Method for diagnosis of high-affinity binders and marker sequences
The present invention relates to a novel method for diagnosing high-affinity binders, in particular antibodies or autoantibodies, and the identification, characterization and selection of marker sequences and diagnostic use thereof, in particular in the form of a panel. The invention also relates to a singleplex assay in which the discovered selection of marker sequences is used in the form of a panel and high-affinity binders are detected using a single signal.
1. A method for the identification and/or selection of marker sequences suitable for use in a singleplex assay for detection and/or diagnosis and/or stratification of a disease in a subject, comprising the following steps:
a) mixing n marker sequences placed on a solid substrate with a sample containing high-affinity binders and detecting a single signal resulting from the interactions between the n marker sequences and the high-affinity binders,
b) mixing at least n−1 marker sequences having statistically sufficient signal intensity identified from a) with said sample containing high-affinity binders and detecting a single signal resulting from the interaction between the at least n−1 marker sequences and the high-affinity binders,
c) optionally repeating step b) with at least n-k marker sequences, wherein k>1, and
d) selecting n−k marker sequences having statistically sufficient signal intensity identified from b) or c) for use in a singleplex assay for detection and/or diagnosis and/or stratification of a disease in a subject,
wherein the n−k marker sequences selected in d) comprise at least 4 different marker sequences.
2. The method of claim 1 , wherein the marker sequences are obtained from biological material.
3. The method of claim 2 , wherein the biological material is selected from the group consisting of tissue, native sources, cells, bacteria, viruses, phages, prions, plants, animals and humans.
4. The method of claim 1 , wherein the marker sequences are mRNA, si-RNA, microRNA, cDNA, peptide, protein, or originate from an expression library.
5. The method of claim 4 , wherein the protein is an antigen or an autoantigen.
6. The method of claim 4 , wherein the expression library is an mRNA, si-RNA, microRNA, cDNA, peptide or protein expression library.
7. The method of claim 1 , wherein the high-affinity binders are antibodies.
8. The method of claim 1 , wherein the high-affinity binders are autoantibodies.
9. The method of claim 1 , wherein the n-k marker sequences selected in d) are applied to a substrate.
10. The method of claim 9 , wherein the substrate is a filter, a membrane, a magnetic or fluorophore labelled bead, a silicon wafer, glass, metal, plastic, a chip, a mass spectrometry target or a matrix.
11. The method of claim 1 , wherein the sample containing high-affinity binders is a bodily fluid or tissue extract.
12. The method of claim 11 , wherein the high affinity binders are antibodies or autoantibodies and the bodily fluid is blood, whole blood, blood plasma, blood serum, patient serum, urine, cerebrospinal fluid, or synovial fluid.
13. The method of claim 1 , wherein the signals are detected with the aid of radioactive or fluorescently labelled antibodies by means of a bioanalytical method or a mass spectrometry method, wherein the bioanalytical method is optionally Western blotting (1D and 2D), immunohistochemistry, antibody arrays, Luminex, ELISA, immunofluorescence, radioimmunoassays, and wherein the mass spectrometry methods are optionally MRM (multi reaction monitoring) or AQUA (absolute quantification).
14. The method of claim 1 , wherein the n-k marker sequences selected in d) comprise at least 10 different marker sequences.
15. The method of claim 1 , wherein the marker sequences are antigens, parts of antigens, haptens or proteins.