Method for the visual detection of specific antibodies by the use of lateral flow assays

Abstract

This disclosure teaches a method and composition for detecting the presence of class specific antibodies reactive with analytes such as bacteria, allergens, autoimmune antigens, viral proteins, haptens, and carbohydrates by lateral flow techniques. In one embodiment of the disclosure, a test sample is added to the test strip, which then migrates to the site of the immobilized allergens, thereby forming a first antibody IgE-allergen complex. A chase release buffer is added upstream to or from the site of the labeled anti-IgE antibodies, which is itself upstream from the sample site. The anti-IgE antibodies migrate downstream to the site of immobilized first complex, thereby forming a second complex indicating the presence of class specific IgE antibodies in the test specimen. In another embodiment of the disclosure, a liquid form of the labeled anti-IgE antibodies can be added to the test strip after the first complex has been formed. These techniques can be used to perform other lateral flow tests.

1 . A lateral flow immunoassay device for detecting immune reactants, said device comprising: at least one test strip, said at least one test strip comprising: a) a sample site for applying a sample comprising antibodies; b) a plurality of reaction sites downstream from said sample site, each said reaction site containing a different allergen such that when IgE antibodies come in contact with an allergen to which the IgE antibodies react, a first complex is formed;
 c) a colorimetric labeled analyte site comprising a colorimetric labeled anti-IgE antibody, said colorimetric labeling site positioned upstream from said sample site; and   d) a location on said test strip to add a chase release buffer for the release of said labeled analyte;   wherein said colorimetric labeled anti-IgE antibody, released by addition of said chase release buffer, migrates to the site of the at least one first complex, forming a colorimetric labeled Anti-IgE antibody—IgE antibody-allergen complex.   
   
   
       2 . The lateral flow immunoassay device of  claim 1 , wherein said immunoassay comprises at least two test strips. 
   
   
       3 . The lateral flow immunoassay device of  claim 1 , wherein said allergens are selected from the groups consisting of pollens, dust mite allergens, molds, animal epithelium, foods, allergen mixes, and combinations thereof. 
   
   
       4 . The lateral flow immunoassay of  claim 1 , wherein said at least one solubilizing agent to immobilize any said allergen, said at least one solubilizing agent being present in an amount such that said allergen protein tertiary structure unfolds to allow for greater binding of said antigen to said test strip, wherein, said solubilizing agent is selected from the group consisting of sugars and alcohol. 
   
   
       5 . The lateral flow immunoassay of  claim 1 , wherein said at least one solubilizing agent is selected from the group consisting of sucrose, mannose, fructose, ethylene glycol, ethanol, methanol, glycerin and dextrans. 
   
   
       6 . The lateral flow immunoassay of  claim 1 , further comprising a solubilizing
 agent wherein said at least one solubilizing agent is a sugar.   
   
   
       7 . The lateral flow immunoassay of  claim 1 , wherein said at least one solubilizing
 agent is an alcohol.   
   
   
       8 . The lateral flow immunoassay device of  claim 6 , wherein said sugar is selected from the group consisting of sucrose, mannose, fructose, dextran, and combinations thereof. 
   
   
       9 . The lateral flow immunoassay device of  claim 7 , wherein said wherein said alcohol is selected from the group consisting of ethylene glycol, glycerol, ethanol, methanol, and combinations thereof. 
   
   
       10 . A method of detecting immune reactants, comprising: a) placing a sample containing antibodies on a lateral flow immunoassay device, said device comprising: at lease one test strip, said at least one test strip comprising: i) a sample site for applying a sample comprising antibodies; ii) a plurality of reaction sites downstream from said sample site, each said reaction site containing a different allergen such that when IgE antibodies come in contact with an antigen to which the IgE antibodies react, a first complex is formed;
 iii) a colorimetric labeled analyte site comprising a colorimetric labeled anti-IgE antibody, said colorimetric labeling site positioned upstream from said sample site; and   iv) a location on said test strip to add a chase release buffer for the release of said labeled analyte;   b) allowing said test sample to migrate from the sample site to the reaction sites;   c) adding said chase release buffer; and   d) reading said lateral flow immunoassay device, wherein a colored line, indicating a positive response will develop when said colorimetric labeled anti-IgE antibodies come in contact with an IgE antibody which has reacted with an antigen.   
   
