What is claimed is:
1. A reagent set for detecting and measuring isoenzymes comprising at least two subunits in a test sample, wherein a first isoenzyme comprises two copies of a first subunit and a second isoenzyme comprises one copy of the first subunit and one copy of a second subunit, the reagent set comprising:
a plurality of sample-insoluble particles having associated therewith immobilized antibody adapted to interact with the first subunit (first capture particles);
a plurality of sample-insoluble particles having associated therewith immobilized antibody adapted to interact with the second subunit (second capture particles), wherein the first capture particles are optically distinguishable from the second capture particles;
a plurality of reporters adapted to interact with the first subunit (first reporters), to thereby produce a first fluorescent signal; and
a plurality of reporters adapted to interact with the second subunit (second reporters), to thereby produce a second fluorescent signal.
2. A reagent set according to claim 1, wherein the first reporters comprise antibodies specific to the first subunit (first antibodies) and wherein the second reporters comprise antibodies specific to the second subunit (second antibodies).
3. A reagent set according to claim 2, wherein the first antibodies and the second antibodies are monoclonal antibodies.
4. A reagent set according to claim 2, wherein the first antibodies are labeled with a first fluorescent dye and the second antibodies are labeled with a second fluorescent dye.
5. A reagent set according to claim 4, wherein the first fluorescent dye is selected from the group consisting of Cy5 and dibenzoCy5.
6. A reagent set according to claim 1, wherein the first reporters comprise sample-insoluble particles having associated therewith immobilized antibody adapted to interact with the first subunit and wherein the reporter particles are labeled with a fluorescent dye.
7. A reagent set according to claim 6, wherein the immobilized antibody is a monoclonal antibody.
8. A reagent set according to claim 6, wherein the fluorescent dye is a lipophilic cyanine dye.
9. A reagent set for measuring creatine kinase (CK) isoenzymes in a test sample, the reagent set comprising:
creatine kinase B (CKB) capture particles comprising a plurality of sample-insoluble particles having associated therewith immobilized antibody adapted to interact with a B subunit of creatine kinase;
creatine kinase M (CKM) capture particles comprising a plurality of sample-insoluble particles having associated therewith immobilized antibody adapted to interact with an M subunit of creatine kinase, wherein the CKM capture particles are optically distinguishable from the CKB capture particles;
a plurality of CKB reporters, adapted to interact with a B subunit of creatine kinase, to thereby produce a first fluorescent signal; and
a plurality of CKM reporters, adapted to interact with M subunit of creatine kinase, to thereby produce a second fluorescent signal.
10. A reagent set according to claim 9, wherein the CKB reporters comprise antibodies specific to the B subunit of creatine kinase and wherein the CKM reporters comprise antibodies specific to the M subunit of creatine kinase.
11. A reagent set according to claim 10, wherein the CKB antibodies and the CKM antibodies are monoclonal antibodies.
12. A reagent set according to claim 10, wherein the CKB antibodies are labeled with a first fluorescent dye and the CKM antibodies are labeled with a second fluorescent dye.
13. A reagent set according to claim 12, wherein the first fluorescent dye is selected from the group consisting of Cy5 and dibenzoCy5.
14. A reagent set according to claim 9, wherein the CKB reporters comprise sample-insoluble particles having associated therewith immobilized antibody adapted to interact with a B subunit of creatine kinase, and wherein the reporter particles are labeled with a first lipophilic fluorescent dye.
15. A reagent set according to claim 14, wherein the immobilized antibody is a monoclonal antibody.
16. A reagent set according to claim 9, wherein the first lipophilic fluorescent dye is a lipophilic cyanine dye.
17. A reagent set according to claim 9, wherein the CKM reporters comprise sample-insoluble particles having associated therewith immobilized antibody adapted to interact with an M subunit of creatine kinase, and wherein the reporter particles are labeled with a second lipophilic fluorescent dye.
