Classifications

• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B30/00—Methods of screening libraries
  • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2458/00—Labels used in chemical analysis of biological material
  • G01N2458/10—Oligonucleotides as tagging agents for labelling antibodies
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

• the detection of complexes formed between the binding agents and their targets, and the identification of the binding agents is carried out, preferably in a plurality of compartments, by pair wise linkage of unique identification sequences of the bound target specific binding agents which are then translated to a nucleotide.
• the alteration of the quantity of complexes and the identity of the binding agents and optionally the target involved can be revealed by sequencing.
• modified nucleotides such as morpholino nucleotides, locked nucleic acids (LNA) and peptide nucleic acids (PNA) can be used.
• Morpholino oligonucleotides are assembled from different morpholino subunits, each of which contains one of the four genetic bases (adenine, cytosine, guanine, and thymine) linked to a 6-membered morpholine ring. The subunits are joined by non-ionic phosphorodiamidate intersubunit linkages to give a morpholino oligonucleotide.
• the binding agents are capable of detecting more than one target, preferably with different apparent affinities.
• the binding agents are capable of detecting a single target using different epitopes or binding sites, preferably with different apparent affinities.
• the binding characteristics of the members of the antibody phage library can be pre-determined. For example it can be determined which epitope is bound by the CDRs encoded and expressed binding agent (antibody) of the phage. This information can be associated with the unique nucleotide sequence which encodes the CDRs. Thus the epitope bound by the antibody expressed by the phage can be identified from the sequence of the unique nucleotide sequence. Once the epitope sequence present in the bound target is known, it may be possible to identify the protein or the group of proteins bound.
• enriched binding agent libraries is generally advantageous as the separation methods (for example microarrays or emulsion based separations) have limited capacity to produce individual complexes within one compartment, so that linked identifiable sequences can be obtained.
• the reduced complexity of an enriched display agent libraries is directly translatable to the number of the separations that need to be carried out to obtain separation based, random paired, joined amplification products.
• binding partners especially when complex protein mixtures are investigated. For example separation may result, on average, in a single unbound binding agent per
• the invention also relates to a method for determining the effects of a compound on protein-protein interaction present in a specimen comprising: (a) obtaining a protein mixture in the presence and absence of a compound;
• a multiplexed form of LGA has been developed.
• an antibody immobilized on a solid support acts as a capture reagent to locally enrich an antigen from a complex mixture of proteins.
• PKA proximity ligation assay
• This is followed by further washes and ligation of oligonucleotides brought in proximity.
• This enables higher specificity on the basis of the need for three binding events.
• This in combination with the use of PCR amplification allows high specificity and sensitivity, and a broad dynamic range for protein quantification.
• This method coupled with next generation sequencing (NGS) to digitally record patterns of protein abundance, and be used to demonstrate simultaneous detection of 36 protein analytes.
• NGS next generation sequencing
• Phage display is an enrichment process that requires multiple cycles to infer protein-protein interaction data. The enrichments are performed in vitro, which biases the interactions and favour high affinity interaction in detection. Certain proteins (particularly larger ones) are not well suited for analysis by phage display.
• a technology has been developed for identifying proteins that specifically bind predicted transcriptional regulatory elements using phage-display library of genomically encoded peptides, which bind to a surface immobilized double-stranded DNA, containing a DNA motif sequence of interest. After enrichment for a specific DNA-protein interaction, the bound phages are amplified, and the inserts from the enriched phage are sequenced to determine the interacting proteins using labelling and hybridization to DNA microarray.
• Example 2 Dimerisation PCR pBluescript II SK(+) phagemid vector (Agilent, 212205) f1 origin in (+) orientation, Sac->Kpn polylinker orientation in host strain: XL1 -Blue MRF' have been used to generate pBluescript II SK(+) phage and M13K07 Helper Phage (NEB, N0315S) was purchased. Phages was labeled with 20 molar excess of biotin according to the manufacturer instruction using EZ-Link Sulfo-NHS-Biotin (Thermo, 21326) and precipitated using isoelectric point precipitation as described (Dong D, Sutaria S, Hwangbo JY, Chen P.
• 5'CCGTCAATATTTACCTTCCC3' (M13K07 specific probe VIC-MGB labeled) were used to amplify the compartmentalized phages, the amplification were recorded in two different channels and droplet generation , PCR and detection were according to the manufacturer protocol. Increased "linkage" counts were expected in the presence of avidin due to biotin-avidin binding.
• the amplified DNA was extracted according to the manufacturer recommendation and subjected to PCR using primers 5TTTTCTGCGCGTAATCTGCT3'
• ACCTCTACAATGGATGCGCAGAGACGGTGACCAGGGTTCC To detect dimerised products a dimer specific real-time PCR reaction is designed: forward - AGTTGGAGTCTTGGGGTCAGG, reverse - AGGTGGGTCGATGTTTGACTACTG and probe - FAM TCTCACCTCTACAATGGAT MGB.
• Example 5 Predetermining the binding characteristics information of an antibody phage library
• the linkage information of bound phages is used to compute the binding characteristics information of the antibody phage library against the cDNA-phage library including determination of significant interactions on the basis of background subtracted linkage information (removal of promiscuous or random or incidental interactions); identification of significant antibody - cDNA bindings on the basis of statistically significant interactions; determination of the binding characteristics information for each detected antibody-phages including detected cDNA fragments, inferred detected proteins; confirmation, calculation of statistical error of the binding characteristics information on the basis of redundant measurements of the detection of several phages with the same binding characteristics information.

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• 2014
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