Method of Attaching a Ligand to a Test Strip

Abstract

The invention features a method of attaching a ligand that has a free carboxyl group to a solid support by adding an amino group to the ligand to form a ligand-amino derivative, converting the ligand amino derivative to a ligand sulfhydryl derivative, attaching the ligand sulfhydryl derivative to a protein to form a ligand-linker-protein conjugate, and applying the ligand-linker-protein conjugate to the solid support. The method is particularly useful for immobilizing small molecule ligands having a free carboxyl group, such as cloxicillin, to a lateral-flow test strip, in order to make a detection zone on the test strip that exhibits a clear signal and enhanced sensitivity.

What is claimed is: 
     
         1 . A method of attaching a ligand to a solid support, the method comprising:
 (a) providing a ligand, said ligand having a free carboxyl group;   (b) adding an amino group to said ligand to form a ligand-amino derivative;   (c) converting said ligand amino derivative to a ligand sulfhydryl derivative;   (d) joining said ligand sulfhydryl derivative to a protein to form a ligand-linker-protein conjugate, wherein said linker ; and   (e) applying said ligand-linker-protein conjugate to said solid support.   
     
     
         2 . The method of  claim 1  wherein said ligand is a small molecule ligand. 
     
     
         3 . The method of  claim 1  wherein said ligand is an antibiotic. 
     
     
         4 . The method of  claim 3 , wherein said ligand is cloxacillin. 
     
     
         5 . The method of  claim 1  wherein said protein is a carrier protein. 
     
     
         6 . The method of  claim 1 , wherein said protein is an albumin. 
     
     
         7 . The method of  claim 6 , wherein said protein is bovine serum albumin (BSA). 
     
     
         8 . The method of  claim 1 , wherein said adding step comprises activating said carboxyl group to form a ligand reactive species, and adding said amino group to said ligand reactive species to form said ligand amino derivative. 
     
     
         9 . The method of  claim 8 , wherein said activating comprises reacting said carboxyl group with a zero-length cross linker 
     
     
         10 . The method of  claim 8 , wherein said activating comprises reacting said carboxyl group with a carbodiimide. 
     
     
         11 . The method of  claim 8 , wherein said activating is in the presence of an N-hydroxysuccinimide ester. 
     
     
         12 . The method of  claim 8 , wherein said activating comprises reacting said carboxyl group with a carbonylating compound. 
     
     
         13 . The method of  claim 1 , wherein said amino group is a diamine. 
     
     
         14 . The method of  claim 13 , wherein said diamine is 1,3-diamino-2-hydroxypropane. 
     
     
         15 . The method of  claim 13 , wherein said diamine is selected from the group consisting of 1,4-diamino-2-butanone, diaminoethylene, ethylene diamine, 1,1-diaminohexane, diaminodipropylamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, and 1,4-diaminobutane. 
     
     
         16 . The method of  claim 1 , wherein said converting step comprises thiolation. 
     
     
         17 . The method of  claim 16 , wherein said thiolation comprises the use of a thio-containing imidoester. 
     
     
         18 . The method of  claim 16 , wherein said thiolation comprises the use of S-acetylmercaptosuccinic  anhydride. 
     
     
         19 . The method of  claim 16 , wherein said thiolation comprises the use of a thiol-containing succinimidyl derivative. 
     
     
         20 . The method of  claim 16 , wherein said thiolation comprises the use of a compound selected from the group consisting of 3-(3-acetylthiopropionyl)thiazolidine-2-thione and 3-(3-p-methoxybenzylthiopropionyll)thiazolidine-2-thione. 
     
     
         21 . The method of  claim 1 , wherein said joining comprises reacting said protein with a heterobifunctional cross-linking reagent to form said linker-protein conjugate, and reacting said linker-protein conjugate with said ligand sulfhydryl derivative. 
     
     
         22 . The method of  claim 21 , wherein said heterobifunctional cross-linking reagent is an amino and sulfhydryl group directed bifunctional reagent. 
     
     
         23 . The method of  claim 21 , wherein said heterobifunctional cross-linking reagent comprises a maleimide and a NHS ester. 
     
     
         24 . The method of  claim 23 , wherein said heterobifunctional cross-linking reagent is sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate. 
     
     
         25 . The method of  claim 1 , wherein said solid support comprises nitrocellulose. 
     
     
         26 . The method of  claim 1 , wherein said solid support comprises polyethylene. 
     
