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 | Screening of monoclonal antibodies using antigens labelled with acetylcholinesterase |
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| In the course of an immunoassay (IA) development, one of the key points is the production of high affinity specific antibodies. Due to the widespread use of im-munometric assays which requires high amounts of antibodies, it is currently necessary to prepare monoclonal antibodies (mAbs). |
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| Among the different steps involved in the production of mAbs, one of the most critical ones is certainly the correct selection of positive clones. An ideal screening method would include i) a good sensitivity allowing the detection of antibodies below the µg/ml range, ii) a high specificity resulting in a strict characterisation of clones secreting the expected antibodies, avoiding positive and negative false and iii) a simple methodology, since cloning primary screening may involve many assays (up to 4 000). |
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| One of the screening procedures proposed is based on the use of a solid-phase containing an universal reagent of mouse immunoglobulins (i.e. a second an-tibody). MAbs bound to this solid-phase are detected by their ability to bind the specific labelled antigen against which they have been raised (see figure). |
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| This has been applied for several molecules (e.g. pe-ripheral proteins of photosystem, Interleukins etc..). Authors namely J. Grassi et al. have shown the differ-ent advantages of this approach (see Methods in Molecular Biology, Vol. 10, 1992, ed. M. Masson). The labelling of antigen with the enzyme acetylcholi-nesterase (AChE) from E. electricus, can be easily carried out by using the avidin/biotin system. The labelling of peptide structure with biotin can be achieved within few hours. In addition, avidin-AChE conjugate is a universal reagent which can be kept for months at -20°C. |
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| Because of its turnover, AChE can be detected at a very low level and therefore provides a greater sensi-tivity compared with other enzymes or 125I-labelled antigens. Consequently, scre-ening requires only small volumes of culture supernatants (50 to 100 µl). |
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| This immuno-enzymatic approach permits to test 2 000 culture supernatants by one person in 4 h (versus approximately 36 hours using RIA). This is the result of the combined use of the microtiter plate, solid-phase separation method and the colorimetric enzyme assay which can readily be automated. J.G.& X.M.. |
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