A fast and simple dot-immunobinding assay for quantification of mouse immunoglobulins in hybridoma culture supernatants
Mouse monoclonal antibodies of IgG subclasses and IgM class in hybridoma culture supernatants were quantified using a dot-immunobinding assay. Immunoglobulins were bound to nitrocellulose (NC) membrane and, after blocking, the membrane was incubated with anti-mouse antibody conjugated to horseradish...
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| Veröffentlicht in: | Journal of immunological methods 2004-06, Vol.289 (1), p.89-95 |
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| container_title | Journal of immunological methods |
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| creator | Sulimenko, T Dráber, P |
| description | Mouse monoclonal antibodies of IgG subclasses and IgM class in hybridoma culture supernatants were quantified using a dot-immunobinding assay. Immunoglobulins were bound to nitrocellulose (NC) membrane and, after blocking, the membrane was incubated with anti-mouse antibody conjugated to horseradish peroxidase (HRP). Binding was revealed by incubation with a sensitive chemiluminiscence reagent. Quantitation was achieved by densitometric comparison with standard curves produced by purified monoclonal antibodies of the same subclass or purified antibodies of the same clone as the antibody to be quantified. These quantitative results were compared with those obtained using purified IgG from mouse serum or purified mouse myeloma IgM as standards. The dot-immunobinding assay requires 1 μl of hybridoma culture sample and takes about 1 h in total. Good linearity between the staining intensity and the amount of immobilized immunoglobulins was observed over the range of 0.05–5 ng/spot. The assay is simple, reproducible and can process simultaneously a large number of samples. |
| doi_str_mv | 10.1016/j.jim.2004.03.010 |
| format | Article |
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Immunoglobulins were bound to nitrocellulose (NC) membrane and, after blocking, the membrane was incubated with anti-mouse antibody conjugated to horseradish peroxidase (HRP). Binding was revealed by incubation with a sensitive chemiluminiscence reagent. Quantitation was achieved by densitometric comparison with standard curves produced by purified monoclonal antibodies of the same subclass or purified antibodies of the same clone as the antibody to be quantified. These quantitative results were compared with those obtained using purified IgG from mouse serum or purified mouse myeloma IgM as standards. The dot-immunobinding assay requires 1 μl of hybridoma culture sample and takes about 1 h in total. Good linearity between the staining intensity and the amount of immobilized immunoglobulins was observed over the range of 0.05–5 ng/spot. 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Immunoglobulins were bound to nitrocellulose (NC) membrane and, after blocking, the membrane was incubated with anti-mouse antibody conjugated to horseradish peroxidase (HRP). Binding was revealed by incubation with a sensitive chemiluminiscence reagent. Quantitation was achieved by densitometric comparison with standard curves produced by purified monoclonal antibodies of the same subclass or purified antibodies of the same clone as the antibody to be quantified. These quantitative results were compared with those obtained using purified IgG from mouse serum or purified mouse myeloma IgM as standards. The dot-immunobinding assay requires 1 μl of hybridoma culture sample and takes about 1 h in total. Good linearity between the staining intensity and the amount of immobilized immunoglobulins was observed over the range of 0.05–5 ng/spot. 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Psychology</subject><subject>Fundamental immunology</subject><subject>Horseradish Peroxidase - chemistry</subject><subject>Hybridoma culture supernatants</subject><subject>Hybridomas - immunology</subject><subject>Immunoblotting - methods</subject><subject>Immunoenzyme Techniques</subject><subject>Immunoglobulin G - analysis</subject><subject>Immunoglobulin M - analysis</subject><subject>Mice</subject><subject>Molecular immunology</subject><subject>Mouse