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

[EN] 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 (RS)-2-(2,4-dichlorophenyl)-3-(1H-1,2,4-triazol-1-yl)propyl-1,1,2,2-tetrafluoroethyl ether (tetraconazole), 2-(4-triazolyl)benzimidazole (thiabendazole), and (RS)-1-(beta-allyloxy-2,4-dichlorophenylethyl)imidazole (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.This work was funded by Ministerio de Educacion y Ciencia (MEC, Spain, Project AGL2002-03266). E. P. was the recipient of a doctoral fellowship from Conselleria d'Educacio (Generalitat Valenciana, Spain).Moreno Tamarit, MJ.; Plana Andani, E.; Manclus Ciscar, JJ.; Montoya Baides, Á. (2014). Comparative Study of Monoclonal and Recombinant Antibody-Based Immunoassays for Fungicide Analysis in Fruit juices. Food Analytical Methods. 7(2):481-489. https://doi.org/10.1007/s12161-013-9655-zS48148972Abad A, Manclús JJ, Moreno M, Montoya A (2001) J AOAC Int 84:1–6Alcocer MJC, Doyen C, Lee HA, Morgan MRA (2000) J Agric Food Chem 48:4053–4059Brichta J, Vesela H, Franek M (2003) Vet Med 48:237–247Brichta J, Hnilova M, Viskovic T (2005) Vet Med 50:231–252Charlton K, Harris WJ, Potter AJ (2001) Biosens Bioelec 16:639–646EU Pesticide Database (2013) Pesticide EU-MRLs. http://ec.europa.eu/sanco_pesticides/public/index.cfm . Accessed Jan 2013Ferrer C, Martínez-Bueno MJ, Lozano A, Fernández-Alba AR (2011) Talanta 83:1552–1561Garret SD, Appleford DJA, Wyatt GM, Lee HA, Morgan MRA (1997) J Agric Food Chem 45:4183–4189Graham BM, Porter AJ, Harris WJ (1995) J Chem Technol Biotech 63:279–289Hiemstra M, de Kok A (2007) J Chromatog A 1154:3–25Kipriyanov SM, Moldenhauer G, Little M (1997) J Immunol Meth 200:69–77Kramer K, Hock B (2007) Recombinant antibodies for agrochemicals: Evolutionary optimization. In: Kennedy IR, Solomon KR, Gee SJ, Crossan AN, Wang S, Sánchez-Bayo F (eds) Rational environmental management of agrochemicals: Risk assessment, monitoring, and remedial action. ACS Symposium Series, vol. 966, pp 155−170Krebber A, Bornhauser S, Burmester J, Honegger A, Willuda J, Bosshard HR, Plückthun A (1997) J Immunol Meth 201:35–55Leong SSJ, Chen WN (2008) Chem Engin Sci 63:1401–1414Li T, Zhang Q, Liu Y, Chen D, Hu B, Blake DA, Liu F (2006) J Agric Food Chem 54:9085–9091Manclús JJ, Moreno M, Plana E, Montoya A (2008) J Agric Food Chem 56:8790–8800Markus V, Janne L, Urpo L (2011) Trends Anal Chem 30:219–226Mersmann M, Schmidt A, Tesar M, Schöneberg A, Welschof M, Kipriyanov S, Terness P, Little M, Pfizenmaier K, Moosmayer D (1998) J Immunol Meth 220:51–58Moreno M, Plana E, Montoya A, Caputo P, Manclús JJ (2007) Food Addit Contam 24:704–712Morozova VS, Levashova AI, Eremin SA (2005) J Anal Chem 60:202–217Nishi K, Imajuku Y, Nakata M, Ohde K, Miyake S, Morimune K, Kawata M, Ohkawa H (2003) J Pest Sci 28:301–309Nishi K, Ishiuchi M, Morimune K, Ohkawa H (2005) J Agric Food Chem 53:5096–5104Scholthof KB, Whang G, Karu AE (1997) J Agric Food Chem 45:1509–1517Sheedy C, MacKenzie CR, Hall JC (2007) Biotech Adv 25:25333–25352Tout NL, Yau KYF, Trevors JT, Lee H, Hall JC (2001) J Agric Food Chem 49:3628–3637Webb SR, Lee H, Hall JC (1997) J Agric Food Chem 45:535–541Yau KYF, Tout NL, Trevors JT, Lee H, Hall JC (1998) J Agric Food Chem 46:4457–4463Yoshioka N, Akiyama Y, Matsuoka T, Mitsuhashi T (2010) Food Control 21:212–21

