Methods and compositions for enhancing immunoassays

Abstract

Embodiments of the methods, compositions, and systems provided herein relate to enzymatic enhancement of immunoassays using photonic sensor arrays.

1 . A method of detecting interleukin-6 (IL-6) comprising:
 (a) obtaining an optical ring resonator having a first anti-cytokine antibody attached thereto, wherein the first anti-cytokine antibody is anti-IL-6 from clone MAB206 antibody;   (b) contacting the first anti-cytokine antibody with a sample comprising IL-6 that selectively binds to the anti-cytokine antibody;   (c) contacting the bound IL-6 with a second anti-cytokine antibody that selectively binds to the IL-6, wherein the second anti-cytokine antibody is a biotinylated anti-IL-6 from clone BAF206;   (d) contacting the biolinylated second anti-cytokine antibody with a streptavidin-horseradish peroxidase conjugate;   (e) contacting the horseradish peroxidase with a solution comprising 4-chloro-1-naphthol and hydrogen peroxide, under conditions that oxidize 4-chloro-1-naphthol to 4-chloro-1-naphthon, whereby the 4-chloro-1-naphthon precipitates on the surface of the optical ring resonator; and   (f) measuring a change in resonance wavelengths of the optical ring resonator, thereby indicating the presence of the IL-6.   
     
     
         2 . The method of  claim 1 , wherein a fluidic cell comprises the optical ring resonator. 
     
     
         3 . The method of  claim 1 , wherein step (e) comprises contacting the horseradish peroxidase with a substantially continuous flow of the solution comprising the 4-chloro-1-naphthol and hydrogen peroxide. 
     
     
         4 . The method of  claim 3 , wherein the 4-chloro-1-naphthon precipitates on the surface of the optical ring resonator during the substantially continuous flow of the solution. 
     
     
         5 . The method of  claim 3 , wherein the substantially continuous flow of the solution is maintained for steps (e) and (f). 
     
     
         6 . The method of  claim 3 , wherein the flow rate of the solution is about 30 μL/min. 
     
     
         7 . The method of  claim 3 , wherein a continuous flow of the solution is maintained for step (e). 
     
     
         8 . The method of  claim 7 , wherein the continuous flow of the solution is maintained for steps (e) and (f). 
     
     
         9 . The method of  claim 7 , wherein the flow rate of the solution is about 30 μL/min. 
     
     
         10 . The method of  claim 3 , wherein a substantially continuous flow of reagents contacting the optical ring resonator having a first anti-cytokine antibody attached thereto is maintained for each of steps (b), (c), (d), and (e). 
     
     
         11 . The method of  claim 10 , wherein the flow rate of the reagents is about 30 μL/min. 
     
     
         12 . The method of  claim 3 , wherein a continuous flow of reagents contacting the optical ring resonator having a first anti-cytokine antibody attached thereto is maintained for each of steps (b), (c), (d), and (e). 
     
     
         13 . The method of  claim 12 , wherein the flow rate of the reagents is about 30 μL/min. 
     
     
         14 . The method of  claim 1 , wherein the concentration of hydrogen peroxide is less than about 0.003%. 
     
     
         15 . The method of  claim 1 , wherein a concentration of the IL-6 less than about 100 pg/ml is detected. 
     
     
         16 . A system for detecting interleukin-6 (IL-6) comprising:
 an optical ring resonator having a first anti-cytokine antibody attached thereto, wherein the first anti-cytokine antibody is anti-IL-6 from clone MAB206 antibody;   a sample comprising IL-6, wherein the IL-6 is selectively bound to the anti-cytokine antibody;   a second anti-cytokine antibody selectively bound to the IL-6, wherein the second anti-cytokine antibody is a biotinylated anti-IL-6 from clone BAF206;   a streptavidin-horseradish peroxidase conjugate in contact with the biolinylated second anti-cytokine antibody;   a solution comprising 4-chloro-1-naphthol and hydrogen peroxide in fluid communication with the horseradish peroxidase, under conditions that oxidize 4-chloro-1-naphthol to 4-chloro-1-naphthon, whereby the 4-chloro-1-naphthon precipitates on the surface of the optical ring resonator; and   a detector adapted to measure a change in resonance wavelengths of the optical ring resonator, thereby indicating the presence of the IL-6.   
     
     
         17 . A system for detecting interleukin-2 (IL-2) comprising:
 an optical ring resonator having a first anti-cytokine antibody attached thereto, wherein the first anti-cytokine antibody is anti-IL-2 from clone MAB206 antibody;   a sample comprising IL-6, wherein the IL-6 is selectively bound to the anti-cytokine antibody;   a second anti-cytokine antibody selectively bound to the IL-6, wherein the second anti-cytokine antibody is a biotinylated anti-IL-6 from clone 555051;   a streptavidin-horseradish peroxidase conjugate in contact with the biolinylated second anti-cytokine antibody;   a solution comprising 4-chloro-1-naphthol and hydrogen peroxide in fluid communication with the horseradish peroxidase, under conditions that oxidize 4-chloro-1-naphthol to 4-chloro-1-naphthon, whereby the 4-chloro-1-naphthon precipitates on the surface of the optical ring resonator; and   a detector adapted to measure a change in resonance wavelengths of the optical ring resonator, thereby indicating the presence of the IL-2.