Methods for detecting and measuring aggregation

Abstract

Methods, compositions, systems, and devices are provided for performing and analyzing agglutination assays. In one aspect, methods for image analysis of agglutination assays are provided. In another aspects, methods for performing agglutination assays are provided. In one aspect, the methods may be used for the detection of various molecules, including viruses or antibodies against a virus. In another aspect, the methods can be used to determine effective immunization of a subject.

1 - 55 . (canceled) 
     
     
         56 . A method for determining the presence of an antibody in a biological sample, where said antibody binds selectively to an agglutinating particle, the method comprising:
 (a) incubating a mixture of visualization particles, the agglutinating particle, and the biological sample suspected of containing said antibody, under conditions permitting agglutination of the visualization particle via interaction with said agglutinating particle; and   (b) capturing with the aid of an optical device an image of said mixture, wherein the presence of a visualization particle-agglutinating particle cluster in said image indicates the occurrence of said agglutination and lack of detectable amount of said antibody, and wherein the absence of said cluster indicates the lack of said agglutination and the presence of detectable amount of said antibody   
     
     
         57 . The method of  claim 56 , wherein the formation of said visualization particle-agglutinating particle cluster is determined by assessing center-to-center distance between individual visualization particles captured in said image. 
     
     
         58 . (canceled) 
     
     
         59 . (canceled) 
     
     
         60 . A method for determining the presence of an agglutinating particle in a biological sample, comprising:
 (a) incubating a mixture of visualization particles and a biological sample suspected of containing said agglutinating particle, under conditions permitting agglutination of the visualization particle via interaction with said agglutinating particle; and   (b) capturing with the aid of an optical device an image of said mixture, wherein the presence of a visualization particle-agglutinating particle cluster in said image indicates the occurrence of said agglutination and the presence of detectable amount of said agglutinating particle, and wherein the absence of said cluster indicates the lack of said agglutination and the lack of detectable amount of said agglutinating particle.   
     
     
         61 . (canceled) 
     
     
         62 . A method for determining the effective immunization of a subject, comprising:
 (a) obtaining a biological sample from a subject that has been immunized with a first dosage of a first vaccine against an agglutinating particle;   (b) incubating a mixture of visualization particles, said agglutinating particle, and said biological sample, under conditions permitting agglutination of the visualization particles via interaction with said agglutinating particle;   (c) capturing with the aid of an optical device an image of said mixture; and   (d) determining the concentration of an antibody against said agglutinating particle in said biological sample based on the clusters formed by the agglutination of the visualization particles,   wherein the presence of a visualization particle-agglutinating particle cluster in said image indicates the occurrence of said agglutination and lack of detectable amount of said antibody, and wherein the absence of said cluster indicates the lack of said agglutination and the presence of detectable amount of said antibody.   
     
     
         63 - 94 . (canceled) 
     
     
         95 . The method of  claim 62 , wherein the determining comprises analyzing the image comprises identifying a region of interest (ROI) in the image. 
     
     
         96 . The method of  claim 95 , wherein the ROI comprises imaged information of the mixture, said mixture comprising visualization particles and agglutinating particles. 
     
     
         97 - 99 . (canceled) 
     
     
         100 . The method of claim  80 , wherein the agglutination is evidenced by the formation of clusters comprising visualization particles and agglutinating particles, and the step of analyzing the image comprises identifying clusters in the image. 
     
     
         101 . The method of  claim 100 , further comprising determining the size or number of clusters in the image. 
     
     
         102 . The method of  claim 100 , wherein the step of identifying clusters in the image comprises determining the distance between visualization particles in the image. 
     
     
         103 . The method of  claim 102 , wherein the step of determining the distance between visualization particles in the image comprises determining the center-to-center distance between visualization particles in the image. 
     
     
         104 . The method of claim  80 , wherein the step of analyzing the image comprises determining the center-to-center distance between a first visualization particle and a second visualization particle in the image. 
     
     
         105 . The method of  claim 104 , further comprising determining the location of the first visualization particle and the second visualization particle in the image. 
     
     
         106 . The method of  claim 100 , wherein the step of identifying the clusters comprises identifying a first visualization particle and a second visualization particle, wherein the center-to-center distance between the first visualization particle and the second visualization particle is no greater than 1.5 times the diameter of either the first or second visualization particle. 
     
     
         107 - 108 . (canceled) 
     
     
         109 . The method of  claim 100 , wherein the step of identifying the clusters comprises selecting a cut-off value distance, wherein visualization particles situated at a distance from each other which is less than the cut-off value distance are considered to be associated with each other. 
     
     
         110 . The method of  claim 109 , wherein the step of selecting a cut-off value distance comprises calculating a radial distribution function for two or more visualization particles in the image. 
     
     
         111 . The method of  claim 100 , wherein the step of analyzing the image comprises extracting a feature from a region of the image of the mixture. 
     
     
         112 . The method of  claim 111 , wherein the step of extracting the feature comprises analyzing the texture of the mixture. 
     
     
         113 . The method of  claim 112 , wherein the step of analyzing the texture of the mixture comprises analyzing the image with a local binary patterns operator. 
     
     
         114 . The method of  claim 112 , wherein the step of extracting the feature comprises analyzing the intensity of a signal in one or more color channels in a region of the image. 
     
     
         115 . The method of  claim 113 , wherein the step of analyzing the texture of the mixture comprises inputting information obtained from the local binary patterns operator into an algorithm which has been previously trained with local binary patterns information from one or more images of a mixture of known agglutination status. 
     
     
         116 - 117 . (canceled)