Specific immunofluorimetric assay detecting the chemotactic epitope of the urokinase receptor (uPAR)
The urokinase plasminogen activator receptor (uPAR) fragments D1 and D2D3 are often found in biological fluids from normal individuals and patients of cancer and other diseases. The D2D3 fragment may possess chemotactic activity depending on its N-terminal sequence. We have developed a sensitive and...
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| creator | Resnati, Massimo Pallavicini, Isabella Daverio, Rita Sidenius, Nicolai Bonini, Pierangelo Blasi, Francesco |
| description | The urokinase plasminogen activator receptor (uPAR) fragments D1 and D2D3 are often found in biological fluids from normal individuals and patients of cancer and other diseases. The D2D3 fragment may possess chemotactic activity depending on its N-terminal sequence. We have developed a sensitive and specific immunoassay for the chemotactic form of D2D3 and show that its level can be measured with high specificity and sensitivity in human serum and urine.
Synthetic peptides (residues 84–92) derived from the linker region between domains 1 and 2 of uPAR were used as immunogens to generate mouse monoclonal antibodies. Recombinant soluble uPAR (D1D2D3
1–277) was used to immunize rabbits to obtain polyclonal antibodies. A sandwich-type immunofluorimetric assay was developed with these antibodies. The assay specifically measures D2D3 containing the 84–88 residues, has a detection limit of 0.25 ng/ml and shows no cross-reactivity with D2D3
93–274. The assay is linear at 0–30 ng/ml, with an intra-assay CV of 10% (
n
=
20), inter-assay CV of 15% (
n
=
9) and a recovery of D2D3
84–274 added to urine samples of between 94% and 105%. A statistically significant difference level of D2D3
84–274 was found in two groups of tumor patients versus healthy volunteers (
p
≤
0.009 in colorectal carcinomas and
p
≤
0.036 in prostatic carcinomas). For the first time, monoclonal antibodies, detecting the chemotactic form of uPAR, D2D3
84–274, have been produced. The immunofluorimetric assay will quantitate uPAR chemotactic fragments in biological samples, including serum and urine, and evaluate their diagnostic or prognostic potential in clinical studies. |
| doi_str_mv | 10.1016/j.jim.2005.10.013 |
| format | Article |
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Synthetic peptides (residues 84–92) derived from the linker region between domains 1 and 2 of uPAR were used as immunogens to generate mouse monoclonal antibodies. Recombinant soluble uPAR (D1D2D3
1–277) was used to immunize rabbits to obtain polyclonal antibodies. A sandwich-type immunofluorimetric assay was developed with these antibodies. The assay specifically measures D2D3 containing the 84–88 residues, has a detection limit of 0.25 ng/ml and shows no cross-reactivity with D2D3
93–274. The assay is linear at 0–30 ng/ml, with an intra-assay CV of 10% (
n
=
20), inter-assay CV of 15% (
n
=
9) and a recovery of D2D3
84–274 added to urine samples of between 94% and 105%. A statistically significant difference level of D2D3
84–274 was found in two groups of tumor patients versus healthy volunteers (
p
≤
0.009 in colorectal carcinomas and
p
≤
0.036 in prostatic carcinomas). For the first time, monoclonal antibodies, detecting the chemotactic form of uPAR, D2D3
84–274, have been produced. The immunofluorimetric assay will quantitate uPAR chemotactic fragments in biological samples, including serum and urine, and evaluate their diagnostic or prognostic potential in clinical studies.