Compound arrays for sample profiling

Abstract

The invention provides arrays of compound for use in profiling samples. The arrays include compounds bind to components of the samples at relatively low affinities. The avidity of compounds binding to components of the samples can be increased by forming arrays such that multivalent components of the samples (e.g., antibodies or cells) can bind to more than one molecule of a compound at the same time. When a sample is applied to an array under such conditions, the compounds of the array bind to component(s) of the sample with significantly different avidities generating a profile characteristic of the sample.

What is claimed is: 
     
         1 . A method of analyzing a sample, comprising:
 (a) contacting the sample with an array of immobilized different compounds occupying different areas of the array, wherein different molecules of the same compound within an area are spaced sufficiently proximate to one another for multivalent binding between at least two of the different molecules in the same area and a multivalent binding partner; and   (b) detecting binding of the different compounds in the array to component(s) of the sample, such as antibodies.   
     
     
         2 . The method of  claim 1 , further comprising characterizing the sample from the relative binding of compounds whose binding to the sample would be difficult to distinguish from each other and nonspecific binding under conditions of monovalent binding but which show significantly different binding from each other and nonspecific binding due to multivalent binding of the sample to the compounds thereby generating a binding profile characteristic of the sample. 
     
     
         3 . The method of  claim 1 , wherein the average spacing between different molecules of a compound in an area of the array is less than 6 nm. 
     
     
         4 . The method of  claim 1 , further comprising characterizing the sample from the relative binding a plurality of the compounds binding to the sample with association constants of 1 mm to 1 μM. 
     
     
         5 . The method of  claim 4 , wherein the plurality of compounds includes at least 10 or 100 compounds binding to the sample with association constants of 1 mm to 1 μM. 
     
     
         6 . The method of  claim 5 , wherein the characterizing comprises comparing a binding profile of the sample that includes the relative binding of the plurality of compounds with a reference binding profile. 
     
     
         7 . The method of  claim 1 , further comprising identifying a component of the sample that binds to the different compounds. 
     
     
         8 . The method of  claim 6 , further comprising detecting the identified component with a binding partner known to bind the component. 
     
     
         9 . The method of  claim 7 , wherein the identified component is detected with a plurality of different binding partners known to bind the component. 
     
     
         10 . The method of  claim 7 , wherein the binding partner is an antibody to the identified component or a peptide known to bind the identified component. 
     
     
         11 . The method of  claim 7 , wherein the binding partner is one of the different compounds detected in step (b). 
     
     
         12 . The method of  claim 7 , wherein the binding partner is a synbody. 
     
     
         13 . The method of  claim 7 , wherein the binding partner is immobilized to a support. 
     
     
         14 . The method of  claim 13 , wherein the binding partner is immobilized to a support in an array. 
     
     
         15 . The method of  claim 14 , further comprising forming a second array, the second array containing one or more of the different compounds in the array binding to identified component not all of the different compounds in the array. 
     
     
         16 . The method of  claim 1 , wherein the second array contains less than 5% of the different compounds in the array. 
     
     
         17 . The method of  claim 1 , further comprising forming an array or other device comprising compounds determined to bind to the sample but not all of the different compounds in the array. 
     
     
         18 . The method of  claim 1 , wherein the detecting step detects binding of the different compounds to an antibody or antibodies in the sample. 
     
     
         19 . The method of  claim 1 , wherein the detecting step detects binding of the different compounds to a biological entity displaying multiple copies of a protein from its outersurface. 
     
     
         20 . The method of  claim 1 , wherein the biological entity is cell displaying multiple copies of receptor from its outersurface. 
     
     
         21 . The method of  claim 1 , wherein the compound having strongest binding to the sample binds to the sample with a dissociation constant of 1 mM to 1 μM. 
     
     
         22 . The method of  claim 1 , wherein the compounds are peptides or small molecules. 
     
     
         23 . The method of  claim 1 , wherein the array has 500-50,000 peptides. 
     
     
         24 . The method of  claim 23 , wherein the peptides are 10−30 amino acid long. 
     
     
         25 . The method of  claim 24 , wherein the sequences of the peptides are randomly selected. 
     
     
         26 . The method of  claim 1 , wherein the different immobilized compounds are selected without regard to the sample and the array further comprises a plurality of compounds known to bind different proteins also occupying different areas of the array. 
     
     
         27 . The method of  claim 26 , wherein the plurality of compounds known to bind different proteins includes compounds known to bind at least 25%, 50 or 75% of different human proteins. 
     
     
         28 . The method of  claim 27 , wherein the different immobilized compounds including a plurality of compounds known to bind at least 25%, 50% or 75% of different human proteins and 500-50,000 random peptides. 
     
     
         29 . The method of  claim 23 , wherein the sequences of the peptides have less than 90% sequence identity to a known binding partner of the target. 
     
