Immuno-gold lateral flow assay

Abstract

A chromatographic lateral-flow assay system for rapid, high sensitivity method of detecting low levels of ligands in body fluids, with few false positives and few false negatives. The lateral-flow assay may have a membrane strip in ribbon form, which increases detection on the order of 2 to 10 fold over the conventional chromatographic specific binding assay techniques by placing a dried or lyophilized conjugate in colloidal spheres opposite side of the lateral flow membrane strip. A chromatographic specific binding assay strip device, comprising: a laminate strip having a first side and an second side; a conjugate pad or membrane disposed on said first side of said laminate; a hinge region connecting sample receiving pad or membrane strip and reservoir pad or membrane disposed on said second side of said laminate; wherein said laminating material isolates the conjugate pad from the sample pad such as to slow the fluid path into the fibrous sample pad, and a detection pad or membrane strip disposed between the sample pad or membrane and the reservoir pad or membrane on said first side of said laminate. The more complete mixing permitted by this temporary obstruction provides an important feature of the invention that gives this 2-step test its superior performance. The assay system comprises a housing device, such as a test tube or cassettes to facilitate the mixing of a sample solution with the dried or lyophilized conjugate, and kits.

1 . A chromatographic specific binding assay strip device, comprising: 
 a laminate strip having a first side and a second side; 
 a sample receiving pad, a fibrous reservoir pad disposed on said first side of said laminate strip; and a fibrous detection pad disposed between said fibrous sample pad, wherein said fibrous reservoir pad is disposed at the distal end of said device facilitating unidirectional capillary migration through the detection pad or membrane;  
 a fibrous conjugate pad disposed on said second side of said laminate;  
 wherein said laminate strip is configured to fold back at a hinge point, such that laminate strip and conjugate pad on the second side are positioned on top of the sample receiving pad;  
 wherein said laminating strip separates the conjugate pad from the sample pad such that the flow path of liquid migration is slowed.  
   
   
   
       2 . The device of  claim 1 , wherein the impeding or obstructing property of said laminate strip is regulated or adjusted by piercing the laminating membrane, or by using a membrane with micropores.  
   
   
       3 . A chromatographic specific binding assay strip device, comprising: 
 two laminate strips; 
 a sample receiving pad, a fibrous reservoir pad disposed on said first laminate strip; and  
   a fibrous detection pad disposed between said fibrous sample pad, wherein said fibrous reservoir pad is disposed at the distal end of said device facilitating unidirectional capillary migration through the detection pad or membrane; 
 a fibrous conjugate pad disposed on said second laminate strip;  
 wherein said conjugate pad on the second laminate strip is positioned on top of the sample receiving pad;  
 wherein said second laminating strip separates the conjugate pad from the sample pad such that the flow path of liquid migration is slowed.  
   
   
   
       4 . The device of  claim 3 , wherein said liquid migration is regulated or adjusted by piercing the laminating membrane, or by using a laminate with micropores.  
   
   
       5 . The device of  claim 1 , comprising a cassette housing further comprising a sample well or opening for receiving the liquid sample or test tube version.  
   
   
       6 . The device of  claim 5 , wherein the sample port is on the same side of the same plane of the test cassette as the visualization window.  
   
   
       7 . The device of  claim 1 , wherein said device contains blocked bovine serum albumin (BSA) and detergent for preventing loss of antibody, by non-specific attachment.  
   
   
       8 . The device of  claim 1 , wherein said binding assay strip is an immunological binding assay strip.  
   
   
       9 . The device of  claim 5 , wherein said cassette contains ridges or channels designed to both pre-mix the sample with conjugate, and facilitates a unidirectional flow path.  
   
   
       10 . The device of  claim 1 , wherein a pre-filter is fitted into a sample application means to remove heavy loads of a particulate matter.  
   
   
       11 . A kit comprised of the device of  claim 1 , further comprising a buffered diluent and wherein said conjugate is a colloidal metal.  
   
   
       12 . A kit comprised of the device of  claim 5 , further comprising a buffered diluent and wherein said conjugate is a colloidal metal.  
   
   
       13 . A method of detecting the presence or absence of an analyte or ligand in a liquid sample comprising the steps of: 
 application of a sample fluid directly unto a dried or lyophilized conjugate in the conjugate pad or membrane region of  claim 1 , wherein said sample fluid rehydrates the conjugate pad or membrane, producing a uniform dispersion of conjugate through the entire applied sample fluid creating a sample-conjugate complex, prior to entering the sample receiving pad or membrane of  claim 1;     allowing the sample and conjugate mixture to flow by capillary action into the sample receiving pad or membrane, permitting said sample conjugate mixture to flow unidirectionally into the lateral flow analyte detection membrane in the direction toward the reservoir pad or membrane; and    detecting the presence or absence of an analyte or ligand in the detection viewing region containing the capture reagent capable of binding the sample-conjugate complex and a control reagent region, both capable of color reactions.    
   
   
       14 . The method of  claim 13 , wherein the analyte or ligand is selected from the group consisting of proteins, modified proteins, hormones, peptides, drugs, carbohydrates, haptens, chemicals, and chemical reaction with an analyte.  
   
   
       15 . The method of  claim 13 , wherein the liquid sample is selected from the group consisting of a bio-fluid, plant extracts, environmental samples, tissue samples and enrichment broths.  
   
   
       16 . The method of  claim 13 , wherein detecting the presence of an analyte or ligand is used as an indicator for the presence of an infectious disease, pregnancy, microbial detection, ovulation, cancer marker identification, autoimmunity, cardiac markers, biowarfare agents, allergy, drugs of abuse or environmental monitoring.  
   
   
       17 . The method of  claim 15 , wherein the bio-fluid is selected from the group consisting of tissue extracts, blood, serum, plasma, tears, perspiration, urine, and saliva.  
   
   
       18 . The method of  claim 13 , wherein said detection is measured by binding selected from the group consisting of: 
 secondary antibody sandwich for measurement of sample antibody; antibody-antigen-antibody sandwich for measurement of antigen in either competitive (inhibition) or non-competitive mode;    antigen-antibody-antigen sandwich for measurement of antibody; and competitive inhibition assay involving antigen bound to the conjugate microparticles, anti-antigen on the membrane, with detectable analyte inhibiting the anti-antigen reaction.    
   
   
       19 . The method of  claim 13 , wherein said conjugate is a colloidal metal.  
   
   
       20 . The method of  claim 19 , wherein said colloidal metal is gold.