Secretory expression and purification of recombinant PLA2R epitopes for the detection of anti-PLA2R autoantibody in serum

Abstract

Most patients with idiopathic membranous nephropathy (IMN) have autoimmune antibodies specifically against the M-type phospholipase A2 receptor (PLA2R). PLA2R acts as a significant biomarker contributing to the clinical diagnosis of...

AQP4-DARPin1: A Chimeric Antigen Based on Scaffold Protein DARPin for Efficient Detection of AQP4-IgG in NMOSD

“…In order to confirm the interaction between recombinant AQP4-DARPins and serum AQP4-IgG, we used indirect ELISA to confirm whether AQP4-DARPins are effectively recognized by serum AQP4-IgG and further identified whether serum AQP4-IgG can bind with AQP4-DARPins by IP. , In the AQP4-IgG ELISA strategy, AQP4-DARPins were coated on microplates (Figure A). Subsequently, serum AQP4-IgG from NMOSD patients was captured by AQP4-DARPins and then recognized by HRP-labeled mouse antihuman IgG.…”

“…The AQP4-IgG ELISA was performed as previously described with slight modifications. 37 First, the recombinant AQP4-DARPins were diluted in coating (PBS) buffer: 10 mM sodium phosphate, pH 7.4, 150 mM NaCl, and then coated on a 96-well microplate (100 μL per well) at 25 °C for 2 h. The plate was then washed with PBST (0.05% v/v Tween-20 in PBS) five times and blocked with blocking buffer (1% m/v BSA in PBS, 200 μL per well) at 25 °C for 2 h. Next, the serum from NMOSD patients and other AID patients was added (50 μL per well), while the serum from healthy individuals was used as a negative control. The plate was then incubated at 25 °C for 1 h and washed with PBST five times.…”

Colloidal gold-based immunochromatographic biosensor for quantitative detection of S100B in serum samples

“…Expression and purification of S100B protein were performed as described previously. 24,25 Briefly, based on the genome sequence of S100B (NCBI reference sequence: NP_006263.1), a codon-optimized gene was designed and synthesized by Genecefe Biotechnology Co., Ltd (Wuxi, China). The fragment was then cloned into the expression vector pCMV3.…”