An assay exhibiting improved sensitivity for achieving a factor of ten times (10×) better sensitivity than current sensitivity levels achieved by prior-art immunoassays, as exemplified by ELISA. Three main implementations are described: IA/MPD, Super-ELISA and Reverse Geometry Immunoassay, including
An assay exhibiting improved sensitivity for achieving a factor of ten times (10×) better sensitivity than current sensitivity levels achieved by prior-art immunoassays, as exemplified by ELISA. Three main implementations are described: IA/MPD, Super-ELISA and Reverse Geometry Immunoassay, including specific panel biomarker relationships for the early detection and recognition patterns of breast and prostate cancers. More sensitive immunoassays are achieved through a better understanding of sources of biological background, and by the rejection of particular sources thereof. Super-sensitive immunoassays permit measurement of blood sample biomarkers, and show distribution of low abundance proteins included relationships between cytokine non-Gaussian distributions, very large dynamic ranges and strong age dependence, and including new algorithms based on 2D correlations of studied biomarkers.
대표청구항▼
What is claimed: 1. A super-sensitive sandwich immunoassay which can quantitate proteins with a detection level at less than 100 fg/ml by reducing biological background interference in the specimen to be tested comprising the steps of: (a) coating a testing substrate with an Abs(capture) solution a
What is claimed: 1. A super-sensitive sandwich immunoassay which can quantitate proteins with a detection level at less than 100 fg/ml by reducing biological background interference in the specimen to be tested comprising the steps of: (a) coating a testing substrate with an Abs(capture) solution and allowing incubation and bonding between the testing substrate and the Abs(capture) solution; (b) super stringent washing the testing substrate; (c) treating the testing substrate with a blocking reagent; (d) super stringent washing the testing substrate; (e) depositing a sample of physiologic fluid on the testing substrate; (f) super stringent washing the testing substrate; (g) applying an Abs(labeling) solution onto the testing substrate and allowing incubation; (h) super stringent washing the testing substrate; (i) applying a streptavidin based reagent onto the testing substrate; (j) super stringent washing the testing substrate; and (k) measuring and generating a signal representative of ultra-low amounts of labeled antibodies at detection levels less than 100 fg/ml, wherein the level of protein is ascertained using one or more of the following: a. multi photon detection (MPD) instrumentation which can quantitate proteins with a limit of detection of less than 10 fg/mg, using 125I radiolabels; b. optical colorimeter readers having a signal capable of quantitating proteins at a limit of detection less than 50 fg/ml; wherein super-sensitivity is obtained by use of super-stringent washing wherein the protein detection ability is diminished by a factor greater than 3; and wherein the supersensitive washing is obtained using one or more of the following procedures: a. application of ultrasound; b. application of shock waves; c. movement of small magnetic actuator, wherein the rotational movement of the said magnetic actuator is induced by a strong external source of magnetic field; d. application of turbulent washing liquid having a temperature higher than room temperature e. application of turbulent washing liquid pH significantly different than pH=7 for at least 10 minutes; and f. washing using an application of a "slow-roll" technique. 2. A super-sensitive sandwich immunoassay according to claim 1, wherein the super-stringent washing is obtained by combination of at least two methods as described therein. 3. A super-sensitive sandwich immunoassay according to claim 1, wherein the super-sensitive immunoassay is performed in 96-or 384-well microtiter plates and capture antibodies are added in considerable excess, incubated for more than 1 hour, and then super-stringently washed. 4. A super-sensitive sandwich immunoassay according to claim 3, wherein cross-talk between capture antibodies and labeling antibodies is diminished by selecting antibodies which have a low interaction probability and wherein said capture antibodies are provided at less than 1 microgram/well. 5. A super-sensitive sandwich immunoassay to claim 1, wherein said immunoassay includes microtiter plates which are blocked with a blocking liquid consisting of one or more of the following products that have been purified by removing biotinylated proteins: a) caseine, caseine byproducts or caseine chemical derivatives; b) plant products imitating milk, including soy milk; c) fine granulated graphite or other simple organic product. 6. A super-sensitive sandwich immunoassay according to claim 5, wherein said microtiter plates are blocked with a graphite powder selected to contain a large spectrum of graphite granules, wherein said large spectrum of graphite granules is obtained by the mixing at least two commercially available graphite based powders wherein graphite granules with a diameter larger than approximately 50 microns are removed by filtration. 7. A super-sensitive sandwich immunoassay according to claim 6, wherein said graphite powder is selected to consist mainly of granules having a diameter smaller than approximately 5 microns, and wherein coagulation of said graphite powder is reduced by placing said graphite powder in a water based liquid with additives which diminish the coagulation of graphite granules or wherein the said graphite powder is charged negatively. 