The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep

Charlton, Keith A.; Harris, William J.; Porter, A.J.

doi:10.1016/s0956-5663(01)00192-0

Cited by 77 publications

(55 citation statements)

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“…Particularly, the production of RAbs to agrochemicals such as paraquat (Graham et al 1995), diuron (Scholthof et al 1997), parathion (Garret et al 1997), cyclohexanedione (Webb et al 1997), picloram (Yau et 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 al. 1998), chlorpyrifos (Alcocer et al 2000), atrazine (Charlton et al 2001), malathion (Nishi et al 2003), 2,4−dichlorophenoxyacetic acid (Brichta et al 2003), simetryn (Nishi et al 2005), and methamidophos (Li et al 2006) has been described. Initially, hapten-specific RAbs were obtained from hybridoma cells; subsequently, immunized lymphocytes were the source of genetic material to construct immune phage-display antibody libraries from which hapten-specific RAbs have been selected (Brichta et al 2005;Markus et al 2011).…”

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Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

et al. 2013

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Abstract:A comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against tetraconazole, thiabendazole, and imazalil were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices. Powered by Editorial Manager® and Preprint Manager® from Aries Systems Corporation AbstractA comparative study of the analytical performance of enzyme-linked immunosorbent assays (ELISAs), based on monoclonal and recombinant antibodies, for the determination of fungicide residues in fruit juices has been carried out. To this aim, three murine hybridoma cell lines secreting specific monoclonal antibodies against tetraconazole, thiabendazole, and imazalil were used as a source of immunoglobulin gene fragments for the production of single-chain variable fragment (scFv) and fusion scFv-pIII recombinant antibodies in Escherichia coli. Selected recombinant antibodies displayed cross-reactivity profiles very similar to those of the parent monoclonal antibodies. Imazalil and tetraconazole recombinant antibodies showed one order of magnitude lower affinity than their respective monoclonal antibodies, whereas the thiabendazole recombinant antibodies showed an affinity similar to that of their parent monoclonal antibody. On the other hand, scFv-pIII fusion fragments showed similar analytical properties as, and occasionally better than, scFv recombinant antibodies. Finally, ELISAs developed from each antibody type showed similar analytical performance when applied to the analysis of the target fungicides in fruit juices.

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Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

et al. 2013

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“…We believe that strategy based in this work on the use of low hapten density in the coated conjugate, together with optimized elution steps, improved selection ability of the library. This approach can be an alternative to the established selection methods of the selection such as affinity separation based on phage particles capture by biotin labeled hapten on magnetic particles and subsequent elution with free hapten (Hawkins et al, 1992) or decreasing concentrations of hapten conjugate in coated tubes used by Charlton et al (2001). The approach suggested in this work allows modulation of the hapten density on the selection surface in order to reduce potential avidity binding formation between phage molecules and hapten protein conjugates.…”

Section: Discussionmentioning

confidence: 99%

“…The authors produced the scFv fragments using direct cloning of variable parts of antibody genes from immunized mice or employed recombinant phage antibody system from RPAS, Pharmacia. The supersensitive antibody against atrazine was produced by Charlton et al (2001) using phage library. The limit of detection (1-2 ppt) established in ELISA is about three orders of magnitude higher compared to the above reported antibody reagents.…”

Section: Discussionmentioning

confidence: 99%

“…The final step, elution of specific phage particles, was performed in this method by non-labeled hapten. Charlton et al (2001) employed for the affinity selection hapten-conjugate coated tubes in combination with elution by triethylamin or hapten molecules. Stringency of selection process was achieved by reduced concentration of the hapten conjugate on the coating surface.…”

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Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naďve phage library

Brichta¹,

Veselá²,

Fránek³

2003

Vet. Med. - Czech

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Three single chain variable fragment (scFv) antibodies against 2,4-dichlophenoxyacetic acid (2,4-D) herbicide were produced by the Griffin1.library. The selection of the scFv from the phage library was carried out by 2,4-D-protein coated tubes with different levels of hapten substitution in the conjugate. The scFv phage clones were isolated within the five round library panning and the antibodies were expressed in Escherichia coli HB2151. The recombinant products were purified by metal affinity chromatography yielding 200 g of pure scFv per 1 liter of bacterial culture. The antibody fragments provided steep curves in conventional indirect ELISA having the IC<sub>50</sub> values from 10.2 to 14.5 ng/ml established for 2,4-D standard. Interestingly enough, the recombinant ScFv E1 antibody exhibited 68% cross-reactivity with 2,4-dichlorphenol (2,4-D = 100%), and 38.0% with methylchlorophenoxyacetic acid (MCPA) whereas reaction with other phenoxyacetic compounds was low. Similar characteristics were obtained for other two recombinant products. Low stability for the isolated scFv antibodies was found in storage buffer even in the presence of stabilizers and protease inhibitors. Factors influencing stability of the recombinant antibodies are discussed.

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“…Prior to 1990, there were few reports of the isolation of rAbs against low molecular weight molecules (haptens) such as drugs of abuse, vitamins, hormones, metabolites, food toxins and environmental pollutants, including heavy metals and pesticides. Hapten-specific antibodies (Charlton et al, 2001), and immunoaffinity chromatography purification columns (Sheedy & Hall, 2001). Commercial immunoassays for haptens such as small environmental contaminants still rely mostly on polyclonal antibodies rather than monoclonal or recombinant antibodies fragments (Sheedy et al, 2007).…”

Section: Applications Of Rabs Against Low Molecular Ligandsmentioning

confidence: 99%

Recombinant Antibodies and Non-Antibody Scaffolds for Immunoassays

Ban¹,

Blake²

2012

Advances in Immunoassay Technology

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The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep

Cited by 77 publications

References 23 publications

Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit Juices

Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naďve phage library

Recombinant Antibodies and Non-Antibody Scaffolds for Immunoassays

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