immunoglobulins</subject><subject>Quantitation</subject><subject>Techniques</subject><issn>0022-1759</issn><issn>1872-7905</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0UFrFDEUB_Agit1WP4AXyUVvM743k2Rm8FSKVqHgRc8hk0lqlkmyTSaW_fam7IKe9BQIv__j8f6EvEFoEVB82Ld759sOgLXQt4DwjOxwHLpmmIA_JzuArmtw4NMFucx5D1CJgJfkAnnHkSHfkcdralXeqAoLzc4fVkOXuDXO-xLi7MLiwj1VOasjtTHRh6LC5qzTanMx0GipjyUbevL3a5zL6kKmLtCfxzm5JXpFdVm3kgzN5WBSUFsdkV-RF1at2bw-v1fkx-dP32--NHffbr_eXN81miFsTc-gF2JSWmg24TLPVpgJRz3asbd8EtYyJgbg89x3qPkwjxMqBV2vtWC6_l6R96e5hxQfismb9C5rs64qmLq5FDUtBIj_QhwGhFHwCvEEdYo5J2PlITmv0lEiyKda5F7WWuRTLRJ6WW9eM2_Pw8vszfInce6hgndnoLJWq00qaJf_chMfGXbVfTw5U2_2y5kks3YmaLO4ZPQml-j-scZvmFusvw</recordid><startdate>20040601</startdate><enddate>20040601</enddate><creator>Sulimenko, T</creator><creator>Dráber, P</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>20040601</creationdate><title>A fast and simple dot-immunobinding assay for quantification of mouse immunoglobulins in hybridoma culture supernatants</title><author>Sulimenko, T ; Dráber, P</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c410t-3403669ac6c491dbbf6e918c8f83f596ff446705bb321c57b891aa023cc64cbb3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Animals</topic><topic>Antibodies, Monoclonal - analysis</topic><topic>Biological and medical sciences</topic><topic>Collodion - chemistry</topic><topic>Dot-immunobinding assay</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Fundamental immunology</topic><topic>Horseradish Peroxidase - chemistry</topic><topic>Hybridoma culture supernatants</topic><topic>Hybridomas - immunology</topic><topic>Immunoblotting - methods</topic><topic>Immunoenzyme Techniques</topic><topic>Immunoglobulin G - analysis</topic><topic>Immunoglobulin M - analysis</topic><topic>Mice</topic><topic>Molecular immunology</topic><topic>Mouse immunoglobulins</topic><topic>Quantitation</topic><topic>Techniques</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sulimenko, T</creatorcontrib><creatorcontrib>Dráber, P</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>Sulimenko, T</au><au>Dráber, P</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A fast and simple dot-immunobinding assay for quantification of mouse immunoglobulins in hybridoma culture supernatants</atitle><jtitle>Journal of immunological methods</jtitle><addtitle>J Immunol Methods</addtitle><date>2004-06-01</date><risdate>2004</risdate><volume>289</volume><issue>1</issue><spage>89</spage><epage>95</epage><pages>89-95</pages><issn>0022-1759</issn><eissn>1872-7905</eissn><coden>JIMMBG</coden><abstract>Mouse monoclonal antibodies of IgG subclasses and IgM class in hybridoma culture supernatants were quantified using a dot-immunobinding assay. Immunoglobulins were bound to nitrocellulose (NC) membrane and, after blocking, the membrane was incubated with anti-mouse antibody conjugated to horseradish peroxidase (HRP). Binding was revealed by incubation with a sensitive chemiluminiscence reagent. Quantitation was achieved by densitometric comparison with standard curves produced by purified monoclonal antibodies of the same subclass or purified antibodies of the same clone as the antibody to be quantified. These quantitative results were compared with those obtained using purified IgG from mouse serum or purified mouse myeloma IgM as standards. The dot-immunobinding assay requires 1 μl of hybridoma culture sample and takes about 1 h in total. Good linearity between the staining intensity and the amount of immobilized immunoglobulins was observed over the range of 0.05–5 ng/spot. 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| ispartof | Journal of immunological methods, 2004-06, Vol.289 (1), p.89-95 |
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| language | eng |
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| source | Elsevier ScienceDirect Journals Complete - AutoHoldings; MEDLINE |
| subjects | Animals Antibodies, Monoclonal - analysis Biological and medical sciences Collodion - chemistry Dot-immunobinding assay Fundamental and applied biological sciences. Psychology Fundamental immunology Horseradish Peroxidase - chemistry Hybridoma culture supernatants Hybridomas - immunology Immunoblotting - methods Immunoenzyme Techniques Immunoglobulin G - analysis Immunoglobulin M - analysis Mice Molecular immunology Mouse immunoglobulins Quantitation Techniques |
| title | A fast and simple dot-immunobinding assay for quantification of mouse immunoglobulins in hybridoma culture supernatants |
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