MULTIPLEX METHOD FOR DETECTING DIFFERENT ANALYTES AND DIFFERENT SUBGROUPS/VARIATIONS OF AN ANALYTE IN A SAMPLE

1 . A multiplex method for detecting different analytes in a sample, the method comprising:

(i) contacting the sample with at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes interacting with a different analyte, wherein if the analyte is a nucleic acid each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically interact with different sub-structures of the same analyte, each analyte-specific probe comprising

(aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and

(bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence),

wherein the analyte-specific probes of a particular set of analyte-specific probes differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T),

wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and

contacting the sample with at least two different sets of analyte-specific probes for at least one analyte and a variation thereof,

wherein the analyte-specific probes comprised in these different sets interacting with the same analyte, but specifically interact with different sub-structures of the same analyte,

wherein the analyte-specific probes of the first set of analyte-specific probes interacts with a sub-structure which is comprised in all variations of an analyte,

wherein the analyte-specific probes of the second set of analyte-specific probes (subgroup-specific probes) interacts with a sub-structure which is comprised only in a specific variation of the analyte,

wherein the analyte-specific probes of the first set of analyte-specific probes comprise the same identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), and

wherein the analyte-specific probes of the second set of analyte-specific probes comprise the same identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence),

wherein the identifier elements (T) of the analyte-specific probes of the first set of analyte-specific probes and the identifier elements (T) of the analyte-specific probes of the second set of analyte-specific probes are different for binding different decoding oligonucleotides and/or non-signal decoding oligonucleotides.

(ii) contacting the sample with at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises:

(aa) an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and

(bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide;

wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the first connect element (t); and

(iii) contacting the sample with at least a set of signal oligonucleotides, each signal oligonucleotide comprising:

(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and

(bb) a signal element;

(iv) detecting the signal caused by the signal element;

(v) selectively removing the decoding oligonucleotides and signal oligonucleotides from the sample, thereby essentially maintaining the specific binding of the analyte-specific probes to the analytes to be encoded; and

(vi) performing at least three (3) further cycles comprising steps (ii) to

(v) to generate an encoding scheme with a code word per analyte.

2 . The method according to claim 1 , wherein the set of analyte-specific probes comprises at least five (5) subgroup-specific probes which specifically interact with different sub-structures of the same variation of an analyte.

3 . The method according to claim 1 , comprising at least one (1) further cycle comprising steps (ii) to (v) to identify the subgroup-specific probes, wherein in particular the cycle may stop with step (iv).

4 .- 5 . (canceled)

6 . The method according to claim 1 , wherein all steps are automated.

7 . (canceled)

8 . The method according to claim 1 , wherein each analyte is associated with a specific code word, wherein said code word comprises a number of positions, and wherein each position corresponds to one cycle resulting in a plurality of distinguishable encoding schemes with the plurality of code words.

9 . (canceled)

10 . The method according to claim 1 wherein the code words obtained for the individual analytes in the performed cycles comprise the detected signals and additionally at least one element corresponding to no detected signal.

11 .- 12 . (canceled)

13 . The method according to claim 1 , wherein the code word zero (0) is generated by using no decoding oligonucleotides having an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for an individual analyte.

14 . The method according to claim 1 , wherein if at least for one individual analyte a position of the code word is zero (0) in this cycle no corresponding decoding oligonucleotides having an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of a corresponding analyte-specific probe for an individual analyte are used.

15 . The method according to claim 1 , wherein the sample is contacted with at least two different sets of signal oligonucleotides, wherein the signal oligonucleotides in each set comprise a different signal element and comprise a different connector element (C).

16 .- 19 . (canceled)

20 . The method according to claim 1 , wherein

the sample is contacted with:

at least two (2) different sets of non-signal decoding oligonucleotides for binding to at least two different identifier elements (T) of analyte-specific probes, each set of non-signal decoding oligonucleotides interacting with a different identifier element (T)

wherein each non-signal decoding oligonucleotide comprises an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of a unique identifier sequence, and does not comprise a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide.

21 .- 22 . (canceled)

23 . The method according to claim 1 , wherein

the sample is contacted with

a set of non-signal oligonucleotides, each non-signal oligonucleotide comprising:

(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of the translator element (c), and

(bb) a quencher (Q), a signal element and a quencher (Q), or does not comprise a signal element.

