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Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes
Published on: July 26, 2019
MAC-CCD system: a novel lymphocyte microwell-array chip system equipped with CCD scanner to generate human monoclonal
T Ozawa1, K Kinoshita, S Kadowaki
1Department of Immunology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Insights
A new MAC-CCD system enables real-time monitoring of intracellular calcium mobilization in over 15,000 B-cells. This advanced system can detect rare antigen-specific B-cells, aiding in antibody discovery.
Area of Science:
- Immunology
- Biotechnology
- Cell Biology
Background:
- Previous lymphocyte microwell-array systems detect antigen-specific B-cells via Ca(2+) mobilization but lack time-lapse capabilities.
- Monitoring intracellular Ca(2+) mobilization is crucial for identifying antigen-specific B-cells.
Purpose of the Study:
- To develop a novel lymphocyte microwell-array chip system with a charge-coupled device (CCD) time-lapse scanner (MAC-CCD system) for enhanced B-cell monitoring.
- To improve the detection sensitivity for rare antigen-specific B-cells.
Main Methods:
- Development of the MAC-CCD system for time-lapse monitoring of intracellular Ca(2+) mobilization in individual B-cells.
- Utilizing a correlation method within the MAC-CCD system to enhance detection sensitivity.
- Analyzing Ca(2+) mobilization in over 15,000 live B-cells every 10 seconds.
Main Results:
- The MAC-CCD system successfully monitored intracellular Ca(2+) mobilization in a large number of B-cells with high temporal resolution.
- The correlation method enabled the detection of B-cells at a low frequency of 0.046%.
- Six influenza nucleoprotein-specific human monoclonal antibodies were successfully obtained from vaccinated volunteers.
Conclusions:
- The MAC-CCD system provides a powerful tool for high-throughput, time-lapse monitoring of single B-cell responses.
- The integration of a correlation method significantly enhances the ability to detect rare antigen-specific B-cells.
- This technology facilitates the discovery of rare B-cells and the development of targeted antibody therapies.
Abstract:
We previously developed a lymphocyte microwell-array system, which effectively detects antigen-specific B-cells by monitoring intracellular Ca(2+) mobilization at the single-cell level with a fluorescent Ca(2+) indicator, fluo-4. However, it is difficult for the system to perform time-lapse monitoring. Here, we developed a novel method, a lymphocyte microwell-array chip system equipped with a charge-coupled device (CCD) time-lapse scanner (MAC-CCD system), for monitoring intracellular Ca(2+) mobilization. The MAC-CCD system is able to monitor intracellular Ca(2+) mobilization of more than 15,000-20,000 individual live B-cells every 10 s. In addition, we adopted a correlation method in a MAC-CCD system, which enabled us to detect B-cells with a frequency of as few as 0.046%. Furthermore, we succeeded in obtaining six influenza nucleoprotein-specific human monoclonal antibodies from the peripheral blood of influenza-vaccinated volunteers. These results demonstrate that the MAC-CCD system with a correlation method could detect very rare antigen-specific B-cells.
