Generation of a Monoclonal Antibody against D-Dimer Using HTS-Based LiCA

Abstract

D-dimer is an essential diagnostic index of thrombotic diseases. Since the existing anti-D-dimer antibodies vary in quality and specificity, a search for alternative anti-D-dimer antibodies is required. The present study aimed to screen a novel monoclonal antibody (mAb) against D-dimer using a light-initiated chemiluminescence assay (LiCA). In this work, mice were immunized with antigen prepared from human plasma by enzyme hydrolysis. After screening, a novel mAb, DD 2G11, was obtained. The results of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis indicated that DD 2G11 could be used as a standard marker for D-dimer. The isotype of DD 2G11 was IgG1, the Ka value was 0.646 nM-1, and the Kd value was 50 nM, indicating that the binding affinity to D-dimer was very high. Furthermore, no cross-reactivity between DD 2G11 and other fibrinogen degradation products (FgDPs) was found. Finally, the correlation between DD 2G11 and the reference antibody (commercial antibody) was investigated by analyzing 56 clinical samples using a latex-enhanced turbidimetric immunoassay (LTIA). The R2 value of the linear regression was 0.94538, indicating that DD 2G11 met clinical requirements. In conclusion, the present study provides a more expeditious protocol to screen mAbs and provides a clinically usable mAb against D-dimer.

SARS-Cov-2 Spike-S1 Antigen Test Strip with High Sensitivity Endowed by High-Affinity Antibodies and Brightly Fluorescent QDs/Silica Nanospheres

“…The affinity of the four MmAbs to SARS-CoV-2 spike-S1 and S1-RBD was determined by indirect ELISA as previously described. 44 Either 2 μg/ mL spike-S1 or 2 μg/mL S1-RBD recombinant protein in 50 mM carbonate buffer (pH 9.0) was used to coat 96-well microtiter plates at 4 °C overnight, followed by washing with PBST (PBS containing 0.05% Tween-20). Then, the microplates were blocked overnight at 4 °C with 200 μL of blocking buffer.…”

Point-of-Care Platform for Diagnosis of Venous Thrombosis by Simultaneous Detection of Thrombin Generation and D-Dimer in Human Plasma

“…For the applications mentioned above, conventional methods for measuring the TG and D-dimer levels were used. Central laboratory quantitative D-dimer assays were initially based on an enzyme-linked immunosorbent assay (ELISA) technology but have more recently been adapted to use coagulation and clinical chemistry analyzers with an endpoint based on immunofluorescence, latex-enhanced immunoturbidimetry, or chemiluminescence . Although these assays are highly sensitive and are economical to perform when analyzing large numbers of specimens, the combination of specimen transportation time and analytical time often results in a prolonged response time of more than 40 min …”