Two distinct antigenic subtypes, RSV A and B, circulate independently or simultaneously to cause illness during annual RSV seasons . using a well-characterized gold standard panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (65 years old). The combined results from the four ECL assays demonstrated good concordance to the gold standard diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population. Introduction Respiratory syncytial virus (RSV) is a worldwide cause of severe lower respiratory tract infections. Two distinct antigenic subtypes, RSV A and B, circulate independently or simultaneously to cause illness during annual RSV seasons . Morbidity and mortality resulting from RSV infection are common in high-risk populations such as infants and young children , the elderly and individuals of all ages with cardiopulmonary disease or compromised immune systems . RSV infection is recognized as the primary cause of hospitalization for acute lower respiratory tract infection among infants worldwide, resulting in an estimated 2.1 million children receiving medical care each year in the U.S. . Among adults over the age of 65, RSV infection contributes to over 170,000 hospitalizations and 14,000 deaths annually in the U.S . Palivizumab, a neutralizing monoclonal antibody which recognizes the RSV fusion (F) protein, is used for prevention of RSV disease in high-risk infants ; however, no prophylactic treatment such as a vaccine or monoclonal antibody is available for other susceptible populations . Sensitive and specific assays to detect recent Voreloxin RSV infection are useful to understand the incidence of RSV infection and potentially identify a correlate of protection from epidemiology studies and vaccine clinical trials . Although serology has been shown to be a more sensitive diagnostic approach than viral culture or RT-PCR in adult populations , existing serology assays, such as ELISA or cell-based microneutralization assays, have limitations. Colorimetric ELISA tests have a narrow dynamic range while cell-based microneutralization assays may have higher variability and are more labor intensive. For these reasons, we evaluated Meso Scale Discovery (MSD)s electrochemiluminescence (ECL) technology platform for its reported wide dynamic range, improved analytical sensitivity and reduced non-specific background signal. Of the eleven proteins encoded by the RSV genome, we selected the fusion (F), nucleocapsid (N) and attachment (G) proteins for assay development using ECL technology. Both F and G antigens elicit neutralizing antibodies that can provide protection against subsequent infection , and RSV vaccines frequently include or express these antigens [9C11]. The use of F, N and G antigens to measure Voreloxin serum antibody levels from RSV exposure is well-documented [12C16]. Although the amino acid sequences of F Rabbit Polyclonal to KAP1 and N are highly conserved between RSV A and B subtypes [17, 18], the sequence of G differs dramatically and provides the principle source of antigenic variation among circulating strains [19C23]. In order to measure G-specific antibodies regardless of the infecting strains subtype, we included G antigen from both RSV subtypes (Ga and Gb) as part of our diagnostic strategy. Four ECL assays (F, N, Ga and Gb IgG) were developed and evaluated for analytical and diagnostic performance . To evaluate the diagnostic sensitivity and specificity of the four ECL assays, we assembled a well-characterized, gold standard panel of acute and convalescent serum samples from eighty-nine elderly (65 years old) participants of an RSV surveillance study . Our results demonstrate that RSV antigen-specific serology assays using ECL technology have several advantages and provide an improved method to detect recent RSV infection in an elderly population. Materials and Methods Reagents RSV antigens were expressed and purified to 90% purity, as determined Voreloxin by SDS-PAGE. Fifty milligrams (50 mg) of a soluble and post-fusion form of the F antigen from the RSV A2 strain was expressed in Chinese Hamster Ovary (CHO) cells and affinity purified with an anti-RSV F monoclonal antibody (palivizumab, MedImmune, Gaithersburg, MD) . The RSV F protein sequence is available in the GenBank database (http://www.ncbi.nlm.nih.gov/GenBank) under accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”KJ155694″,”term_id”:”594191634″,”term_text”:”KJ155694″KJ155694. One hundred milligrams (100 mg) of full length nucleocapsid protein.