Self-contained biological analysis

Abstract

Devices, containers, and methods are provided for performing biological analysis in a closed environment. Illustrative biological analyses include nucleic acid amplification and detection and immuno-PCR.

1 . (canceled) 
     
     
         2 . A method of performing immuno-PCR on a sample to detect an antigen present in the sample, comprising the steps of
 providing a container having a plurality of fluidly connected reaction blisters and one or more sealable ports, the sealable ports providing the only access from an exterior of the container to reaction blisters,   injecting the sample into the container via a first of the sealable ports, and sealing the first port subsequent to injecting the sample,   moving the sample to a first of the plurality of blisters, the first blister comprising a capture antibody sample comprising a first set of capture antibodies having specificity for the antigen, the first set of capture antibodies restrained in the first blister, and incubating the sample with the capture antibody mixture to bind the antigen to the capture antibodies,   providing a reporter antibody sample, the reporter antibody sample comprising a first set of reporter antibodies having specificity for the antigen, each of the first set of reporter antibodies coupled to an oligonucleotide, and incubating the reporter antibody sample with the captured antigen to bind some of the first set of reporter antibodies,   washing all unbound reporter antibodies from the bound sample and moving the washed unbound reporter antibodies to a second blister within the container, the second blister configured as a waste receptacle, and   amplifying the oligonucleotides coupled to the captured reporter antibodies and detecting the amplified oligonucleotides,   wherein all steps subsequent to injecting the sample into the container are all performed within the container.   
     
     
         3 . The method of  claim 2 , wherein
 the capture antibody sample comprises additional sets of capture antibodies, each additional set of antibodies having specificity for an additional antigen,   the reporter antibody sample comprises additional sets of reporter antibodies having specificity for the additional antigens, wherein the antibodies in each additional set of reporter antibodies is coupled to a unique oligonucleotide for that set of antibodies, and   the amplifying step includes dividing the captured oligonucleotides into a plurality of individual amplification chambers, wherein each individual amplification chambers comprises a pair of primers for amplifying one of the oligonucleotides.   
     
     
         4 . The method of  claim 3 , wherein the amplifying step includes multiplex amplifying all captured oligonucleotides, and then dividing the amplified oligonucleotides into the plurality of individual amplification chambers. 
     
     
         5 . The method of  claim 2 , wherein the capture antibodies are coupled to a magnetic particle, and the capture antibodies are magnetically restrained in the first blister. 
     
     
         6 . The method of  claim 5 , wherein the reporter antibodies are incubated with the captured antigen in the first blister. 
     
     
         7 . The method of  claim 5 , wherein the capture antibodies are released from the magnetic restraint during incubation of the captured antigen with the reporter antibodies and are magnetically recaptured prior to the washing step. 
     
     
         8 . The method of  claim 2 , wherein the washing step is repeated multiple times to move additional unbound reporter antibodies to the waste receptacle. 
     
     
         9 . The method of  claim 2 , further comprising a washing step prior to providing the reporter antibody sample, the washing step to remove unbound antigen, and moving the unbound antigen to the second blister. 
     
     
         10 . The method of  claim 2 , wherein the capture antibody sample and the reporter antibody sample are injected into the container via additional sealable ports and the additional sealable ports are sealed prior to mixing the sample with the capture antibodies. 
     
     
         11 . The method of  claim 2 , wherein the capture antibody sample and the reporter antibody sample are provided in reservoirs during manufacture of the sample container. 
     
     
         12 . The method of  claim 2 , wherein the container comprises a plurality of additional fluidly connected reaction blisters configured for extraction of nucleic acids in the sample, and the amplifying step includes amplifying the extracted nucleic acids. 
     
     
         13 . The method of  claim 12 , wherein the amplifying step includes first-stage multiplex amplification and second-stage singleplex amplification. 
     
     
         14 . A method of performing immuno-PCR on a sample to detect an antigen that may be present in the sample, comprising the steps of
 providing a container having a plurality of fluidly connected reaction blisters and one or more sealable ports, the sealable ports providing the only access from an exterior of the container to reaction blisters,   injecting the sample into the container via a first of the sealable ports, and sealing the first port subsequent to injecting the sample,   moving the sample to a first of the plurality of blisters and allowing the sample to conjugate with
 a capture antibody sample comprising a set of capture antibodies, a first one of the capture antibodies having specificity for tie antigen, and 
 a reporter antibody sample comprising a set of reporter antibodies, a first one of the reporter antibodies having specificity for the antigen, each of the set of reporter antibodies coupled to a unique oligonucleotide, 
 and incubating the sample, the capture antibody sample, the reporter antibody sample to form a ternary complex of the capture antibody, the antigen and the reporter antibody, 
   washing all unbound reporter antibodies from the ternary complex and moving the washed unbound reporter antibodies to a second blister within the container, the second blister configured as a waste receptacle,   amplifying the oligonucleotides coupled to the captured reporter antibodies,   detecting an amplified oligonucleotide, and   identifying the antigen based on the detected oligonucleotide,   wherein all steps subsequent to injecting the sample into the container are all performed within the container.   
     
     
         15 . The method of  claim 14 , wherein the amplifying step includes a first-stage multiplex amplification reaction and a plurality of second-stage singleplex amplification reactions. 
     
