Luminescent oxygen channeling immunoassay utilizing three antibodies and methods of production and use thereof

Abstract

A chemiluminescent detection system, as well as kits and microfluidics devices containing same, are disclosed. Methods of using the system, kits, and devices are also disclosed. The first, second, and third antibodies or binding fragments thereof may be provided in any form that allows these antibodies/binding fragments to function in accordance with the presently disclosed and claimed inventive concept(s). For example, each of the first, second, and third antibodies/binding fragments may be a polyclonal antibody/binding fragment or a monoclonal antibody/binding fragment.

1 . A kit containing a chemiluminescent detection system for a specific analyte, the kit comprising:
 (a) a first composition comprising a singlet-oxygen activatable chemiluminescent compound and a first antibody or binding fragment thereof associated therewith, wherein the first antibody or binding fragment thereof is a detection antibody that specifically binds to a first epitope of the analyte whereby the singlet-oxygen activatable chemiluminescent compound is capable of indirectly binding to the analyte via the first antibody or binding fragment thereof;   (b) a second composition comprising a singlet-oxygen activatable chemiluminescent compound and a second antibody or binding fragment thereof associated therewith, wherein the second antibody or binding fragment thereof is a detection antibody that specifically binds to a second epitope of the analyte whereby the singlet-oxygen activatable chemiluminescent compound is capable of indirectly binding to the analyte via the second antibody or binding fragment thereof, and wherein the first and second epitopes at least partially overlap such that the first and second antibodies or binding fragments thereof cannot both bind to a single analyte molecule; and   (c) a third composition comprising a third antibody or binding fragment thereof, the third antibody or binding fragment thereof being a capture antibody that specifically binds to a third epitope of the analyte that does not overlap with the first and second epitopes, whereby a single analyte molecule can bind the third antibody or binding fragment thereof and one of the first and second antibodies or binding fragments thereof, and wherein the third antibody or binding fragment thereof is capable of association with a sensitizer capable of generating singlet oxygen in its excited state, whereby association of the third antibody or binding fragment thereof with the sensitizer allows for the indirect binding of the sensitizer to the analyte.   
     
     
         2 . The kit of  claim 1 , further comprising the sensitizer and wherein third composition further comprises the sensitizer associated with the third antibody or binding fragment thereof. 
     
     
         3 . The kit of  claim 1 , wherein at least one of the first and second compositions further comprises at least one fluorescent molecule that is excited by the activated chemiluminescent compound. 
     
     
         4 . The kit of any of  claim 1 , wherein at least one of (a)-(c) is further defined as being in the form of a lyophilized reagent. 
     
     
         5 . The kit of  claim 4 , further comprising an excipient for the reconstitution of the lyophilized reagent. 
     
     
         6 . A microfluidics device, comprising:
 at least one compartment containing:
 (i) a first composition comprising a singlet-oxygen activatable chemiluminescent compound and a first antibody or binding fragment thereof associated therewith, wherein the first antibody or binding fragment thereof is a detection antibody that specifically binds to a first epitope of a specific analyte whereby the singlet-oxygen activatable chemiluminescent compound is capable of indirectly binding to the analyte via the first antibody or binding fragment thereof; 
 (ii) a second composition comprising a singlet-oxygen activatable chemiluminescent compound and a second antibody or binding fragment thereof associated therewith, wherein the second antibody or binding fragment thereof is a detection antibody that specifically binds to a second epitope of the analyte whereby the singlet-oxygen activatable chemiluminescent compound is capable of indirectly binding to the analyte via the second antibody or binding fragment thereof, and wherein the first and second epitopes at least partially overlap such that the first and second antibodies or binding fragments thereof cannot both bind to a single analyte molecule; and 
 (iii) a third composition comprising a third antibody or binding fragment thereof, the third antibody or binding fragment thereof being a capture antibody that specifically binds to a third epitope of the analyte that does not overlap with the first and second epitopes, whereby a single analyte molecule can bind the third antibody or binding fragment thereof and one of the first and second antibodies or binding fragments thereof; and 
 (iv) a sensitizer capable of association with the third antibody or binding fragment thereof, the sensitizer being capable of generating singlet oxygen in its excited state, and wherein association of the third antibody or binding fragment thereof with the sensitizer allows for the indirect binding of the sensitizer to the analyte. 
   
