Immunoassay and method of use

Abstract

A method for performing an immunoassay is described. The method is particularly useful for detecting extracellular polysaccharide (EPS) and/or lipopolysaccharide (LPS) producing microorganisms. The method is particularly useful for detecting microorganisms which produce extracellular polysaccharides (EPS) also known as exocellular polysaccharides, capsule, and/or lipopolysaccharides (LPS). In a preferred method for detecting microorganisms which produce EPS, LPS, or both, the EPS and/or LPS is extracted from a sample with cetyltrimethylammonium bromide (CTAB) to produce molecular aggregates which are then preferentially bound to colored polystyrene latex particles over other components in the sample, and the bound EPS and/or LPS detected using a lateral flow immunoassay apparatus which has immobilized thereon antibodies specific for the EPS and/or LPS. The method can also be used to detect particular viruses, for example viruses of the potyviridae or tobamoviridae group.

1 . A method for determining whether a material contains a microorganism by detecting the presence of an extracellular polysaccharide (EPS), a lipopolysaccharide (LPS), or both, produced by the microorganism, which comprises:
 (a) providing a detection apparatus which includes mounted on a support member an elongated membrane having a first end and a second end wherein in lateral contact with the first end of the membrane is a sample pad for receiving a liquid sample and in lateral contact with the second end of the membrane is a wicking pad which allows the liquid sample to flow through the membrane from the sample pad to the wicking pad and wherein the membrane further comprises at least one detection zone laterally spaced from the sample pad in which is immobilized an antibody which is specific for the EPS or LPS of the microorganism;   (b) mixing the material with an extraction solution to produce a mixture including the EPS, LPS, or both;   (c) mixing an aliquot of the mixture in step (b) with particles at room temperature for a time sufficient to bind the EPS, LPS, or both, to the particles without substantial binding of other components of the mixture;   (d) mixing a particle blocking solution including a blocking agent with the mixture in step (c) at room temperature for a time sufficient to block sites on the particles not bound to the EPS, LPS, or both; and   (e) applying the mixture in step (d) to the sample pad in the detection apparatus at room temperature, wherein presence of the microorganism in the material is indicated by a visible signal produced by binding of the EPS or LPS bound to the particles by the antibody specific for the EPS or LPS immobilized in the detection zone.   
   
   
       2 . The method of  claim 1  wherein the extraction solution includes a salt in a buffer. 
   
   
       3 . The method of  claim 1  wherein the extraction solution contains about 2% NaCl in the buffer. 
   
   
       4 . The method of  claim 1  wherein the material is boiled in the extraction solution for a time sufficient to extract the EPS, LPS, or both, into the extraction solution. 
   
   
       5 . The method of  claim 1  wherein the extraction solution includes cetyltrimethylammonium bromide (CTAB) in a high salt buffer wherein the CTAB and the high salt selectively extracts the EPS, LPS, or both from the microorganism and material into the extraction solution. 
   
   
       6 . The method of  claim 5  wherein the CTAB is at a concentration between about 0.25% and 2% and the salt is NaCl at a concentration of about 2%. 
   
   
       7 . The method of  claim 1  wherein the particle blocking agent is bovine serum albumin. 
   
   
       8 . The method of  claim 1  wherein the particle blocking solution includes bovine serum albumin and polysorbate 20 in a buffer. 
   
   
       9 . The method of  claim 1  wherein the particle blocking solution includes bovine serum albumin, polysorbate 20, and n-dodecyl-N,N-dimethyl glycine in a buffer. 
   
   
       10 . The method of  claim 1  wherein the membrane further includes a reference zone laterally spaced between the detection zone and the wicking pad in which is immobilized therein a control antibody and the particle blocking solution further includes particles which have bound thereon an antigen which binds the control antibody. 
   
   
       11 . The method of  claim 1  wherein the particles are naked, unmodified, visible polystyrene latex particles which bind to the EPS, LPS, or both at room temperature for a time sufficient to bind a sufficient amount the EPS, LPS, or both to the particles to enable visualization of the particles bound to the EPS, LPS, or both and without substantial binding of other components of the mixture in step (c) to the particles. 
   
   
       12 . The method of  claim 11  wherein the polystyrene latex particles have a diameter from about 0.06 μm to 1.0 μm. 
   
   
       13 . The method of  claim 1  wherein the membrane is a nitrocellulose membrane. 
   
   
       14 . The method of  claim 13  wherein the nitrocellulose membrane has a pore size from about 10 to 20 μm. 
   
   
       15 . The method of  claim 1  wherein the membrane has been treated with a blocking agent. 
   
   
       16 . The method of  claim 15  wherein the blocking agent is bovine serum albumin. 
   
   
       17 . The method of  claim 1  wherein the sample pad includes a polyester or glass fibers. 
   
   
       18 . The method of  claim 1  wherein the sample pad has been treated with a blocking agent. 
   
   
       19 . The method of  claim 18  wherein the blocking agent is selected from the group consisting of bovine serum albumin, non-fat dry milk, and mixtures thereof. 
   
   
       20 . The method of  claim 1  wherein the wicking pad includes a cellulosic material. 
   
   
       21 . The method of  claim 1  wherein the material is a plant material. 
   
   
       22 . The method of  claim 1  wherein the material is serum or tissue from an animal or human. 
   
