Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies Sheikh M. Talha 1 *, Jukka Hyto ¨ nen 2 , Adam Westhorpe 3 , Sushil Kumar 4 , Navin Khanna 4 , Kim Pettersson 1 1 Department of Biotechnology, University of Turku, Turku, Finland, 2 Department of Medical Microbiology and Immunology, University of Turku, Turku, Finland, 3 University of Surrey, Guildford, Surrey, United Kingdom, 4 Recombinant Gene Products Group, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi, India Abstract Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies. Citation: Talha SM, Hyto ¨ nen J, Westhorpe A, Kumar S, Khanna N, et al. (2013) Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies. PLoS ONE 8(12): e84050. doi:10.1371/journal.pone.0084050 Editor: Jianming Tang, University of Alabama at Birmingham, United States of America Received September 13, 2013; Accepted November 15, 2013; Published December 26, 2013 Copyright: ß 2013 Talha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work has been carried out under a bilateral Indo-Finnish collaborative research programme, and was supported by funding from the Finnish Funding Agency for Technology and Innovation (TEKES), Finland, and Department of Biotechnology (DBT), Government of India. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: talshe@utu.fi Introduction Treponema pallidum subspecies pallidum (Tp), a spirochete bacte- rium, is responsible for syphilis which is a sexually transmitted infection in humans. In 2008 there were an estimated 10.6 million new cases of syphilis in adults worldwide [1]. Worldwide nearly 1.4 million pregnant women, with the disease burden of 44% in Asia and 39% in Africa, had active syphilis infection and were at the risk of transmitting the disease to their unborn babies [2]. Syphilis infection is well known for its multiple stages of pathogenicity, split by stages of latency. As the stages of infection proceed, so does the complication of the disease, thus it is essential that the infection is diagnosed and treated as early as possible [3–6]. Syphilis is also one of the transfusion-transmissible infections, which is of a particular concern for the developing countries. To curtail the spread of syphilis through the transfusion of blood and blood- products meticulous screening of the donated blood is paramount [7–9]. Serological diagnosis of syphilis includes the detection of two different kinds of molecules, namely treponemal and non- treponemal antibodies. Treponemal antibodies are raised against specific antigens of Tp, and non-treponemal antibodies are raised against cardiolipin [10]. Treponemal antibodies last throughout the life, whereas non-treponemal antibodies are present only in an on-going infection, thus non-treponemal antibody tests are useful to distinguish between convalescent cases and active infections. However, non-treponemal antibody tests usually suffer from low specificity, thus a combination of treponemal and non-treponemal antibodies tests are required for the clinical diagnosis of syphilis. According to WHO’s recommendation, in a blood-bank setting screening of only treponemal antibodies should be performed in a population with low incidence of syphilis [11]. Three Tp membrane proteins Tp15, Tp17 and Tp47, named after their respective sizes in kDa, are known to be highly immunogenic, and varying titers of the (treponemal) antibodies against these proteins can be detected in individuals during primary, secondary and latent stages of syphilis infections [12–16]. We have developed a PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e84050