Saturation binding ratiometric assay

Abstract

Methods, devices, and reagents are described for performing ratiometric assays for hemoglobin A1c. The methods involve a direct ratio determination between Hb A1c and normalized total hemoglobin utilizing a saturating amount of hemoglobin so that Hb A1c binds proportionately to a substrate. In some applications, the assay utilizes a proximity label system for signal generation and/or labeled magnetic beads. The methods can be configured as homogeneous or heterogeneous assays.

What is claimed is: 
     
         1 . A method for determining the fraction of a glycated hemoglobin in a sample, comprising
 contacting optionally labeled particles comprising a solid phase substrate bearing first binding members specific for total hemoglobin with
 a) a sample containing total hemoglobin in sufficient volume whereby binding of total hemoglobin on said solid phase surface is saturated; and 
 b) second binding member specific for hemoglobin A1c of said total hemoglobin and labeled with a second label; 
   detecting at least one signal from said second label from specific binding member bound to said particles, wherein the level of said at least one signal is indicative of the fraction of hemoglobin A1c relative to total hemoglobin.   
     
     
         2 . The method of  claim 1 , wherein said solid phase substrate is labeled with a first label, and said method further comprises detecting the distinguishable signal from first label as an indication of the number of particles present. 
     
     
         3 . The method of  claim 1 , wherein the binding member specific for total hemoglobin binds within beta chain amino acids 7-25 and preferentially binds to HbA rather than to HbF. 
     
     
         4 . The method of  claim 3 , wherein binding of said first specific binding member with said hemoglobin under assay conditions causes exposure of the amino terminus of bound hemoglobin. 
     
     
         5 . The method of  claim 4 , wherein said second specific binding member preferentially binds with hemoglobin bound with said first specific binding member over binding with unbound hemoglobin. 
     
     
         6 . The method of  claim 1 , wherein said method is performed as a homogenous assay. 
     
     
         7 . A method for determining the fraction of Hb A1c in a sample containing hemoglobin, comprising
 contacting labeled magnetic beads linked with a first specific binding agent which binds with total hemoglobin with
 a) a sample containing total hemoglobin in sufficient volume and concentration whereby binding of total hemoglobin on said particles is saturated; and 
 b) second specific binding member specific for Hb A1c subpopulation of said total hemoglobin and labeled with a second label; 
   thereby forming sandwich complexes comprising labeled magnetic beads with bound total hemoglobin, and second specific binding member with a second label bound to Hb A1c,   separating second specific binding member in said sandwich complexes from second specific binding member not in said sandwich complexes,   detecting second signal from said second label from second specific binding member bound in said sandwich complexes, wherein the level of said signal is indicative of the fraction of Hb A1c,   detecting first signal from said labeled magnetic beads, wherein the level of said signal is indicative of the number of bears and therefore indicative of the amount of total hemoglobin,   wherein said amount of Hb A1c compared to the level of total hemoglobin indicates the fraction of total hemoglobin which is glycated hemoglobin.   
     
     
         8 . The method of  claim 7 , wherein said binding member specific for total hemoglobin binds within beta chain amino acids 7-25 and preferentially binds to HbA over binding to HbF. 
     
     
         9 . The method of  claim 8 , wherein said second specific binding member preferentially binds with hemoglobin bound with said first specific binding member over binding with unbound hemoglobin. 
     
     
         10 . The method of  claim 7 , wherein method further comprises detecting the presence and/or level of hemoglobin S, C, E or D. 
     
     
         11 . The method of  claim 7 , wherein said method is performed as a homogeneous assay. 
     
     
         12 . A method for one-step measurement of fraction of glycated hemoglobin in a sample, comprising
 (a) lysing a blood sample;   (b) contacting said lysed blood sample with a predefined quantity of solid phase media modified with antibody binding total hemoglobin thereby saturation binding total hemoglobin to said solid phase media, wherein said antibody binds hemoglobin beta chain within amino acids 7-25;   (c) contacting hemoglobin bound on said solid phase with a reagent specifically binding hemoglobin A1c; and   (d) determining the level of said reagent specifically bound to hemoglobin A1c as an indication of the fraction of bound total hemoglobin which is glycated,   wherein said method does not require denaturation of hemoglobin beta chain.   
     
     
         13 . The method of  claim 12 , wherein said solid phase media is particles or planar surface. 
     
     
         14 . The method of  claim 12 , wherein said reagent is antibody, aptamer, molecularly imprinted polymers, polypeptides, or a boronic acid derivative. 
     
     
         15 . The method of  claim 12 , wherein said reagent is labeled with a detectable label and said detecting comprises detecting signal from said label. 
     
     
         16 . The method of  claim 15 , wherein said label is a TRF label. 
     
     
         17 . The method of  claim 12 , wherein said reagent is not labeled with a detectable label, and said method further comprises contacting reagent bound to hemoglobin A1c bound on said solid phase with labeled second reagent linked with a detectable label. 
     
     
         18 . The method of  claim 17 , wherein said detecting comprises detecting signal from said detectable label. 
     
     
         19 . The method of  claim 17 , wherein hemoglobin beta chain in said blood sample is not denatured. 
     
     
         20 . The method of  claim 12 , wherein said method is performed as a homogeneous assay.