Modified ELISA with hemoglobin correction apparatus and methods thereof
A disposable bioassay diagnostic cartridge for performing enzyme-linked immunosorbent assay for a single patient in whole blood and plasma where the cartridge has a plurality of wells. The cartridge includes a pipette tip with internal sidewalls disposed in one well of the plurality of wells, the internal sidewalls being pretreated and coated with antibodies, a reagent mixture disposed in another well of the plurality of wells, the reagent mixture comprising at least one reagent selected from the group consisting of pH stabilizer, ionic catalyst control agent, ionic intracellular inhibitory control agent, detergent, negative control agent, chelating agent, and biocide preservative, antibody, and conjugate stabilizing agent. At least a chromogenic substrate and visualizing reagent disposed in an integrated cuvette of the plurality of wells. It also includes the detection of the hemoglobin amount in the blood sample so a correction for plasma amount in a sample can be performed.
1 . A disposable bioassay diagnostic cartridge for performing an enzyme-linked immunosorbent assay (ELISA) for a single patient, wherein the disposable bioassay diagnostic cartridge has a plurality of wells, the disposable bioassay diagnostic cartridge comprising:
a pipette tip with internal sidewalls disposed in a first well of the plurality of wells, the internal sidewalls being pretreated and coated with antibodies;
a reagent mixture disposed in a second well of the plurality of wells, the reagent mixture comprising at least one reagent selected from the group consisting of a pH stabilizer, an ionic catalyst control agent, an ionic intracellular inhibitory control agent, a detergent, a negative control agent, a chelating agent, a biocide preservative, an antibody, and a conjugate stabilizing agent; and
at least a chromogenic substrate and a visualizing reagent disposed in an integrated cuvette of the plurality of wells.
2 . The disposable bioassay diagnostic cartridge according to claim 1 , wherein the antibodies coating the internal sidewalls are selected from the group consisting of anti-c-reactive protein antibodies, anti-cystatin-c antibodies, anti-insulin-like growth factor 1 (anti-IGF-1) antibodies, anti-alanine aminotransferase antibodies, and anti-aspartate aminotransferase antibodies.
3 . The disposable bioassay diagnostic cartridge according to claim 1 , wherein the chromogenic substrate and the antibodies are present in a ratio of approximately 1:1.
4 . The disposable bioassay diagnostic cartridge according to claim 1 , wherein the chromogenic substrate is 3,3′,5,5′-tetramethylbenzidine.
5 . The disposable bioassay diagnostic cartridge according to claim 1 , wherein the at least one reagent is present in an amount corresponding to an amount of the chromogenic substrate and the visualizing reagent.
6 . The disposable bioassay diagnostic cartridge according to claim 1 , wherein the at least one reagent consists of at least 150 uL of a mixture of a lysing solution with 0 .002% saponin, 0.01% low-foaming surfactant, and 0.02% sodium azide.
7 . The disposable bioassay diagnostic cartridge according to claim 1 , further comprising a third well of the plurality of wells having phosphate buffered saline (PBS), 0.05% polysorbate-type nonionic surfactant, and 0.03% biocide preservative.
8 . The disposable bioassay diagnostic cartridge according to claim 1 , further comprising a third well of the plurality of wells having antibodies selected from the group consisting of anti-c-reactive protein antibodies, anti-cystatin-c antibodies, anti-insulin-like growth factor 1 (anti-IGF-1) antibodies, anti-alanine aminotransferase antibodies, anti-aspartate aminotransferase antibodies, a conjugate stabilizing agent, and 0.5% antifoaming/defoaming agent.
9 . The disposable bioassay diagnostic cartridge according to claim 1 , wherein the antibodies coating the sidewalls of the pipette tip are anti-cystatin C antibodies and the at least one reagent in the second well consists of a lysing solution with 0.002% saponin, 0.01% low-foaming surfactant, and 0.02% sodium azide.
