An ultrasensitive and long-lasting chemiluminescence immunoassay for IP-10 detection based on a 4-bromophenol-reinforced bienzymatic system

ABSTRACT

The 10-kDa chemokine interferon-gamma-inducible protein 10 (IP-10) is considered one of the most promising biomarkers for diagnosing both tuberculosis and COVID-19 infections. The blood samples of patients at different disease states contain different levels of IP-10, which need to be detected in a rapid, specific and ultrasensitive manner. Here, we report a bienzymatic chemiluminescence sandwich immunoassay (BCSI) assay for the ultrasensitive and stable detection of IP-10. In this assay, IP-10 is first efficiently captured using a double-antibody sandwich strategy. The detection antibody is linked to catalase (CAT) via a streptavidin-biotin signal amplification system to achieve highly efficient conversion of hydrogen peroxide (H2O2) to oxygen and water. In the chemiluminescence (CL) reaction, horseradish peroxidase (HRP) acts as an efficient catalyst, and 4-bromophenol acts as an enhancer for the cyclic transition of HRP, which results in a strong and durable CL signal. The bienzymatic catalysis with CAT and HRP and the potentiation of 4-bromophenol enables the assay to be ultrasensitive and stable. The CL intensity was found to be well correlated with the detection of IP-10 at levels in the range of 0.71 to 125,000 pg/mL, which covers more than 6 orders of magnitude, with a detection limit of 0.63 pg/mL. The coefficient of variation was 1.49%, and the recovery range of IP-10 in serum was 86.21%-104.57%. This assay provides a wide linear range and high sensitivity and may be a promising method for the high-throughput detection of IP-10 in the diagnosis of tuberculosis and COVID-19.