Multiple hybrid immunoassay
The invention relates to compositions and methods for the immunoassay of an analyte of interest. The analyte is detected in an immunoassay using three or more antibodies, where in each antibody specifically binds to a different epitope on the analyte. When the analyte of interest in a clinical marker for an acute disease, the detection of the analyte by immunoassay is a diagnosis of the occurrence of the disease.
1. A cartridge for sensing at least one analyte in a sample, said cartridge comprising:
a housing;
at least one sensor located in a sensor region within the housing;
sample retainer within the housing, for retaining the sample out of contact with the sensor, prior to sensing;
sample collector within the housing including an orifice for drawing the sample into the sample retainer;
a sample conduit connecting the sample retainer with the sensor;
sample displacer for automatically displacing the sample by actively forcing the sample through the sample conduit and into contact with the sensor to permit sensing, the automatic displacement of the sample being under the control of a reading apparatus; and
an immunoassay composition for detecting said analyte, said immunoassay composition comprising at least three different antibodies, the at least three different antibodies being capable of binding to at least three different epitopes on said analyte, in which at least two of the at least three different antibodies are capable of binding to at least two different epitopes on a subform of the analyte, and in which at least one of the at least three different epitopes on the analyte is unavailable for binding on the subform.
2. The cartridge of claim 1 , wherein at least one of the antibodies comprises a Fab fragment.
3. The cartridge of claim 1 , wherein the immunoassay composition comprises a common member conjugated to at least one of the antibodies.
4. The cartridge of claim 3 , wherein at least one of the antibodies is covalently attached to the common member and wherein the covalent attachment comprises a crosslinker, which is derived from a crosslinking agent.
5. The cartridge of claim 4 , wherein the crosslinking agent comprises succinimidyl-4-[N-maleimidomethyl]-cyclohexane-1-carboxy[6-amidocaproate], glutaraldehyde, adipic acid dihydrazide, bis-diazotized benzidine, 1,4-butane diglycidyl ether, bis-maleimido hexane, sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate, or N-hydroxysuccinimidyl 4-azidosalicylic acid.
6. The cartridge of claim 3 , wherein the common member is a microparticle comprising latex beads, polystyrene beads, polystyrene/acrylic acid beads, or combinations thereof.
7. The cartridge of claim 3 , wherein the common member comprises a signal-generating element comprising a radiolabel, metal particle, fluorescent dye, chromogenic dye, labeled protein, enzyme, or combinations thereof.
8. The cartridge of claim 7 , wherein the enzyme comprises peroxidase, glucose oxidase, phenol oxidase, β-galactosidase, alkaline phosphatase, or combinations thereof.
9. The cartridge of claim 7 , wherein the molar ratio of the at least one of the antibodies conjugated to the signal-generating element, to the signal-generating element is from 1.001:1 to 10:1.
10. The cartridge of claim 7 , wherein the molar ratio of the antibodies conjugated to the signal-generating element, to the signal-generating element is about 3:1.
11. The cartridge of claim 7 , wherein at least two of the antibodies are conjugated to the signal-generating element.
12. The cartridge of claim 11 , wherein the molar ratio of the at least two of the antibodies conjugated to the signal-generating element, to the signal-generating element is from 1.001:1 to 10:1.
13. The cartridge of claim 1 , wherein at least one of the antibodies is selected from the group consisting of G129C, G130C, A34720, 8E10, A34500, A34440 and A34730.
14. The cartridge of claim 1 , wherein at least one of the antibodies is selected from the group consisting of G129C and G130.
15. The cartridge of claim 1 , wherein said at least three different antibodies comprise:
(a) G129C antibody;
(b) G130C antibody; and
(c) at least one additional troponin I antibody different from (a) and (b).
16. The cartridge of claim 15 , wherein (c) is selected from the group consisting of 19C7, A34720, 8E10, A34500, A34440, A34730, and 34503228P.
17. The cartridge of claim 15 , wherein (c) is selected from the group consisting of 19C7 and 34503228P.
18. The cartridge of claim 1 , wherein the analyte is human cTnI and wherein the sample is a whole blood sample.