     
         30 . The method of  claim 23 , wherein the sequences of the peptides have less than 90% sequence identity to known proteins. 
     
     
         31 . The method of  claim 1 , wherein the contacting of the sample to the array is performed in the presence of a potential competitor of binding of the sample to the array. 
     
     
         32 . The method of  claim 31 , wherein the competitor is a known binding partner of a suspected component of the sample. 
     
     
         33 . The method of  claim 1 , wherein the sample is a patient sample, and the competitor is a protein known to be associated with a disease affecting the patient. 
     
     
         34 . The method of  claim 1 , wherein the sample is a patient sample. 
     
     
         35 . The method of  claim 1 , wherein the sample contains a plurality of antibodies. 
     
     
         36 . The method of  claim 34 , wherein the patient is known or suspected to be suffering from a disease. 
     
     
         37 . The method of  claim 34 , wherein the patient is known to be at risk of a disease but is not showing symptoms of the disease. 
     
     
         38 . The method of  claim 36  or  37 , wherein the disease is an autoimmune disease. 
     
     
         39 . The method of  claim 36  or  37 , wherein the disease is an infectious disease. 
     
     
         40 . The method of  claim 36  or  37 , wherein the disease is a disease of the CNS. 
     
     
         41 . The method of  claim 33 , wherein the sample is a blood, urine, or CNS sample. 
     
     
         42 . The method of  claim 1 , wherein component(s) of the sample are labeled. 
     
     
         43 . The method of  claim 1 , wherein binding of the peptides to component(s) of the sample is detected using a, secondary antibody. 
     
     
         44 . The method of  claim 43 , wherein the secondary antibody is an isotype-specific antibody. 
     
     
         45 . The method of  claim 1 , wherein binding of the peptides to component(s) of the sample is detected by spr or mass spectrometry. 
     
     
         46 . The method of  claim 1 , further comprising affinity purifying a component of the sample using a peptide determined to bind to the sample. 
     
     
         47 . The method of  claim 1 , further comprising washing unbound component(s) of the sample from the array, and dissociating bound component(s) from the array. 
     
     
         48 . The method of  claim 47 , further comprising preparing an antibody library from the patient and using a peptide to which an antibody in the sample binds as an affinity reagent to screen the library. 
     
     
         49 . The method of  claim 48 , further comprising identifying a natural binding partner of the affinity purified antibody. 
     
     
         50 . The method of  claim 23 , further comprising comparing the sequence(s) of peptide(s) binding to the component(s) of the sample to a database of natural sequences to identify natural binding partner(s) of components of the sample. 
     
     
         51 . The method of  claim 50 , further comprising comparing a profile of different compounds binding to the sample with profiles of the different compounds associated with different diseases or different stages of a disease to diagnose a patient as having one of the diseases or stages of disease. 
     
     
         52 . The method of  claim 50 , further comprising comparing a profile of different compounds binding to the sample with a profile of the different compounds associated with lack of a disease to determine whether a disease is present. 
     
     
         53 . The method of  claim 50 , further comprising repeating the method for different samples from a plurality of patients with the same disease to develop a binding profile characteristic of the disease. 
     
     
         54 . The method of  claim 50 , further comprising repeating the method for different samples from a plurality of patients with different disease to develop a plurality of binding profiles characteristic of different diseases. 
     
     
         55 . An array of immobilized different compounds occupying different areas of the array, wherein different molecules of the same compound within an area are spaced sufficiently proximate to one another for multivalent binding between at least two of the different molecules in the same area and a multivalent binding partner. 
     
     
         56 . A method of analyzing a sample, comprising:
 contacting the sample with an array of immobilized different peptides occupying different areas of the array;   detecting binding of the different peptides in the array to component(s) of the sample; and   characterizing the sample from the relative binding of a plurality of peptides with dissociation constants between 1 mM and 1 μM for the sample.   
     
     
         57 . The method of  claim 56 , wherein the sample is analyzed from the relative binding of at least ten peptides with dissociation constants between 1 mM and 1 μM. 
     
     
         58 . A method of analyzing a sample, comprising contacting the sample with an array of immobilized different compounds occupying different areas of the arrays;
 detecting binding of the different compounds in the array to component(s) of the sample; and   characterizing the sample from the relative binding of a plurality of compounds having binding strengths to the sample greater than but within three orders of magnitude of the mean plus three standard deviations of the binding strength of empty areas in the array.   
     
     
         59 . The method of  claim 58 , wherein the characterizing comprising comparing a binding profile of the sample including the plurality of compounds with a reference binding profile including the plurality of compounds. 
     
     
         60 . The method of  claim 58 , wherein the sample is characterized from the relative binding of at least 10 or 100 compounds having binding strengths to the sample greater than but within three orders of magnitude of the mean plus three standard deviations of the binding strength of empty areas in the array. 
     