     
         16 . The method of  claim 15 , further comprising a dilution step between the first-stage multiplex amplification reaction and the second-stage singleplex amplification reactions. 
     
     
         17 . The method of  claim 14 , wherein the amplification step includes generating an amplification curve for a positive sample that is distinguishable from an amplification curve from non-specifically bound reporter antibodies. 
     
     
         18 . The method of  claim 14 , wherein the set of capture antibodies includes at least one capture antibody having specificity for each of a plurality of additional antigens that may be in the sample and the set of reporter antibodies includes at least one reporter antibody having specificity for each of the plurality of additional antigens. 
     
     
         19 . The method of  claim 18 , wherein an additional one of the reporter antibodies is not specific for any of the antigens that may be present in the sample, and amplification of an additional oligonucleotide coupled to the additional reporter antibody is used as a negative control. 
     
     
         20 . The method of  claim 14 , wherein the capture antibodies are coupled to a magnetic particle, and the capture antibodies are magnetically restrained in the first blister. 
     
     
         21 . The method of  claim 14 , wherein the washing step is repeated multiple times to move additional unbound reporter antibodies to the waste receptacle. 
     
     
         22 . A method of identifying antigens in a sample, comprising the steps of
 (a) providing a container having
 i. a plurality of fluidly connected reaction blisters including a capture blister, 
 ii. an amplification blister fluidly connected to the capture blister, 
 iii. an additional set of second-stage amplification chambers fluidly connected to the amplification blister, each additional second-stage amplification chamber configured for further amplifying a specific nucleic acid that may be present in the amplification blister, and 
 iv. one or more sealable ports fluidly connected to the reaction blisters, the sealable ports providing the only access from an exterior of the container to the reaction blisters such that when all of the one or more sealable ports is sealed, the container is fully closed, 
   (b) injecting the sample into the container via a first of the sealable ports, and sealing the first port subsequent to injecting the sample,   (c) binding each of the antigens to a specific pair of antibodies to form a ternary complex, wherein each pair of antibodies comprises a capture antibody and a reporter antibody wherein each reporter antibody is conjugated to a unique oligonucleotide,   (d) subjecting the oligonucleotides to amplification conditions to generate amplicons,   (e) fluidly moving a portion of the amplicons to each of the additional second-stage amplification chambers, and   (f) performing second-stage amplification in the additional second-stage amplification chambers and identifying the antigens based on which second-stage amplification chambers have detectable amplification.   
     
     
         23 . The method of  claim 22 , wherein step (c) further comprises one or more wash steps. 
     
     
         24 . The method of  claim 23 , wherein the capture antibodies are conjugated to magnetic beads, and the one or more wash steps includes deploying a magnet to retain the ternary complex in the capture blister. 
     
     
         25 . The method of  claim 22 , further comprising a dilution step between step (d) and step (f). 
     
     
         26 . A method of performing immuno-PCR on a sample to detect an antigen that may be present in the sample, comprising the steps of
 providing a container having a plurality of fluidly connected reaction blisters and one or more sealable ports, the sealable ports providing the only access from an exterior of the container to reaction blisters,   injecting the sample into the container via a first of the sealable ports, and sealing the first port subsequent to injecting the sample,   moving the sample to a first of the plurality of blisters and allowing the sample to conjugate with
 a capture antibody sample comprising a set of capture antibodies, a first one of the capture antibodies having specificity for the antigen, and 
 a reporter antibody sample comprising a set of reporter antibodies, a first one of the reporter antibodies having specificity for the antigen, each of the set of reporter antibodies coupled to a unique oligonucleotide, 
 and incubating the sample, the capture antibody sample, the reporter antibody sample to form a ternary complex of the capture antibody, the antigen and the reporter antibody, 
   amplifying the oligonucleotides coupled to the captured reporter antibodies,   detecting an amplified oligonucleotide, and   identifying the antigen based on the detected oligonucleotide,   wherein all steps subsequent to injecting the sample into the container are all performed within the container.   
     
     
         27 . The method of  claim 26 , wherein the amplifying step includes a first-stage multiplex amplification reaction and a plurality of second-stage singleplex amplification reactions. 
     
     
         28 . The method of  claim 27 , further comprising a dilution step between the first-stage multiplex amplification reaction and the second-stage singleplex amplification reactions. 
     
     
         29 . The method of  claim 26 , wherein the amplification step includes generating an amplification curve for a positive sample that is distinguishable from an amplification curve from non-specifically bound reporter antibodies. 
     
     
         30 . The method of  claim 26 , wherein the set of capture antibodies includes at least one capture antibody having specificity for each of a plurality of additional antigens that may be in the sample and the set of reporter antibodies includes at least one reporter antibody having specificity for each of the plurality of additional antigens. 
     
     
         31 . The method of  claim 30 , wherein an additional one of the reporter antibodies is not specific for any of the antigens that may be present in the sample, and amplification of an additional oligonucleotide coupled to the additional reporter antibody is used as a negative control. 
     
     
         32 . The method of  claim 26 , wherein the capture antibodies are coupled to a magnetic particle, and the capture antibodies are magnetically restrained in the first blister.