1. A method for detecting activation of immune cells or platelets comprising:
(a) immobilizing one or more adhesion proteins or extracellular matrix molecules on the surface of a colorimetric resonance reflectance biosensor;
(b) adding one or more types of immune cells or platelets to the surface of the biosensor;
(c) adding a potential activator of the immune cells or the platelets to the surface of the biosensor;
(d) detecting adhesion of the immune cells to the adhesion proteins or the adhesion of the platelets to the extracellular matrix molecules on the surface of the biosensor by illuminating the biosensor and detecting changes in peak wavelength values over time; wherein an increase in peak wavelength values over time indicates that the potential activator of the immune cells has activated the immune cells or that the potential activator of the platelets has activated the platelets.
2. The method of claim 1, wherein the immune cells are lymphocytes or granulocytes.
3. The method of claim 1, wherein the adhesion protein is ICAM-1 or VCAM-1.
4. A method for detecting blocking or enhancing properties of a test reagent or stimuli on activation of one or more immune cells or platelets comprising:
(a) immobilizing one or more adhesion proteins or extracellular matrix molecules on the surface of a colorimetric resonance reflectance biosensor;
(b) adding one or more types of immune cells or platelets to the surface of the biosensor;
(c) adding an activator of the immune cells or platelets to the surface of the biosensor;
(d) adding a test reagent or stimuli to the surface of the biosensor; wherein the one or more types of immune cells, platelets, activator, and test reagent or stimuli can be added to the biosensor surface sequentially in any order, or at the same time;
(e) detecting adhesion of the immune cells to the adhesion proteins or adhesion of the platelets to the extracellular matrix molecules on the surface of the biosensor by illuminating the biosensor and detecting changes in peak wavelength values over time for each immune cell or platelet;
(f) comparing the changes in peak wavelength values for each immune cell or platelet to a control that does not comprise the test reagent or stimuli, wherein an increase in peak wavelength values over time indicates that the test reagent or stimuli enhances immune cell or platelet activation, and wherein a decrease in peak wavelength values over time indicates that the test reagent or stimuli blocks activation of the immune cells.
5. The method of claim 4, wherein the adhesion protein is ICAM-1 or VCAM-1.
6. A method of selecting hybridomas producing antibodies to an antigen for highest strength of binding to the antigen comprising:
(a) adding one or more hybridomas to the surface of colorimetric resonance reflectance biosensor having one or more integrin ligands immobilized to the biosensor surface;
(b) adding the antigen to which the hybridomas are specific to the surface of the biosensor;
(c) illuminating the biosensor and detecting changes in peak wavelength value over time for each hybridoma; and
(d) selecting and isolating the hybridomas with the largest increases in peak wavelength value over time;
wherein hybridomas are selected that produce antibodies to the antigen with the highest strength of binding to the antigen.
7. The method of claim 6, further comprising:
(e) adding the individual selected hybridomas to a colorimetric resonant reflectance biosensor, wherein capture molecules for the antibodies produced by the hybridoma are immobilized on the biosensor surface and allowing the hybridoma to multiply to form a hybridoma population;
(f) illuminating the biosensor and detecting changes in peak wavelength value for each hybridoma;
(g) selecting hybridomas having the greatest increase in peak wavelength value;
whereby hybridomas are selected that produce the greatest quantity of antibodies.
8. The method of claim 6, wherein the one or more integrin ligands are VCAM-1 or ICAM-1.
9. A method of determining if a subject has had an immune response to an immunogen comprising:
(a) obtaining test B cells from a subject;
(b) adding the test B cells to the surface of a colorimetric resonance reflectance biosensor having one or more integrin ligands immobilized to the biosensor surface;
(c) adding the immunogen to the surface of the biosensor;
(d) illuminating the biosensor and detecting changes in peak wavelength values over time for the test B cells; and
(e) comparing the changes in peak wavelength values for the test B cells to a control B cell population that does not react with the immunogen;
wherein an increase in peak wavelength values over time for the test B cells as compared to the control B cells indicates that the subject has had an immune response to the immunogen.
10. The method of claim 9, wherein the one or more integrin ligands are VCAM-1 or ICAM-1.
11. A method of isolating neutralizing antibodies for an immunogen comprising:
(a) obtaining test B cells from a subject that has had an immune response to the immunogen;
(b) adding the test B cells to the surface of a colorimetric resonance reflectance biosensor having one or more integrin ligands immobilized to the biosensor surface;
(c) adding the immunogen to the surface of the biosensor;
(d) illuminating the biosensor and detecting changes in peak wavelength values over time for each test B cell;
(e) comparing the changes in peak wavelength values for each test B cell to a control B cell that does not react with the immunogen or that is not exposed to the immunogen;
(f) isolating the test B cells having peak wavelength values higher than the control B cell; and
(g) isolating antibodies produced by the isolated test B cells;
wherein neutralizing antibodies for the immunogen are isolated.
12. The method of claim 11, wherein the one or more integrin ligands are VCAM-1 or ICAM-1.
13. A method of classifying a B cell lymphoma comprising:
(a) obtaining a B cell sample from a patient with an unclassified B cell lymphoma;
(b) adding the B cell sample to the surface of a colorimetric resonance reflectance biosensor having one or more adhesion proteins immobilized to the biosensor surface;
(c) adding one or more chemokines to the biosensor;
(c) optionally adding one or more specific inhibitors of GTPases to the biosensor;
(d) illuminating the biosensor and detecting changes in peak wavelength values over time for each B cell sample; and
(e) comparing the responses of the B cell samples to known responses of B cell samples from patients with classified B cell lymphoma,
wherein the B cell lymphoma is classified.
