Determination of analytes in particle-containing medium

Abstract

Methods and compositions are provided for concentrating particles in a minute area on a solid surface. The method permits the detection of small amounts of analytes by providing for an observable signal in relation to the concentration of particles in the area.

What is claimed is: 
     
       1. A method for detecting the presence of a member of a specific binding pair ("sbp member") in a .[.liquid assay medium.]. .Iadd.sample.Iaddend., said specific binding pair consisting of ligand and homologous receptor, where said method involves particles to which are bound at least one member of a specific binding pair, a solid bibulous member and a signal-producing system which involves at least one label which is bound to said particles or an sbp member, said method comprising:   combining in an aqueous assay medium, .[.said.]. a sample .Iadd.suspected of containing an sbp .Iaddend.member and at least one .Iadd.of the group consisting .Iaddend.of said particles and .Iadd.said .Iaddend.labeled sbp members, with the proviso that particles are added when said sample lacks particles having an sbp member; under conditions where particles only within a predetermined size.[.,.]. range and charge will concentrate in an area on said bibulous member adjacent the air/liquid interface, when said bibulous member is contacted with said assay medium;   contacting said bibulous member with said assay medium whereby said assay medium is wicked past said area and particles within said predetermined size .Iadd.range .Iaddend.and charge concentrate at a small site in said area; and   detecting the signal as a result of said signal-producing system, wherein said signal is related to the amount of label in said area and the amount of label in said area is related to the amount of said sbp member in said sample.   
     
     
       2. A method according to claim 1, wherein said sample has particles within said predetermined size .Iadd.range .Iaddend.to which are bound an sbp member. 
     
     
       3. A method according to claim 1, wherein said sample has particles below said predetermined size .Iadd.range .Iaddend.to which are bound an sbp member and said label is joined to a polyvalent homologous sbp member. 
     
     
       4. A method according to claim 1 wherein said particles are labeled with a member of said signal producing system. 
     
     
       5. A method for detecting cells that have at least one predetermined determinant site, where said method involves labeled receptors which bind to said determinant site or to receptors binding to said determinant site, and a solid bibulous member, where the label of said labeled receptor by itself or in combination with other members of a signal producing system produces a detectable signal, said method comprising: combining in an aqueous assay medium, a sample suspected of containing said cells, labeled receptors to said determinant site or receptors for said determinant site and labeled receptors for said receptors, under conditions where individual cells migrate along a bibulous member away from an air-liquid interface when said medium is contacted with said bibulous member, but receptor linked cells concentrate at .[.the.]. .Iadd.an .Iaddend.area of .Iadd.said .Iaddend.bibulous member at the air-liquid interface;   contacting said bibulous member with said assay medium, whereby linked cells concentrate in said area; and   detecting the signal as a result of said signal producing system wherein said signal is related to the amount of label in said area.   
     
     
       6. A method according to claim 5, wherein said cell is a bacterial cell, fungal .Iadd.cell.Iaddend., protozoan .Iadd.cell.Iaddend., or other parasitic or infectious agent. 
     
     
       7. A method according to claim 5, wherein said label is an enzyme. 
     
     
       8. A method according to claim 5, wherein said bibulous member has a small orifice surrounded .[.on one side of said bibulous member.]. with a water impermeable layer and said assay medium is contacted with said bibulous member at said orifice. 
     
     
       9. A method according to claim 5, wherein said bibulous member is supported by a plastic support having a centrally located orifice. .Iadd.10. A method according to claim 1 wherein after said contacting of said bibulous member with said assay medium, said area on said bibulous member is contacted with an aqueous medium containing a labeled sbp member when said labelled sbp member is not included in said assay medium. .Iaddend. .Iadd.11. A method according to claim 1 wherein said contacting is carried out by introducing a portion of said bibulous member into said assay medium. .Iaddend. .Iadd.12. A method according to claim 1 wherein said contacting is carried out by introducing a portion of said assay medium onto said bibulous member. .Iaddend. .Iadd.13. A method according to claim 1 wherein said combining is carried out by combining said sample in an aqueous medium with said particles and said contacting is carried out by contacting said assay medium with a portion of said bibulous member. .Iaddend. .Iadd.14. A method for detecting the presence of an analyte, said method comprising: combining a medium suspected of containing said analyte, which is a member of a specific binding pair (sbp), with particles having bound thereto an sbp member reciprocal to said analyte,   contacting said medium with an area of a bibulous member under conditions where said medium wicks away from said area and at least a portion of said particles concentrate in a localized site on said bibulous member, and   examining said site to determine the presence of said analyte. .Iaddend.   
     
