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Recently, several oligonucleotides have been launched for clinical use and a number of therapeutic oligonucleotides are under clinical trials. Aptamer is one of the oligonucleotide therapeutics and has received a lot of attention as a new technology and an efficacious pharmaceutical compound comparable to antibody. Aptamer could be used for various purposes, not only therapeutics but also diagnostics, and applicable to affinity chromatography as a carrier molecule to purify proteins of interest. Here we demonstrate the usage and advantages of RNA aptamer to Fc region of human IgG (i.e., IgG aptamer) for purification of human antibodies. IgG aptamer requires divalent cations for binding to IgG and bound IgG dissociates easily upon treatment with chelating reagent, such as EDTA, under neutral conditions. This elution step is very mild and advantageous for maintaining active conformations of therapeutic antibodies compared to the widely used affinity purification with Protein A/G, which requires acidic elution that often damages the active conformation of antibodies. In fact, of several monoclonal antibodies tested, three antibodies were prone to aggregate on acidic elution from the Protein A/G resin, while remained fully active upon neutral elution from the IgG aptamer resin. The IgG aptamer was fully manipulated to alkaline resistant by ribose 2'-modifications, and thereby reusable numerous times with 1 N NaOH washing. The capacity of the aptamer resin to bind IgG was equivalent to that of the Protein A/G resin. Therefore, the IgG aptamer will provide us with a unique tool to uncover and purify human monoclonal antibodies, which hold therapeutic potential but lose the activity upon acidic elution from Protein A/G-based affinity resin., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Over the last decade, isolation and purification of monoclonal antibodies, for diagnostic analysis, have been carried out using the hybridoma expression system. The present study describes a novel example of a detection system using hybridoma cells containing antibody against O1 antigen directly for V. cholerae diagnosis, which is a major health problem in many parts of the world, especially in developing countries. This method has advantages such as simplicity, ease of process, and it does not require manipulation of hybridoma cell. For this approach, an efficient amount of fluorescence calcium indicator, fura 2-AM, was utilized, which emitted light when the intracellular calcium concentration increased as result of antigen binding to specific antibody. More reliable results are obtained via this method and it is considerably faster than other methods, which has the response time of less than 45 s for detection of V. Cholerae O1. Also, the limit of detection was computed to be 50 CFU/mL (<13 CFU per assay). In addition, no significant responses were observed in the presence of other bacteria with specific hybridoma or other cell lines exposed to V. cholerae O1. Furthermore, this method was successfully applied to V. cholerae O1 detection in spiked environmental samples, including water and stool samples without any pretreatment. All results reveal that hybridoma cells can provide a valuable, simple, and ready to use tool for rapid detection of other pathogenic bacteria, toxins, and analytes.