ANTIGEN DETECTION USING PHOTOCLEAVABLE LABELS
Provided herein are methods of using photocleavable labels for multiplex and serial antigen detection. The methods comprise detecting the presence of photocleavable labels, which are conjugated through functional linkers to antigen-binding complexes, which in turn non-covalently bind to antigens. The presence of a photocleavable label is indicative of the presence of an antigen specifically or selectively bound by an antigen-binding complex. Also provided are apparatuses for using photocleavable labels for multiplex and serial antigen detection.
1 . A method for detecting the presence of at least a first antigen and a second antigen on or in a sample, the method comprising:
(i) contacting the sample with a first antigen-binding complex that is conjugated to a first photocleavable label through a first functional linker, wherein the first antigen-binding complex is capable of non-covalently binding to the first antigen, thereby forming an antigen-bound antigen-binding complex of formula (I):
(ii) detecting the presence of the first photocleavable label in the sample, wherein the presence of the first photocleavable label is indicative of the presence of the first antigen;
(iii) exposing the sample to ultraviolent light in order to cleave the first photocleavable label;
(iv) contacting the sample with a second antigen-binding complex that is conjugated to a second photocleavable label through a second functional linker, wherein the second antigen-binding complex is capable of non-covalently binding to the second antigen, thereby forming an antigen-bound antigen-binding complex of formula (I-a):
(v) detecting the presence of the second photocleavable label in the sample, wherein the presence of the second photocleavable label is indicative of the presence of the second antigen,
wherein the first functional linker and the second functional linker are each a partially double-stranded oligonucleotide, wherein each oligonucleotide of the partially double-stranded oligonucleotide is 34 nucleotides in length and wherein the partially double-stranded oligonucleotide has a double-stranded section that is 28 nucleotides in length, wherein each oligonucleotide in the partially double-stranded oligonucleotide has a biotin on its 5′ end.
2 . The method of claim 1 , wherein each oligonucleotide in the partially double-stranded oligonucleotide has a photocleavable label on its 3′ end.
3 . The method of claim 1 , further comprising detecting the presence of a third antigen in the sample by contacting the sample with a third antigen-binding complex that is conjugated to a third photocleavable label and detecting the presence of the third photocleavable label in the sample, wherein the third antigen-binding complex is capable of non-covalently binding to the third antigen, wherein the presence of the third photocleavable label is indicative of the presence of the third antigen.
4 . The method of claim 3 , wherein detecting the first photocleavable label and detecting the third photocleavable label is performed simultaneously.
5 . The method of claim 4 , wherein the first antigen-binding complex and the third antigen-binding complex each comprise a unique photocleavable label.
6 . The method of claim 3 , wherein detecting the second photocleavable label and detecting the third photocleavable label is performed simultaneously.
7 . The method of claim 6 , wherein the second antigen-binding complex and the third antigen-binding complex each comprise a unique photocleavable label.
8 . The method of claim 3 , wherein detecting the third photocleavable label is performed sequentially after detecting the second photocleavable label, wherein the second photocleavable label is cleaved prior to contacting the sample with the third antigen-binding complex.
9 . The method of claim 1 , wherein the first antigen-binding complex and the second antigen-binding complex each comprise the same photocleavable label.
10 . The method of claim 1 , wherein the first antigen-binding complex and the second antigen-binding complex each comprise a unique photocleavable label.
11 . The method of claim 1 , wherein the photocleavable label comprises a reporter moiety covalently bound to a photocleavable moiety.
12 . The method of claim 11 , wherein the photocleavable moiety is a 2 -nitrobenzyl group.
13 . The method of claim 12 , wherein the 2-nitrobenzyl group comprises a substitution on the α-carbon and/or a 5-methoxy substitution on the benzene ring.
14 . The method of claim 11 , wherein the reporter moiety is a colorimetric dye, a fluorescent dye, a radioactive label, a chemiluminescent group, or a bioluminescent group.
15 . The method of claim 1 , wherein the first antigen-binding complex and the second antigen-binding complex are each defined by formula (VIII):
wherein the first antigen-binding complex and the second antigen-binding complex each comprise a primary antibody bound by a secondary antibody, wherein the secondary antibody is covalently bound to a first ligand, wherein the first ligand is non-covalently bound to a first anti-ligand, wherein the antigen-bound antigen-binding complex is further defined by formula (IX):
16 . The method of claim 1 , wherein the sample is a tissue section, biopsy sample, cell culture sample, cell smear, or protein lysate.