Use this URL to cite or link to this record in EThOS:
Title: Development of a novel immunoassay format for haptens using atrazine as a model molecule
Author: Carvou, Nicholas
ISNI:       0000 0001 3524 5401
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2001
Availability of Full Text:
Access from EThOS:
Access from Institution:
The herbicide atrazine was chosen as a model molecule for the development of a novel non-competitive immunoassay for small, monoepitopic molecules (haptens). This assay involves the use of a monoclonal antibody which can specifically detect a primary antiatrazine monoclonal antibody bound to atrazine, i.e. a monoclonal anti-immune complex (anti-IC) antibody. In order to raise such an antibody, a covalent complex between the mouse monoclonal anti-atrazine antibody P01-93-41M and the atrazine derivative 3-{{4-(ethylamino)-6- [( 1 -methylethyl)amino] -1,3,5-triazin-2-yl} thio} propanoic-acid ( At-COOH) was produced for immunisation purposes. A generic method for the preparation of covalent immune complexes (CICs) between hapten and anti-hapten antibody was entirely developed in house. At-COOH was covalently linked to 4[pazidosalicylamido]butylamine (ASBA), the latter compound being a photo-reactive arylazide cross-linker which would allow covalent cross-linking of At-COOH to its specific antibody binding site under UV light. One group of BALB/c mice was immunised with CIC, in order to produce monoclonal anti-IC antibodies. Another group of BALB/c mice was immunised with non-covalent immune complex (NCIC) in order to compare the efficiency of the two immunogens in raising an appropriate anti-IC immune response. BALB/c splenocytes were also immunised in vitro with NCIC. Hybridomas were raised using splenocytes (a) from mice immunised with CIC, (b) and mice immunised in vivo with NCIC and, (c) splenocytes exposed in vitro to NCIC. A blocked Fc ELISA was developed to overcome the problem of syngeneicity of IC and anti-IC Ab during screening of the fusions. Dual and triple screening procedures were developed to establish the specificity of hybridoma supernatants using anti-atrazine Ab, NCIC and CIC as immobilised antigens. Unfortunately, no truly specific anti-IC antibody was raised from any of the fusions performed. As an alternative RNA extracted from the spleens of two BALB/c mice immunised with either CIC or NCIC was used as a template to produce two phage displayed scFv libraries. The two libraries were panned using a de-selection strategy whereby nonspecific anti-Ab and anti-atrazine scFvs were eliminated from the library prior to selecting specific anti-IC scFvs. Selected scFvs were then screened using a triple screening strategy similar to the one adopted in the hybridoma work. Eventually, two specific anti-IC clones were isolated from the CIC library; however their affinities for IC were low and only three fold greater than their affinities for anti-atrazine Ab. Future approaches to the selection of anti-IC scFvs are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Bioengineering & biomedical engineering