Immunoassay standards and measurement of clinical biomarkers using intra-assay calibration standards

Abstract

The present invention provides novel compositions and methods for creating quantitative standards to calibrate analytes. These compositions and methods enable the creation of standards and calibrators for analyzing analytes and measuring clinical biomarkers. Also provided are kits comprising the novel compositions for use in assays, for example sandwich immunoassays.

1 . A composition comprising an N-terminal immunoreactive region, a C-terminal immunoreactive region and a linker region. 
     
     
         2 . A composition comprising an N-terminal immunoreactive region, a C-terminal immunoreactive region and a linker region, wherein the composition is used as a reference standard in an immunoassay. 
     
     
         3 . The composition of  claim 2  wherein the immunoassay is selected from the group consisting of a sandwich immunoassay, a single antibody assay, a double sandwich immunoassay and a competition assay. 
     
     
         4 . The composition of  claim 1 , wherein the N-terminal immunoreactive region binds Aβ 42 . 
     
     
         5 . The composition of  claim 1 , wherein the C-terminal immunoreactive region binds Aβ 42 . 
     
     
         6 . The composition of  claim 1 , wherein the linker region is a non-immunoreactive domain. 
     
     
         7 . The composition of  claim 6 , wherein the linker region comprises a linker selected from the group consisting of polyethylene glycol, a glutamine residue, an alanine residue, a lysine residue, a lipid, a globular protein, a nucleic acid and an alkyl chain. 
     
     
         8 . An isolated modified peptide molecule having an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23, 24, 25, 26 and 27. 
     
     
         9 . A method of measuring the quantity of an analyte in a biological sample, the method comprising:
 (a) attaching a reference standard to at least two beads thereby forming a first bead set and a second bead set, wherein the reference standard comprises an epitope recognized by a first detection antibody and wherein each bead set comprises a different concentration of the reference standard;   (b) attaching a capture antibody specific to the analyte to a third bead set;   (c) mixing all of the bead sets together to form a suspension array;   (d) applying the biological sample to the suspension array whereby the analyte binds to the capture antibody on the third bead set;   (e) adding a first detection antibody to the suspension array, wherein the first detection antibody binds the reference standard and analyte bound to the capture antibody;   (f) measuring a first signal from the first detection antibody bound to the reference standard in the first bead set;   (g) measuring a second signal from the first detection antibody bound to the reference standard in the second bead set;   (h) generating a standard curve based upon the first and second signals; and   (i) quantitating the amount of the analyte in the third bead set by measuring a third signal from the first detection antibody and comparing the third signal to the first and second signal measurements on the standard curve.   
     
     
         10 . The method of  claim 9 , wherein the reference standard comprises the composition of  claim 1 . 
     
     
         11 . The method of  claim 9 , wherein the biological sample is selected from the group consisting of blood, serum, plasma, peripheral blood mononuclear cells, peripheral blood lymphocytes, tissue, cerebrospinal fluid and cells. 
     
     
         12 . The method of  claim 9 , wherein the analyte is selected from the group consisting of Aβ 42 , Aβ 40 , Aβ 38 , tau and insulin growth factor receptor 1. 
     
     
         13 . The method of  claim 9 , wherein the method is performed in a multi-well plate, nitrocellulose filter, glass fiber or on a glass slide. 
     
     
         14 . The method of  claim 9 , wherein the first signal and second signal is a signal selected from the group consisting of phycoerythrin, alexa 532, streptavidin-phycoerythrin and streptavidin-Alexa 532. 
     
     
         15 . A kit for conducting an immunoassay to detect Aβ 42  peptide, the kit comprising a composition of  claim 1 . 
     
     
         16 . The composition of  claim 2 , wherein the N-terminal immunoreactive region binds Aβ 42 . 
     
     
         17 . The composition of  claim 2 , wherein the C-terminal immunoreactive region binds Aβ 42 . 
     
     
         18 . The composition of  claim 2 , wherein the linker region is a non-immunoreactive domain. 
     
     
         19 . The method of  claim 9 , wherein the reference standard comprises the composition of  claim 2 . 
     
     
         20 . The method of  claim 9 , wherein the reference standard comprises the composition of  claim 8 . 
     
     
         21 . A kit for conducting an immunoassay to detect Aβ 42  peptide, the kit comprising a composition of  claim 2 . 
     
     
         22 . A kit for conducting an immunoassay to detect Aβ 42  peptide, the kit comprising a composition of  claim 8 .