Two-Site Expression Immunoassay Using a Firefly Luciferase-coding DNA Label
We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase. The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA l...
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| Veröffentlicht in: | Clinical chemistry (Baltimore, Md.) Md.), 1999-11, Vol.45 (11), p.1954-1959 |
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| container_end_page | 1959 |
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| container_issue | 11 |
| container_start_page | 1954 |
| container_title | Clinical chemistry (Baltimore, Md.) |
| container_volume | 45 |
| creator | Chiu, Norman H.L Christopoulos, Theodore K |
| description | We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase.
The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction.
The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-microL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 microg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 microg/L; r = 0.971; n = 44).
The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA. |
| doi_str_mv | 10.1093/clinchem/45.11.1954 |
| format | Article |
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The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction.
The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-microL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 microg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 microg/L; r = 0.971; n = 44).
The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA.</description><identifier>ISSN: 0009-9147</identifier><identifier>EISSN: 1530-8561</identifier><identifier>DOI: 10.1093/clinchem/45.11.1954</identifier><identifier>PMID: 10545065</identifier><identifier>CODEN: CLCHAU</identifier><language>eng</language><publisher>Washington, DC: Am Assoc Clin Chem</publisher><subject>Animals ; Biological and medical sciences ; Coleoptera - chemistry ; DNA - biosynthesis ; DNA - chemistry ; Firefly Luciferin ; Humans ; Immunoassay - methods ; Investigative techniques, diagnostic techniques (general aspects) ; Luciferases - chemistry ; Luciferases - genetics ; Luminescent Measurements ; Medical sciences ; Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques ; Prostate-Specific Antigen - blood ; Prostate-Specific Antigen - chemistry ; Protein Biosynthesis ; Sensitivity and Specificity ; Transcription, Genetic ; Urinary system</subject><ispartof>Clinical chemistry (Baltimore, Md.), 1999-11, Vol.45 (11), p.1954-1959</ispartof><rights>2000 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c473t-32bd6ca47f6f085b00ddd0ff367f52df82350ca8ca4dbdff5e943de73bf792de3</citedby><cites>FETCH-LOGICAL-c473t-32bd6ca47f6f085b00ddd0ff367f52df82350ca8ca4dbdff5e943de73bf792de3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>309,310,314,776,780,785,786,23909,23910,25118,27901,27902</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=1223936$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/10545065$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Chiu, Norman H.L</creatorcontrib><creatorcontrib>Christopoulos, Theodore K</creatorcontrib><title>Two-Site Expression Immunoassay Using a Firefly Luciferase-coding DNA Label</title><title>Clinical chemistry (Baltimore, Md.)</title><addtitle>Clin Chem</addtitle><description>We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase.
The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction.
The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-microL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 microg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 microg/L; r = 0.971; n = 44).
The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA.</description><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Coleoptera - chemistry</subject><subject>DNA - biosynthesis</subject><subject>DNA - chemistry</subject><subject>Firefly Luciferin</subject><subject>Humans</subject><subject>Immunoassay - methods</subject><subject>Investigative techniques, diagnostic techniques (general aspects)</subject><subject>Luciferases - chemistry</subject><subject>Luciferases - genetics</subject><subject>Luminescent Measurements</subject><subject>Medical sciences</subject><subject>Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques</subject><subject>Prostate-Specific Antigen - blood</subject><subject>Prostate-Specific Antigen - chemistry</subject><subject>Protein Biosynthesis</subject><subject>Sensitivity