     
         61 . A method of characterizing a plurality of different samples, comprising
 contacting the different samples with the same array or copies of the same   array of immobilized different peptides occupying different areas of the array; and   
       detecting different binding profiles of the different peptides to the different samples; wherein the samples are characterized from their respective binding patterns. 
     
     
         62 . The method of  claim 61 , wherein the plurality of different samples includes samples from patients with different disease symptoms. 
     
     
         63 . The method of  claim 61 , wherein the plurality of different samples includes samples of patients presenting with disease and lack of disease 
     
     
         64 . A method of analyzing a sample, comprising:
 contacting the sample with an array of immobilized different compounds occupying different areas of the array, wherein different molecules of the same compound within an area are spaced at an average distance of less than 4 nm apart in the same area; and   detecting binding of the different compounds in the array to component(s) of the sample.   
     
     
         65 . A method of analyzing a sample, comprising
 (i) contacting a sample with a known binding partner of a component of the sample; and   (ii) determining whether the binding partner binds to the sample compared with a control lacking the component;   wherein the known binding partner is identified by a process comprising   (a) contacting an initial sample with an array of immobilized different compounds occupying different areas of the array, wherein different molecules of the same compound within an area are spaced sufficiently proximate to one another for multivalent binding between at least two of the different molecules in the same area and a multivalent binding partner; and   (b) detecting binding of the different compounds in the array to component(s) of the sample;   (c) identifying a component of the sample that binds to the different compounds; and   (d) identifying a known binding partner of the component for use in step (i).   
     
     
         66 . The method of  claim 65 , wherein the component in step (i) is detected with a plurality of different binding partners known to bind the component. 
     
     
         67 . The method of  claim 65 , wherein the binding partner is an antibody to the component. 
     
     
         68 . The method of  claim 65 , wherein the binding partner is a peptide known to bind the component. 
     
     
         69 . The method of  claim 65 , wherein the binding partner is one of the different compounds detected in step (b). 
     
     
         70 . The method of  claim 65 , wherein the binding partner is a synbody. 
     
     
         71 . The method of  claim 65 , wherein the binding partner is immobilized to a support in step (i). 
     
     
         72 . The method of  claim 65 , wherein the binding partner is immobilized to a support in an array in step (i). 
     
     
         73 . A method of manufacturing a device for use in detecting a component of a sample comprising;
 (a) contacting the sample with an array of immobilized different compounds occupying different areas of the array, wherein different molecules of the same compound within an area are spaced sufficiently proximate to one another for multivalent binding between at least two of the different molecules in the same area and a multivalent binding partner; and   (b) detecting binding of the different compounds in the array to component(s) of the sample.   (c) forming a device including a known binding partner of a component to which binding of the different compounds is detected in step (b).   
     
     
         74 . The method of  claim 73 , further comprising identifying the component of the sample that binds to the different compounds. 
     
     
         75 . The method of  claim 73 , wherein step (c) comprising forming the device including a plurality of different binding partners known to bind the component. 
     
     
         76 . The method of  claim 73 , wherein the binding partner is an antibody to the component. 
     
     
         77 . The method of  claim 73 , wherein the binding partner is a peptide known to bind the component. 
     
     
         78 . The method of  claim 73 , wherein the binding partner is one of the different compounds detected in step (b). 
     
     
         79 . The method of  claim 73 , wherein the binding partner is a synbody. 
     
     
         80 . The method of  claim 73 , wherein the binding partner is immobilized to a support. 
     
     
         81 . The method of  claim 73 , wherein the binding partner is immobilized to a support in an array. 
     
     
         82 . The method of  claim 73 , wherein step (c) comprising forming a second array, the second array containing one or more of the different compounds in the array binding to component(s) of the sample and not all of the different compounds in the array. 
     
     
         83 . The method of  claim 82  wherein the second array contains less than 5% of the different compounds in the array. 
     
     
         84 . An array of immobilized different compounds occupying different areas of the array, wherein the different compounds include a plurality of compounds known to bind at least 25, 50 or 75% of the known human proteins and 500-50,000 random peptides, wherein different molecules of the same peptide within an area are spaced sufficiently proximate to one another for multivalent binding between at least two of the different molecules in the same area and a multivalent binding partner. 
     
     
         85 . A method of testing a vaccine, comprising
 contacting a blood sample of a subject immunized with a vaccine against a pathogenic microorganism with an array of immobilized different compounds occupying different areas of the array;   detecting a pattern of binding of the sample to the different compounds in the array; and   comparing the pattern of binding to the pattern of binding of one or more reference samples, wherein the reference samples are from subjects who have survived an infection with the virus, similarity of binding profile between the subject and reference samples providing an indication the vaccine is effective against the pathogenic microorganism.   
     
     
         86 . The method of  claim 85 , wherein the subjects have been immunized with a vaccine before exposure to the virus.