Kits, methods and their uses for detecting cell-cell interactions in a sample

Abstract

The present invention provides an in vitro method for detecting cell-cell interactions and associated kits and uses, where the method comprises: (a) at least two primary binding agents, wherein the first primary binding agent binds to a first molecule on a first cell and the second primary binding agent binds to a second molecule on a second cell, and wherein the first and second primary binding agents are immunologically distinct; (b) at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds the second primary binding agent, wherein the first secondary binding agent does not bind the second primary binding agent and the second secondary binding agent does not buy the first primary binding agent; and wherein: (i) the first secondary binding agent is labelled with a FRET donor and the second secondary binding agent is labelled with a FRET acceptor; (ii) the first and second secondary binding agents are conjugated or fused to a DNA sequence, wherein the DNA sequences are different and ligate to form a circle and are amplified by rolling circle DNA amplification, and are bound by externally applied fluorescently labelled DNA probes complimentary to that of the amplified DNA sequences; or (iii) the first secondary binding agent is labelled with a FRET donor and the second secondary binding agent is fused to an enzyme which reacts with a conjugate comprising a FRET acceptor and a substrate specific enzyme to form an activated conjugate that binds to electron rich moieties on a molecular surface adjacent to the enzyme; wherein the method comprises: i. contacting an isolated sample containing cells with the at least two primary binding agents; ii. contacting the sample with the at least two secondary binding agents; iii. performing a wash step; iv. detecting the interaction between the secondary binding agents.