     
     
         7 . The microfluidics device of  claim 6 , further comprising an inlet channel through which a sample may be disposed, wherein the at least one compartment is capable of being in fluidic communication with the inlet channel. 
     
     
         8 . The microfluidics device of  claim 7 , further defined as comprising at least two compartments, wherein a first compartment contains (i), (ii), and (iii), and wherein a second compartment contains (iv). 
     
     
         9 . The microfluidics device of  claim 8 , further comprising an inlet channel through which a sample may be disposed, wherein the first compartment is capable of being in fluidic communication with the inlet channel, and wherein the second compartment is capable of being in fluid communication with at least one of the inlet channel and the first compartment. 
     
     
         10 . The microfluidics device of any of  claims 6 - 9 , further comprising at least one additional compartment capable of being in fluidic communication with at least one of the inlet channel and the at least one compartment, and wherein the at least one additional compartment contains a diluent. 
     
     
         11 . A method for detecting the presence and/or concentration of a specific analyte in a sample, comprising the steps of:
 (a) combining, either simultaneously or wholly or partially sequentially:
 (i) a sample suspected of containing the specific analyte; 
 (ii) a first composition comprising a singlet-oxygen activatable chemiluminescent compound and a first antibody or binding fragment thereof associated therewith, wherein the first antibody or binding fragment thereof is a detection antibody that specifically binds to a first epitope of a specific analyte; 
 (iii) a second composition comprising a singlet-oxygen activatable chemiluminescent compound and a second antibody or binding fragment thereof associated therewith, wherein the second antibody or binding fragment thereof is a detection antibody that specifically binds to a second epitope of the analyte, and wherein the first and second epitopes at least partially overlap such that the first and second antibodies or binding fragments thereof cannot both bind to a single analyte molecule; 
 (iv) a third composition comprising a third antibody or binding fragment thereof, the third antibody or binding fragment thereof being a capture antibody that specifically binds to a third epitope of the analyte that does not overlap with the first and second epitopes, whereby a single analyte molecule can bind the third antibody or binding fragment thereof and one of the first and second antibodies or binding fragments thereof; and 
 (v) a sensitizer capable of association with the third antibody or binding fragment thereof, the sensitizer being capable of generating singlet oxygen in its excited state; 
   (b) allowing the binding of (ii), (iii) and/or (iv) to analyte within the sample, wherein a first sandwich complex comprising an analyte molecule and (ii) and (iv) is formed, and a second sandwich complex comprising another analyte molecule and (iii) and (iv) is formed, and wherein (v) associates with (iv) in the first and second sandwich complexes, thus bringing the sensitizer into close proximity to the chemiluminescent compounds of (ii) and (iii);   (c) activating the sensitizer to generate singlet oxygen, wherein activation of the sensitizer present in the first and second sandwich complexes causes the activation of the chemiluminescent compounds present in the first and second sandwich complexes;   (d) determining the amount of chemiluminescence generated by the activated chemiluminescent compounds present in the first and second sandwich complexes;   (e) optionally repeating steps (b)-(d); and   (f) detecting the presence and/or concentration of the analyte by analyzing the amount of chemiluminescence so produced, wherein the amount of chemiluminescence is directly proportional to the amount of analyte in the sample.   
     
     
         12 . The method of  claim 11 , wherein the sensitizer is a photosensitizer, and wherein step (c) is further defined as activating the photosensitizer via irradiation with light. 
     
     
         13 . The method of  claim 12 , further comprising a step of exposing the sample to a separation step prior to combining with any of (ii)-(v). 
     
     
         14 . The method of any of  claim 11 , wherein the singlet-oxygen activatable chemiluminescent compounds of the first and second compositions are the same. 
     
     
         15 . The method of any of  claim 11 , wherein the singlet-oxygen activatable chemiluminescent compounds of the first and second compositions are different.