</description><identifier>ISSN: 0022-1759</identifier><identifier>EISSN: 1872-7905</identifier><identifier>DOI: 10.1016/j.jim.2005.10.013</identifier><identifier>PMID: 16386755</identifier><identifier>CODEN: JIMMBG</identifier><language>eng</language><publisher>Amsterdam: Elsevier B.V</publisher><subject>Amino Acid Sequence ; Animals ; Antibodies, Monoclonal - biosynthesis ; Biological and medical sciences ; Blotting, Western ; Cancer ; Cell Line ; Cercopithecus aethiops ; Chemotactic Factors - analysis ; Chemotactic Factors - genetics ; Chemotactic Factors - immunology ; Chemotaxis ; CHO Cells ; COS Cells ; Cricetinae ; ELISA ; Enzyme-Linked Immunosorbent Assay ; Epitopes - analysis ; Epitopes - genetics ; Flow Cytometry ; Fluoroimmunoassay - methods ; Fluoroimmunoassay - statistics & numerical data ; Fundamental and applied biological sciences. Psychology ; Fundamental immunology ; Humans ; Immunoprecipitation ; Mice ; Molecular immunology ; Monoclonal antibodies ; Peptide Fragments - analysis ; Peptide Fragments - genetics ; Peptide Fragments - immunology ; Rabbits ; Receptors, Cell Surface - analysis ; Receptors, Cell Surface - genetics ; Receptors, Cell Surface - immunology ; Receptors, Urokinase Plasminogen Activator ; Recombinant Proteins - genetics ; Recombinant Proteins - immunology ; Sensitivity and Specificity ; Techniques ; Urokinase receptor</subject><ispartof>Journal of immunological methods, 2006-01, Vol.308 (1), p.192-202</ispartof><rights>2005 Elsevier B.V.</rights><rights>2006 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c412t-9597c16e721e1b7e1d17c35554453e54df711b2711dda210a788184b1e97932c3</citedby><cites>FETCH-LOGICAL-c412t-9597c16e721e1b7e1d17c35554453e54df711b2711dda210a788184b1e97932c3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S0022175905003947$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=17448185$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/16386755$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Resnati, Massimo</creatorcontrib><creatorcontrib>Pallavicini, Isabella</creatorcontrib><creatorcontrib>Daverio, Rita</creatorcontrib><creatorcontrib>Sidenius, Nicolai</creatorcontrib><creatorcontrib>Bonini, Pierangelo</creatorcontrib><creatorcontrib>Blasi, Francesco</creatorcontrib><title>Specific immunofluorimetric assay detecting the chemotactic epitope of the urokinase receptor (uPAR)</title><title>Journal of immunological methods</title><addtitle>J Immunol Methods</addtitle><description>The urokinase plasminogen activator receptor (uPAR) fragments D1 and D2D3 are often found in biological fluids from normal individuals and patients of cancer and other diseases. The D2D3 fragment may possess chemotactic activity depending on its N-terminal sequence. We have developed a sensitive and specific immunoassay for the chemotactic form of D2D3 and show that its level can be measured with high specificity and sensitivity in human serum and urine.
Synthetic peptides (residues 84–92) derived from the linker region between domains 1 and 2 of uPAR were used as immunogens to generate mouse monoclonal antibodies. Recombinant soluble uPAR (D1D2D3
1–277) was used to immunize rabbits to obtain polyclonal antibodies. A sandwich-type immunofluorimetric assay was developed with these antibodies. The assay specifically measures D2D3 containing the 84–88 residues, has a detection limit of 0.25 ng/ml and shows no cross-reactivity with D2D3
93–274. The assay is linear at 0–30 ng/ml, with an intra-assay CV of 10% (
n
=
20), inter-assay CV of 15% (
n
=
9) and a recovery of D2D3
84–274 added to urine samples of between 94% and 105%. A statistically significant difference level of D2D3
84–274 was found in two groups of tumor patients versus healthy volunteers (
p
≤
0.009 in colorectal carcinomas and
p
≤
0.036 in prostatic carcinomas). For the first time, monoclonal antibodies, detecting the chemotactic form of uPAR, D2D3
84–274, have been produced. The immunofluorimetric assay will quantitate uPAR chemotactic fragments in biological samples, including serum and urine, and evaluate their diagnostic or prognostic potential in clinical studies.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Antibodies, Monoclonal - biosynthesis</subject><subject>Biological and medical sciences</subject><subject>Blotting, Western</subject><subject>Cancer</subject><subject>Cell Line</subject><subject>Cercopithecus aethiops</subject><subject>Chemotactic Factors - analysis</subject><subject>Chemotactic Factors - genetics</subject><subject>Chemotactic Factors - immunology</subject><subject>Chemotaxis</subject><subject>CHO Cells</subject><subject>COS Cells</subject><subject>Cricetinae</subject><subject>ELISA</subject><subject>Enzyme-Linked Immunosorbent Assay</subject><subject>Epitopes - analysis</subject><subject>Epitopes - genetics</subject><subject>Flow Cytometry</subject><subject>Fluoroimmunoassay - methods</subject><subject>Fluoroimmunoassay - statistics & numerical data</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Fundamental immunology</subject><subject>Humans</subject><subject>Immunoprecipitation</subject><subject>Mice</subject><subject>Molecular immunology</subject><subject>Monoclonal antibodies</subject><subject>Peptide Fragments - analysis</subject><subject>Peptide Fragments - genetics</subject><subject>Peptide Fragments - immunology</subject><subject>Rabbits</subject><subject>Receptors, Cell Surface - analysis</subject><subject>Receptors, Cell Surface - genetics</subject><subject>Receptors, Cell Surface - immunology</subject><subject>Receptors, Urokinase Plasminogen Activator</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - immunology</subject><subject>Sensitivity and Specificity</subject><subject>Techniques</subject><subject>Urokinase receptor</subject><issn>0022-1759</issn><issn>1872-7905</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU9v1DAQxS0EotvCB-CCcgHBIcuMY8eJOFUVBaRKIP6cLa89oV6SONgOUr89Xnal3uDikd_8ZmS_x9gzhC0Ctm_2272fthxAlvsWsHnANtgpXqse5EO2AeC8RiX7M3ae0h4AEFp4zM6wbbpWSblh7utC1g_eVn6a1jkM4xqinyjHIpmUzF3lKJPNfv5R5Vuq7C1NIZsi2IoWn8NCVRj-ttYYfvrZJKoiWVpyiNWr9fPll9dP2KPBjImenuoF-3797tvVh_rm0_uPV5c3tRXIc93LXllsSXEk3ClCh8o2UkohZENSuEEh7ng5nDMcwaiuw07skHrVN9w2F-zlce8Sw6-VUtaTT5bG0cwU1qQVKEAB8F8QFYLoOl5APII2hpQiDXop7ph4pxH0IQO91yUDfcjgIJUMyszz0_J1N5G7nziZXoAXJ8Aka8Yhmtn6dM8pIcq_DtzbI0fFs9-eok7W02zJ-WJw1i74fzzjD6igo5g</recordid><startdate>20060120</startdate><enddate>20060120</enddate><creator>Resnati, Massimo</creator><creator>Pallavicini, Isabella</creator><creator>Daverio, Rita</creator><creator>Sidenius, Nicolai</creator><creator>Bonini, Pierangelo</creator><creator>Blasi, Francesco</creator><general>Elsevier B.V</general><general>Elsevier</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>7T5</scope><scope>H94</scope><scope>7X8</scope></search><sort><creationdate>20060120</creationdate><title>Specific immunofluorimetric assay detecting the chemotactic epitope of the urokinase receptor (uPAR)</title><author>Resnati, Massimo ; Pallavicini, Isabella ; Daverio, Rita ; Sidenius, Nicolai ; Bonini, Pierangelo ; Blasi, Francesco</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c412t-9597c16e721e1b7e1d17c35554453e54df711b2711dda210a788184b1e97932c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Antibodies, Monoclonal - biosynthesis</topic><topic>Biological and medical sciences</topic><topic>Blotting, Western</topic><topic>Cancer</topic><topic>Cell Line</topic><topic>Cercopithecus aethiops</topic><topic>Chemotactic Factors - analysis</topic><topic>Chemotactic Factors - genetics</topic><topic>Chemotactic Factors - immunology</topic><topic>Chemotaxis</topic><topic>CHO Cells</topic><topic>COS Cells</topic><topic>Cricetinae</topic><topic>ELISA</topic><topic>Enzyme-Linked Immunosorbent Assay</topic><topic>Epitopes - analysis</topic><topic>Epitopes - genetics</topic><topic>Flow Cytometry</topic><topic>Fluoroimmunoassay - methods</topic><topic>Fluoroimmunoassay - statistics & numerical data</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fundamental immunology</topic><topic>Humans</topic><topic>Immunoprecipitation</topic><topic>Mice</topic><topic>Molecular immunology</topic><topic>Monoclonal antibodies</topic><topic>Peptide Fragments - analysis</topic><topic>Peptide Fragments - genetics</topic><topic>Peptide Fragments - immunology</topic><topic>Rabbits</topic><topic>Receptors, Cell Surface - analysis</topic><topic>Receptors, Cell Surface - genetics</topic><topic>Receptors, Cell Surface - immunology</topic><topic>Receptors, Urokinase Plasminogen Activator</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - immunology</topic><topic>Sensitivity and Specificity</topic><topic>Techniques</topic><topic>Urokinase receptor</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Resnati, Massimo</creatorcontrib><creatorcontrib>Pallavicini, Isabella</creatorcontrib><creatorcontrib>Daverio, Rita</creatorcontrib><creatorcontrib>Sidenius, Nicolai</creatorcontrib><creatorcontrib>Bonini, Pierangelo</creatorcontrib><creatorcontrib>Blasi, Francesco</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>Immunology