14. The method of claim 13, wherein the one or more adhesion proteins are VCAM-1 or ICAM-1.
15. A method for selecting activated B-cells expressing an antibody to one or more antigens comprising:
(a) adding B-cells expressing antibody libraries on their cell surfaces to the surface of a colorimetric resonance reflectance biosensor having one or more integrin ligands immobilized to the biosensor surface;
(b) adding the one or more antigens to the surface of the colorimetric resonance reflectance biosensor;
(c) determining the amount of activation of each B-cell on the biosensor surface by illuminating the biosensor and determining the changes in peak wavelength values over time for each B cell, wherein an increase in peak wavelength values over time indicates B cell activation, and wherein a decrease or no change in peak wavelength values over time indicates no B cell activation; and
(d) isolating activated B cells;
wherein a B-cell that expresses an antibody to the one or more antigens is selected.
16. The method of claim 15, wherein polynucleotide sequences corresponding to the expressed antibodies are amplified.
17. The method of claim 15, wherein the one or more integrin ligands are VCAM-1 or ICAM-1.
The claims below are in addition to those above.
All refrences to claims which appear below refer to the numbering after this setence.
1. A valve assembly, comprising:
a fitting on a liquid holding container, the fitting having a through-hole therethrough between an upper end and a lower end, the lower end having a ledge portion;
a valve member having a lower protruding telescopic end with a raised ridge edge, the lower protruding end for telescoping into the upper end of the fitting so that the raised ridge edge protrudes underneath and below the ledge portion of the lower end of the fitting, wherein the valve assembly is used for dispensing liquid having different viscosities, densities and textures from the liquid holding container, wherein the raised ridge edge of the telescopic end further includes:
an inwardly sloping angled lower surface portion for directing the telescopic end of the valve member into the through-hole of the fitting; and
an upper catch surface portion for snapping outward under the lower end of the fitting for locking the valve member to the fitting.
2. The valve assembly of claim 1, wherein the liquid holding container includes:
a bag in a box wherein the fitting is between the bag and the box.
3. The valve assembly of claim 1, wherein the lower protruding telescopic end is at least approximately \xbd inch in length.
4. The valve assembly of claim 1, the valve member further comprising:
an upper chamber housing a valve, the upper chamber having a continuously cylindrical and uniform diameter interior diameter; and
an annular rim about the valve member for separating the upper chamber from the lower protruding end of the valve member.
5. The valve assembly of claim 1, wherein the valve member includes:
a ball check valve end opposite to the lower protruding end, and
a single O-ring adjacent to an upper edge of the ball check valve end.
6. The valve assembly of claim 5, wherein the valve member includes:
an annular rim having a ledge portion which rests against the upper end of the fitting.
7. The valve assembly of claim 6, further comprising:
a seal member between the ledge portion and the upper end of the fitting.
8. The valve assembly of claim 7, wherein the seal member includes:
an O-ring.
9. The valve assembly of claim 1, further comprising:
a seal member for sealing the valve member to the fitting.
10. The valve assembly of claim 9, wherein the seal member includes:
an O-ring.
11. The valve assembly of claim 1, further comprising:
a removable cap cover for covering an open end of the valve member.
12. A telescopic snap valve assembly for a bag in a box, comprising:
a fitting on the bag, the fitting having a through-hole therethrough between an upper end and a lower end, the lower end having a ledge portion;
a valve member having a dispensing end and a lower protruding telescopic end;
a raised ridge portion along a bottom outer edge of the telescopic end of the valve member, the raised ridge portion having an angled lower surface portion for directing the telescopic end of the valve member into the through-hole of the fitting, and an upper retainer portion for snapping outward underneath and below the ledge portion of the lower end of the fitting for locking the valve member to the fitting, wherein the valve assembly is used for dispensing liquid from the dispensing end of the bag.
13. The telescopic snap valve assembly of claim 12, wherein the valve member includes an annular rim having a ledge portion which rests against the upper end of the fitting, and a seal member being located between the ledge portion and the upper end of the fitting.
14. The telescopic snap valve assembly of claim 12, wherein the telescopic end of the valve member includes a length of at least approximately \xbd inch long.
15. The telescopic snap valve assembly of claim 14, wherein the dispensing end of the valve member includes:
a ball check valve with a single O-ring adjacent to the valve.
16. A method of assembling a valve member into a bag in a box fitting, comprising the steps of:
telescopically inserting a protruding portion of a valve member into a through-hole opening of the fitting to the bag located in the box, the fitting having an upper end and a lower end, the lower end having a ledge portion; and
expanding a raised edge portion on a lower end of the protruding portion of the valve member underneath the ledge portion of the fitting to lock the valve member to the fitting;
directing the telescopic end of the valve member into the through-hole of the fitting by an inwardly sloping angled lower surface portion on the raised ridge edge; and
snapping outward an upper catch surface portion of the raised ridge edge portion under the lower end of the fitting for locking the valve member to the fitting.
17. The method of claim 16, further comprising the step of:
sealing the valve member to the fitting by compressing an O-ring between a ledge portion on the valve member and a shoulder portion on the fitting.