Rapid chemiluminescent sandwich enzyme immunoassay capable of consecutively quantifying multiple tumor markers in a sample.

Using the role of p-iodophenol in enzyme assay, enhanced 1,1'-oxalyldiimidazole chemiluminescent enzyme immunoassays (ODI-CLEIAs) were developed to consecutively quantify trace levels of triple tumor markers, such as alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) in a sample. Due to the high sensitivity of enhanced ODI-CLEIAs, it was possible to fix the incubation times (1) to capture a tumor marker with two antibodies, which are primary antibody immobilized on the surface of polystyrene strip-well and detection antibody-conjugated horseradish peroxidase (HRP), and (2) to form resorufin with the addition of substrates (e.g., Amplex Red, H2O2) in order to quantify triple markers in human serum. Enhanced ODI-CLEIAs capable of consecutively and rapidly quantifying triple markers with the same incubation time were more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) capable of separately and slowly quantifying them with different incubation times. In addition, accuracy, precision, and recovery of enhanced ODI CLEIAs in the presence of p-iodophenol were acceptable within statistical error range.

Role of Triton X-100 in chemiluminescent enzyme immunoassays capable of diagnosing genetic disorders.

The use of Triton X surfactants in developing 1,1'-oxalylimidazole chemiluminescent enzyme immunoassays (ODI CEIs) with extended linear response range for the quantification of unconjugated estriol (uE3), alpha-fetoprotein (AFP), and human chorionic gonadotropin (hCG) is reported for the first time. The wider linear dynamic range in ODI CLEIA results from Triton X series (e.g., Triton X-100, -114, -405, -705) acting as an inhibitor in the interaction between Amplex Red (hydrophobic substrate) and horseradish peroxidase (hydrophilic enzyme) to produce resorufin (hydrophobic fluorescent dye). Triton X-100 acts as the appropriate inhibitor in ODI CLEIA. The maximum concentrations of AFP and hCG quantified with sandwich ODI CLEIA in the presence of Triton X-100 were 8 times higher than when analyzed with the same system in the absence of Triton X-100. In addition, the lowest concentration of uE3 determined using competitive ODI CLEIA in the presence of Triton X-100 was 20 times lower than that measured with competitive ODI CLEIA in the absence of Triton X-100. These results indicate that rapid quantification of AFP, uE3, and hCG using cost effective and highly sensitive ODI CLEIAs in the presence of Triton X-100 can be applied as an accurate, precise, and reproducible method to diagnose genetic disorders (e.g., trisomy 18 and trisomy 21) in fetuses.

1,1'-Oxalyldiimidazole chemiluminescent enzyme immunoassay capable of simultaneously sensing multiple markers.

In order to rapidly and simultaneously quantify and screen trace levels of multiple biomarkers in a single sample, rapid 1,1'-oxalyldiimidazole chemiluminescence (ODI CL) was applied as a biosensor of immunoassays using various enzymes such as alkaline phosphatase (ALP) and horseradish peroxidise (HRP). (1) Fluorescein was formed from the reaction of fluorescein diphosphate (FDP) and immuno-complex conjugated with ALP. (2) Resorufin was formed from the reaction between Amplex Red and H(2)O(2) in the presence of immuno-complex conjugated with HRP. When ODI CL reagents (H(2)O(2) in isopropyl alcohol, ODI in ethyl acetate) were injected in a test tube or strip-well containing fluorescein and resorufin formed from above two reactions a bright CL emission spectrum having two peaks (518 nm for fluorescein and 602 nm for resorufin) was observed. The two peaks can be independently quantified with an appropriate statistical tool capable of deconvoluting multiple emission peaks. In conclusion, we expect that ODI chemiluminescent enzyme immunoassays (CLEIAs) using a couple of enzymes conjugated with antigen or antibody and substrates can rapidly and simultaneously quantify and screen multiple biomarkers in a single sample.

Evidence of multiple pathways capable of emitting peroxyoxalate chemiluminescence using a charge coupled device spectrometer.

Peroxyoxalate chemiluminescence (PO-CL) spectra obtained simultaneously and continuously using a CCD spectrometer provide evidence of the complexity of PO-CL reactions.

Advent of innovative chemiluminescent enzyme immunoassay.

Using 1,1'-oxalyldiimidazole (ODI) chemiluminescence detection, a new chemiluminescent enzyme immunoassay (CLEIA) was developed to quantify prostate specific antigen (PSA) in human serum. The results observed in ODI CLEIA were compared with those obtained in commercially available enzyme linked immunosorbent assay (ELISA), fluorescence enzyme immunoassay (FEIA), and luminol CLEIA. PSA complex-conjugated horseradish peroxidase (HRP) was formed from one-step sandwich immunoreaction of PSA, PSA primary antibody and PSA secondary antibody-conjugated HRP for 15 min in a strip-well at 36.5°C. CL substrate solution (Amplex Red and H2O2 in PBS buffer, pH 7.4) was added in the washed strip-well and incubated for 10 min at room temperature. When resorufin formed in this process was mixed with 1,1'-oxalyldi-4-methylimidazole and H2O2 in a testing tube, rapid and bright CL was observed. Detection limit (0.035 ng/ml) of PSA in ODI CLEIA was much lower than those (0.50 and 0.25 ng/ml) in commercially available ELISA and luminol CLEIA even though total incubation time of the former (25 min) was shorter than those of the latter (45 and 35 min). Also, the dynamic range (0-100 ng/ml, R2=0.9996) of ODI CLEIA was wider than those of other EIAs. In conclusion, the excellent correlation (r=0.9767) between ODI CLEIA and Advia Centaur XP Immunoassay System indicates that the accurate, precise, and rapid ODI CLEIA can be applied as a novel CLEIA capable of diagnosing and monitoring various diseases.