Abstracts</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of immunological methods</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Resnati, Massimo</au><au>Pallavicini, Isabella</au><au>Daverio, Rita</au><au>Sidenius, Nicolai</au><au>Bonini, Pierangelo</au><au>Blasi, Francesco</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Specific immunofluorimetric assay detecting the chemotactic epitope of the urokinase receptor (uPAR)</atitle><jtitle>Journal of immunological methods</jtitle><addtitle>J Immunol Methods</addtitle><date>2006-01-20</date><risdate>2006</risdate><volume>308</volume><issue>1</issue><spage>192</spage><epage>202</epage><pages>192-202</pages><issn>0022-1759</issn><eissn>1872-7905</eissn><coden>JIMMBG</coden><abstract>The urokinase plasminogen activator receptor (uPAR) fragments D1 and D2D3 are often found in biological fluids from normal individuals and patients of cancer and other diseases. The D2D3 fragment may possess chemotactic activity depending on its N-terminal sequence. We have developed a sensitive and specific immunoassay for the chemotactic form of D2D3 and show that its level can be measured with high specificity and sensitivity in human serum and urine.
Synthetic peptides (residues 84–92) derived from the linker region between domains 1 and 2 of uPAR were used as immunogens to generate mouse monoclonal antibodies. Recombinant soluble uPAR (D1D2D3
1–277) was used to immunize rabbits to obtain polyclonal antibodies. A sandwich-type immunofluorimetric assay was developed with these antibodies. The assay specifically measures D2D3 containing the 84–88 residues, has a detection limit of 0.25 ng/ml and shows no cross-reactivity with D2D3
93–274. The assay is linear at 0–30 ng/ml, with an intra-assay CV of 10% (
n
=
20), inter-assay CV of 15% (
n
=
9) and a recovery of D2D3
84–274 added to urine samples of between 94% and 105%. A statistically significant difference level of D2D3
84–274 was found in two groups of tumor patients versus healthy volunteers (
p
≤
0.009 in colorectal carcinomas and
p
≤
0.036 in prostatic carcinomas). For the first time, monoclonal antibodies, detecting the chemotactic form of uPAR, D2D3
84–274, have been produced. The immunofluorimetric assay will quantitate uPAR chemotactic fragments in biological samples, including serum and urine, and evaluate their diagnostic or prognostic potential in clinical studies.</abstract><cop>Amsterdam</cop><pub>Elsevier B.V</pub><pmid>16386755</pmid><doi>10.1016/j.jim.2005.10.013</doi><tpages>11</tpages></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0022-1759 |
| ispartof | Journal of immunological methods, 2006-01, Vol.308 (1), p.192-202 |
| issn | 0022-1759 1872-7905 |
| language | eng |
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| source | MEDLINE; ScienceDirect Journals (5 years ago - present) |
| subjects | Amino Acid Sequence Animals Antibodies, Monoclonal - biosynthesis Biological and medical sciences Blotting, Western Cancer Cell Line Cercopithecus aethiops Chemotactic Factors - analysis Chemotactic Factors - genetics Chemotactic Factors - immunology Chemotaxis CHO Cells COS Cells Cricetinae ELISA Enzyme-Linked Immunosorbent Assay Epitopes - analysis Epitopes - genetics Flow Cytometry Fluoroimmunoassay - methods Fluoroimmunoassay - statistics & numerical data Fundamental and applied biological sciences. Psychology Fundamental immunology Humans Immunoprecipitation Mice Molecular immunology Monoclonal antibodies Peptide Fragments - analysis Peptide Fragments - genetics Peptide Fragments - immunology Rabbits Receptors, Cell Surface - analysis Receptors, Cell Surface - genetics Receptors, Cell Surface - immunology Receptors, Urokinase Plasminogen Activator Recombinant Proteins - genetics Recombinant Proteins - immunology Sensitivity and Specificity Techniques Urokinase receptor |
| title | Specific immunofluorimetric assay detecting the chemotactic epitope of the urokinase receptor (uPAR) |
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