This article presents a simple chronoamperometric immunosensor for the quantitative assessment of creatine kinase MB (CK-MB) in 50 µL undiluted serum samples. The immunosensor consists of gold working and counter electrodes patterned onto a glass chip by thin-film photolithography and an external Ag|AgCl reference electrode. The detection limit (DL) of the chronoamperometric method is 13 ng mL −1 (DL = 2×RMSD/S, where RMSD is the residual mean standard deviation of the measured points around a calibration curve with a slope of S). In spiked serum samples, the response was linear up to 300 ng mL −1 of CK-MB. A surface plasmon resonance (SPR) system with simultaneous electrochemical detection (EC-SPR) aided the development of the sandwich immunoassay. Real-time monitoring of the SPR signal was used to optimize the capture antibody immobilization, CK-MB and detection antibody binding, as well as to minimize the nonspecific adsorption of serum proteins to the sensor surface. The detection antibody has been labeled with alkaline phosphatase (ALP) enzyme for sensitive electrochemical detection. ALP catalyzes the hydrolysis of ascorbic acid phosphate and generates ascorbic acid, which is measured chronoamperometrically. The electrochemical immunoassay for CK-MB was less sensitive to nonspecific adsorption related interferences, had a better detection limit, and required a lower volume of sample than the SPR method.

Toward an understanding of biomarkers for acute viral diseases we have examined Influenza serum in comparison to healthy donors. We determined the levels of Interferon (IFN) Alpha, Beta, Gamma as well as Neopterin and Beta-2 Microglobulin (B2M) by ELISA in the Serum of 38 Influenza and 65 healthy donors using commercial assays. We measured chemokines and proinflammatory cytokines as biomarkers for IFN Activity by electrochemiluminescence. Assays were performed according to the manufacturer’s protocol. IFN-Omega levels were determined using a newly developed ultra-sensitive single molecule assay sensitive to 0.04 pg/ml. Percentile stratification was by Mann-Whitney non-parametric testing and correlations were determined under Spearman conditions. None of the healthy donors exhibited quantifiable IFN-Alpha or Beta and only two had detectable levels of IFN-Omega. Within the Flu cohort, 68.5% of samples contain quantifiable IFN-Alpha and Omega and 39.5% had IFN-Beta. 79% of the Flu samples displayed IFN-Gamma levels above the 95th percentile determined from the healthy donor distribution. Neopterin, B2M, and CXCL-10 were all significantly elevated in the Flu cohort versus the healthy subclass. Other chemokines either showed no correlation or a mild negative correlation. There was a striking concordance between IFN-Alpha and Omega levels. To our knowledge this is the first study to measure IFN-Omega and a broad panel of other analytes in Flu samples. As such this study may provide a beginning to discriminate between different viral diseases.

Interleukin-22 (IL-22), an IL-10 cytokine family member, is produced by a variety of immune cells including Th1, Th17 and Th22 T helper cells, CD8+ T-cells, NK cells, ILC3, neutrophils and macrophages. Interleukin-22 crucially participates in regulating the integrity of epithelial barriers by modulating inflammation, mucus production, additional aspects of wound healing and has been noted to be substantially induced in patients with various chronic inflammatory conditions. Upregulation of IL-22 in psoriasis (PS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) inflammatory bowel disease, Crohn’s disease, cystic fibrosis and atopic dermatitis (AD) has been well documented, with IL-22 often considered a hallmark of IL-1-driven immune responses. Furthermore, IL-22 protein has been proposed as a stratification biomarker for psoriatic arthritis therapeutic choice between IL-17 and TNF inhibitions, to correlate with the severity of clinical sequelae of Sjogren’s Syndrome, and IL-22 mRNA expression stratifies AD responses to fezakinumab. In healthy individuals, IL-22 activity resulting from low level expression may be functionally neutralized, at least in selected tissues such as the intestinal tract, by binding to soluble IL-22 binding protein (IL-22BP) expressed locally by dendritic cells. However, in these pathologies, inhibition of IL-22 by IL-22BP is presumably overcome by elevated expression of IL-22 protein. Explore ranges of IL-22 serum concentrations to differentiate between healthy donors (HD) and select autoimmune disease patients with a simple-to-use, high sensitivity ELISA. A commercial sandwich ELISA (PBL Assay Science, 41701-1) quantifying human IL-22 was characterized and used to assess IL-22 concentration in sera from HD and patients exhibiting one of several autoimmune diseases. This high sensitivity ELISA, having a manufacturer-stated LLOQ of 0.78 pg/ml, exhibited good assay precision, dilutional linearity and spike recovery using HD serum. Parallelism was demonstrated using serum samples from four patients with AD and three HD. Addition of a 10- to 100-fold mass excess of exogenous recombinant IL-22BP reduced the ability of the assay to quantify IL-22 suggesting that at least one antigenic region recognized by ELISA antibodies may be sterically hindered by IL-22BP binding to IL-22. However, such high levels of 10 and 100 ng/ml of IL-22BP are unlikely to be achieved in vivo, and therefore may not adversely affect quantitation of physiological levels of IL-22 protein by this assay. Commercially sourced HD sera concentrations of IL-22 exceeded the assay LLOQ in 23 of 24 samples (96%) with a mean IL-22 concentration of 1.84 ± 1.33 pg/ml. Commercial sera from patients with AD (n = 10, 100%), PS (n = 10, 100%), RA (n = 10, 100%), SLE (n = 10, 100%) were similarly analysed and yielded mean levels of 14.8 ± 11.2, 5.1 ± 2.4, 5.6 ± 0.91 and 3.1 ± 0.74 pg/mL, respectively. Interleukin-22 measured in AD (and PS) sera exhibited sufficiently broad ranges of concentrations such that statistical differences between AD and all other groups were not readily achievable with this number of samples. Notably, these commercially obtained serum samples were not controlled for overall severity of any disease at the time of sample collection. Sample sets were randomly obtained from a population of individuals self-identifying as AD sufferers, as were the other patient samples. With mean serum IL-22 in AD appearing to trend as substantially elevated, an opportunity exists to explore IL-22 trends along with other biomarkers in clinically identified and possibly symptom-stratified populations of patients with AD. Should this trend toward IL-22 elevation be fully substantiated in AD, therapeutic stratification and precision medicine approaches may be conceivable based on serum IL-22 protein concentrations, supporting other investigators’ earlier work examining IL-22 mRNA. The baseline readability of endogenous IL-22 in HD sera using this ELISA is also highly advantageous.

We examined Interferon (IFN)-Alpha and -Beta levels in pooled sera and individual bleeds from C57/Bl, Balb/C and CD-1 mice. 20 Individuals from each strain from two independent colonies were evaluated in commercial assays. The IFN-Beta ELISA detected IFN in 1 of the 60 individuals while the IFN-Alpha assay which detects all 14 subtypes measured IFN in 13 of 60. 5 individuals exhibited greater than 4 pg/ml of IFN-Alpha detected. Specificity was demonstrated by knockdown of the signal using non-biotinylated detection antibody. 14 of 54 porcine sera samples from 4 independent sources were positive for IFN alpha by an ELISA under development, and similar knock-down experiments suggest specificity. This is in contrast to human sera/plasma samples where less than 3% of healthy donor samples have quantifiable IFN-Alpha or -Beta above 0.5 pg/ml. This study may raise questions about the regulation of the IFN system in different species and the potential of these differences to affect experimental results in a variety of models.