     
         27 . A lateral-flow test strip comprising the solid support of  claim 25 . 
     
     
         28 . A lateral-flow test strip comprising the solid support of  claim 26 . 
     
     
         29 . A method of attaching a ligand to a solid support, the method comprising:
 (a) providing a ligand having a free carboxyl group;   (b) adding an amino group to said ligand to form a ligand-amino derivative;   (c) joining said ligand amino derivative to a protein having a free amino group through an amino group directed homobifunctional cross-linker, to form a ligand-spacer-protein conjugate; and   (d) applying said ligand-spacer-protein conjugate to said solid support.   
     
     
         30 . The method of  claim 29 , wherein said adding step comprises activating said carboxyl group to form a ligand reactive species, and adding said amino group to said ligand reactive species to form said ligand amino derivative. 
     
     
         31 . The method of  claim 29 , wherein said amino group directed homobifunctional cross-linker is a NHS ester reagent. 
     
     
         32 . The method of  claim 29 , wherein said amino group directed homobifunctional cross-linker is a N-hydroxysuccinimide ester derivative reagent. 
     
     
         33 . The method of  claim 29 , wherein said solid support comprises nitrocellulose. 
     
     
         34 . The method of  claim 29 , wherein said solid support comprises polyethylene. 
     
     
         35 . A lateral-flow test strip comprising the solid support of  claim 33 . 
     
     
         36 . A lateral-flow test strip comprising the solid support of  claim 34 . 
     
     
         37 . A lateral flow test strip comprising a solid support and a capture agent attached to said solid support, said attachment prepared by:
 (a) providing a ligand, said ligand having a free carboxyl group;   (b) adding an amino group to said ligand to form a ligand-amino derivative;   (c) converting said ligand amino derivative to a ligand sulfhydryl derivative;   (d) joining said ligand sulfhydryl derivative to a protein to form a ligand-linker-protein conjugate; and   (e) applying said ligand-linker-protein conjugate to said solid support.   
     
     
         38 . The lateral flow test strip of  claim 37 , wherein said ligand is a small molecule ligand. 
     
     
         39 . The lateral flow test strip of  claim 37 , wherein said ligand is an antibiotic. 
     
     
         40 . The lateral flow test strip of  claim 39 , wherein said ligand is cloxacillin. 
     
     
         41 . The lateral flow test strip of  claim 37 , wherein said protein is a carrier protein. 
     
     
         42 . The lateral flow test strip of  claim 37 , wherein said protein is an albumin. 
     
     
         43 . The lateral flow test strip of  claim 37 , wherein said protein is bovine serum albumin (BSA). 
     
     
         44 . The lateral flow test strip of  claim 37 , wherein said adding step comprises activating said carboxyl group to form a ligand reactive species, and adding said amino group to said ligand reactive species to form said ligand amino derivative. 
     
     
         45 . The lateral flow test strip of  claim 44 , wherein said activating comprises reacting said carboxyl group with a zero-length cross linker 
     
     
         46 . The lateral flow test strip of  claim 44 , wherein said activating comprises reacting said carboxyl group with a carbodiimide. 
     
     
         47 . The lateral flow test strip of  claim 44 , wherein said activating is in the presence of an N-hydroxysuccinimide ester. 
     
     
         48 . The lateral flow test strip of  claim 44 , wherein said activating comprises reacting said carboxyl group with a carbonylating compound. 
     
     
         49 . The lateral flow test strip of  claim 37 , wherein said amino group is a diamine. 
     
     
         50 . The lateral flow test strip of  claim 49 , wherein said diamine is 1,3-diamino-2-hydroxypropane. 
     
     
         51 . The lateral flow test strip of  claim 49 , wherein said diamine is selected from the group consisting of 1,4-diamino-2-butanone, diaminoethylene, ethylene diamine, 1,1-diaminohexane, diaminodipropylamine, 1,3-diaminopropane, 1,3-diamino-2-hydroxypropane, and 1,4-diaminobutane. 
     
     
         52 . The lateral flow test strip of  claim 37 , wherein said converting step comprises thiolation. 
     
     
         53 . The lateral flow test strip of  claim 52 , wherein said thiolation comprises the use of a thio-containing imidoester. 
     
     
         54 . The lateral flow test strip of  claim 52 , wherein said thiolation comprises the use of S-acetylmercaptosuccinic  anhydride. 
     
     
         55 . The lateral flow test strip of  claim 52 , wherein said thiolation comprises the use of a thiol-containing succinimidyl derivative. 
     
