Tachyzoites of a high-virulence RH T. gondii strain (ATCC PRA-310) were maintained in vitro by infection of the APRE19 cell line (ATCC CRL-2302) and cultured in DMEM/F-12 medium (LM002-04, Welgene, Korea) containing 2% of fetal bovine serum (FBS) (16000-044, Gibco, Thermo Fisher Scientific) and 1% penicillin–streptomycin (15140–122, Gibco Thermo Fisher Scientific). Infected cells were cultured at 37°C in a 5% CO₂ atmosphere, and the fresh medium was changed every 2–3 days until tachyzoites were harvested.

The supernatant and scraped infected cells were centrifuged at 1,200g for 10 min at room temperature (RT) to collect the parasites, which were then repeatedly passed through a 25 gauge needle. Tachyzoites were subsequently isolated by gradual and careful layering them on a 40% Percoll (GE17089101, MERK) solution and centrifuging at 1,200g for 20 min at RT. Following the removal of Percoll, the parasite was washed three times in PBS (ML 008-01, Welgene, Korea) to remove cell debris.

8–12-week-old female BALB/c mice were purchased from Orient Bio (Seongnam, Gyeonggi, Korea) and were maintained under conventional conditions. Mice from each group (n = 5) were intraperitoneally (i.p.) inoculated with 10¹–10⁶ tachyzoites of RH strains per mouse. After 2–10 days of infection, mouse blood was collected and sera were obtained for further experiments (Greenfield, 2017). Negative controls included sera from uninfected BALB/c mice of the same age and sex (n = 10), and additional serum groups of P. yoelii (n = 10) were used to assess cross-reactivity. P. yoelii was i.p. injected in the mouse belly with 10⁴ infected RBCs per mouse, and sera from blood were collected on day 5 post-infection at 20–30% parasitemia.

The WHO International Standard 4th IS for Antibodies to Toxoplasma gondii in human plasma (NIBSC code 13/132) is a reference reagent comprising 160 IU (international unit)/ 0.5 mL of pooled plasma obtained from six donors who were recently infected with T. gondii. This freeze-dried preparation contains a significant amount of both IgG and IgM antibodies and is used as a benchmark for diagnostic testing (Rijpkema et al., 2016; NIBSC, 2020).

Healthy individual samples (toxoplasmosis-negative sera) (n = 10), P. vivax-infected samples (n = 10), and a Dengue Mixed Titer Accuset Performance Panel (0845-0074, SeraCare) (n = 10) were used as additional comparable serum samples.

TLA of T. gondii tachyzoites and bradyzoites mixture compared with APRE19 mock-infected cells was prepared and dissolved in 500 μL of sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT, and 2% IPG buffer, pH 3–10 NL (GE17-6000-88, MERK)) and then incubated on ice for 60 min. Freezing and thawing the sample with liquid N₂ were repeated three times, followed by a sonication step for 5 min on ice (1 s on, 5 s off, 40 Amp) and centrifugation at 17,000 rpm for 45 min at 4°C to collect the supernatant. Subsequently, the protein supernatant was subjected to the Readyprep 2D cleanup kit (163-2130, Bio-Rad), following the manufacturer’s instructions. The final purified pellet was re-suspended in 200 μL of sample buffer containing 0.04% w/v bromophenol blue and loaded onto either a ReadyStrip IPG Strip 7 cm pH 3–10 NL (163-2002, Bio-Rad) or Readystrip IPG strip, 7 cm, pH 5–8 (163-2004, Bio-Rad) for rehydration at 50 V for 12–16 h at 20°C using the PROTEAN IEF Cell system (165-4000, Bio-Rad).

All bubbles were removed from underneath the IPG strip before adding 1 mL of strip cover fluid mineral oil (163-2129, Bio-Rad). Subsequently, isoelectric focusing was carried out using a multistep protocol on a 7-cm strip: 250 V for 15 min in linear voltage mode, followed by 1,000 V for 30 min, 4,000 V for 2 h, and then 4,000 V for 5 h in rapid voltage mode. An optional step at 50 V for 15 h was also included.

After the focusing step was completed, individual strips were removed from the IEF machine and then equilibrated for 25 min with equilibrated buffer (1.5 M Tris HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS, and 0.04% bromophenol blue) containing 65 mM DTT followed by additional equilibration for 25 min with the same buffer as used for DTT with 135 mM iodoacetamide. For the second dimension, proteins were separated by SDS-PAGE in a 10% resolving gel. Electrophoresis was performed at 50–70 V for 3 h.