   
       11 . The method of  claim 10 , wherein said allergens are selected from the group consisting of pollens, dust mite allergens, molds, animal epithelium, foods, allergen mixes, and combinations thereof. 
   
   
       12 . A lateral flow assay device for detecting immune reactants, said device comprising: at least one test strip, said at least one test strip comprising: a) a sample site for applying a sample comprising at least one analyte for which the device is designed to detect; b) at least one reactant downstream from said sample site, such that when said at least one analyte comes in contact with said reactant, a first complex is formed;
 c) a colorimetric label site comprising a colorimetric label, said colorimetric labeling site positioned upstream from said sample site; said colorimetric label capable of reacting or attaching to the analyte, said colorimetric labeled having the ability to be released from said colorimetric label site by a chase release buffer; and   d) a location on said test strip to add said chase release buffer,   wherein said colorimetric label, released by addition of said chase release buffer, migrates to the site of the at least one first complex, forming a colorimetric labeled analyte reactant.   
   
   
       13 . A lateral flow immunoassay device kit for detecting immune reactants, said kit comprising:
 I) a chase release buffer; II) at least one test strip, said at least one test strip comprising: a) a sample site for applying a sample comprising antibodies;
 b) a plurality of reaction sites downstream from said sample site, each said reaction site containing a different allergen such that when IgE antibodies come in contact with an allergen to which the IgE antibodies react, a first complex is formed; 
 a colorimetric site for labeling the first complex, forming a colorimetric labeled Anti-IgE antibody-IgE antibody-allergen-complex, said colorimetric labeling site comprising a colorimetric labeled anti-IgE antibody, said colorimetric labeling site positioned upstream from said sample site, said colorimetric labeled anti-IgE antibody being released by said chase release buffer; and 
 d) a location on said test strip to add said chase release buffer; 
   
     wherein said allergens are immobilized to said test strip using at least one solubilizing agent, said at least one solubilizing agent being present in an amount such that said allergen protein tertiary structure unfolds to allow for greater binding of said allergen to said test strip, wherein said at least one solubilizing agent is selected from the group consisting of sugars and alcohols; and
 III) a plastic casing holding said at least one test strip in place, said plastic casings having
 a) a sample opening in which to add the sample: 
 b) a second opening in which to add the chase release buffer, said second opening positioned upstream from said sample opening and upstream from said colorimetric site; and 
 c) a third opening in which to view the labeling sites after the sample and the chase release buffer have been added. 
 
 
   
   
       14 . The lateral flow immunoassay device kit of  claim 13 , wherein said immunoassay comprises at least two test strips. 
   
   
       15 . The lateral flow immunoassay device kit of  claim 13 , wherein said allergens are selected from the groups consisting of pollens, dust mite allergens, molds, animal epithelium, foods, allergen mixes, and combinations thereof. 
   
   
       16 . The lateral flow immunoassay device kit of  claim 13 , wherein said at least one solubilizing agent to immobilize any said allergen is present in an amount such that said allergen protein tertiary structure unfolds to allow for greater binding of said antigen to said test strip, wherein, said solubilizing agent is selected from the group consisting of sugars and alcohol. 
   
   
       17 . The lateral flow immunoassay device kit of  claim 13 , wherein said at least one solubilizing agent is selected from the group consisting of sucrose, mannose, fructose, ethylene glycol, ethanol, methanol, glycerin and dextrans. 
   
   
       18 . The lateral flow immunoassay device kit of  claim 13 , wherein said alcohol of said solubilizing agent is selected from the group consisting of ethylene glycol, glycerol, ethanol, methanol, and combinations thereof.