18. A reagent set according to claim 17, wherein the immobilized antibody is a monoclonal antibody.
19. A reagent set according to claim 17, wherein the second lipophilic fluorescent dye is a lipophilic cyanine dye.
20. A method for detecting and measuring isoenzymes comprising at least two subunits in a test sample, wherein a first isoenzyme comprises two copies of a first subunit and a second isoenzyme comprises one copy of the first subunit and one copy of a second subunit, the method comprising the steps of:
(a) mixing the test sample with a reagent set comprising:
(1) first capture particles comprising a plurality of sample-insoluble particles having associated therewith immobilized antibody adapted to interact with the first subunit and further comprising coding indicia which confer uniquely identifying optical properties on the first capture particles;
(2) second capture particles comprising a plurality of sample-insoluble particles having associated therewith immobilized antibody adapted to interact with the second subunit and further comprising coding indicia which confer uniquely identifying optical properties on the second capture particles;
(3) a plurality of first reporters, each adapted to interact with the first subunit, to thereby produce a first fluorescent signal; and
(4) a plurality of second reporters, each adapted to interact with the second subunit, to thereby produce a second fluorescent signal;
(b) incubating the resulting mixture for a period of time sufficient for:
(1) the first capture particles to interact with one of the first subunits of the first isoenzyme to form a first capture particlefirst isoenzyme capture particle complex andor with the first subunit of the second isoenzyme to form a first capture particlesecond isoenzyme capture particle complex (collectively, first capture particle complex);
(2) the second capture particles to interact with the second subunit of the second isoenzyme to form a second capture particlesecond isoenzyme capture particle complex (second capture particle complex);
(3) the first reporters to interact with the first subunit of the first isoenzyme on the first capture particlefirst isoenzyme capture particle complex andor with the first subunit of the second isoenzyme on second capture particle complex; and
(4) the second reporters to interact with second subunit of the second isoenzyme on second capture particle complex;
(c) reading both the coding optical properties and the fluorescent signal or signals of each capture particle complex individually;
(d) storing the measured fluorescent signal of both the first capture particle complexes and the second capture particle complexes according to the coding optical properties read from the complexes; and
(e) processing the stored measurements for the first capture particle complexes to obtain an assay result for the first isoenzyme and for the second isoenzyme and processing the stored measurements for the second capture particle complexes to obtain an assay result for the second isoenzyme;
whereby a complete chemical analysis of the isoenzyme content of the test sample is obtained.
21. A method according to claim 20, wherein the first reporters comprise antibodies specific to the first subunit (first antibodies) and wherein the second reporters comprise antibodies specific to the second subunit (second antibodies).
22. A method according to claim 21, wherein the first antibodies and second antibodies are monoclonal.
23. A method according to claim 21, wherein the first antibodies are labeled with a first fluorescent dye and the second antibodies are labeled with a second fluorescent dye.
24. A method according to claim 23, wherein the first fluorescent dye is selected from the group consisting of Cy5 and dibenzoCy5.
25. A method according to claim 20, wherein the first reporters comprise sample-insoluble particles having associated therewith immobilized antibody adapted to interact with the first subunit, and wherein each reporter particle is labeled with a lipophilic fluorescent dye.
