Multiplex immunoassays for hemoglobin, hemoglobin variants, and glycated forms
Hemoglobin, its variants, and glycated forms of each are determined individually in a multiplex assay that permits correction of the measured level of HbA1c to account for glycated variants and other factors related to the inclusion of the variants in the sample. New antibodies that are particularly well adapted to the multiplex assay are also provided.
1. A method for individually detecting a plurality of hemoglobin-containing analytes comprising HbA 1c and a hemoglobin variant, if present, in a single sample of blood cell lysate, said method comprising:
(a) incubating said sample with a population of beads in a common mixture, said population comprising a subpopulation to detect HbA1c and one or more further subpopulations, each of which detects a hemoglobin variant, wherein:
(i) each bead of said subpopulation to detect HbA1c has bonded thereto a classifier dye and each bead of said one or more subpopulations to detect a hemoglobin variant has bonded thereto a classifier dye where the classifier dyes are selected such that the classifier dyes, and thereby said subpopulations, are differentiable from each other by fluorescent emissions emitted by said classifier dyes upon excitation, and
(ii) each bead of said subpopulation to detect HbA1c further has bonded thereto an antibody having selective binding affinity towards HbA1c and each bead of said one or more subpopulations to detect a hemoglobin variant has bonded thereto a monoclonal antibody that has selective binding affinity towards the variant and the glycated form of the variant to cause each analyte to bind to the different bead subpopulation through the antibody bonded to said subpopulations, wherein:
one subpopulation of the one or more further subpopulations detects HbC, if present in the sample, and has bonded thereto a monoclonal antibody having selective binding affinity towards HbC and glycated HbC; or
one subpopulation of the one or more further subpopulations detects HbS, if present in the sample, and has bonded thereto a monoclonal antibody having selective binding affinity towards HbS and glycated HbS;
(b) with said analytes bound to the beads of said subpopulations, incubating said population with a labeled binding member that binds to all of said analytes, thereby labeling said analytes thus bound; and
(c) detecting the labeled binding member that binds to all of said analytes and detecting classifier dyes from the subpopulations of beads and differentiating the labels of the subpopulations by fluorescent emissions, thereby individually detecting HbA1 c and if present, the hemoglobin variant.
2. The method of claim 1 , wherein the population of beads further comprises a subpopulation that detects total hemoglobin, wherein each bead of the subpopulation to detect total hemoglobin has bonded thereto a classifier dye that is selected such that the classifier dye, and thereby said subpopulation, is differentiable from the other classifier dyes; and each member of said subpopulation that detects total hemoglobin having bonded thereto a monoclonal antibody having selective binding affinity towards all hemoglobin-containing analytes.
3. The method of claim 2 , wherein said monoclonal antibody that has selective binding affinity towards all hemoglobin-containing analytes binds to a beta globin chain minimal epitope 9 SAVTALWGKVNV 20 (SEQ ID NO:15), 8 KSAVTALWGKVNV 20 (SEQ ID NO:16), or 11 VTALW 15 (SEQ ID NO:17).
4. The method of claim 1 , wherein the antibody having selective binding affinity for HbA 1c is a monoclonal antibody.
5. The method of claim 1 , wherein one subpopulation of the one or more further subpopulations of (ii) detects HbC.
6. The method of claim 5 , wherein the monoclonal antibody having selective affinity for HbC and glycated HbC is a monoclonal antibody that binds to a HbC minimal epitope 4 TPKEKSAVT 12 (SEQ ID NO:1).
7. The method of claim 1 , wherein one subpopulation of the one or more further subpopulations of (ii) detects HbS.
8. The method of claim 7 , wherein monoclonal antibody having selective affinity for HbS and glycated HbS is a monoclonal antibody that binds to a HbS minimal epitope 3 LTPVEKSAVT 12 (SEQ ID NO:3).
9. The method of claim 1 , wherein the population comprises:
a subpopulation that detects HbE, if present in the sample, wherein each bead of the subpopulation to detect HbE has bonded thereto a classifier dye that is selected such that the classifier dye, and thereby said subpopulation, is differentiable from the other classifier dyes; and each member of said subpopulation that detects HbE has bonded thereto a monoclonal antibody having selective binding affinity towards HbE and glycated HbE; or
a subpopulation that detects HbD, if present in the sample, wherein each bead of the subpopulation to detect HbD has bonded thereto a classifier dye that is selected such that the classifier dye, and thereby said subpopulation, is differentiable from the other classifier dyes; and each member of said subpopulation that detects HbD has bonded thereto a monoclonal antibody having selective binding affinity towards HbD and glycated HbD.
10. The method of claim 9 , wherein the monoclonal antibody that has selective binding affinity towards HbE and glycated HbE binds to a HbE minimal epitope 22 EVGGK 26 (SEQ ID NO:6) or 21 DEVGGK 26 (SEQ ID NO:7).
11. The method of claim 9 , wherein the monoclonal antibody that has selective binding affinity towards HbD and glycated HbD binds to a HbD minimal epitope 121 QFTPP 125 (SEQ ID NO:8) or 119 GKQFTPP 125 (SEQ ID NO:46).
12. The method of claim 1 , wherein the population comprises a subpopulation that detects HbC, if present in the sample, and a subpopulation that detects HbS, if present in the sample.
13. The method of claim 12 , wherein the monoclonal antibody having selective binding affinity towards HbC and glycated HbC binds to a HbC minimal epitope 4 TPKEKSAVT 12 (SEQ ID NO:1); and the monoclonal antibody having selective affinity for HbS and glycated HbS binds to a HbS minimal epitope 3 LTPVEKSAVT 12 (SEQ ID NO:3).
14. The method of claim 12 , wherein the population comprises:
a subpopulation that detects HbE, if present in the sample, wherein each bead of the subpopulation to detect HbE has bonded thereto a classifier dye that is selected such that the classifier dye, and thereby said subpopulation, is differentiable from the other classifier dyes; and each member of said subpopulation that detects HbE has bonded thereto a monoclonal antibody having selective binding affinity towards HbE and glycated HbE; and
a subpopulation that detects HbD, if present in the sample, wherein each bead of the subpopulation to detect HbD has bonded thereto a classifier dye that is selected such that the classifier dye, and thereby said subpopulation, is differentiable from the other classifier dyes; and each member of said subpopulation that detects HbD has bonded thereto a monoclonal antibody having selective binding affinity towards HbD and glycated HbD.
15. The method of claim 14 , wherein the monoclonal antibody that has selective binding affinity towards HbE and glycated HbE binds to a HbE minimal epitope 22 EVGGK 26 (SEQ ID NO:6) or 21 DEVGGK 26 (SEQ ID NO:7); and the monoclonal antibody that has selective binding affinity towards HbD and glycated HbD binds to a HbD minimal epitope 121 QFTPP 125 (SEQ ID NO:8) or 119 GKQFTPP 125 (SEQ ID NO:46).
16. The method of claim 1 , wherein said method further comprises determining total hemoglobin in a non-immunoassay method.
17. The method of claim 1 , wherein the blood cell lysate is a denatured blood cell lysate.
18. The method of claim 1 , wherein the sample is from a diabetic patient.
19. A monoclonal antibody that selectively binds to hemoglobin variant HbC and glycated HbC, wherein the antibody binds to a HbC minimal epitope 4 TPKEKSAVT 12 (SEQ ID NO:1).
20. A monoclonal antibody that selectively binds to hemoglobin variant HbS and glycated HbS, wherein the antibody binds to a HbS minimal epitope 3 LTPVEKSAVT 12 (SEQ ID NO:3).