Establishing an immunological profile of 96 cytokines, chemokines, and growth factors in human biofluids via MILLIPLEX® multiplex immunoassay

Abstract

Abstract Multiplex immunoassays are well-established research tools enabling the simultaneous measurement of several proteins in biological samples. In the field of immunology, these immunoassay kits have been critically important for profiling the expression of a wide array of immunomodulatory proteins, including cytokines, chemokines, and growth factors. To this end, we recently developed two MILLIPLEX® multiplex immunoassay kits, each capable of the simultaneous measurement of 48 unique proteins in human samples. In this study, our objective was to profile the relative abundance of 96 cytokines, chemokines, and growth factors in a diverse collection of human biofluids, including serum, plasma, cerebrospinal fluid, saliva, sputum, urine, tears, milk, synovial fluid, and bronchoalveolar lavage (BAL). For each analyte, the fold increase in the average sample measurement relative to the limit of detection was determined. Some biofluids were generally rich in these immunological factors, such as BAL samples, whereas others were sparse, as noted in milk samples. Additionally, the reagent compatibility of the two immunoassay kits used to interrogate the sample cohort was examined to define opportunities for developing novel custom multiplex immunoassays containing analytes from each of the parent kits. The result of this effort was the generation of a set of rules guiding the acceptable combinations amongst the 96 proteins as well as the identification of mutually exclusive protein targets.