HCV antigen-antibody combination assay and methods and compositions for use therein
First Claim
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1. An immunoassay for the combined detection of HCV antigen and HCV antibody in a test sample, the method comprising:
- a) simultaneously providing the following reagents;
i. a solid phase capable of binding to biotin;
ii. a biotinylated anti-HCV antibody for the capture of an HCV antigen present in said test sample;
iii. a first biotinylated HCV antigen for the capture of an anti-HCV antibody in said test sample, wherein the first biotinylated HCV antigen comprises the amino acid sequence as set forth in SEQ ID NO;
101; and
iv. a first detectably labeled HCV antigen for binding to the anti-HCV antibody captured by the first biotinylated HCV antigen of (iii);
b) incubating the reagents of step (a) under conditions to produce a reaction mixture, wherein(i) the biotinylated anti-HCV antibody of (a)(ii) binds to said solid phase through said biotin and specifically binds to the HCV antigen present in said test sample to produce a biotinylated anti-HCV antibody-HCV antigen complex captured on said solid phase;
(ii) the first biotinylated HCV antigen of (a)(iii) binds to said solid phase through said biotin and specifically binds to the anti-HCV antibody present in said test sample to produce a first biotinylated HCV antigen-anti-HCV antibody complex captured on said solid phase; and
(iii) said first detectably labeled HCV antigen of (a)(iv) specifically binds to the anti-HCV antibody in the first biotinylated HCV antigen-anti-HCV antibody complex captured on said solid phase;
c) isolating solid phase that comprises attached captured anti-HCV antibody and captured HCV antigen from unreacted test sample and reagents;
d. contacting the isolated solid phase with a detectably labeled conjugate antibody that binds to said HCV antigen captured in the biotinylated anti-HCV antibody-HCV antigen complex of (b)(i); and
e. detecting(i) a first signal generated from the detectable label of the detectably labeled conjugate antibody, wherein presence of the first signal indicates presence of the HCV antigen in the test sample; and
(ii) a second signal generated from the detectable label of the first detectably labeled HCV antigen, wherein presence of the second signal indicates presence of the anti-HCV antibody in the test sample.
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Abstract
The present invention generally relates to combination immunoassays, reagents and kits for simultaneous detection of HCV antigens and anti-HCV antibodies in a test sample.
41 Citations
13 Claims
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1. An immunoassay for the combined detection of HCV antigen and HCV antibody in a test sample, the method comprising:
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a) simultaneously providing the following reagents; i. a solid phase capable of binding to biotin; ii. a biotinylated anti-HCV antibody for the capture of an HCV antigen present in said test sample; iii. a first biotinylated HCV antigen for the capture of an anti-HCV antibody in said test sample, wherein the first biotinylated HCV antigen comprises the amino acid sequence as set forth in SEQ ID NO;
101; andiv. a first detectably labeled HCV antigen for binding to the anti-HCV antibody captured by the first biotinylated HCV antigen of (iii); b) incubating the reagents of step (a) under conditions to produce a reaction mixture, wherein (i) the biotinylated anti-HCV antibody of (a)(ii) binds to said solid phase through said biotin and specifically binds to the HCV antigen present in said test sample to produce a biotinylated anti-HCV antibody-HCV antigen complex captured on said solid phase; (ii) the first biotinylated HCV antigen of (a)(iii) binds to said solid phase through said biotin and specifically binds to the anti-HCV antibody present in said test sample to produce a first biotinylated HCV antigen-anti-HCV antibody complex captured on said solid phase; and (iii) said first detectably labeled HCV antigen of (a)(iv) specifically binds to the anti-HCV antibody in the first biotinylated HCV antigen-anti-HCV antibody complex captured on said solid phase; c) isolating solid phase that comprises attached captured anti-HCV antibody and captured HCV antigen from unreacted test sample and reagents; d. contacting the isolated solid phase with a detectably labeled conjugate antibody that binds to said HCV antigen captured in the biotinylated anti-HCV antibody-HCV antigen complex of (b)(i); and e. detecting (i) a first signal generated from the detectable label of the detectably labeled conjugate antibody, wherein presence of the first signal indicates presence of the HCV antigen in the test sample; and (ii) a second signal generated from the detectable label of the first detectably labeled HCV antigen, wherein presence of the second signal indicates presence of the anti-HCV antibody in the test sample. - View Dependent Claims (2, 3)
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4. An immunoassay for the simultaneous detection of both HCV antigens and HCV antibodies in a test sample, wherein said combination assay comprises:
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a. a first capture antigen comprising the amino acid sequence as set forth in SEQ ID NO;
101;b a first detection antigen comprising a peptide sequence of a first HCV protein and further comprising a first detectable label; c. a second capture antigen comprising a peptide sequence of a second HCV protein; d. a second detection antigen comprising a peptide sequence of a second HCV protein and further comprising a second detectable label; e. a third capture antigen comprising a peptide sequence of a third HCV protein; f. a third detection antigen comprising a peptide sequence of a third HCV protein and further comprising a third detectable label; g. a first capture antibody; and h. a conjugate antibody comprising a fourth detectable label, wherein said first capture antibody and said conjugate antibody specifically bind a fourth HCV protein from said test sample, and said combination assay is performed by; (i) contacting said test sample with the first, second, and third capture antigens, the first, second, and third detection antigens, said first capture antibody and said conjugate antibody under conditions to allow; a) formation of a first sandwich complex between said first capture antigen, the first detection antigen and a first anti-HCV antibody present in said test sample; b) formation of a second sandwich complex between said second capture antigen, the second detection antigen and a second anti-HCV antibody present in said test sample; c) formation of a third sandwich complex between said third capture antigen, the third detection antigen and a third anti-HCV antibody present in said test sample; and d) formation of a fourth sandwich complex between said capture antibody, said conjugate antibody and the fourth HCV antigen present in said test sample; and (ii) measuring first, second, third, and fourth signals generated from said first, second, third, and fourth detectable labels as a result of formation of said first, second, third, and fourth sandwich complexes, thereby simultaneously detecting the first, second, and third anti-HCV antibodies and the fourth HCV antigen present in said test sample. - View Dependent Claims (5, 6, 7, 8, 9, 10, 11, 12)
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13. A kit for the simultaneous detection of HCV antigens and antibodies in a sample comprising:
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a first pair of capture antigen and detection antigen for detecting a first anti-HCV antibody against a first HCV protein, wherein said detection antigen is detectably labeled, and wherein the capture antigen comprises the amino acid sequence as set forth in SEQ ID NO;
101;a second pair of capture antigen and detection antigen for detecting a second anti-HCV antibody against a second HCV protein, wherein said detection antigen is detectably labeled; a third pair of capture antigen and detection antigen for detecting a third anti-HCV antibody against a third HCV protein, wherein said detection antigen is detectably labeled; and a first pair of capture antibody and conjugate antibody for detecting a fourth HCV protein, wherein said conjugate antibody is detectably labeled.
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Specification