     
        .Iadd.15.  The method of claim 14 wherein said particles have a label. .Iaddend. .Iadd.16. The method of claim 14 wherein said examining is carried out by (1) contacting said site with a medium containing a labeled receptor for said analyte and (2) determining the presence of said labeled receptor at said site to determine the presence of said analyte. .Iaddend. .Iadd.17. The method of claim 14 wherein said examining is carried out by (1) contacting said site with a medium containing a labeled receptor for said reciprocal sbp member and (2) determining the presence of said labeled receptor at said site to determine the presence of said analyte. .Iaddend. .Iadd.18. The method of claim 14 wherein said site is examined by contacting said site with members of a signal producing system and then examining said site for the presence of a detectable signal to determine said analyte. .Iaddend. .Iadd.19. The method of claim 14 wherein said medium wicks away from said area by migrating outward along said bibulous member. .Iaddend. .Iadd.20. A method for detecting the presence of an epitopic site on particles, said method comprising: contacting a spot on a bibulous member with a medium containing a labelled antibody reciprocal to said epitopic site and particles suspected of having said epitopic site under conditions where excess of said labelled antibody will wick away from said spot; and   detecting the presence of said labelled antibody at said spot. .Iaddend. .Iadd.21. The method of claim 20 wherein said antibody is labelled with an enzyme or a fluorescer. .Iaddend. .Iadd.22. A method for detecting the presence of an analyte, said method comprising:   providing in combination an aqueous medium suspected of containing said analyte, which is a member of a specific binding pair (sbp), particles having bound thereto an sbp member reciprocal to said analyte, and an area of a bibulous member under conditions where said medium wicks away from said area and at least a portion of said particles concentrate in a localized site on said bibulous member, and   examining said site to determine the presence of said analyte. .Iaddend. .Iadd.23. The method of claim 22 wherein said particles have a label. .Iaddend. .Iadd.24. The method of claim 22 wherein said examining is carried out by (1) contacting said site with a medium containing a labeled receptor for said analyte and (2) determining the presence of said labeled receptor at said site to determine the presence of said analyte. .Iaddend. .Iadd.25. The method of claim 22 wherein said examining is carried out by (1) contacting said site with a medium containing a labeled receptor for said reciprocal sbp member and (2) determining the presence of said labeled receptor at said site to determine the presence of said analyte. .Iaddend. .Iadd.26. The method of claim 22 wherein said examining is carried out by (1) contacting said site with members of a signal producing system and then (2) determining the presence of a detectable signal at said site to determine the presence of said analyte. .Iaddend. .Iadd.27. The method of claim 22 wherein said medium wicks away from said area by migrating outward along said bibulous member. .Iaddend. .Iadd.28. A device for conducting an assay, said device comprising a non-bibulous member having an orifice and serving as a support for a bibulous member, said bibulous member having an orifice that extends through said bibulous member, said orifices corresponding in location. .Iaddend. .Iadd.29. The device of claim 28 wherein said orifice of said bibulous member and said orifice of said support are centrally located. .Iaddend. .Iadd.30. The device of claim 28 wherein said bibulous member is a circular disc.   
     