26. A method according to claim 25, wherein the immobilized antibody is monoclonal antibody.
27. A method according to claim 25, wherein the lipophilic fluorescent dye is a lipophilic cyanine dye.
28. A method for measuring creatine kinase isoenzymes CK-1, CK-2, CK-3 and total creatine kinase content in a test sample, the method comprising the steps of:
(a) mixing the test sample with a reagent set comprising:
(1) CKB capture particles comprising a plurality of sample-insoluble particles having associated therewith immobilized antibody adapted to interact with a B subunit of creatine kinase and further comprising coding indicia which confer uniquely identifying optical properties on the CKB capture particles;
(2) CKM capture particles comprising a plurality of sample-insoluble particles having associated therewith immobilized antibody adapted to interact with an M subunit of creatine kinase and further comprising coding indicia which confer uniquely identifying optical properties on the CKM capture particles;
(3) a plurality of CKB reporters, each adapted to interact with a CKB subunit, to thereby produce a first fluorescent signal; and
(4) a plurality of CKM reporters, each adapted to interact with a CKM subunit, to thereby produce a second fluorescent signal;
(b) incubating the resulting mixture for a period of time sufficient for:
(1) the CKB capture particles to interact with B subunit of CK-1 to form a CKBCK-1 capture particle complex andor with B subunit of CK-2 to form a CKBCK-2 capture particle complex;
(2) the CKM capture particles to interact with M subunit of CK-2 to form a CKMCK-2 capture particle complex andor with M subunit of CK-3 to form a CKMCK-3 capture particle complex;
(3) the CKB reporters to interact with B subunit of CK-1 on CKBCK-1 capture particle complexes andor with B subunit of CK-2 on CKMCK-2 capture particle complexes; and
(4) the CKM reporters to interact with M subunit of CK-2 on CKBCK-2 capture particle complexes andor with M subunit of CK-3 on CKMCK-3 capture particle complexes;
(c) reading both the coding optical properties and the fluorescent signal or signals of each capture particle complex individually;
(d) storing the measured fluorescent signal of both the CKB capture particle complexes and the CKM capture particle complexes according to the coding optical properties read from the complexes; and
(e) processing the stored measurements for the CKB capture particle complexes to obtain an assay result for CK-1 and for CK-2 and processing the stored measurements for the CKM capture particle complexes to obtain an assay result for CK-2 and for CK-3;
whereby a complete chemical analysis of the creatine kinase content of the test sample is obtained.
29. A method according to claim 28, wherein the CKB reporters comprise antibodies specific to the B subunit of creatine kinase and wherein the CKM reporters comprise antibodies specific to the M subunit of creatine kinase.
30. A method according to claim 29, wherein the CKB antibodies and the CKM antibodies are monoclonal antibodies.
31. A method according to claim 29, wherein the CKB antibody is labeled with a first fluorescent dye and the CKM antibody is labeled with a second fluorescent dye.
32. A method according to claim 31, wherein the first fluorescent dye is selected from the group consisting of Cy5 and dibenzoCy5.
33. A method according to claim 28, wherein the CKB reporters comprise sample-insoluble particles having associated therewith immobilized antibody adapted to interact with a B subunit of creatine kinase, and wherein each reporter particle is labeled with a first lipophilic fluorescent dye.
34. A method according to claim 33, wherein the immobilized antibody is monoclonal antibody.
35. A method according to claim 33, wherein the first lipophilic fluorescent dye is a lipophilic cyanine dye.
36. A method according to claim 28, wherein the CKM reporters comprise sample-insoluble particles having associated therewith immobilized antibody adapted to interact with an M subunit of creatine kinase, and wherein each reporter particle is labeled with a second lipophilic fluorescent dye.
37. A method according to claim 36, wherein the immobilized antibody is monoclonal antibody.
38. A method according to claim 36, wherein the second lipophilic fluorescent dye is a lipophilic cyanine dye.
39. A kit comprising:
an apparatus for assaying multiple isoenzymes in a test sample, and
instructions for use of the apparatus setting forth the method of claim 20.
40. A kit comprising:
an apparatus for assaying creatine kinase isoenzymes and total creatine kinase content in a test sample, and
instructions for use of the apparatus setting forth the method of claim 28.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A method for aluminizing an internal passage of a metal substrate comprising:
injecting a slurry composition that comprises a powder comprising aluminum, a binder selected from the group consisting of colloidal silica, an organic resin, and a combination thereof, and inert organic pyrolysable thickener particles comprising poly(methyl methacrylate) microbeads, into the internal passage;
applying compressed air to the internal passage to facilitate distribution of the slurry composition throughout the internal passage; and,
heat treating the slurry composition under conditions effective to remove volatile components from the composition, and to promote diffusion of aluminum into a surface of the internal passage.
2. The method of claim 1, wherein the injecting of the slurry composition is performed at a temperature of about room temperature to about 60\xb0 C.
3. The method of claim 1, further comprising stirring the slurry prior to injection.
4. The method of claim 1, further comprising agitating the metal substrate after the injection of the slurry composition.