8. A super-sensitive sandwich immunoassay according to claim 1, that includes the step of incubation of labeling antibodies in an assay buffer with pH substantially different than PH=7. 9. A super-sensitive sandwich immunoassay which can quantitate proteins with detection level at less than 100 fg/ml by reducing biological background interference in the specimen to be tested comprising the steps of: (a) coating a testing substrate with an Abs(capture) solution and allowing incubation and bonding between the testing substrate and the Abs(capture) solution; (b) super stringent washing the testing substrate; (c) treating the testing substrate with a blocking reagent; (d) super stringent washing the testing substrate; (e) depositing a sample of physiologic fluid on the testing substrate; (f) super stringent washing the testing substrate; (g) applying an Abs(labeling) solution onto the testing substrate and allowing incubation; (h) super stringent washing the testing substrate; (i) applying a streptavidin based reagent onto the testing substrate; (j) super stringent washing the testing substrate; and (k) measuring and generating a signal representative of ultra-low amounts of labeled antibodies at detection levels less than 100 fg/ml, wherein super-sensitive assay incorporates a buffer having greater than 10% per mass component of fraction IV human serum. 10. A super-sensitive sandwich immunoassay which can quantitate proteins with a detection level at less than 100 fg/ml by reducing biological background interference the specimen to be tested comprising the steps of: (a) coating a testing substrate with an Abs(capture) solution and allowing incubation and bonding between the testing substrate and the Abs(capture) solution; (b) super stringent washing the testing substrate; (c) treating the testing substrate with a blocking reagent; (d) super stringent washing the testing substrate; (e) depositing a sample of physiologic fluid on the testing substrate; (f) super stringent washing the testing substrate; (g) applying an Abs(labeling) solution onto the testing substrate and allowing incubation; (h) super stringent washing the testing substrate; (i) applying a streptavidin based reagent onto the testing substrate; (j) super stringent washing the testing substrate; and (k) measuring and generating a signal representative of ultra-low amounts of labeled antibodies at detection levels less than 100 fg/ml, wherein the super-sensitive assay incorporates a buffer having greater than 10% per mass component of bird, reptile or fish serum. 11. A super-sensitive sandwich immunoassay which can quantitate proteins with a detection level at less than 100 fg/ml by reducing biological background interference in the specimen to be tested comprising the steps of: (a) coating a testing substrate with an Abs(capture) solution and allowing incubation and bonding between the testing substrate and the Abs(capture) solution; (b) super stringent washing the testing substrate; (c) treating the testing substrate with a blocking reagent; (d) super stringent washing the testing substrate; (e) depositing a sample of physiologic fluid on the testing substrate; (f) super stringent washing the testing substrate; (g) applying an Abs(labeling) solution onto the testing substrate and allowing incubation; (h) super stringent washing the testing substrate; (i) applying a streptavidin based reagent onto the testing substrate; (j) super stringent washing the testing substrate; and (k) measuring and generating a signal representative of ultra-low amounts of labeled antibodies at detection levels less than 100 fg/ml, wherein the super-sensitive assay incorporates a buffer having greater than 10% per mass component of milk, milk byproducts or plant extracted proteins or soy byproducts. 12. A super-sensitive sandwich immunoassay which can quantitate proteins with a detection level at less than 100 fg/ml by reducing biological background interference in the specimen to be tested comprising the steps of: (a) coating a testing substrate with an Abs(capture) solution and allowing incubation and bonding between the testing substrate and the Abs(capture) solution; (b) super stringent washing the testing substrate; (c) treating the testing substrate with a blocking reagent; (d) super stringent washing the testing substrate; (e) depositing a sample of physiologic fluid on the testing substrate; (f) super stringent washing the testing substrate; (g) applying an Abs(labeling) solution onto the testing substrate and allowing incubation; (h) super stringent washing the testing substrate; (i) applying a streptavidin based reagent onto the testing substrate; (j) super stringent washing the testing substrate; and (k) measuring and generating a signal representative of ultra-low amounts of labeled antibodies at detection levels less than 100 fg/ml, which includes the steps of labeling an antibody, incubating said antibody and performing a super-stringent wash to remove more than 50% of incubated labeled antibody, wherein super-stringent washing is performed according to one or more of the following procedures: a. application of ultrasound; b. application of shock waves; c. movement of small magnetic actuator, wherein the rotational movement of said magnetic actuator is induced by a strong external source of magnetic field; d. application of turbulent washing liquid having a temperature higher than room temperature e. application of turbulent washing liquid with pH significantly different than pH=7 for at least 10 minutes; and f. washing using an application of a "slow-roll" technique. 