Preparation of Anti-Human Podoplanin Monoclonal Antibody and its application in Immunohistochemical Diagnosis

Abstract

Podoplanin (PDPN), a 38 kDa transmembrane sialoglycoprotein from human, is expressed in lymphatic endothelial cells but not in vascular endothelial cells, and has been considered as a specific marker of lymph. In this study, the gene encoding the extracellular part of PDPN (ePDPN) was synthesized and used to expressed fusion protein ePDPN-His and GST-ePDPN, respectively, in E.coli. The purified GST-ePDPN fusion protein was mixed with QuickAntibody-Mouse5W adjuvant to immune mice, and the antiserum titer was determined by indirect ELISA. A stable cell line named 5B3 generating anti-PDPN monoclonal antibody (mAb) was obtained by hybridoma technology. The isotype of 5B3 cell line was IgG2b, and the chromosome number was 102 ± 4. The 5B3 mAb was purified successfully from ascites fluid through Protein G column, and its affinity constant was 2.94 × 108 L/mol. Besides, excellent specificity of the 5B3 mAb was further demonstrated in ELISA, western blot and immunohistochemistry experiments, suggesting that 5B3 mAb displays similar application value to D2-40, a commercial available antibody. Hence, the current study provides conclusive guidelines for preparation of other mAbs and their applications in immunohistochemistry diagnosis.

A sensitive lateral flow immunoassay for the multiple residues of five adamantanes

“…After three days, cell fusion between splenocytes from the immunized mouse and SP2/0 myeloma cells was carried out under sterile conditions (Xie et al, 2018;Zhao et al, 2017). The culture supernatants were collected and monitored by ELISA and ic-ELISA after 7 days.…”

“…Then, the cell lines were injected intraperitoneally into three paraffin-primed BALB/c mice for producing ascites fluid. The mAb was purified from ascites fluid using the caprylic acidammonium sulfate precipitation method (Chunsheng et al, 2018;Xie et al, 2018;Xie, Chen, & Yang, 2012) and the mAb was stored at −20°C until needed for further experiments.…”

Application of Aptamer-Based Biosensor for Rapid Detection of Pathogenic Escherichia coli

“…Therefore, the analysis and quantification of E. coli in water and food by ELISA method has received widespread attention [ 9 , 10 ]. However, the shortcomings of ELISA method cannot be ignored, e.g., it is difficult, antibody preparation takes a long time and it has a high detection limit in protein analysis [ 11 , 12 ]. Moreover, some samples with small molecular weight have no immunogenic activity, and need be coupled to macromolecular proteins for ELISA detection.…”

Development of an Electrochemical Biosensor for Rapid and Effective Detection of Pathogenic Escherichia coli in Licorice Extract

“…Owing to the immune activity between antigen and antibody, ELISA method has superior specificity, high sensitivity and short detection time, and has been used in detection of different subtypes of E. coli [11][12][13]. However, due to the costly and cumbersome preparation process of antibodies, the detection cost of ELISA has increased greatly [14,15]. Another disadvantage is their limited shelf lives, which limits the viability of antibody-based biosensors in ELISA technology [16].…”

“…Recently, great efforts have been devoted to the development of novel modified electrodes for E. coli rapid detection, because the direct oxidation current on the bare electrode was not attractive owing to the high overpotential and sluggish electron transfer processes [22,23]. To date, three kinds of materials, including enzymes [14,24], aptamers [25] and nano-composites [26,27], have been used to modify the electrode surface for better analytical properties. Among them, aptamers are expected to be a promising candidate for the design of electrochemical biosensors for E. coli for their easy preparation, low cost, high stability and strong affinity.Aptamers are single-stranded oligonucleotides screened by systematic evolution of ligands by exponential enrichment (SELEX) [28].…”