中国生物工程杂志

With the development of flow cytometry instruments, the accuracy and stability of flow cytometry are continuously improved. An increasing number of studies suggest the clinical value of quantitative detection of protein molecules expressed on cells. The demand for quantitative flow cytometry is increasing, but the traditional method cannot meet the needs of polychromatic antibody quantification due to its own limitations that antibody binding quantification is still limited to using of a single fluorescent labeled antibody. Quantum simply cellular (QSC) beads could be used for antibody binding quantification, but the quantification method using QSC beads was not widely adopted because of the huge difference between the results using this method and using the classical methods. Considering that the reason for the failure of the QSC method was the poor specificity of the QSC beads, this paper proposes a new method that has improved the staining technique by using a protein free microsphere staining buffer and is combined with a suitable specific capture microsphere (the anti-mouse IgG k beads for human cell analysis) as a calibrate for the value of QSC standards. In the test of CD3, CD4, CD8, CD25, CD27, CD28, CD45RA, CD127 and CD197 on the surface of human T cells, reasonable results were obtained. Although the accuracy of this technique still needs more tests, it has many possible uses, including calculating the absolute number of antibody binding, as a reference for polychromatic fluorescence compensation, visualizing the compensated spillover spread, calculating the amount of fluorescence produced by a single antibody under specific detection conditions, indicating the quality of antibody, and tracking the instrument performance. The calibrated fluorescence value of antibody-bound microspheres can be references to design a multicolor panel and help to achieve a comparable detection system among different platforms. The quantification of polychromatic flow cytometry is of great significance for single cell multi-omics technology. Our study suggests that a specific binding antibody light chain of a capture beads series can be used to achieve accurate and simple multiple quantification of cell surface proteins.