Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society

Satsuki Chikura; Takafumi Kimoto; Satoru Itoh; Hisakazu Sanada; Shigeharu Muto; Katsuyoshi Horibata

doi:10.1186/s41021-021-00181-7

Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society

Genes Environ. 2021 Mar 20;43(1):10. doi: 10.1186/s41021-021-00181-7.

Authors

Satsuki Chikura¹, Takafumi Kimoto², Satoru Itoh³, Hisakazu Sanada⁴, Shigeharu Muto⁵, Katsuyoshi Horibata⁶

Affiliations

¹ Toxicology Research Department, Teijin Institute for Bio-medical Research, Teijin Pharma Limited, 4-3-2 Asahigaoka, Hino-shi, Tokyo, 191-8512, Japan.
² Toxicology Research Department, Teijin Institute for Bio-medical Research, Teijin Pharma Limited, 4-3-2 Asahigaoka, Hino-shi, Tokyo, 191-8512, Japan. t.kimoto@teijin.co.jp.
³ Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo, 134-8630, Japan.
⁴ Development ADMET Department, Translational Research Division, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba-shi, 412-8513, Japan.
⁵ Safety Research Laboratories, Mitsubishi Tanabe Pharma Corporation, Shonan Health Innovation Park, 2-26-1, Muraoka-Higashi, Fujisawa, Kanagawa, 251-8555, Japan.
⁶ Division of Genetics and Mutagenesis, National Institute of Health Sciences, 3-25-26 Tonomachi, Kawasaki-ku, Kawasaki-shi, Kanagawa, 210-9501, Japan. horibata@nihs.go.jp.

Abstract

The PIGRET assay is one of the Pig-a assays targeting reticulocytes (RETs), an in vivo genotoxicity evaluation method using flow cytometry with endogenous reporter glycosylphosphatidylinositol anchor protein. The PIGRET assay with RETs selectively enriched with anti-CD71 antibodies has several desirable features: high-throughput assay system, low background frequency of mutant cells, and early detection of mutation. To verify the potential and usefulness of the PIGRET assay for short-term testing, an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society was conducted. The collaborating laboratories assessed the mutagenicities of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on the total red blood cell assay and the PIGRET assay. Here the standard protocol for the PIGRET assay was described in detail.

Keywords: CD59; CD71; Flow cytometry; Glycosylphosphatidylinositol; HIS49; Immunomagnetics; In vivo gene mutation; Pig-a assay; Reticulocytes.