Establishment of a novel homogeneous nanoparticle-based assay for sensitive procalcitonin detection of ultra low-volume serum samples

Abstract

PurposeSepsis is a potentially fatal systemic body infection with a significant mortality rate worldwide. Although C-reactive protein (CRP), interleukin-6 (IL-6), and procalcitonin (PCT) might be biomarkers for sepsis diagnosis, PCT is more sensitive and specific than CRP or IL-6. We aimed to establish an efficient immunoassay that precisely detects PCT in human serum for the early diagnosis of sepsis.Materials and methodsWe developed a novel amplified luminescent proximity homogeneous assay (AlphaLISA) for the quantitative detection of PCT in serum. In this assay, a pair of antibodies was used to capture PCT in serum and to form sandwich complexes after incubating for 15 minutes at 37°C.ResultsPCT concentrations were determined within a linear range of 0.016–100 ng/mL. The limit of detection was 18.6 pg/mL. The results demonstrate that the reproducibility, recovery, and specificity of this assay for PCT meet the requirements of clinical detection. The coefficient of determination (R2) between this method and commercially available enzyme-linked fluorescent assay (ELFA) kits was estimated to be 0.93045 in clinical serum testing.ConclusionThe novel assay for PCT detection was robust with high sensitivity and a broad dynamic range. Compared with conventional heterogeneous detection methods such as ELISA, this assay measured the concentration of the homogeneous form of PCT and provided results that are more accurate within a shorter detection time. We expect that this novel method will be useful for the early screening and prognosis evaluation of patients with sepsis.

Spiegelmer-Based Sandwich Assay for Cardiac Troponin I Detection

“…Although dissociation constants of applied antibodies of the kit are not provided, they are most likely inferior to those of the here presented spiegelmers. Furthermore, it is known that the limit of detection of AlphaLISA is determined by many factors, such as buffer composition, concentration of receptors, and acceptor and donor beads, and even the order of the addition of various components may affect the assay sensitivity [ 22 ]; thus, the low pg/mL sensitivity of the kit has been achieved by thorough optimization of assay conditions…”

MicroRNA-155: Regulation of Immune Cells in Sepsis

“…Therefore, it is very important to find biomarkers for the early diagnosis of sepsis. The laboratory indexes of the clinical diagnosis of sepsis include C-reactive protein (CRP), procalcitonin (PCT), and IL-6 [ 62 ]. As a single biomarker has limited value, the use of new biomarkers and the combination of several markers is recommended in improving diagnostic accuracy [ 63 , 64 ].…”

Comparison of Bead-Based Fluorescence Versus Planar Electrochemiluminescence Multiplex Immunoassays for Measuring Cytokines in Human Plasma

“…Whereas both assays showed clearly distinct cytokine expression differences between the high PCT group and the other two groups, results varied considerably among both analytes and distinct samples. Although general patterns emerged, such as higher relative cytokine expression in high-PCT (12,16,17) and possibly diabetic subjects (9) versus healthy control subjects within the LMX data, these observations were not consistent, possibly due to the low levels of cytokines measured in these samples.…”

“…Sixty-two EDTA-anticoagulated human plasma samples were evaluated by both assays. Samples comprised 20 from healthy donors, 21 from subjects who had previously been diagnosed with diabetes mellitus, and 21 from individuals with elevated PCT levels (>3 ng/mL), an indicator of sepsis (12,16,17). All samples were purchased from Central BioHub GmbH (Hennigsdorf, Germany), and were stored at −80 • C until used.…”