Multifaceted Regulation of Translational Readthrough by RNA Replication Elements in a Tombusvirus

Translational readthrough of stop codons by ribosomes is a recoding event used by a variety of viruses, including plus-strand RNA tombusviruses. Translation of the viral RNA-dependent RNA polymerase (RdRp) in tombusviruses is mediated using this strategy and we have investigated this process using a variety of in vitro and in vivo approaches. Our results indicate that readthrough generating the RdRp requires a novel long-range RNA-RNA interaction, spanning a distance of ∼3.5 kb, which occurs between a large RNA stem-loop located 3'-proximal to the stop codon and an RNA replication structure termed RIV at the 3'-end of the viral genome. Interestingly, this long-distance RNA-RNA interaction is modulated by mutually-exclusive RNA structures in RIV that represent a type of RNA switch. Moreover, a different long-range RNA-RNA interaction that was previously shown to be necessary for viral RNA replicase assembly was also required for efficient readthrough production of the RdRp. Accordingly, multiple replication-associated RNA elements are involved in modulating the readthrough event in tombusviruses and we propose an integrated mechanistic model to describe how this regulatory network could be advantageous by (i) providing a quality control system for culling truncated viral genomes at an early stage in the replication process, (ii) mediating cis-preferential replication of viral genomes, and (iii) coordinating translational readthrough of the RdRp with viral genome replication. Based on comparative sequence analysis and experimental data, basic elements of this regulatory model extend to other members of Tombusviridae, as well as to viruses outside of this family

Sequence characterization, molecular phylogeny reconstruction and recombination analysis of the large RNA of Tomato spotted wilt virus (Tospovirus: Bunyaviridae) from the United States

BACKGROUND: Tomato spotted wilt virus (TSWV; Tospovirus: Bunyaviridae) has been an economically important virus in the USA for over 30 years. However the complete sequence of only one TSWV isolate PA01 characterized from pepper in Pennsylvania is available. RESULTS: The large (L) RNA of a TSWV WA-USA isolate was cloned and sequenced. It consisted of 8914 nucleotides (nt) encoding a single open reading frame of 8640 nts in the viral-complementary sense. The ORF potentially codes for RNA-dependent RNA polymerase (RdRp) of 330.9 kDa. Two untranslated regions of 241 and 33 nucleotides were present at the 5′ and 3′ termini, respectively that shared conserved tospoviral sequences. Phylogenetic analysis using nucleotide sequences of the complete L RNA showed that TSWV WA-USA isolate clustered with the American and Asian TSWV isolates which formed a distinct clade from Euro-Asiatic Tospoviruses. Phylogeny of the amino acid sequence of all tospoviral RdRps used in this study showed that all the known TSWV isolates including the USA isolate described in this study formed a distinct and a close cluster with that of Impateins necrotic spot virus. Multiple sequence alignment revealed conserved motifs in the RdRp of TSWV. Recombination analysis identified two recombinants including the TSWV WA-USA isolate. Among them, three recombination events were detected in the conserved motifs of the RdRp. CONCLUSIONS: Sequence analysis and phylogenetic analysis of the L RNA showed distinct clustering with selected TSWV isolates reported from elsewhere. Conserved motifs in the core polymerase region of the RdRp and recombination events were identified. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13104-016-1999-1) contains supplementary material, which is available to authorized users