13. A super-sensitive sandwich immunoassay according to claim 12, wherein said labeled antibody is labeled with EC emitter, wherein said labeled antibody is derivatized with biotin, and wherein a reagent containing at least one streptavidin labeled moiety having an enzymatic or 125-I radioactive label is used to provide a measurable signal via binding to said biotinylated labeled antibody. 14. A super-sensitive sandwich immunoassay according to claim 13 wherein the said streptavidin is labeled with the enzymatic label horseradish peroxidase (HRP) and wherein the biological stickiness of streptavidin base reagent is diminished by: shortening of incubation time; use of optimal pH and/or temperature; super-stringent wash, wherein said super-stringent washing is implemented in accordance with one or more of the following procedures: a. application of ultrasound; b. application of shockwaves; c. movement of small magnetic actuator, wherein the rotational movement of said magnetic actuator is induced by a strong external source of magnetic field; d. application of turbulent washing liquid having a temperature higher than room temperature e. application of turbulent washing liquid with pH significantly different than pH=7for at least 10 minutes; and f application of a "slow-roll" washing technique. 15. A super-sensitive sandwich immunoassay according to claim 14 wherein biological background due to stickiness of 125I-streptavidin is diminished by application of 125I-streptavidin with activity no larger than 10,000 dpm per well and wherein biological background is diminished by application of 125I-streptavidin for no longer than 5 minutes. 16. A super-sensitive sandwich immunoassay according to claim 13 wherein multiple iodination is obtained by modifying utilizing genetically engineered streptavidin having multiple additional thyrosines in the tetramer of streptavidin. 17. A super-sensitive sandwich immunoassay according to claim 16 wherein the genetically engineered steptavidin is streptavidin-HRP or streptavidin-polyHRP, and wherein biological background due to stickiness of streptavidin-HRP or streptavidin-polyHRP is diminished at least five fold by super-stringent washing. 18. A super-sensitive sandwich immunoassay according to claim 17, having a labeled steptavidin wherein said labeled steptavidin is streptavidin HRP(i) with 1<i<80, and the signal is obtained by change of color induced by HRP or polyHRP e; or streptavidin, streptavidin-HRP or streptavidin-polyHRP derivitized with appropriate fluorescent tag. 19. A super-sensitive sandwich immunoassay according to claim 13 wherein said streptavidin moiety is used in two steps signal amplification techniques of amplified immunoassay/multi photon detection (IA/MPD), amplified Super-ELISA or amplified reverse geometry immunoassay (RGIA). 20. A super-sensitive sandwich immunoassay according to claim 19 wherein biotinylated anti-HRP is used to bind to streptavidin-polyHRP which streptavidin-polyHRP is first subjected to a super-stringent wash, and then used to implement amplified Super-ELISA. 21. A super-sensitive sandwich immunoassay which can quantitate proteins with a detection level at less than 100 fg/ml by reducing biological background interference in the specimen to be tested comprising the steps of: (a) coating a testing substrate with an Abs(capture) solution and allowing incubation and bonding between the testing substrate and the Abs(capture) solution; (b) super stringent washing the testing substrate; (c) treating the testing substrate with a blocking reagent; (d) super stringent washing the testing substrate; (e) depositing a sample of physiologic fluid on the testing substrate; (f) super stringent washing the testing substrate; (g) applying an Abs(labeling) solution onto the testing substrate and allowing incubation; (h) super stringent washing the testing substrate; (i) applying a streptavidin based reagent onto the testing substrate; (j) super stringent washing the testing substrate; and (k) measuring and generating a signal representative of ultra-low amounts of labeled antibodies at detection levels less than 100 fg/ml, wherein said sandwich immunoassay is performed in 96-well microtiter plates having the volume of a biological sample to be placed in said plates increased above 300 microliter by multiloading or "slow-roll" technique and including a superstringent wash. 22. A super-sensitive sandwich immunoassay according to claim 21 wherein glass tubes tightly fitting the microtiter plate include glass tubes tightly fitting said plate which are used to implement "slow roll" movement and wherein the amount of biological sample is selected to provide at least 100 microliter of air in said glass tubes and wherein the rotational speed of "slow roll" is below 20 rotations per minute and the time of rotation in "slow roll" technique is at least 10 minutes. 23. A super-sensitive sandwich immunoassay according to claim 21, wherein the sandwich immunoassay is performed with capture antibodies coated on a substrate surface different than the microtiter plates, whereby the volume of said biological sample may be larger than 200 microliters by us of a granulated substrate. 24. A super-sensitive sandwich immnunoassay according to claim 23 wherein said substrate surface is one of the group consisting of diverse plastics not including polypropylene; diverse glasses; diverse magnetic materials; and diverse metals. 25. A super-sensitive sandwich immunoassay according to claim 23, wherein the sandwich immunoassay is performed using diverse glasses as a substitute surface and HRP or other enzymes are used as a label. 26. A super-sensitive sandwich immunoassay according to claim 23 wherein the granulated, substrate comprises microscopic or macroscopic beads.
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이 특허에 인용된 특허 (4)
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