Broad-specificity immunoassay of low molecular weight food contaminants: new paths to Utopia!

Spinks, Caroline A

doi:10.1016/s0924-2244(01)00009-7

Cited by 63 publications

(56 citation statements)

References 33 publications

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“…Further development in agri-food analytical immunochemistry depends almost exclusively on the ability to develop novel antibodies in a more cost-effective manner (Spinks, 2000). Phage antibodies produced by naïve recombinant phage libraries represent an alternative to the conventional and monoclonal antibodies.…”

Section: Discussionmentioning

confidence: 99%

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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Section: Discussionmentioning

confidence: 99%

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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“…By nature, mAb originate from one specific clone with higher specificity, purity, reliability and identify only one epitope of the antigen. However, these antibodies have need of secondary antibodies which are used in two analyses (Spinks, 2000). Such methods are used in laboratories for common techniques in many medical research and diagnostics (Hawkins et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

The Profile of the Expression of IBMR3 Antigens 3T3 and HT29 Cancer Cell Line after Analysis

Alrawi¹

2014

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Abstract:Aims: The aim of this study was to evaluate the effectiveness of specific monoclonal IBMR3 antibodies expression in the cancer cell lines of 3T3 and HT29, in order to recognize specific antigen and make evaluation, using immunoblotting technique which is widely use in this filed. Methods: Protein extracts were extracted from these cancer cell lines and used for western bloat. The Immunobloting were consequently subjected to densitometry analysis using bioimaging machine. This bioimaging process will facilitate to measure the molecular weights, peak height and raw volume of the protein bands for the 3T3 and HT29 cancer cell lines, which helps in diagnosis of any pathogenic antigen. Results: The bands obtained from bioimaging were exposed on the PVDF membrane. In the 3T3 bio imaging process revealed four bands and molecular weight were 299. IgM serotype was not indicated that means no specific antigens for negative control IgM. Conclusion: The bioimaging revealed different results of the expression profile in molecular weight, peak height and raw volume between the two cancer cell lines. The results from this study suggest that the IBMR3 antigens were differentially expressed in 3T3 and HT29 and the molecular weight is higher in the 3T3 than HT29 cancer cell lines. In future it will be beneficial to categorize and study the character analysis of the IBMR3 antigen and its prospective role in cancer cells.

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“…1,2 However, because these compounds are hapten substances and have no immunogenesis, they have to be conjugated to macromolecular carriers such as proteins before immunizing animals to generate specific immunoglobulin. Therefore, preparation of high-quality antibodies appears to be one of the most important techniques when developing an immunoassay method for the low-molecular-weight contaminants.…”

Section: Introductionmentioning

confidence: 99%

Cloning, expression, and identification of anti‐carbofuran single chain Fv gene

Wang

Yang

Liu

et al. 2009

Biotechnology Progress

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Phage display method was used to clone anti-carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF-specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly(4)Ser)(3). The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBF antibody libraries were then constructed. After one round of panning with CBF-ovalbumin (CBF-OVA) as a conjugate, antigen-binding positive recombinant phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS-PAGE and showed HRP-anti-E-tag antibody-recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC(50) value of 1.07 ng/mL. The cross reactivity studies showed that the anti-CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF.

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Broad-specificity immunoassay of low molecular weight food contaminants: new paths to Utopia!

Cited by 63 publications

References 33 publications

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

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

The Profile of the Expression of IBMR3 Antigens 3T3 and HT29 Cancer Cell Line after Analysis

Cloning, expression, and identification of anti‐carbofuran single chain Fv gene

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