Flow cytometry evaluation for virus-size particles with antibody stain having low fluorophore ratio
Inventors
Artinger, Michael A. • Kohlmeier, Francis Kevin • Olszowy, Michael W. • Gates, Tyler Donald
Assignees
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Publication Number
US-11585813-B2
Publication Date
2023-02-21
Expiration Date
Abstract
A method for evaluating a biological material for unassociated virus-size particles having a particular epitope uses a fluorescent antibody stain specific for binding with the epitope and a fluid sample with the virus-size particles and fluorescent antibody stain is subjected to flow cytometry with identification of fluorescent emission detection events indicative of passage through a flow cell of a flow cytometer of unassociated labeled particles of virus size including such a virus-size particle and fluorescent antibody stain.
Core Innovation
The invention relates to a flow cytometry method for evaluating a biological material for the presence of unassociated virus-size particles having a target epitope. A fluid sample is subjected to flow cytometry with a fluorescent antibody stain comprising an antibody targeted to bind directly or indirectly with the target epitope to form unassociated labeled particles including a virus-size particle. The flow cytometry detects radiation from the flow cell within a wavelength range of fluorescent emission and evaluates detected radiation to identify detection events indicative of passage through the flow cell of the unassociated labeled particles of virus size.
The unassociated labeled particles have a maximum cross dimension in a range of from 10 nanometers to 2 microns. The fluorescent antibody stain comprises a fluorophore attached to an antibody molecule with an average of from 3 to 8 fluorophores per antibody molecule, and detection is based on excitation radiation causing a fluorescent emission response from the fluorescent antibody stain.
The invention further encompasses a stained fluid sample in an aqueous liquid medium comprising unassociated labeled particles of virus size having a maximum cross dimension in a range of from 10 nanometers to 2 microns. Each unassociated labeled particle includes a virus-size particle having a target epitope and a fluorescent antibody stain with an antibody targeted to bind with the epitope, where the fluorescent antibody stain has a fluorescent emission response in a wavelength range in response to excitation by excitation radiation.
The document describes fluorescent antibody staining applied to virus-size particles that include viruses, virus-like particles, and exosomes/nanoparticles, with detection events evaluated from flow cell fluorescence rather than light-scatter identification. It also describes evaluation approaches that can use multiple fluorescent stains with different fluorescent emission responses and that can incorporate a fluorogenic nucleic acid stain in the stained fluid sample.
Claims Coverage
The document provides two independent claims: one directed to a flow cytometry method and one directed to a stained fluid sample. Across the independent claims, the inventive features focus on detecting fluorescent emission events from unassociated virus-size particles with a target epitope, within a defined virus-size cross dimension range, using a fluorescent antibody stain with defined fluorophore-to-antibody loading.
Detecting fluorescent emission events for unassociated virus-size particles with target epitope
A flow cytometry method evaluating a biological material for the presence of unassociated virus-size particles having a target epitope by subjecting a fluid sample to excitation radiation to cause a fluorescent emission response from a fluorescent antibody stain and detecting radiation from the flow cell within a wavelength range of the fluorescent emission to identify detection events indicative of passage of unassociated labeled particles of virus size with the target epitope bound directly or indirectly to the antibody of the fluorescent antibody stain.
Virus-size cross dimension window and fluorophore-per-antibody loading for fluorescent antibody stain
The unassociated labeled particles have a maximum cross dimension in a range of from 10 nanometers to 2 microns, and the fluorescent antibody stain comprises a fluorophore attached to an antibody molecule with an average of from 3 to 8 of the fluorophore attached per antibody molecule.
Aqueous stained fluid sample with unassociated labeled virus-size particles and fluorescent antibody stain
A stained fluid sample comprising an aqueous liquid medium containing unassociated labeled particles of virus size having a maximum cross dimension in a range of from 10 nanometers to 2 microns, where each unassociated labeled particle includes a virus-size particle having a target epitope and a fluorescent antibody stain with an antibody targeted to bind directly or indirectly with the epitope, the fluorescent antibody stain producing a fluorescent emission response in a wavelength range in response to excitation by an excitation radiation.
Fluorophore-per-antibody loading in the stained fluid sample fluorescent antibody stain
The fluorescent antibody stain comprises a fluorophore attached to an antibody molecule with an average of from 3 to 8 of the fluorophore attached per antibody molecule.
Across the independent claims, the coverage centers on flow cytometry detection of fluorescent emission response from a fluorescent antibody stain bound to unassociated virus-size particles bearing a target epitope, with a defined virus-size maximum cross dimension range (10 nanometers to 2 microns) and a defined average fluorophore-to-antibody loading (3 to 8). The second independent claim additionally recites the corresponding stained fluid sample structure in an aqueous medium.
Stated Advantages
The document describes detection events evaluated from flow cell fluorescence rather than light-scatter identification.
Documented Applications
Fluorescent antibody staining applied to virus-size particles that include viruses, virus-like particles, and exosomes/nanoparticles.
Evaluation approaches that can use multiple fluorescent stains with different fluorescent emission responses.
Evaluation approaches that can incorporate a fluorogenic nucleic acid stain in the stained fluid sample.
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