IMMUNOASSAY FOR CROSS-REACTING SUBSTANCES
The present disclosure provides an immunoassay involving a multiplex of antibodies that recognize the same analyte but that have a different cross-reactivity to structurally similar compounds. Data obtained from the immunoassay involving observed analyte concentrations is input into an algorithm to determine the true concentration of the analyte in a sample.
1 . A method for determining the concentration of an analyte in a test sample comprising the analyte and a plurality of competitive ligands, the method comprising:
contacting the test sample with at least two different anti-analyte antibodies, wherein each of the antibodies bind the analyte and have a different level of cross-reactivity for the competitive ligands;
detecting binding of the analytes and competitive ligands to the antibodies, thereby determining an observed analyte binding amount for each antibody; and
performing a regression analysis on the observed analyte binding amount for each antibody to determine the concentration of the analyte in the test sample.
2 . The method of claim 1 , wherein the analyte is cortisol and the competitive ligands are non-cortisol steroids.
3 . The method of claim 1 , wherein the regression analysis is linear regression.
4 . The method of claim 1 , wherein the regression analysis is non-linear regression.
5 . The method of claim 1 , wherein the regression analysis is displayed graphically.
6 . The method of claim 1 , wherein the regression analysis comprises solving the formula:
Y=Σβ n x n +c
wherein,
Y is the cortisol concentration;
n is the number of antibodies;
x is the observed steroid amount for each antibody;
β is the level of cross-reactivity for each antibody;
c is a calibration constant; and
Σ is the sum of βx for all antibodies.
7 . The method of claim 1 , wherein the test sample is plasma, serum, saliva, or urine.
8 . The method of claim 2 , wherein the non-cortisol steroids are selected from the group consisting of prednisolone, cortisone, 6-α methylprednisolone (6-AMP), progesterone, prednisone, fludrocortisone and dexamethasone.
9 . The method of claim 2 , wherein the test sample is contacted with cortisol comprising a label or prednisolone comprising a label prior to the detecting step.
10 - 15 . (canceled)
16 . The method of claim 9 , wherein the cortisol comprising a label is
wherein,
R is a label.
17 . The method of claim 9 , wherein the prednisolone comprising a label is:
wherein,
R is a label.
18 - 20 . (canceled)
21 . The method of claim 1 , wherein the antibodies are monoclonal antibodies.
22 . The method of claim 1 , wherein the antibodies are polyconal antibodies.
23 . The method of claim 1 , wherein the antibodies are immobilized on a solid support.
24 - 29 . (canceled)
30 . The method of claim 1 , wherein the test sample is contacted with at least three antibodies.
31 . The method of claim 1 , wherein the test sample is contacted with at least five antibodies.
32 - 46 . (canceled)
47 . A method for detecting cortisol levels in an individual, the method comprising:
contacting a test sample from the individual with at least two different anti-steroid antibodies, wherein each of the antibodies bind cortisol and have a different level of cross-reactivity with non-cortisol steroids;
detecting binding of steroids to each of the antibodies, thereby determining an observed steroid binding amount for each of the antibodies;
performing a regression analysis on the observed steroid binding amount for each antibody to determine the concentration of cortisol in the test sample; and
comparing the concentration of cortisol in the test sample from the individual with cortisol levels in a control sample to detect cotisol levels in the individual.
48 - 50 . (canceled)
51 . A method for determining the concentration of cortisol in a test sample, the
method comprising:
contacting the test sample with at least two different anti-steroid antibodies, wherein each of the antibodies bind cortisol and have a different level of cross-reactivity with non-cortisol steroids;
detecting binding of steroids to the antibodies, thereby determining an observed steroid binding amount for each antibody; and
performing a regression analysis on the observed steroid binding amounts for each antibody to determine the concentration of cortisol in the test sample.