US 7,604,956 B2 · Supersensitive immunoassays

Abstract

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.

Claims26

Dependent claims

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.

22 more elements

(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.

16 more elements

(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.

9 more elements

Dependencies are inferred from the claim text.

Supersensitive immunoassays

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Supersensitive immunoassays

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Abstract

Claims26