Long-Term Measurements of Human Inflammatory Cytokines Reveal Complex Baseline Variations between Individuals.

Comprehensive characterization of the healthy human proteome baseline is essential for personalized medicine. Baseline data are necessary to understand the variation between individuals, as well as longitudinal variation within individuals. Many important protein biomarkers, such as cytokines, exist at extremely low or undetectable levels in the healthy state. This paper describes results from a 14-week study of healthy human subjects using ultrasensitive single-molecule array (Simoa) assays to measure both intra and intersubject variation of 15 cytokines. The results show a wide variation in the ranges of some cytokines between individuals and demonstrate that individual baseline values will be essential for predicting disease presence and progression. Although all of the studied cytokines demonstrated high temporal stability (or low intrasubject variation) over the entire study period, there were two distinct groups of cytokines that demonstrated either high (IL-8, IFN-γ, IL-2, IL-6, and IL-1ß) or low (IL-15, TNF-α, IL-12 p70, IL-17A, GM-CSF, IL-12 p40, IL-10, IL-7, IL-1α, and IL-5) subject-to-subject variation. This work demonstrates that ultrasensitive assays are essential for characterizing human cytokines in healthy subjects. The results show that some cytokines vary by more than two orders of magnitude between individuals, making it an imperative to obtain individual baseline measurements if they are to play a role in health and disease diagnosis.

Using Antigen-antibody Binding Kinetic Parameters to Understand Single-Molecule Array Immunoassay Performance.

This paper provides insights into the performance of single-molecule array (Simoa) immunoassays by examining the effects of various capture and detector antibody-antigen binding kinetic parameters. Simoa is similar to other immunoassays in that the overall Simoa performance is heavily dependent on the choice of antibodies; however, little is known about how the different properties of the antibodies result in the wide variations in assay performance. Here, we focus on antibody-antigen binding kinetics and demonstrate how the association (kon) and dissociation (koff) rate constants of the capture and detection antibodies affect Simoa performance. We compared six different antibodies with over a four-log range of equilibrium dissociation constants (KD) and found that Simoa assay performance had an inverse relationship to the koff value of the detection antibody. The Simoa fluorescent signals were highest when the koff of the detection antibody was less than 10-5 s-1. The capture antibody koff did not have as significant an effect, but a koff of less than 10-3 s-1 was preferred. We also found that the kon values of the capture and detection antibodies were not important factors for Simoa performance. Therefore, the assay optimization process could be accelerated by choosing detection antibodies with the slowest koff values.

Ultrasensitive Detection of Ricin Toxin in Multiple Sample Matrixes Using Single-Domain Antibodies.

Ricin is an extremely potent ribosomal inactivating protein listed as a Category B select agent. Although ricin intoxication is not transmittable from person to person, even a single ricin molecule can lead to cell necrosis because it inactivates 1500 ribosomes/min. Since there is currently no vaccine or therapeutic treatment for ricin intoxication, ultrasensitive analytical assays capable of detecting ricin in a variety of matrixes are urgently needed to limit exposure to individuals as well as communities. In this paper, we present the development and application of a single-molecule array (Simoa) for the detection of ricin toxin in human urine and serum. Single-domain antibodies (sdAbs), among the smallest engineered binding fragments, were chemically coupled to the surface of paramagnetic beads for the sensitive detection of ricin toxin. The Simoa was able to detect ricin at levels of 10 fg/mL, 100 fg/mL, and 1 pg/mL in buffer, urine and serum, respectively, in a fraction of the assay time need using immuno-polymerase chain reaction (IPCR). Using a fully automated state-of-the-art platform, the Simoa HD-1 analyzer, the assay time was reduced to 64 min.