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. 2022 Oct 22;12:17792. doi: 10.1038/s41598-022-20560-6

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© The Author(s) 2022

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Screening and selection strategy for hybridoma clones producing anti-PD-1 antibodies with PD-1/PD-L1 binding blockade activity. (a) Pearl chains of splenocyte-myeloma cells after electrofusion. (b) Hybridoma colonies in semisolid HAT medium after selection for 7 days. (c) Illustration of the anti-PD-1 NAb screening strategy using high-throughput flow cytometry. The lower left quarter (no binding) displayed no fluorescence signals detected. The upper-left quarter (hPD-L1 detected only) displayed the detection of fluorescence 647-labeled secondary antibody that bound to the hPD-L1 human Fc chimera proteins. The upper-right quarter (non-NAb detected) displayed the detection of both 647-labeled and 488-labeled fluorescence signals that bound to mouse anti-PD-1 antibody and hPD-L1 Fc chimeric protein. The lower-right quarter (NAb detected) displayed only the detection of fluorescence 488-labeled secondary antibody that bound to mouse anti-PD-1 antibody with PD-1/PD-L1 binding blockade activity. (d) The dot-plot results of selected hybridoma mini-pools producing anti-PD-1 antibody with various properties. The percentage of the NAb detected population is indicated in the lower-right corner. (e) Summary results of hybridoma clones derived from each screening step. Altogether, of these efficient strategies, we successfully isolated 5 hybridoma clones with high PD-1 binding and PD-1/PD-L1 binding blockade activities.