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.

Description

REFERENCE TO PRIOR APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 12 / 080,044, filed Mar. 31, 2008, which is a divisional of U.S. application Ser. No. 10 / 289,089, filed Nov. 6, 2002, now abandoned, which claims the benefit of provisional application No. 60 / 332,877, filed Nov. 6, 2001, each of which is hereby incorporated by reference.FIELD OF THE INVENTION[0002]The invention relates to attaching small molecule ligands to a solid support, e.g., a lateral-flow test strip.BACKGROUND OF THE INVENTION[0003]Lateral-flow test strips for detecting one or more analytes in a fluid sample generally include a reagent, or ligand, immobilized within a defined region of the test strip, variously referred to as a detection zone, a test zone, and / or a control zone. The ligand of choice has binding affinity for another reagent in the mobile phase of the test strip. If the mobile-phase reagent is detectably labeled, a detectable signal is generated within the region o...

AI Technical Summary

Benefits of technology

[0017]One aspect of achieving uniform and consistent test zone development is efficient attachment of the conjugate to the nitrocellulose. Another aspect of uniform and consistent test zone development is optimizing the conformation of the test zone conjugate so that it both attaches consistently to the nitrocellulose and maximizes the availability of the receptor-binding portion to the receptor. It is desirable to maximize the percentage of the conjugate that attaches to the nitrocellulose and assure even spacing across the test zone. At the same time, the capture portion of the binder conjugate molecule must remain readily available for binding unbound labeled receptor. If attachment to the nitrocellulose is inefficient and non-uniform, then some conjugate may remain unattached to the nitrocellulose. The unattached conjugate will interfere by competing with attached conjugate for binding to the labeled receptor. That is, some receptor that would otherwise bind in a clearly defined area will either bind in a poorly defined or wider area, or not at all, resulting in diffuse and generally poor, test zone development. Such a poor test zone is a problem whether or not an instrument, such as a reflectance reader, for example, a spectrophotometer, is used to analyze the results of a test. The problem, however, is more acute when reflectance reading is used to read and analyze test results. It is, therefore, an object of this invention to improve the quality of the test zone. We have found that adding a spacer linkage improves test zone quality.

[0019]In one embodiment, a labeled beta-lactam binding protein, unlabeled antibodies to ceftiofur, ampicillin and cephapirin, and labeled antibodies to cloxacillin are combined in a solution and applied, for example, by spraying, onto a porous surface. When exposed to a sample, for example of fluid milk, the antibodies and the binding protein bind to their related analytes to form receptor-analyte complexes. Unlabeled antibody reduces the amount of related analyte available to bind to the labeled binding protein, decreasing test sensitivity to that analyte. Similarly, labeled antibody will bind its related analyte thereby increasing test sensitivity to the analyte, above that of the binding protein alone. In the specific example of detecting beta-lactams in fluid milk, a labeled antibody to cloxacillin binds cloxacillin from the sample preferentially to beta-lactam binding protein providing increased test sensitivity to cloxacillin. Lateral capillary flow occurs carrying the analyte-receptor complex, and any unbound labeled receptor, along a membrane, such as nitrocellulose, to one or more test lines or zones positioned on the membrane.

Problems solved by technology

In the case of a multianalyte receptor, the affinity of certain analytes to the receptor may be different than others, and may not reflect the desired detection level for one or more of the analytes requiring detection.

In the case of gold, the amount used is limited by the necessity of rapid, consistent flow through the porous test strip.

A high percentage of gold in the spray solution may clog pores and inhibit flow.

The above-described limitation on the use of gold is more pronounced when a second test zone is employed, for example, a second test zone for cloxacillin.

Adding a second test zone to a test strip presents special challenges when trying to optimize signal development for each zone.

If there is a second test zone, which would require a second gold sol label, the technique of adding additional gold label to improve line quality is limited.

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