Inside a cohort of kidney transplant individuals (CMV donor positive, recipient negative) who acquired primary CMV infection, all developed powerful interferon (IFN-) responses to a pool of CMV peptides that included UL144. and optimization. A cutoff optical denseness (OD) that distinguishes UL144 antibody-positive from antibody-negative sera was founded. UL144 A, B, C, and mixtures of these antigens were recognized in sera. An assay DNM3 threshold of 0.1 was established and, from a total of 303 sera, the overall sensitivity, specificity, and positive and negative predictive ideals of the multiplex ELISA were 86.72% (95% confidence interval [CI] 79.59% to 92.07%), 96.57% (92.69% to 98.73%), 94.40% (88.45% to 97.38%), and 91.60% (87.50% to 94.44%), respectively. The inter- and intraassay median coefficients of variance were 0.06 (interquartile range [IQR] 0.56, 0.2) and 0.171 (IQR 0.038, 0.302), respectively. No cross-reactivity was observed with HSV-positive CMV-negative sera. This ELISA gives simple and reproducible results for detection of anti-CMV UL144 IgG. It may assist in differentiating Gosogliptin natural illness from CMV vaccines that lack UL144, and may provide an important tool for epidemiological studies of CMV strains. strong class=”kwd-title” KEYWORDS: ELISA, UL144, cytomegalovirus, multiplex, serotypes Intro The advancement of molecular assays for monitoring cytomegalovirus (CMV) disease offers improved clinical end result, allowing for timely therapeutic decisions. However, creating CMV serostatus in pregnant women and organ donors/recipients remains critical for further diagnostic and restorative considerations (1). Antibody measurements are particularly useful in determining risk of CMV acquisition in seronegative individuals (1, 2). Dedication of CMV acquisition or exposure in previously CMV seronegative individuals is based on interval screening. CMV DNAemia is definitely detectable for a short time in plasma, although CMV DNA can be recognized in urine and saliva for up to several months (3). An antibody-based assay may provide a practical and accurate assessment of CMV serostatus. The commercially available ELISAs are based on detection of antibody reactions to whole disease preparations prepared from attenuated laboratory-adapted strains such as AD169. Realizing the importance of CMV strain variance, several ELISA methods were developed to identify strain-specific antibodies against the CMV glycoproteins gH and gB (4, 5). Furthermore, the development of CMV vaccines resulted in the need for sensitive and specific assays for follow up of infections after immunization. For that reason, concomitant with the development of CMV glycoprotein B vaccines, an IgG ELISA was developed using UL32 (pp150) as capture antigen, to eliminate the need for gB preabsorption and verification (6). Immunization with CMV vaccines based on the AD169 strain may elicit neutralizing antibody responses that are indistinguishable from neutralizing antibodies to natural infection. To address this limitation, we aimed to develop a new multiplex ELISA for CMV protein UL144. The UL144 gene, present in the UL/b boundary of the CMV genome, is present in clinical isolates but absent in AD169 and other laboratory-adapted strains (7). We as well as others defined three major, unique UL144 genotypes from multiple initial samples and clinical isolates (8,C11). The three major UL144 genotypes (A, B, and C) and recombinants have similar distribution in different geographical locations (9, 12, 13), and specific genotypes may be associated with severe sequelae of congenital CMV contamination (8, 12). UL144 is usually a membrane-anchored glycoprotein that is largely intracellular, but is also found on the cell surface when overexpressed (14, 15). This statement characterizes an ELISA method for Gosogliptin measurement of the serum antibody response to the three UL144 types. MATERIALS AND METHODS UL144 protein expression and purification. UL144 A, B, and C genotypes were codon optimized for expression and secretion by mammalian cells, and a C-terminal 6 histidine tag was added (Genscript Biotech Corp., Piscataway, NJ). Gene sequences were subcloned into pcDNA3.4. The producing plasmids were transfected into Expi293F cells produced in serum-free Expi293 expression medium (Thermo Fisher Scientific, Waltham, MA) and managed at 37C with 8% CO2 on an orbital shaker. DNA and ExpiFectamine 293 reagent (Thermo) were mixed and added into the cells. Cell culture supernatants were collected, Gosogliptin centrifuged, and purified on a Ni-NTA column. Sample purity was evaluated using a 4 to 20% gradient SDS-PAGE gel, and protein concentration was determined by Bradford assay. Protein sequences are provided in Fig. S1 in the supplemental material. Serum samples and ethics statement. CMV seropositive and seronegative sera were collected from your Childrens Wisconsin Clinical Microbiology Laboratory and the Wisconsin Diagnostic Laboratories (WDL, March 2019 to March 2021). The Institutional Review Table.