24 .- 43 . (canceled)

44 . The method according to claim 1 , wherein the binding element (S) comprises moieties which are affinity moieties from affinity substances or affinity substances in their entirety selected from the group consisting of antibodies, antibody fragments, anticalin proteins, receptor ligands, enzyme substrates, lectins, cytokines, lymphokines, interleukins, angiogenic or virulence factors, allergens, peptidic allergens, recombinant allergens, allergen-idiotypical antibodies, autoimmune-provoking structures, tissue-rejection-inducing structures, immunoglobulin constant regions and combinations thereof.

45 . The method according to claim 1 , wherein the binding element (S) is an antibody or an antibody fragment selected from the group consisting of Fab, scFv; single domain, or a fragment thereof, bis scFv, Fab2, Fab3, minibody, diabody, triabody, tetrabody and tandab.

46 . The method according to claim 1 , wherein the signal caused by the signal element, therefore in particular the binding of the signal oligonucleotides to the decoding oligonucleotides, interacting with the corresponding analyte probes, bound to the respective analyte is determined by at least one approach selected from the list consisting of

Imaging at least a portion of the sample;

Using an optical imaging technique;

Using a fluorescence imaging technique;

Multi-color fluorescence imaging technique; and

Super-resolution fluorescence imaging technique.

47 . The method according to claim 1 , wherein the decoding oligonucleotides in at least one set of decoding oligonucleotides are multi-decoders comprising

(aa) an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and

(bb) at least two translator elements (c), wherein the translator elements comprising different nucleotide sequences allowing a specific hybridization of a different signal oligonucleotide.

48 . The method according to claim 47 , wherein the different signal oligonucleotides comprise a different signal element and comprise a different connector element (C).

49 . A kit for multiplex analyte encoding, comprising

(i) at least twenty (20) different sets of analyte-specific probes for encoding of at least 20 different analytes, each set of analyte-specific probes interacting with a different analyte, wherein if the analyte is a nucleic acid each set of analyte-specific probes comprises at least five (5) analyte-specific probes which specifically interact with different sub-structures of the same analyte, each analyte-specific probe comprising

(aa) a binding element (S) that specifically interacts with one of the different analytes to be encoded, and

(bb) an identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence),

wherein the analyte-specific probes of a particular set of analyte-specific probes differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T),

wherein the analyte-specific probes in each set of analyte-specific probes binds to the same analyte and comprises the same nucleotide sequence of the identifier element (T) which is unique to said analyte; and

(ii) at least one set of decoding oligonucleotides per analyte, wherein in each set of decoding oligonucleotides for an individual analyte each decoding oligonucleotide comprises:

(aa) an identifier connector element (t) comprising a nucleotide sequence which is essentially complementary to at least a section of the unique identifier sequence of the identifier element (T) of the corresponding analyte-specific probe set, and

(bb) a translator element (c) comprising a nucleotide sequence allowing a specific hybridization of a signal oligonucleotide;

wherein the decoding oligonucleotides of a set for an individual analyte differ from the decoding oligonucleotides of another set for a different analyte in the identifier connect element (t); and

(iii) a set of signal oligonucleotides, each signal oligonucleotide comprising:

(aa) a translator connector element (C) comprising a nucleotide sequence which is essentially complementary to at least a section of the nucleotide sequence of a translator element (c) comprised in a decoding oligonucleotide, and

(bb) a signal element.

50 . The kit according to claim 49 , wherein the kit comprises at least two different sets of analyte-specific probes for an analyte,

wherein the analyte-specific probes comprised in these different sets interacting with the same analyte, but specifically interact with different sub-structures of the same analyte,

wherein the analyte-specific probes of the first set of analyte-specific probes interacts with a sub-structure which is comprised in all variations of an analyte,

wherein the analyte-specific probes of the second set of analyte-specific probes (subgroup-specific probes) interacts with a sub-structure which is comprised only in a specific variation of the analyte,

wherein the analyte-specific probes of the first set of analyte-specific probes comprise the same identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence), and

wherein the analyte-specific probes of the second set of analyte-specific probes comprise the same identifier element (T) comprising a nucleotide sequence which is unique to the analyte to be encoded (unique identifier sequence),

wherein the identifier elements (T) of the analyte-specific probes of the first set of analyte-specific probes and the identifier elements (T) of the analyte-specific probes of the second set of analyte-specific probes are different.

51 . The kit according to claim 50 , wherein the kit comprises at least five (5) sets of subgroup-specific probes that differ from the analyte-specific probes of another set of analyte-specific probes in the nucleotide sequence of the identifier element (T).

52 .- 54 . (canceled)

55 . The kit according to claim 49 , wherein the kit comprises at least two different sets of signal oligonucleotides, wherein the signal oligonucleotides in each set comprise a different signal element and comprise a different connector element (C).

56 .- 132 . (canceled)