Analytica Chimica Acta 651 (2009) 98–104 Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca Development of a surface plasmon resonance-based assay for the detection of Corynebacterium pseudotuberculosis infection in sheep Sharon Stapleton a,b , Bernard Bradshaw c , Richard O’Kennedy a,b,∗ a School of Biotechnology, Dublin City University, Dublin 9, Ireland b National Centre for Sensor Research, Dublin City University, Dublin 9, Ireland c Department of Agriculture and Food, Central Veterinary Research Laboratory, Backweston Campus, Young’s Cross, Celbridge, Co. Kildare, Ireland article info Article history: Received 26 April 2009 Received in revised form 3 August 2009 Accepted 11 August 2009 Available online 13 August 2009 Keywords: Corynebacterium pseudotuberculosis Caseous lymphadenitis Surface plasmon resonance (SPR) Serum Non-specific binding abstract Caseous lymphadenitis (CLA), a disease affecting sheep and goats, is caused by Corynebacterium pseu- dotuberculosis and is difficult to detect, especially at early stages in its development. A surface plasmon resonance-based biosensor assay for the detection of antibodies to the phospholipase D (PLD) exotoxin of C. pseudotuberculosis in sheep serum was successfully generated. It employed a recombinant form of PLD, which was immobilised, and all aspects of the assay including minimisation of non-specific binding, and the regeneration of the chip, were optimised. The applicability of the assay was initially demon- strated using sera collected from experimentally infected sheep and from sheep with no prior history of infection. The assay was then evaluated on a panel of clinical samples and the results obtained compared very favourably to those obtained by a double sandwich ELISA (over 90% similarity) and clearly verified its analytical value. © 2009 Published by Elsevier B.V. 1. Introduction Caseous lymphadenitis (CLA) is a disease affecting sheep and goats caused by C. pseudotuberculosis infection [1] and is charac- terised by the enlargement and suppuration of one or more lymph nodes [2]. The bacteria can infect other animal species, such as horses, causing ulcers and abscesses [3] and cows, causing masti- tis and visceral lesions [4]. Human infection has also been reported, although it is usually associated with those occupationally exposed to farm animals [5]. CLA is endemic in many countries worldwide, being most preva- lent in nations where intensive husbandry is practiced. It is also emerging in European countries previously ‘CLA-free’ as border controls are removed and the movement of animals becomes less restricted. The disease is considered to be one of the most econom- ically important diseases of sheep and goats in the U.S., Canada and Australia [6]. Significant losses can occur when the internal lesions go undetected causing decreased milk and wool production and reproductive performance [7]. Other losses may result from the disruption of shearing time to treat a ruptured lesion or disinfect shearing equipment. ∗ Corresponding author at: School of Biotechnology, Dublin City University, Dublin 9, Ireland. Tel.: +353 1 7005319; fax: +353 1 7005412. E-mail address: richardokennedy@gmail.com (R. O’Kennedy). CLA was first recorded in Northern Ireland in 1999, where the source of infection was traced back to imported Scottish sheep [8]. A year later the first case was reported in the Republic of Ireland [9]. Since then there have been a number of cases of the disease reported but it is possible, due to its insidious nature, that many other cases have gone undetected. CLA is a difficult disease to control within a flock due to a num- ber of factors. The subclinical nature of the infection, the long incubation period and recurring nature of the disease make it extremely difficult to distinguish animals that carry the disease from those that do not. Once C. pseudotuberculosis has established itself within a flock, eradication is problematic, as the thick walls of the abscesses characteristic of the bacterial infection are virtu- ally impossible to penetrate with medication [10,11]. Vaccination is used in both Australia and the U.S. as a method of disease control but commercial vaccines are not licensed in the UK and Ireland. However, vaccination would not essentially eradicate CLA from a flock but it would merely reduce the prevalence and severity of the disease. The optimal method of control of CLA is eradication of infection by identification and removal of infected carrier animals [12,13]. A variety of diagnostic tests have been described in the litera- ture. These include an anti-beta-haemolysin test [14], a synergistic haemolysis test [12,15,16], an immunodiffusion test [17], a com- plement fixation assay [18], an indirect haemagglutination test [18], a microagglutination assay [19], and diagnosis using poly- merase chain reaction (PCR) [20]. However, although useful, the 0003-2670/$ – see front matter © 2009 Published by Elsevier B.V. doi:10.1016/j.aca.2009.08.004