Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus Ann Nordengrahn, Sigrun. M. Gustafsdottir, Katja Ebert, Scott M. Reid, Donald P. King, Nigel P. Ferris, Emiliana Brocchi, Santina Grazioli, Ulf Landegren, Malik Merza To cite this version: Ann Nordengrahn, Sigrun. M. Gustafsdottir, Katja Ebert, Scott M. Reid, Donald P. King, et al.. Eval- uation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth dis- ease virus. Veterinary Microbiology, Elsevier, 2008, 127 (3-4), pp.227. 10.1016/j.vetmic.2007.08.026. hal-00532312 HAL Id: hal-00532312 https://hal.archives-ouvertes.fr/hal-00532312 Submitted on 4 Nov 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Accepted Manuscript Title: Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus Authors: Ann Nordengrahn, Sigrun. M. Gustafsdottir, Katja Ebert, Scott M. Reid, Donald P. King, Nigel P. Ferris, Emiliana Brocchi, Santina Grazioli, Ulf Landegren, Malik Merza PII: S0378-1135(07)00414-2 DOI: doi:10.1016/j.vetmic.2007.08.026 Reference: VETMIC 3805 To appear in: VETMIC Received date: 29-6-2007 Accepted date: 15-8-2007 Please cite this article as: Nordengrahn, A., Gustafsdottir, Sn.M., Ebert, K., Reid, S.M., King, D.P., Ferris, N.P., Brocchi, E., Grazioli, S., Landegren, U., Merza, M., Evaluation of a novel proximity ligation assay for the sensitive and rapid detection of foot-and-mouth disease virus, Veterinary Microbiology (2007), doi:10.1016/j.vetmic.2007.08.026 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Manuscript 1 Evaluation of a novel proximity ligation assay for the sensitive and rapid 2 detection of foot-and-mouth disease virus 3 4 Ann Nordengrahn1+*, Sigrun. M Gustafsdottir2+, Katja Ebert3, Scott M. Reid3, Donald P. 3 3 4 4 2 5 King , Nigel P. Ferris , Emiliana Brocchi , Santina Grazioli , Ulf Landegren and 6 Malik Merza1 7 8 1 Svanova Biotech AB, Uppsala Science Park, S-751 83 Uppsala, Sweden 9 2 The Beijer Laboratory, Department of Genetics and Pathology, Rudbeck Laboratory, 10 Uppsala Sweden 11 3Institute of Animal Health, Ash Road, Pirbright, GU24 0NF, United Kingdom 12 4Instituto Zooprofilattico Sperimentale della Lombardia e dell´Emilia Romagna, Via A. 13 Bianchi 7/9, 25124 Brescia, Italy 14 15 * Address correspondence to this author: Svanova Biotech AB, Uppsala Science Park, 16 S-751 83 Uppsala, Sweden email [email protected], tel +4618654907, fax 17 +4618654999 18 + These authors contributed equally to this work 19 20 Abstract 21 A novel proximity ligation assay (PLA) using a pan-serotype reactive monoclonal antibody 22 was developed andAccepted evaluated for the detection of foot-and-mouthManuscript disease virus (FMDV) in 23 clinical samples collected from field cases of disease. The FMDV-specific PLA was found to 24 be 100 times more sensitive for virus detection than the commonly used antigen capture- 25 ELISA (AgELISA). As few as 5 TCID50 were detected in individual assays, which was 1 Page 1 of 24 26 comparable with the analytical sensitivity of real-time RT-PCR. Although this assay was 27 capable of detecting diverse isolates from all 7 FMDV serotypes, the diagnostic sensitivity of 28 the PLA assay was lower than real-time RT-PCR mainly due to a failure to detect some SAT 29 1, SAT 2 and SAT 3 FMDV strains. In conclusion, this new PLA format has high analytical 30 sensitivity for the detection of FMDV in clinical samples and may prove valuable as a rapid 31 and simple tool for use in FMD diagnosis. 32 33 Keywords 34 Proximity ligation assay (PLA), Foot-and-mouth disease (FMD), Diagnostic test, Pathogen 35 detection. 36 37 Abbreviations 38 Proximity Ligation assay (PLA), monoclonal antibody (mAb), Signal to noise (S/N), Tissue 39 culture infectious dose (TCID50), Phosphate-buffered saline (PBS), Reverse Transcriptase 40 PCR (RT-PCR), threshold cycle (Ct) 41 42 Introduction 43 Foot-and-mouth disease virus (FMDV) is a member of the family Picornaviridae and exists as 44 seven immunologically distinct serotypes (A, O, C, SAT 1, SAT 2, SAT 3, and Asia 1). The 45 disease caused by this virus is endemic in many regions of Africa, Asia and South America, 46 often causing extensive epidemics in domesticated cloven-hoofed livestock. In addition, more 47 than 70 species of wild mammals belonging to more than 20 families are susceptible to 48 infection. The virusAccepted can also cause persistent infection Manuscript of the pharynx in cattle, sheep, goats, 49 and the other ruminants which can complicate the epidemiology and control of the disease. 