   
       23 .- 40 . (canceled) 
   
   
       41 . A kit for detecting an extracellular polysaccharide (EPS), a lipopolysaccharide (LPS), or both, produced by a microorganism, which comprises:
 (a) providing a detection apparatus which includes mounted on a support member an elongated membrane having a first end and a second end wherein in lateral contact with the first end of the membrane is a sample pad for receiving a liquid sample and in lateral contact with the second end of the membrane is a wicking pad which allows the liquid sample to flow through the membrane from the sample pad to the wicking pad and wherein the membrane further comprises at least one detection zone laterally spaced from the sample pad in which is immobilized an antibody which is specific for the EPS or LPS;   (b) a first container containing an extraction solution;   (c) a second container containing a particle blocking solution; and   (d) a third container containing a suspension including particles which are capable of binding of the EPS and LPS.   
   
   
       42 . The kit of  claim 41  wherein the membrane further includes a reference zone laterally spaced between the detection zone and the wicking pad in which is immobilized therein a control antibody and the particle blocking solution further includes particles which have bound thereon an antigen which binds the control antibody. 
   
   
       43 . The kit of  claim 41  wherein the particles are naked, unmodified, visible polystyrene latex particles which are capable of binding to the EPS, LPS, or both at room temperature for a time sufficient to bind a sufficient amount the EPS, LPS, or both to the particles to enable visualization of the particles bound to the EPS, LPS, or both and without substantial binding of other components of a sample mixture to the particles. 
   
   
       44 . The kit of  claim 41  wherein the extraction solution includes cetyltrimethylammonium bromide (CTAB) in a high salt buffer. 
   
   
       45 . The kit of  claim 41  wherein the second and third containers are dropper bottles. 
   
   
       46 . The kit of  claim 41  wherein the membrane is a nitrocellulose membrane. 
   
   
       47 . The kit of  claim 46  wherein the membrane has been treated with a blocking agent. 
   
   
       48 . The kit of  claim 41  wherein the sample pad includes a polyester or glass fibers. 
   
   
       49 . The kit of  claim 41  wherein the particle blocking solution includes bovine serum albumin. 
   
   
       50 . The kit of  claim 41  wherein the wicking pad includes a cellulosic material. 
   
   
       51 .- 112 . (canceled) 
   
   
       113 . The method of  claim 11  wherein the naked, unmodified, visible polystyrene latex particles are colored and enable visualization by the human eye in visible light. 
   
   
       114 . The method of  claim 11  wherein the naked, unmodified, visible polystyrene latex particles are fluorescently labeled and enable visualization by the human eye or a machine reader upon illumination with an ultraviolet light. 
   
   
       115 . The method of  claim 11  wherein the naked, unmodified, visible polystyrene latex particles are free of conjugated antibodies. 
   
   
       116 . A method for determining whether a material contains a microorganism by detecting the presence of an extracellular polysaccharide (EPS), a lipopolysaccharide (LPS), or both, produced by the microorganism, which comprises:
 (a) providing a detection apparatus which includes mounted on a support member an elongated membrane having a first end and a second end wherein in lateral contact with the first end of the membrane is a sample pad for receiving a liquid sample and in lateral contact with the second end of the membrane is a wicking pad which allows the liquid sample to flow through the membrane from the sample pad to the wicking pad and wherein the membrane further comprises at least one detection zone laterally spaced from the sample pad in which is immobilized an antibody which is specific for the EPS or LPS of the microorganism;   (b) mixing the material with an extraction solution to produce a mixture including the EPS, LPS, or both;   (c) mixing an aliquot of the mixture from step (b) with naked, unmodified, visible polystyrene latex particles which bind to the EPS, LPS, or both at room temperature for a time sufficient to bind a sufficient amount the EPS, LPS, or both to the particles to enable visualization of the particles bound to the EPS, LPS, or both and without substantial binding of other components of the mixture to the particles;   (d) mixing a particle blocking solution including a blocking agent with the mixture in step (c) at room temperature for a time sufficient to block sites on the particles not bound to the EPS, LPS, or both; and   (e) applying the mixture from step (d) to the sample pad in the detection apparatus at room temperature, wherein binding of (i) the EPS, LPS, or both bound to the particles by (ii) the antibody specific for the EPS, LPS, or both that is immobilized in the detection zone indicates the presence of the microorganism in the material.   
   
   
       117 . The method of  claim 116  wherein the extraction solution includes cetyltrimethylammonium bromide (CTAB) in a high salt buffer wherein the CTAB and the high salt selectively extracts the EPS, LPS, or both from the microorganism and material into the extraction solution. 
   
   
       118 . The method of  claim 117  wherein the CTAB is at a concentration between about 0.25% and 2% and the salt is NaCl at a concentration of about 2%. 
   
   
       119 . The method of  claim 116  wherein the membrane further includes a reference zone laterally spaced between the detection zone and the wicking pad in which is immobilized therein a control antibody and the particle blocking solution further includes particles which have bound thereon an antigen which binds the control antibody. 
   
   
       120 . The method of  claim 116  wherein the naked, unmodified, visible polystyrene latex particles are colored and enable visualization by the human eye in visible light. 
   
   
       121 . The method of  claim 116  wherein the naked, unmodified, visible polystyrene latex particles are fluorescently labeled and enable visualization by the human eye or a machine reader upon illumination with an ultraviolet light. 
   
   
       122 . The method of  claim 116  wherein the naked, unmodified, visible polystyrene latex particles are free of conjugated antibodies.