Evaluating biological material for unassociated virus-size particles with influenza virus epitope
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.
1. A flow cytometry method for evaluating a biological material sample for unassociated virus-size particles having an influenza virus epitope, the method comprising:
subjecting to flow cytometry a fluid sample comprising at least a portion of the biological material sample, wherein the fluid sample comprises a fluorescent antibody stain capable of binding, directly or indirectly, with an influenza virus epitope, the fluorescent antibody stain comprising a fluorescent component or components attached to an antibody molecule that is specific to binding to the epitope and the fluorescent antibody stain having a fluorescent emission response when subjected to an excitation radiation, the flow cytometry comprising:
flowing the fluid sample through a flow cell of a flow cytometer;
subjecting the fluid sample flowing through the flow cell to the excitation radiation causing the fluorescent emission response from the fluorescent antibody stain; and
detecting radiation from the flow cell within a wavelength range of the fluorescent emission response and evaluating the detected radiation within the wavelength range to identify fluorescent emission detection events indicative of passage through the flow cell of unassociated labeled particles of virus size, each said unassociated labeled particle including a said virus-size particle with the influenza virus epitope bound with a portion of the fluorescent antibody stain;
wherein, the fluid sample as fed to the flow cytometer comprises:
a concentration of the unassociated labeled particles in a range of from 1×10 5 to 1×10 9 particles per milliliter; and
a concentration of the fluorescent antibody stain not bound in the unassociated labeled particles in a range of from 0.5 microgram per milliliter to 8 micrograms per milliliter.
2. The flow cytometry method according to claim 1 , wherein the fluid sample as fed to the flow cytometer comprises a concentration of the fluorescent antibody stain not bound in the unassociated labeled particles in a range of from 0.5 microgram per milliliter to 5 micrograms per milliliter.
3. The flow cytometry method according to claim 1 , wherein the fluid sample as fed to the flow cytometer includes a total concentration of the fluorescent antibody stain in a range of from 0.5 microgram per milliliter to 8 micrograms per milliliter.
4. The flow cytometry method according to claim 3 , wherein the fluorescent antibody stain comprises a fluorophore attached to antibody molecules and the fluorescent antibody stain in the fluid sample comprises an average of from 3 to 8 of the fluorophore attached per antibody molecule in the fluorescent antibody stain.
5. The flow cytometry method according to claim 3 , wherein the flow cytometry is in the absence of detecting for light scatter.
6. The flow cytometry method according to claim 3 , wherein the flow cytometry comprises detecting only for fluorescent emission response.
7. The flow cytometry method according to claim 3 , wherein the influenza virus epitope is selected from the group consisting of type A influenza virus epitope, type B influenza virus epitope and combinations thereof.
8. The flow cytometry method according to claim 3 , wherein the fluorescent antibody stain comprises fluorescently-labeled polyclonal antibody.
9. The flow cytometry method according to claim 8 , wherein the fluorescently-labeled polyclonal antibody comprises fluorescently-labeled antibody molecules that bind with multiple different influenza virus serotypes including of type A influenza virus and of type B influenza virus.
10. The flow cytometry method according to claim 9 , wherein the fluorescently-labeled polyclonal antibody comprises anti-human influenza A, B rabbit polyclonal antibody.
11. The flow cytometry method according to claim 3 , wherein the fluorescent antibody stain comprises fluorescently-labeled monoclonal antibody.
12. The flow cytometry method according to claim 3 , wherein the virus-size particles are virus-like particles with the influenza virus epitope.
13. The flow cytometry method according to claim 3 comprising:
preparing the fluid sample, comprising mixing biological material to be evaluated for presence of the unassociated virus-size particles with the fluorescent antibody stain; and
after the mixing, not removing fluorescent antibody stain not bound in the unassociated labeled particles from the fluid sample prior to the flow cytometry.
14. The flow cytometry method according to claim 3 , wherein the fluorescent antibody stain is a first fluorescent stain and the fluid sample comprises a second fluorescent stain having a second fluorescent emission response, different than the first fluorescent emission response of the first fluorescent stain, caused by the excitation radiation.
15. The flow cytometry method according to claim 14 , wherein the second fluorescent stain comprises a fluorescent nucleic acid stain not specific to particle type.
16. The flow cytometry method according to claim 14 , wherein:
the influenza virus epitope is a first epitope, the virus-size particles are first virus-size particles and the unassociated labeled particles are first unassociated labeled particles;
the second fluorescent stain is a second fluorescent antibody stain that is different than the first fluorescent stain and is capable of binding with second virus-size particles having a second epitope that is different than the first epitope to form second unassociated labeled particles of virus size; and
the method comprises detecting radiation from the flow cell within a wavelength range of the second fluorescent emission and evaluating the detected radiation to identify second fluorescent emission detection events indicative of passage of the second unassociated labeled particles through the flow cell.
17. The flow cytometry method according to claim 16 , wherein the second epitope is a baculovirus epitope and the second fluorescent antibody stain includes monoclonal antibody for binding with baculovirus gp64 envelope glycoprotein as the second epitope.
