Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.662124
Title: Monoclonal antibodies to vasopressin and atrial natriuretic peptide : preparation, characterization and use in two-site immunometric assays
Author: Smith, Shirley Carolyn
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1995
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Abstract:
Two-site immunometric assays for AVP were possible with a range of antibody combinations, despite the small size of the peptide, although steric hindrance severely limited antibody binding and none of the assays were sufficiently sensitive to measure normal physiological AVP concentrations (1-5 pg/ml plasma). The two IgG1 MAbs, ESVP 1 and ESVP 2, were specific for the AVP ring and the tail tripeptide, respectively, and could therefore bind simultaneously to AVP, although their relatively low binding affinities (2.8 x 108 and 2.2 x 108 1/mole, respectively) limited the sensitivity of a direct IRMA to 1ng AVP/ml. An immunofiltration assay with these MAbs could be carried out in only 1 hour, but its detection limit was 10 ng AVP/ml. The most sensitive assay was an IRMA which used 125I-ESVP-1 with an affinity-purified tail-specific polyclonal antibody, and had a detection limit of 250 pg AVP/ml after a 4 hour incubation period. A two-site ELISA, using biotin-ESVP 1 with the affinity-purified polyclonal antibody, was less sensitive because of high non-specific binding, but was used to analyse the separation of 125I-AVP from unlabelled AVP on a Sephadex G-25 column and, therefore to determine the specific activity of the purified 125I-AVP. The four anti-ANP MAbs which were produced in this project (with binding affinities of 8.3 x 107 to 2.4 x 109 1/mole), and three other anti-ANP MAbs, were tested for compatibility in two-site assays but there were no pairs of MAbs which could bind to ANP simultaneously. Two-site assays could only be carried out using 125I-MAbs with a polyclonal antibody preparation in the IRMA format, although steric hindrance limited antibody binding. The detection limit of the most sensitive assay was 1 ng ANP/ml, whereas normal physiological ANP concentrations are in the range 5-70 pg/ml plasma.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.662124  DOI: Not available
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