10 . The disposable bioassay diagnostic cartridge according to claim 1 , further comprising:
a third well of the plurality of wells having 10 mM phosphate buffered saline (PBS), 0.05% polysorbate-type nonionic surfactant, and 0.03% biocide preservative; and
a fourth well of the plurality of wells having antibodies selected from the group consisting of anti-c-reactive protein antibodies, anti-cystatin-c antibodies, anti-insulin-like growth factor 1 (anti-IGF-1) antibodies, anti-alanine aminotransferase antibodies, anti-aspartate aminotransferase antibodies, a conjugate stabilizing agent, and 0.5% antifoaming/defoaming agent.
11 . The disposable bioassay diagnostic cartridge according to claim 1 , wherein the integrated cuvette has a first wall and a second wall capable of facilitating a first optical detection reading via a first light-emitting diode (LED) at 530 nm and a second optical detection reading via a second LED at 660 nm.
12 . An enzyme-linked immunosorbent bioassay diagnostic kit comprising:
a fingerstick device; and
a cartridge having a plurality of wells and an optical cuvette, the cartridge having a a pipette tip disposed in one of the plurality of wells and bioassay components disposed in one or more remaining wells of the plurality of wells;
wherein the pipette tip has sidewalls coated with an antibody; and
wherein the bioassay components comprise at least one reagent being chosen from a group consisting of a pH stabilizer, an ionic catalyst control agent, an ionic intracellular inhibitory control agent, a detergent, a negative control agent, a chelating agent, a biocide preservative, an antibody, a conjugate stabilizing agent, a chromogenic substrate, and a visualizing reagent.
13 . A method of preparing a disposable pipette for use in performing enzyme-linked immunosorbent assays while reducing washing time, the method comprising steps of:
pretreating a pipette tip by coating an internal surface of the pipette tip, wherein the pretreating step includes:
disposing a first coating containing an affinity agent along the internal surface of the pipette tip;
disposing a second coating containing a blocking solution over the first coating along the internal surface of the pipette tip; and
disposing a third coating containing an activity preservative over the second coating along the internal surface of the pipette tip, forming a coated pipette tip for use in performing the enzyme-linked immunosorbent assays that reduces washing time by facilitating direct pipetting into the coated pipette tip with at least one reagent mixture being selected from the group consisting of a pH stabilizer, an ionic catalyst control agent, an ionic intracellular inhibitory control agent, a detergent, a negative control agent, a chelating agent, a biocide preservative, a first antibody, and a conjugate stabilizing agent; and
providing one well containing a chromogenic substrate and a visualizing reagent within a cartridge for directly reacting the chromogenic substrate and the visualizing reagent with the affinity agent in the coated pipette tip.
14 . The method according to claim 13 , further comprising:
selecting the affinity agent from a group consisting of a second antibody, an antibody fragment, a recombinant protein, protein A, protein G, protein A/G, streptavidin, a recombinant protein fragment, a nucleic acid, a polysaccharide, a glycoprotein, peptidoglycan, a molecular imprinted polymer, and an aptamer molecule.
15 . The method according to claim 13 , further comprising:
selecting a coating buffer from a group consisting of a carbonate/bicarbonate buffer, a sucrose buffer, phosphate buffered saline, and tris (hydroxymethyl) aminomethane saline.
16 . The method according to claim 13 , wherein the step of disposing the first coating along the internal surface of the pipette tip with the affinity agent comprises:
selecting the affinity agent;
selecting a coating buffer;
preparing an antibody solution by mixing the selected first antibody and the selected coating buffer;
filling the pipette tip with the mixed antibody solution; and
incubating the pipette tip for a predefined time.
17 . The method according to claim 13 , wherein the step of disposing the second coating over the first coating along the internal surface of the pipette tip with the blocking solution comprises:
selecting the blocking solution;
filling the pipette tip with the blocking solution; and
incubating the pipette tip for a predefined time.
18 . The method according to claim 13 , wherein the step of disposing the third coating over the second coating along the internal surface of the pipette tip with the activity preservative comprises:
selecting the activity preservative;
filling the pipette tip with the activity preservative; and
incubating the pipette tip for a predefined time.