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. 2017 Oct 25;7:13979. doi: 10.1038/s41598-017-14277-0

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Two days protocol of Ecobody technology. SP, signal peptide sequence; T7P, T7 promoter; T7T, T7 terminator; SKIK, SKIK tag. (1) Collection of lymphocytes by density gradient centrifugation from a few millilitres of animal blood. (2) Fluorescent staining of plasma cells with endoplasmic reticulum (ER)-tracker. (3) Sorting of stained cells by fluorescence-activated cell sorting (FACS). (4) Adsorption of cells binding to the antigen using antigen-coated magnetic beads. (5) Separation of one cell per 10 µL into 384-well plate. (6) Selection and confirmation of cell-bead complexes under an inverted phase-contrast microscope. (7) Reverse transcription using gene specific primers and SuperScript IV reverse transcriptase. (8) First PCR, using primers annealing to the signal peptide (SP) sequence and constant region of antibody genes. (9) Second PCR, using primers with tails required for the subsequent Gibson assembly step. (10) Gibson assembly to combine T7 promoter and terminator with antibody genes. (11) PCR to amplify DNA fragments for cell-free protein synthesis. HA and His tags are present downstream of leucine zipper A (LZA) and B (LZB), respectively. (12) Expression of Zipbody with SKIK tag by cell-free protein synthesis (CFPS) with DsbC and oxidised glutathione (GSSG). (13) Enzyme-linked immunosorbent assay (ELISA) evaluation. DNA sequences were analysed as required using plasmids constructed in step (10) In this protocol, steps 1 to 9 are conducted for the first day and steps 10 to 13 are next day.