Properties of Polyclonal, Monoclonal, and Recombinant Antibodies Recognizing the Organophosphorus Pesticide Chlorpyrifos-ethyl

Abstract

A rabbit polyclonal antiserum and two murine monoclonal antibodies recognizing the organophosphorus pesticide chlorpyrifos-ethyl were produced. The two hybridoma cell lines were then used as sources of immunoglobulin genes for the generation of recombinant scFv antibodies in Escherichia coli. The two scFvs showed either similar or improved limits of detection in an ELISA when compared with the monoclonal antibodies. Cross-reactivity studies showed that all of the antibodies were specific toward the chlorinated aromatic ring. Furthermore, scFv gene sequences were linked directly to sequences coding for either a c-Myc tag, a His-tag, or alkaline phosphatase. The fusion products generated were functional, and their properties were determined. The problems associated with producing scFvs and scFv derivatives for detection of pesticide residues from hybridoma are addressed and discussed.

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

“…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).…”

“…Different fusion protein partners have been evaluated to increase scFv solubility and stability, and to improve the detection, purification or immobilization of scFv (Leong and Chen 2008). Thus, short peptides as c-myc, poly-histidine, FLAG or HSV and larger polypeptides as the truncated pIII protein of phage M13 or alkaline phosphatase have been used for detection and purification; on the other hand, engineered single-amino acid residues have been introduced for site-specific chemical coupling of RAbs (Alcocer et al 2000;Krebber et al 1997;Markus et al 2011;Nishi et al 2005). …”

“…As previously argued, advances in RAb production and selection, the introduction at convenience of functional molecules for detection or immobilization, and the possibility of modifying RAb properties afford a powerful technology to obtain tailor-made binders for analytical purposes in food (Markus et al 2011). However, few studies dealing with the application of recombinant-based immunoassays to the analysis of agrochemicals in food and environmental samples have been reported to date (Alcocer et al 2000;Nishi et al 2005). Consequently, understanding the potential, limitations and opportunities of RAbs in food analysis as compared to conventional antibodies is an important issue to find out the future role of RAbs as immunochemical tools.…”

Selección y caracterización de anticuerpos recombinantes para fungicidas. Aplicación al desarrollo de técnicas inmunoanalíticas

“…Aunque la molécula scFv presenta grandes ventajas frente a Fab para aplicaciones biomédicas (Leong y Chen, 2008), para aplicaciones analíticas de haptenos, no parece haber un criterio claro al respecto. Así, aunque un gran número de investigadores como Webb et al (1997), Alcocer et al (2000), Charlton et al (2001), Li et al (2000), Choi et al (2004), Navarro-Teulon et al (1995), Garret et al (1997), Pan et al (2006), Nishi et al (2005), Graham et al (1995) Yuan et al (1997), Tout et al (2001) y Li et al (2006) han optado por el fragmento scFv; otros como Yau et al (1998), Sholthof et al (1997), Lee et al (1998), Gerdes et al (1999) y Chiu et al (2000 han preferido el fragmento Fab. No obstante, se han descrito más ventajas asociadas al fragmento scFv que al fragmento Fab .…”

“…Por tanto, el fago recombinante no tiene por qué presentar las mismas características analíticas que el AcRb, lo que puede interferir a la hora de seleccionar anticuerpos en función de su afinidad. Por este motivo, algunos autores han optado por la expresión del fragmento soluble y su posterior determinación mediante ELISA como método de cribado y selección de AcRbs (Kramer y Hoch, 1996;Garret et al, 1997;Alcocer et al, 2000;Nishi et al, 2005).…”