First identified as a high-affinity kinase-deficient receptor for class-3 semaphorins and vascular endothelial growth factor (VEGF) families, Neuropilin2 (NRP2) is a transmembrane non-tyrosine-kinase glycoprotein that has a vital function in neuronal patterning. Furthermore, NRP2 expression is often upregulated in cancer tissues and correlated with poor prognosis. In the present study, we report the establishment of a monoclonal antibody specific for NRP2b1b2 domain (NRP2 MAb) through hybridoma method. NRP2 MAb is measured to have a titer of 5.12 × 10(5) against NRP2b1b2 in indirect ELISA. Western blotting, flow cytometry, and immunofluorescence analysis indicate that NRP2 MAb can combine full-length NRP2 in LoVo and SW480 cells. Besides helping further understand NRP2-related pathological mechanisms and cell-signaling pathways, NRP2 MAb may act as a therapeutic agent for cancer in the future.
To solve processing problems caused by bacterial activity in sugar industry, a sensitive method for dextran detection is expected. In this study, monoclonal antibodies (mAbs) against dextran were prepared using dextran T40 conjugated with bovine serum albumin as immunogens. Basing on the mAbs, a competitive enzyme-linked immunosorbent assay (ELISA) method to measure dextran quantitatively was developed. Results demonstrated that this competitive ELISA method had a calibration curve with correlation coefficient of 0.9986, IC 50 value of 204.2 ng/mL and detection value ranging from 26.3 ng/mL to 1174.9 ng/mL. In addition, cross-reaction assay suggested the method had no significant crossreaction with dextran-related substances such as b-glucan, starch, sucrose, sugar and glucose. Besides, spike recovery assay indicated this quantitative method for dextran detection had a perfect accuracy.
PRACTICAL APPLICATIONSWith wide detection range, high specificity and good calibration curve, this developed competitive ELISA method will be reliable to detect dextran quantitatively in sugar industry, and even may be commercially applied into kit development.
Dextran as anti-nutritional factor is usually a result of bacteria activity and has associated serial problems during the process stream in the sugar industry and in medical therapy. A sensitive method is expected to detect dextran quantitatively. Here we generated four monoclonal antibodies (MAbs) against dextran using dextran T40 conjugated with bovine serum albumin (BSA) as immunogen in our lab following hybridoma protocol. Through pairwise, an MAb named D24 was determined to be conjugated with horseradish peroxidase (HRP) and was used in the establishment of a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method for determination of dextran, in which MAb D9 was chosen as a capture antibody. The detection limit and working scope of the developed sandwich ELISA method were 3.9 ng/mL and 7.8-500 ng/mL with a correlation coefficient of 0.9909. In addition, the cross-reaction assay demonstrated that the method possessed high specificity with no significant cross-reaction with dextran-related substances, and the recovery rate ranged from 96.35 to 102.00%, with coefficient of variation ranging from 1.58 to 6.94%. These results indicated that we developed a detection system of MAb-based sandwich ELISA to measure dextran and this system should be a potential tool to determine dextran levels.
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