HTRF: a technology tailored for biomarker determination—novel analytical detection system suitable for detection of specific autoimmune antibodies as biomarkers in nanogram level in different body fluids

Abstract

BackgroundClassical methods of gene product analysis such as binding assays (e.g., ELISA, protein chip technology) are generally time-consuming, lab-intensive, less sensitive, and lack high-throughput capacity. In addition, all existing methods used to measure proteins necessitate multiple divisions of the original sample and individual tests carried out for each substance, with an associated cost for each test.MethodTogether with a small biotech company, we developed a new and innovative analytical detection system based on homogenous time-resolved fluorescence (HTRF) technology. Our system facilitates the development of immune assays that measure selective different analytes such as selected biomarkers in a small sample volume at less than 20 min with a much higher sensitivity compared to common binding assay systems such as enzyme-linked immunosorbent assay (ELISA). Recent advances of the application of this novel detection system combine the power of miniaturization, microfluidics, better linear range, and faster quantification.ResultsThe power of the HTRF technology offers great promise for point-of-care clinical testing and monitoring of many important analytes such as disease-specific biomarkers in the nanogram level in different human body fluids such as CSF, blood, serum, plasma, and saliva. The linear dynamical range of our HTRF assay was determined between 2.5 and 100 ng/mL. Precision and accuracy calculated for inter- as well as intra-assays was less than ± 10 %. Intra-assay and inter-assay precision for high, medium, and low analyte concentrations show mean CV values less than ± 10 %. Intra- and inter-assay accuracy for all three concentrations show mean recovery values of 80–120 %.ConclusionThe aim of this work is to describe the development and establishment of this novel HTRF system that allows the very fast detection and quantification of biomarkers in different human body fluids. Furthermore, a specific antibody combination that assures a specific binding of the correct refolded autoimmune IgG is evaluated.

Pathology-specific molecular profiles of saliva in patients with multiple dental caries—potential application for predictive, preventive and personalised medical services

“…The focus is predominantly on socially significant diseases with dental caries-related diseases being one of the most important ones due to their huge impact on people's social lives [2]. The analysis of modern publication on the development of cutting-edge medical technologies [3,4], i.e. on the approaches to early caries prevention [5][6][7], suggests that the topic is a national top priority.…”

Bladder cancer hunting: A microfluidic paper‐based analytical device

“…In other words, the formation of a double-antibody sandwich structure requires a suitable ratio of antigen to the antibody. Meanwhile, it is found that the antigen concentration is not as high as possible, and it may lead to the occurrence of HOOK effect and produce false-negative results caused by inappropriate proportion of antigen and antibody [47]. Through ELISA and standard antigen, the optimum ratio of antigen and antibody is screened out.…”

Oily formulations challenge: how to evaluate their beneficial effects in hydrophilic cell-based models?

“…it is more convenient for minimal sample volume; it is able to measure 384 samples simultaneously, and; process time is quicker than ELISA (Einhorn and Krapfenbauer, 2015).…”