     
         56 . The lateral flow test strip of  claim 52 , wherein said thiolation comprises the use of a compound selected from the group consisting of 3-(3-acetylthiopropionyl)thiazolidine-2-thione and 3-(3 -p-methoxybenzylthiopropionyll)thiazolidine-2-thione. 
     
     
         57 . The lateral flow test strip of  claim 37 , wherein said joining comprises reacting said protein with a heterobifunctional cross-linking reagent to form said linker-protein conjugate, and reacting said linker-protein conjugate with said ligand sulfhydryl derivative. 
     
     
         58 . The lateral flow test strip of  claim 57 , wherein said heterobifunctional cross-linking reagent is an amino and sulfhydryl group directed bifunctional reagent. 
     
     
         59 . The lateral flow test strip of  claim 57 , wherein said heterobifunctional cross-linking reagent comprises a maleimide and a NHS ester. 
     
     
         60 . The lateral flow test strip of  claim 59 , wherein said heterobifunctional cross-linking reagent is sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate. 
     
     
         61 . The lateral flow test strip of  claim 37 , wherein said solid support comprises nitrocellulose. 
     
     
         62 . The lateral flow test strip of  claim 37 , wherein said solid support comprises polyethylene. 
     
     
         63 . A lateral flow test strip comprising a solid support, and a capture agent attached to said solid support, said attachment between said solid support and said capture agent prepared by:
 (a) providing a ligand having a free carboxyl group;   (b) adding an amino group to said ligand to form a ligand-amino derivative;   (c) joining said ligand amino derivative to a protein having a free amino group through an amino group directed homobifunctional cross-linker, to form a ligand-spacer-protein conjugate;   and (d) applying said ligand-spacer-protein conjugate to said solid support.   
     
     
         64 . The lateral flow test strip of  claim 63 , wherein said adding step comprises activating said carboxyl group to form a ligand reactive species, and adding said amino group to said ligand reactive species to form said ligand amino derivative. 
     
     
         65 . The lateral flow test strip of  claim 63 , wherein said amino group directed homobifunctional cross-linker is a NHS ester reagent. 
     
     
         66 . The lateral flow test strip of  claim 63 , wherein said amino group directed homobifunctional cross-linker is a N-hydroxysuccinimide ester derivative reagent. 
     
     
         67 . The lateral flow test strip of  claim 63 , wherein said solid support comprises nitrocellulose. 
     
     
         68 . The lateral flow test strip of  claim 63 , wherein said solid support comprises polyethylene. 
     
     
         69 . A lateral flow test strip for detecting one or more analytes in a test sample, the test strip comprising:
 (a) a test zone, the test zone comprising a test zone capture agent having affinity to a labeled analyte receptor that is unbound by analyte from the sample but not to a labeled analyte receptor that is bound by analyte from the sample and further comprising an attachment to the test strip through a spacer linkage to a protein, said protein being attached to both the test strip and the spacer linkage,   (b) a control zone, the control zone located on the test strip spaced apart from the test zone and configured to provide a signal for comparison to a signal in the test zone,   c) a sample application area, the sample application area comprising unbound labeled analyte receptor, the unbound labeled receptor having affinity to an analyte to be detected so that the unbound labeled receptor reacts with the analyte to form analyte bound labeled receptor, the analyte bound labeled receptor having reduced affinity for the test zone capture agent due to said analyte being bound thereto,   wherein when the sample is added to the sample application area the analyte present can compete with the test zone capture agent for binding to the analyte receptor and wherein when the test zone capture agent binds analyte a visible result is observed in the test zone,   wherein the test is negative for the detection of the one or more analytes when the signal from the test zone is more intense than the signal from the control zone and the test is positive for the one or more analytes when the signal from the control zone is more intense than the signal from the test zone.   
     
     
         70 . A lateral-flow test method for detecting one or more analytes in a test sample, the method comprising:
 (a) attaching a binder for unbound labeled analyte receptor to a test strip;   (b) contacting the sample with the unbound labeled receptor;   (c) allowing the unbound labeled receptor to react with target analyte;   (d) contacting the unbound labeled receptor, previously allowed to react with target analyte, with the binder for unbound labeled receptor, wherein a portion of the receptor, unbound to analyte from the sample, will react with binder in the test zone generating a visible result, characterized in that the binder is attached to the test strip through a spacer linkage to a protein and wherein the protein is attached to both the membrane and the linkage.