to one month did not impact analyte stability. Antibody blockage of highly concentrated proteins in BALF (SP-D, ICAM-1, albumin) resulted in complete signal depletion. Inflammatory cytokines were significantly elevated in BALF after allergen and endotoxin challenge compared with baseline and saline control.

Conclusion: Validated ELISA and MSD assays reliably measured multiple biomarkers in BALF. Short- and long-term storage did not impact protein measurement. Assay specificities were demonstrated by blockade of respective analytes and spiking of recombinant proteins allowing for comprehensive biomarker analysis of human BALF following inflammatory challenge.