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GSJ: Received June 5, 2005:
http://wbabin.net/saba/saba50.htm
Molecules Which Only Recognize Ligand-Bound Receptors
James Saba
Herein is a means of isolating and utilizing molecules, especially proteins, which only bind to a receptor if it is bound to a ligand.
A 'ligand' as defined herein could be relatively large, and even be a virus or cell. "Receptors" are preferably proteins and polynucleotides. A "protein" comprises two or more amino acids, and thus includes peptides.
In essence the method utilizes negative selection of encoded libraries, as exemplified in Figure 1 wherein an antibody-display phage which only binds to ligand-bound receptor is isolated.
Other encoded libraries could be utilized, for example cell surface display, cis-expression, or bead-based libraries. Conceivably, even nonencoded libraries could be utilized, such as those whose members can be distinguish by mass spectrometry.
Once the encoded library member which selectively binds ligand-bound receptor is identified is has considerable utility.
For example, it may have the capacity to stabilize or make more specific a drug-receptor interaction (1,2).
Another, probably more important application is in development of assays for a virtually limitless number of ligands. Importantly, an advantage of this method relative to assays which target the ligand, is that the washing step after contacting the ligand with the support-affixed receptor is avoided.
The assay could be made completely one-step using antibody and complement C1 or rolling circle amplification (RCA) as previously described (3,4).
Of course, arrays (including microarrays) of different receptors are of considerable utility in multiplex analyses and assays.
This invention is considered valuable and a US patent application is anticipated to be filed in the very near future. However, it is hoped that others with laboratory facilities will investigate its full potential, perhaps even to establish a collaboration with the inventor.
The following condensed provisional claims are an attempt to encompass important aspects of this invention.
2) The method of 1, which utilizes negative selection of an encoded library.
3) A molecule which only binds a ligand-bound receptor, isolated by the method of either 1 or 2.
4) A process for detecting a ligand, which comprises contacting the ligand with a support-affixed receptor, and generating a signal independent of removing unbound ligand.
4) A kit for assaying one or more ligands which utilizes one or more molecules as defined in claim 3.
5) The kit of claim 4 which also comprises an array of different receptors.
2) Allosteric Drug Discovery Utilizing Microarrayed
Ligands.
Saba, JA Gen Sci J 2005 June 1
3) Homogeneous One-Pot Immunoassays Utilizing
Complement C1 and Fluorogenic Peptide Substrate.
Saba, JA Gen Sci J 2005 May 8
4) Immunoassays and Arrayassays Utilizing
Oligonucleotide-conjugated Antibodies.
Saba, JA Gen
Sci J 2005 May 16
Regarding Figure 1, it has been recognized that the phage specific for the ligand-bound target could be labeled. So doing a homogenous assay could be designed wherein the proximity and/or concentration of label at the surface of the target-affixed support would indicat the presence of the ligand, as exemplified in Figure 2.
A more eleaborate process would perhaps involve FRET, both the phage and target (or support) where labeled.
Figure 3 is yet another of what appear to be several derivations, this time the phage being affixed, perhaps arrayed, on the support.
Furthermore, in contrast to Figures 1 & 2, the phage in this example does not recognize an allosteric change, but an epitope formed by the combination of the target and ligand.
Recently disclosed were new and interesting methods of making high density targets, phage or antibodies (1).
Conceivably, labeling is not a necessity, as exemplified by the following references.
Label-free detection of nucleic acid and protein microarrays by scanning Kelvin nanoprobe. Thompson, et al Biosens Bioelectron. 2005 Feb 15;20(8):1471-81
Readout of protein microarrays using intrinsic time resolved UV fluorescence for label-free detection. Striebel, et al Proteomics. 2004 Jun;4(6):1703-11
High-density miniaturized thermal shift assays as a general strategy for drug discovery. Pantolianno, et al J Biomol Screen. 2001 Dec;6(6):429-40
A label-free optical technique for detecting small molecule interactions. Lin, et al Biosens Bioelectron. 2002 Sep;17(9):827-34
Pattern-Based Detection of Different Proteins Using an Array of Fluorescent Protein Surface Receptors Baldini, et al J. Am. Chem. Soc., 126 (18), 5656 -5657, 2004