Interrogation of the plasma proteome with differential scanning calorimetry.

as analyte concentrations increased, within-and between-day imprecision decreased. The CVs of these samples, when assay dilution was accounted for, were consistent with the low (ng/L) LOQ estimated for each SMIA. Highly sensitive and precise SMIAs with excellent recoveries for several other analytes were also developed (Table 1 and see Fig. S1C in the online Data Supplement). All LODs were Ͻ1 ng/L, with the exception of the RANTES SMIA (LOD of 1.2 ng/L). Such LODs compare favorably with those reported for ELISAs and cytometric bead immunoassays and suggest utility in clinical research applications on the basis of their relationships to reference interval blood concentrations: 0 –14 ng/L (IL-8), 73–737 ng/L (IFN-inducible protein-10), 0 to Ͻ7.8 ng/L (IL-5), 1.7–18 ng/L (MIP-1␣), and 580 – 48 000 ng/L (RAN-TES) (see Table S1 in the online Data Supplement). Of the SMIAs tested, recoveries of analyte added to plasma were between 80% and 129% (Table 1), and in most cases CVs were Ͻ20%. These results illustrate that highly sensitive SMIAs can be developed for human plasma to precisely quantify cytokines and chemokines with good recoveries. Although comparisons with other detection platforms are difficult to make, the sensitivities, recovery, and precision of the SMIAs reported here rival those achieved in mul-tiplex cytometric bead immunoassays and conventional ELISAs for cytokines (see (9 –11) and Table S1 in the online Data Supplement). Efforts are currently underway to optimize assay performance and to capitalize on the miniaturization made possible by the single-molecule approach. On the basis of the molecular weights of the analytes tested here (7.8 –17 kDa), the observed LODs of 0.10 –1.2 ng/L correspond to 7.1–154 fmol/L of analyte, values well above the detection limits of the instrument for reporter molecules (Ͻ3 fmol/ L). Thus, there is considerable room for improving the LODs, which could be achieved by reducing assay background or by increasing sample enrichment during the assay, as follows. First, because nonspecific binding of reporter molecules resulted in a background of approximately 2–20 bins/s above the typical buffer background, which corresponds to approximately 10 –100 fmol/L of SA-A647 molecules (see Fig. S1A in the online Data Supplement), background could be decreased by as much as 10-fold. Second, the 2-to 5-fold enrichment of sample, which is brought about by eluting reporter molecules into 20 ␮L, could be increased approximately 10-fold by reducing the elution volume to match the few microliters required for a typical 36-s sample …