18. The flow cytometry method according to claim 14 , wherein the second fluorescent emission has a peak wavelength at least 20 nanometers different than a peak wavelength of the first fluorescent emission and the flow cytometry comprises separately detecting for at least the first and second fluorescent emissions and not detecting for light scatter.
19. The flow cytometry method according to claim 1 , wherein;
the fluorescent antibody stain comprises a fluorophore attached to the antibody molecules and the fluorescent antibody stain in the fluid sample comprises an average of from 3 to 7 of the fluorophore attached per antibody molecule in the fluorescent antibody stain;
the fluid sample as fed to the flow cytometer includes a total concentration of the fluorescent antibody stain and a concentration of the fluorescent antibody stain not bound in the unassociated labeled particles each in a range of from 0.5 microgram per milliliter to 5 micrograms per milliliter; and
the method comprises preparing the fluid sample, the preparing the fluid sample comprising:
mixing biological material to be evaluated for presence of the unassociated virus-size particles with the fluorescent antibody stain; and
after the mixing, not removing fluorescent antibody not bound in the unassociated labeled particles from the fluid sample prior to flow cytometry.
20. A method for manufacturing a product comprising virus-size particles having an influenza virus epitope, the method comprising:
production processing to prepare a purified product including the virus-size particles having the influenza virus epitope, the production processing including:
generating the virus-size particles in a biological production operation;
harvesting from the biological production operation crude product comprising the virus-size particles generated during the generating;
purifying at least a portion of material of the crude product to prepare a purified product including the virus-size particles;
collecting a biological material sample from a stage of the production processing during which the virus-size particles would be expected to be present in the biological material;
evaluating the biological material sample for presence of unassociated virus-size particles having the influenza epitope;
performing the collecting multiple times to collect different said biological material samples to be evaluated from multiple different said stages and performing the evaluating on each said collected biological material sample to be evaluated, wherein the multiple different said stages include at least a first stage during the generating or the harvesting and a second stage during or after the purifying; and
the evaluating comprising:
subjecting to flow cytometry a fluid sample comprising at least a portion of the biological material sample, wherein the fluid sample comprises a fluorescent antibody stain capable of binding, directly or indirectly, with the influenza virus epitope, the fluorescent antibody stain comprising a fluorescent component or components attached to an antibody molecule that is specific to binding to the influenza virus epitope and the fluorescent antibody stain having a fluorescent emission response when subjected to an excitation radiation, the flow cytometry comprising:
flowing the fluid sample through a flow cell of a flow cytometer;
subjecting the fluid sample flowing through the flow cell to the excitation radiation to cause the fluorescent emission response from the fluorescent antibody stain; and
detecting radiation from the flow cell within a wavelength range of the fluorescent emission response and evaluating the detected radiation to identify fluorescent emission detection events indicative of passage through the flow cell of unassociated labeled particles of virus size including a said virus-size particle having the influenza virus epitope bound with a portion of the fluorescent antibody stain;
wherein, the fluid sample as fed to the flow cytometer comprises:
a concentration of the unassociated labeled particles in a range of from 1×10 5 to 1×10 9 particles per milliliter; and
a concentration of the fluorescent antibody stain not bound in the unassociated labeled particles in a range of from 0.5 microgram per milliliter to 8 micrograms per milliliter.
21. The method according to claim 20 , wherein the evaluating comprises:
preparing the fluid sample, comprising mixing the at least a portion of the biological material sample with the fluorescent antibody stain; and
after the mixing, not removing fluorescent antibody stain not bound in the unassociated labeled particles from the fluid sample prior to the flow cytometry.
22. The method according to claim 21 , wherein the fluorescent antibody stain comprises a fluorophore attached to antibody molecules and the fluorescent antibody stain in the fluid sample comprises an average of from 3 to 8 of the fluorophore attached per antibody molecule in the fluorescent antibody stain.
23. The method according to claim 21 , wherein the flow cytometry is in the absence of detecting for light scatter.
24. The method according to claim 21 , wherein the flow cytometry comprises detecting only for fluorescent emission response.
25. The flow cytometry method according to claim 20 , wherein the fluorescent antibody stain is a first fluorescent stain and the fluid sample comprises a second fluorescent stain having a second fluorescent emission response, different than the first fluorescent emission response of the first fluorescent stain, caused by the excitation radiation.
26. The flow cytometry method according to claim 25 , wherein the second fluorescent stain comprises a fluorescent nucleic acid stain not specific to particle type.
27. The flow cytometry method according to claim 25 , wherein:
the influenza virus epitope is a first epitope, the virus-size particles are first virus-size particles and the unassociated labeled particles are first unassociated labeled particles;
the second fluorescent stain is a second fluorescent antibody stain that is different than the first fluorescent stain and is capable of binding with second virus-size particles having a second epitope that is different than the first epitope to form second unassociated labeled particles of virus size; and
the method comprises detecting radiation from the flow cell within a wavelength range of the second fluorescent emission and evaluating the detected radiation to identify second fluorescent emission detection events indicative of passage of the second unassociated labeled particles through the flow cell.