Production and characterization of a monoclonal antibody specific for the polyamine spermidine and its application in ELISA.
To enable the immunoassay of spermidine in tissue extracts, a monoclonal antibody, JAC-1, specific for free spermidine was raised, using a spermidine-ovalbumin conjugate as immunogen. This antibody was characterized, and found to possess a high degree of specificity for spermidine, with only 4% molar cross-reactivity with spermine. A competitive ELISA using this antibody was developed. This assay is able to detect as little as 10 pmol spermidine extracted from small, circa 10 mg, tissue samples. The assay is unaffected by the presence of up to a 4-fold molar ratio of spermine, whereas the corresponding spermine competitive ELISA is adversely affected by a 0.5-fold molar ratio of spermidine. The spermidine competitive ELISA using JAC-1 was used to estimate the spermidine content of several plant and animal tissue extracts and the results compared with HPLC data. This novel assay is a useful development in the assay of polyamines since, compared to routinely employed HPLC methods, it offers increased convenience, rapidity, and capacity for large numbers of samples.
Catcheside
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JA
Division of Biochemistry, Royal Holloway and Bedford New College, University of London, Surrey, UK.
Stead
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Rider
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CC
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D051379:Mice; D008807:Mice, Inbred BALB C; D013095:Spermidine / Q000276:immunology*; D013096:Spermine / Q000276:immunology
D000911:Antibodies, Monoclonal; D013096:Spermine; D013095:Spermidine