50 The highly contagious nature of FMD motivates great urgency in laboratory diagnostic 2 Page 2 of 24 51 analysis, especially when the virus is introduced into countries previously classified as FMD- 52 free. Such was the case in the United Kingdom in 2001 when the FMD epidemic resulted in 53 devastating losses to the food, farming, tourism and leisure industries. (Thompson et al., 54 2002). 55 56 Diagnosis of FMD depends upon early recognition of the clinical signs of disease in the field, 57 followed by confirmation of the presence and serotype-specificity of FMDV in the laboratory. 58 Established laboratory assays for the detection of FMDV include virus isolation (VI) in cell 59 culture (Snowden, 1966), antigen capture ELISA (Ferris and Dawson, 1988), and reverse 60 transcription polymerase chain reaction (RT-PCR; Reid et al., 2002, 2003). VI has high 61 sensitivity and is considered the gold standard method but it can be slow and laborious. In 62 contrast, antigen ELISA is more rapid but has lower sensitivity and therefore cannot be 63 reliably used to confirm negative cases. RT-PCR has recently been shown to be a rapid 64 method with still higher diagnostic sensitivity than VI (Shaw et al., 2004; King et al., 2006). 65 Furthermore, since FMD cannot be differentiated clinically from the other vesicular viral 66 diseases of swine e.g. swine vesicular disease (SVD), vesicular stomatitis (VS) and vesicular 67 exanthema of swine (VES), differential diagnosis is an important aspect of laboratory 68 investigation. 69 70 The aim of this study was to evaluate the proximity ligation assay (PLA) for detection of 71 FMDV in clinical samples. This is a new technique that has been used for detection of 72 proteins and microorganisms in complex biological samples and has already been shown to be 73 as sensitive as moreAccepted established nucleic acid detection Manuscript assays such as PCR (Gustafsdottir 74 et.al., 2006). The basis of the PLA is that FMDV specific-antibodies binding target proteins 75 are coupled to oligonucleotide strands. These oligonucleotides can be joined by ligation when 3 Page 3 of 24 76 two or more such reagents are brought into proximity by binding to the same target molecule 77 or target molecule complex (Figure.1). The DNA ligation products are subsequently detected 78 by PCR amplification using fluorogenic probes to detect the amplified product. 79 80 Material and Methods 81 Propagation of FMDV cell culture-grown antigen and determination of TCID50 82 TCID50 values for cell culture-grown viruses were calculated according to the method 83 described by Kärber (1979). 84 85 Monoclonal antibody (mAb) 1F10 for use as a binding ligand in the PLA 86 The mAb 1F10 was chosen for this study due to its capability to recognise all seven FMDV 87 serotypes in both a trapping ELISA (Samuel et al, 1991) and a sandwich ELISA (Brocchi et 88 al. 1993). In principle, in the trapping ELISA, the mAb 1F10 reacted with each of the seven 89 serotypes preliminarily immune-captured onto the solid phase by an homologous rabbit 90 antiserum; in the sandwich ELISA, the mAb 1F10 was used either as antigen-capture 91 antibody or as the second antibody conjugated with peroxidase. 92 The mAb 1F10 was obtained from a mouse immunised with the FMD virus type O, strain 93 UK31/2001. The mAb belongs to IgG1 isotype, does not neutralise virus infectivity and is 94 presumably directed against a conformation-dependent epitope since it does not recognise 95 isolated viral proteins in immuno-blotting test (data not shown). 96 97 Biotinylation of mAb and preparation of proximity ligation probes 98 Biotinylation wasAccepted performed according to the manufacturer’s Manuscript instructions (Roche Diagnostics 99 Corp, Germany). Briefly, D-biotin-N-hydroxysuccinimide ester was mixed with the antibody 100 in a 10-fold molar excess and with a volume ratio of 1:10. The solution was incubated for 4 4 Page 4 of 24 101 hours at room temperature with continuous agitation. The biotinylated mAbs were then 102 dialysed thoroughly in phosphate buffered saline (PBS, pH 7.4) to remove unbound biotin.