and Specificity</subject><subject>Transcription, Genetic</subject><subject>Urinary system</subject><issn>0009-9147</issn><issn>1530-8561</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1999</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNpNkMtOwzAQRS0EoqXwBUgoCwSrtHZs57FEpQVEBQtgbTl-UCMnKZlGoX-PqxbR1Wg0Z-6MDkKXBI8JLuhEeVerpakmjI8JGZOCsyM0JJziOOcpOUZDjHERF4RlA3QG8BValuXpKRoQzBnHKR-i5_e-id_c2kSzn1VrAFxTR09V1dWNBJCb6ANc_RnJaO5aY_0mWnTKWdNKMLFq9HZ2_3IXLWRp_Dk6sdKDudjXEfqYz96nj_Hi9eFpereIFcvoOqZJqVMlWWZTi3NeYqy1xtbSNLM80TZPKMdK5gHRpbaWm4JRbTJa2qxItKEjdLPLXbXNd2dgLSoHyngva9N0INIiSSgmSQDpDlRtAxD-F6vWVbLdCILF1qH4cygYF4SIrcOwdbWP78rK6IOdnbQAXO8BCUp628paOfjnwvWCpgG73WFL97nsgz4BlfQ-pBLR9_3BxV9L8on0</recordid><startdate>19991101</startdate><enddate>19991101</enddate><creator>Chiu, Norman H.L</creator><creator>Christopoulos, Theodore K</creator><general>Am Assoc Clin Chem</general><general>American Association for Clinical Chemistry</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19991101</creationdate><title>Two-Site Expression Immunoassay Using a Firefly Luciferase-coding DNA Label</title><author>Chiu, Norman H.L ; Christopoulos, Theodore K</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c473t-32bd6ca47f6f085b00ddd0ff367f52df82350ca8ca4dbdff5e943de73bf792de3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1999</creationdate><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Coleoptera - chemistry</topic><topic>DNA - biosynthesis</topic><topic>DNA - chemistry</topic><topic>Firefly Luciferin</topic><topic>Humans</topic><topic>Immunoassay - methods</topic><topic>Investigative techniques, diagnostic techniques (general aspects)</topic><topic>Luciferases - chemistry</topic><topic>Luciferases - genetics</topic><topic>Luminescent Measurements</topic><topic>Medical sciences</topic><topic>Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques</topic><topic>Prostate-Specific Antigen - blood</topic><topic>Prostate-Specific Antigen - chemistry</topic><topic>Protein Biosynthesis</topic><topic>Sensitivity and Specificity</topic><topic>Transcription, Genetic</topic><topic>Urinary system</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Chiu, Norman H.L</creatorcontrib><creatorcontrib>Christopoulos, Theodore K</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Clinical chemistry (Baltimore, Md.)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Chiu, Norman H.L</au><au>Christopoulos, Theodore K</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Two-Site Expression Immunoassay Using a Firefly Luciferase-coding DNA Label</atitle><jtitle>Clinical chemistry (Baltimore, Md.)</jtitle><addtitle>Clin Chem</addtitle><date>1999-11-01</date><risdate>1999</risdate><volume>45</volume><issue>11</issue><spage>1954</spage><epage>1959</epage><pages>1954-1959</pages><issn>0009-9147</issn><eissn>1530-8561</eissn><coden>CLCHAU</coden><abstract>We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase.
The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction.
The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-microL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 microg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 microg/L; r = 0.971; n = 44).
The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA.</abstract><cop>Washington, DC</cop><pub>Am Assoc Clin Chem</pub><pmid>10545065</pmid><doi>10.1093/clinchem/45.11.1954</doi><tpages>6</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 0009-9147 |
| ispartof | Clinical chemistry (Baltimore, Md.), 1999-11, Vol.45 (11), p.1954-1959 |
| issn | 0009-9147 1530-8561 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_69223012 |
| source | Oxford University Press Journals All Titles (1996-Current); MEDLINE |
| subjects | Animals Biological and medical sciences Coleoptera - chemistry DNA - biosynthesis DNA - chemistry Firefly Luciferin Humans Immunoassay - methods Investigative techniques, diagnostic techniques (general aspects) Luciferases - chemistry Luciferases - genetics Luminescent Measurements Medical sciences Pathology. Cytology. Biochemistry. Spectrometry. Miscellaneous investigative techniques Prostate-Specific Antigen - blood Prostate-Specific Antigen - chemistry Protein Biosynthesis Sensitivity and Specificity Transcription, Genetic Urinary system |
| title | Two-Site Expression Immunoassay Using a Firefly Luciferase-coding DNA Label |
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