     
        .Iaddend. .Iadd.31.  A kit comprising in packaged form: a device comprising a non-bibulous member having an orifice and serving as a support for a bibulous member such that, when said device is used in conducting an assay, liquid containing particles applied to said bibulous member through said orifice migrates away from point of application and at least a portion of the particles present in said liquid concentrate at said joint of application and in a separate container particles which have bound irreversibly thereto a member of a specific binding pair. .Iaddend. .Iadd.32. The kit of claim 31 which further comprises in a separate container a member of a signal producing system. .Iaddend. .Iadd.33. A method for detecting the presence of a member of a specific binding pair ("sbp member") in a sample, said specific binding pair consisting of ligand and homologous receptor, where said method involves particles to which are bound at least one member of a specific binding pair, a solid bibulous member and a signal-producing system which involves at least one label which is bound to an sbp member,   said method comprising:   combining in an aqueous assay medium, a sample suspected of containing an sbp member and said particles and said labeled sbp member under conditions where said particles concentrate in an area on said bibulous member adjacent the air/liquid interface, when said bibulous member is contacted with said assay medium;   contacting said bibulous member with said assay medium whereby said assay medium is wicked past said area and particles concentrate at a small site in said area; and   detecting the signal as a result of said signal-producing system, wherein said signal is related to the amount of label in said area and the amount of label in said area is related to the amount of said sbp member in said sample. .Iaddend. .Iadd.34. The method of claim 33 wherein said label is an enzyme. .Iaddend. .Iadd.35. The method of claim 33 wherein said sbp member in said sample is an antigen, said sbp member bound to said particles is an antibody for said antigen and said sbp member bound to said label is an antibody. .Iaddend. .Iadd.36. The method of claim 33 wherein said sbp member in said sample is a hapten, said sbp member bound to the particles is an antibody for said hapten and said sbp member bound to said label is said hapten. .Iaddend. .Iadd.37. A method for detecting the presence of an analyte, said method comprising:   combining a medium suspected of containing said analyte, which is a member of a specific binding pair (sbp), with particles having bound thereto an sbp member reciprocal to said analyte and with a labeled sbp member wherein the amount of labeled sbp member which binds to said particles is related to the amount of analyte in said medium,   contacting said medium with an area of a bibulous member under conditions where said medium wicks away from said area and said particles concentrate in a localized site on said bibulous member, and   examining said site for the presence of labeled sbp member to determine the presence of said analyte. .Iaddend. .Iadd.38. The method of claim 37 wherein said site is contacted with members of a signal producing system and then is examined for the presence of a detectible signal to determine the analyte. .Iaddend. .Iadd.39. The method of claim 37 wherein said medium wicks away from said area by migrating outward along said bibulous member. .Iaddend. .Iadd.40. A method for performing an immunoassay for an analyte in a sample, which method comprises providing a chromatographic medium, forming a reaction mixture containing a specific binder for the analyte, said specific binder being immobilized by attachment to a particulate solid phase, and a labeled reagent partly in a form which is mobile on said medium and partly in the form of a labeled analyte/specific binder reaction product insolubilized so as to be immobile on the medium, the proportions of the two forms being determined by the amount of analyte in the sample, applying the reaction mixture to a spot on the chromatographic medium, causing the mobile form of the labeled reagent to migrate from the spot, and thereafter, observing one or both of the mobile and immobile, insolubilized forms of the labeled reagent. .Iaddend. .Iadd.41. The method as claimed in claim 40, wherein the chromatographic medium is in sheet form, with the reaction mixture being applied to a spot on the sheet and the mobile form of the labeled reagent being caused to migrate radially from the spot. .Iaddend. .Iadd.42. The method as claimed in claim 40, wherein migration is assisted by subsequent application to the spot of a solvent which causes mobile species, but not immobile species, to migrate through the chromatographic medium. .Iaddend. .Iadd.43. The method as claimed in claim 42, wherein the solvent is an aqueous liquid. .Iaddend. .Iadd.44. The method as claimed in claim 42 wherein the reaction mixture is formed by causing the analyte in the sample, and a labeled version of the analyte, to compete for reaction with a limited amount of a specific binder for the analyte, the analyte/specific binder complex being rendered insoluble. .Iaddend. .Iadd.45. The method as claimed in claim 44, wherein the analyte/specific binder complex is rendered insoluble by reaction of the specific binder with its antibody, said reaction being effected before, during and after incubation with the analyte. .Iaddend. .Iadd.46. The method as claimed in claim 40, wherein the reaction mixture is formed by causing the analyte in the sample to bind to an excess of an immobilized specific binder and a labeled specific binder for the analyte is caused to bind to vacant binding sites of the analyte. .Iaddend.