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.

Lukas Einhorn,Kurt Krapfenbauer

doi:10.1186/s13167-015-0046-y

Lukas Einhorn, Kurt Krapfenbauer

Open Access

https://doi.org/10.1186/s13167-015-0046-y

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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.

Highlights

Classical methods of gene product analysis such as binding assays (e.g., enzyme-linked immunosorbent assay (ELISA), protein chip technology) are generally time-consuming, lab-intensive, less sensitive, and lack high-throughput capacity
Samples for analysis are routinely generated in aqueous solutions of suspension from different body fluids that may contain a range of non-volatile salts, solvents, albumins, immune globulins, and lipids
ELISA normally requires 4–5 h until the result can be validated. Such ELISA immunoassays for quantification of diagnostic relevant immunoproteins as biomarkers have already been developed and described, but most of them are too unspecific, expensive, and time-consuming

Summary

Objectives

The 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

Methods

Results

Conclusion