== Characterization of ROP4 antigen. ELISA, rhoptry protein 4, virus-like particle, IgG == INTRODUCTION == Toxoplasma gondii, the causative agent of toxoplasmosis, is a parasite capable of evading both phagocytic and non-phagocytic cells of warm-blooded vertebrates, and approximately a third of the worlds population are estimated to be affected by these pathogens. Their infection can have fatal consequences in pregnant women or immunocompromised individuals [1]. Therefore,T. gondiiis a pathogenic protozoan organism of global importance [1].T. gondiiinfections can be subdivided into acute and chronic stages, which are characterized by the rapid growth of tachyzoites and slow-growing cyst formations predominantly in the brain and musculature. There are 3 distinct clonal lineages ofT. gondiiand these are classified as type I (RH strain), type II (ME49), or type III based on the parasites virulence. While type II and III strains are relatively avirulent and can establish chronic infections, type I strains are highly virulent and uniformly lethal in mice [2,3]. To successfully treat and manage toxoplasmosis patients, as well as estimating disease prevalence, economic loss avoidance, food safety risk evaluation, and establishing epidemic prevention policies at a national level, developing an effectiveT. gondiidiagnostic method for both humans and animals is necessary [4]. Although various direct detection methods are available, such as bioassay, microscopy, or molecular-based assays, these methods are reported to be time-consuming, costly, and possess limited sensitivity. By contrast, multiple serologic tests including indirect haemagglutination test (IHAT), modified agglutination test (MAT), latex agglutination test (LAT), indirect fluorescent antibody test (IFAT), and enzyme-linked immunosorbent assay (ELISA) are more optimized and practical. ELISA, in particular, is widely utilized for its convenience in antigen-specific antibody response detection across various laboratories [5,6]. However, test performance depends mostly on diagnostic antigens. While most conventional tests used today are based on theT. gondiitachyzoite antigens, standardizing this method is difficult due to the presence of nonparasitic components from eukaryotic host cells. Consequently, numerous studies have reported the acquisition of false-positive results along with poor specificity and sensitivity forT. gondiifor this method [7]. In the past decade, multiple recombinant antigens with serodiagnostic potential were identified, which includes dense granule AS8351 proteins (GRA) [810], the surface antigens (SAG) [5,11], microneme proteins (MIC), cyst matrix antigen (MAG1), apical membrane antigen (AMA) [12], and the rhoptry proteins (ROP) [13,14]. In the aforementioned studies, recombinant protein antigens were tested using the sera acquired fromT. gondii-infected humans and animals. To date, not a single study investigated the relationship between RH or ME49 strain infection dose and the resulting antibody induction in murine models. Rather, in majority of the studies performing recombinant antigen-based ELISA using humanT. gondiiinfection sera, several variables remain unknown such as the duration post-infection, infection dose, AS8351 and the strain information. Additionally, most studies only assessed IgG response against recombinant protein along with either IgM or IgA. None CANPml of them evaluated the responses of all 3 antibody isotypes under identical experimental conditions. ROP4, belonging to the ROP2 protein family, is expressed at all AS8351 3 infective stages of theT. gondii, which includes the tachyzoite, bradyzoite, and the sporozoite. Previously, we have demonstrated that ROP4-expressing VLPs were highly immunogenic and as such, these could be exploited as a potentialT. gondiidiagnostic marker [15]. In the present study, sera ofT. gondiiRH and ME49-infected mice were collected and used to evaluate the diagnostic antigen potential ofT. gondiiROP4 VLPs by comparing the IgG, IgM, and IgA antibody responses with those of TLA. Findings herein suggest that ROP4 VLP antigen were highly sensitive and enables the detection ofT. gondiiat an early stage of infection, thus signifying their potential for further development. == MATERIALS AND METHODS == == Ethics statement == Seven-week-old female BALB/c mice were purchased from NARA Biotech (Seoul, Korea) and maintained under specific-pathogen-free conditions. All animal experiments were carried out in accordance with the regulations of the Kyung Hee University IACUC (institutional approval number: KHSASP-20-165). == Parasites and cells == Parasites and cells were maintained and harvested as described previously [4]. Briefly, mice were intraperitoneally infected withT. gondiiRH strain and ME49 strains. Tachyzoites of RH strain were separated from peritoneal fluids at 5 days PI and resuspended in phosphate-buffered saline (PBS), which were subsequently purified by discontinuous Percoll density gradient centrifugation..