5. The method of claim 4, wherein the agitating is performed under conditions sufficient to expel excess injected slurry composition.
6. The method of claim 4, wherein the agitating is performed at a temperature of about room temperature to about 60\xb0 C.
7. The method of claim 4, wherein the agitating is performed for about one minute to about two hours.
8. The method of claim 4, wherein the agitating is performed on a two-axis rotator.
9. The method of claim 1, further comprising draining excess injected slurry composition.
10. The method of claim 1, wherein the amount of aluminum in the slurry composition exceeds the amount of aluminum present in the substrate by up to about 65 atomic percent.
11. The method of claim 1, wherein the amount of powder comprising aluminum in the slurry composition is about 10 weight percent to about 90 weight percent.
12. The method of claim 1, wherein the powder comprising aluminum further comprises a metal selected from the group consisting of platinum group metals, rare earth metals, scandium, yttrium, iron, chromium, cobalt, and a combination comprising at least one of the foregoing metals.
13. The method of claim 1, wherein the powder comprising aluminum has an average particle size of about 0.5 micrometer to about 200 micrometers measured across the longest axis of the particle.
14. The method of claim 1, wherein the powder comprising aluminum comprises particles that are spherical, hollow, porous, rod, plate, flake, fibrous, or a combination comprising at least one of the foregoing particles.
15. The method of claim 1, wherein the powder comprising aluminum comprises an alloy of aluminum and silicon.
16. The method of claim 1, wherein the slurry composition further comprises a liquid carrier.
17. The method of claim 1, wherein the binder comprises colloidal silica.
18. The method of claim 1, wherein the colloidal silica is present at a level in the range of about 5% by weight to about 20% by weight, based on silica solids as a percentage of the entire composition.
19. The method of claim 1, wherein the silica in the colloidal silica has an average particle size of about 10 nanometers to about 100 nanometers measured across the longest axis of the particle.
20. The method of claim 1, wherein the colloidal silica comprises particles that are spherical, hollow, porous, rod, plate, flake, fibrous, or a combination comprising at least one of the foregoing particles.
21. The method of claim 1, wherein the heat treating is performed under conditions that are sufficient to cause decomposition of the inert organic pyrolysable thickener particles.
22. The method of claim 1, wherein the removing of the volatile components is further accomplished by mechanically removing the excess material, dissolving the excess material, or a combination thereof.
23. The method of claim 1, wherein the slurry composition further comprises a liquid carrier selected from the group consisting of water, alcohols, halogenated hydrocarbon solvents, and compatible mixtures thereof.
24. The method of claim 1, wherein the slurry composition further comprises an organic stabilizer that comprises two or more hydroxyl groups.
25. The method of claim 1, wherein the slurry composition further comprises an organic stabilizer that comprises three or more hydroxyl groups.
26. The method of claim 1, wherein the slurry composition further comprises an organic stabilizer selected from the group consisting of an alkane diol, glycerol, pentaerythritol, a fat, a carbohydrate, and a combination comprising at least one of the foregoing organic compounds.
27. The method of claim 1, wherein the slurry composition further comprises glycerol.
28. The method of claim 1, wherein the slurry composition further comprises an organic stabilizer present in an amount effective to chemically stabilize the powder comprising aluminum during contact with any aqueous component present in the composition.
29. The method of claim 28, wherein the organic stabilizer is present in an amount of about 0.1% by weight to about 20% by weight, based on the total weight of the composition.
30. The method of claim 1, wherein the heat treatment comprises a preliminary heat treatment to remove the volatile components, and a final heat treatment to diffuse the aluminum into the substrate.
31. The method of claim 1, wherein the heat treatment is carried out at a temperature of about 650\xb0 C. to about 1100\xb0 C.
32. The method of claim 1, wherein the heat treatment comprises a graduated heat treatment.
33. The method of claim 1, wherein the surface of the substrate extends to a depth of about 200 micrometers into the substrate.
34. The method of claim 1, further comprising removing excess material from the internal passage.
35. The method of claim 1, wherein the substrate is a turbine engine component.