Validation of assays for inflammatory mediators in sputum

Commercially available immune assays are being used with increasing frequency in the study of lung inflammation. However, their performance in complex biological fluids is rarely assessed. The authors wished to assess their reliability to determine whether the results obtained in sputum samples can be easily interpreted. The reproducibility of several such assays was therefore determined together with their ability to recover known amounts of pure reagent. Sputum sol phase was obtained from several patients with chronic lung disease and used together with the reagents in a series of "spiking" and dilutional experiments. Results confirmed that the enzyme assay for myeloperoxidase and the immune assays for interleukin-8, leukotriene B4 and secretory leukoproteinase inhibitor were all reproducible (intra-assay coefficient of variation 3.8-7.7%). Furthermore, each of these assays gave >85% recovery of a "spike" with pure reagent. However, the immune assay for myeloperoxidase (although reproducible) gave poor recovery and was dependent on the degree of sample dilution and elastase content. These studies confirm that the reliabilities of fluid phase measurements should be assessed before being widely applied to complex biological samples.