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A flow cytometric immunoassay for beta2-microglobulin in whole blood
1Becton Dickinson Immunocytometry Systems, San Jose, CA 95131, USA.
Insights
This study presents a novel homogeneous immunoassay for soluble beta2-microglobulin (beta2M) in whole blood. The assay uses flow cytometry and bead immobilization, enabling accurate detection without sample washing.
Area of Science:
- Biomedical Engineering
- Immunology
- Analytical Chemistry
Background:
- Flow cytometry offers particle discrimination in unwashed whole blood.
- Soluble beta2-microglobulin (beta2M) is a clinically relevant biomarker.
- Existing assays for beta2M often require sample manipulation.
Purpose of the Study:
- To develop a homogeneous bead-immobilized sandwich immunoassay for soluble beta2M in whole blood.
- To leverage flow cytometry's particle discrimination capabilities for assay development.
- To create an assay that minimizes sample handling and processing steps.
Main Methods:
- Utilized flow cytometry with a 4-micrometer bead that fluoresces in the FL3 channel for triggering.
- Employed a capture antibody (clone A7801) on the bead specific for soluble beta2M, distinguishing it from cellular beta2M.
- Incorporated a signal antibody (PE conjugate of clone L376) for detection, optimized assay parameters including antibody concentrations and blood volume.
Main Results:
- Successfully developed a bead-immobilized sandwich immunoassay for soluble beta2M in unwashed whole blood.
- Optimized bead size, antibody concentrations, and blood volume for assay performance, achieving low blank and minimal hook effect.
- Demonstrated a working range of 2.5 decades compatible with physiological beta2M concentrations.
Conclusions:
- The developed homogeneous flow cytometric immunoassay is effective for detecting soluble beta2M in whole blood.
- The assay's design minimizes sample handling, making it suitable for clinical applications.
- This method offers a sensitive and efficient alternative for beta2M quantification.
Abstract:
Flow cytometers can discriminate a single particle type in an unwashed whole blood sample. Utilizing this capability. we devised a homogeneous bead-immobilized sandwich immunoassay for soluble beta2M (beta2-microglobulin) in whole blood, utilizing an antibody that discriminates soluble from cellular beta2M. A 4 microm bead was chosen that fluoresces only in a FACScan flow cytometer's FL3 channel. thus allowing triggering on this bead to the exclusion of the many blood cell events. The bead was adsorbed with a capture antibody (clone A7801) which binds only to soluble and not to cellular beta2M. This antibody appears to recognize an epitope on beta2M which interfaces to the heavy chain of cellular Class I MHC molecules. The signal antibody (PE conjugate of clone L376, emitting in the FL2 channel) binds to both soluble and cellular beta2M (present in roughly equal amounts in normal blood). The various parameters required for a flow cytometric immunoassay were optimized. The 4 microm sized bead was adequately large to give a near full scale signal at saturation. The relative amounts of signal antibody and capture beads were balanced to give a low blank, minimal 'hook effect', and reasonable event rate on the flow cytometer. The amount of blood added was selected to give a signal near the bottom of the immunassay range for normals with the higher range available for clinical samples. The assay requires no washing, minimizes blood handling, and has a working range (2.5 decades) that is compatible with the biological range of beta2M concentrations with a single blood dilution.
