Simultaneous Chemiluminescence Immunoassay Of Dual Biomarkers For Early Diagnosis Of Brain Diseases

With human genome sequencing having been done, the focus of malignant disease research was transferred to proteomics from genomics.Simultaneous detection of numerous proteins has gained considerable interest in various field including clinical diagnosis, environmental protection, etc.Compared with parallel single-analyte immunoassays, a multi-analyte immunoassay offers some remarkable advantages, such as higher sample throughput, improved efficiency, lower sample consumption, and reduced overall cost per assay. Biomarkers are loosely defined as biomolecules that are differentially expressed during disease, which indicate disease stage and are detected for evaluating the extent of disease, monitoring the response of tumors to therapy, and predicting recurrence.Accurate detection diagnosis requires simultaneous detection of a suite of biomarkers.An ideal biomarker-based detection strategy should be capable of the simultaneous and ultra-sensitive detection of multiple biomolecules in complex biological samples.Chemiluminescence (CL) immunoassay, combining good specificity of immunoreactions with high sensitivity of the CL assay, has been proved to be a powerful analytical tool. In this paper, we developed a novel CL duplex immunoassay strategy for the simultaneous determination of two biomarkers of brain diseases, based on horseradish peroxidase (HRP) and alkaline phosphatase (ALP) catalyzed CL reactions.(1)Review of the current multiple detection technologies.We first introduce multi-label resolution based detection techniques, and then review spatially resolution mode including micro arrays technique and encoding micro carriers, etc.(2) A novel CL substrate-resolved immunoassay platform for the determination of two proteins (NSE and CA15-3)is proposed, based on ALP and HRP as CL labels.The dose-response curves showed the linear ranges from 0.2 to 10 ng/mL (R2>0.99) for NSE and 0.2 to 10 U/mL (R2>0.99) for CA15-3 with the limits of detection 0.2 ng/mL and 0.2 U/mL, respectively. Spiked serum samples with different amounts of antigens (high, middle, low) were prepared and detected to show an acceptable recovery. Two proteins were determined in a single run and no cross-reaction was detected. Overall, this proposed method may be used for the simultaneous determination of dual biomarkers in a single vessel and will find applications in the early-stage diagnosis of human golima.(3) We established a magnetic-separation based duplex immunoassay of S100βand NSE for early-stage diagnosis of stroke, where magnetic beads were used as the carrier for the special biomolecules immobilization. Goat-anti-rabbit IgG-HRP and anti-FITC-ALP were utilized to react with two reporter antibodies respectively in this homogeneous sandwich CL immunoassay. There is no cross reaction between two antigens and two pairs of antibodies.The dose-response curves showed the linear ranges from 0.02 to lng/mL (R2>0.99) for S100βand 1 to 20 ng/mL (R2>0.99) for NSE with the limits of detection 0.005 ng/mL and 0.2 ng/mL, respectively. The effect of serum on sensitivity of two antigens was investigated and the results showed this work is suitable for the early-stage diagnosis of stroke patients with elevated levels of biomarker S100βand NSE.