The protein samples obtained from the 2DE gels were transferred onto PVDF membranes, which were previously activated with 100% methanol to enable the optimal transfer of proteins. The membrane was treated with a blocking solution containing 5% skim milk in PBS with 0.1% Tween 20 (PBST) for 2 h at RT, followed by three 5-min washes to minimize non-specific binding. Subsequently, the membranes were incubated at RT for 1 h with a 1:100 dilution of pooled sera obtained either from a normal mouse or 10⁶ tachyzoites of T. gondii RH-infected mice, which were collected 5 days post-infection and prepared in 5% BSA. After washing three times with PBST, the blots were incubated with the corresponding goat anti-mouse IgM (heavy chain) secondary antibody, HRP (Thermo Fisher 62-6820), and diluted 1:5000 in blocking buffer for 1 h at RT. The membranes were washed five times before detection using Clarity Western ECL Substrate (Bio-Rad Cat# 170-5060).

Consistent spots containing the aligned immunoreactive proteins that detected only in T. gondii-infected mouse sera were excised from the five 2DE gels of tachyzoite samples of IPG strip pH3-10 and sent to the LC/MS–MS service for analysis.

To characterize the antigenicity of the proteins, the Linear B cell epitopes of the predicted proteins were determined using ABCprep, BCPreds, and BepiPred-2.0, while the peptides expected to be subjected to T-cell epitope processing and bind to MHC class I and II molecules were analyzed using the EIDB analysis resource tool.¹ The 3D structure was generated using the I-TASSER server, and the 3D structure was modeled and visualized using the PyMOL molecular graphics system.

DNA fragments of three discovered proteins of T. gondii, EF1γ, PGKI, and GAP50, with lengths of 1,182 bp, 1,248 bp, and 1,293 bp, respectively, were sub-cloned into the plasmid pGEM-T Easy Vector (A1360, Promega, Madison, WI, United States) before being cloned into the pET21b (+) plasmid vector. Next, the recombinant plasmid encoding T. gondii EF1γ was transformed into Escherichia coli BL21 DE3 PlysS, while the plasmid DNAs harboring T. gondii PGKI and GAP50 were transduced into the E. coli BL21 DE3 strain, followed by induction with 1 mM isopropyl-b-D-thiogalactopyranoside (IPTG) (Sigma). PGKI was mainly expressed as soluble form in PBS (pH 7.4) lysis buffer. EF1γ and GAP50 proteins were predominantly expressed as inclusion bodies (IB), and then, the protein pellet forms were solubilized and refolded according to previously reported methods (Nguyen et al., 2019). In summary, the induced culture was centrifuged to isolate the cell pellet containing the protein target inclusion bodies. The pellet was washed with Buffer A [10 mM Tris–HCl (pH 7.5)/10 mM EDTA/100 mM NaCl] and resuspended in Buffer A containing 1 mM freshly prepared PMSF. After sonication, the lysate was stirred for 1 h at 4°C with an equal volume of 8 M urea solution. Following centrifugation at 10,000 rpm by a A650TC Cryste rotor, the IB pellet was washed with Buffer B [10 mM Tris–HCl (pH 7.5)/1 mM EDTA/1 M NaCl] and distilled water. The IBs were then re-suspended in solubilization buffer, [100 mM Tris–HCl (pH 7.5)/0.2 mM EDTA/6 M GuHCl], stirred on a magnetic stirrer for 2 h at room temperature (RT), and clarified by centrifugation for 1 h at 4°C. The supernatant containing the solubilized IBs was collected by centrifugation at 17,000 rpm by a Labogene 1730R machine, and then, the refolding was initiated by rapid dilution of the denatured IBs in freshly prepared refolding buffer (100 mM Tris–HCl/0.5 M L-arginine/0.2 mM EDTA, pH 7.5) and incubated for 36–48 h at 10°C. The refolded preparation was dialyzed against 20 mM phosphate buffer (pH 7.5), containing 300 mM NaCl, 10 mM imidazole, and 100 mM freshly prepared urea for 48 h, with buffer changes every 12 h. The dialyzed sample was centrifuged at 10,000 rpm and clarified further by filtration through a 0.45 μm membrane. Finally, batch purification on Ni–NTA resin was conducted for the His-tagged three recombinant proteins according to the manufacturer’s instructions, followed by concentration using a Centricon filter unit.

Western blotting analysis was performed to confirm protein expression. The purified proteins were loaded onto a 10% SDS-PAGE gel and then transferred onto activated PVDF membranes. A blocking buffer (5% non-fat milk in PBS) was used to block the membrane for 2 h at RT. After washing the membrane with PBS-T (PBS containing 0.1% Tween 20), the first anti-mouse 6× His-tag antibody (dilution 1:10000) was incubated with the membrane in a blocking buffer for 1 h at RT. The membranes were then washed three times with PBS-T and incubated with the secondary goat anti-mouse antibody conjugated with HRP (Ab97046, Abcam) in blocking buffer and diluted (1,30,000) for 45 min at RT. The protein bands were visualized using a Bio-Rad ChemiDoc XRS+.

The purified T. gondii proteins such as EF1γ, PGKI, and GAP50, in comparison to BSA, along with TLA of T. gondii RH tachyzoites in uninfected APRE19 cells as a reference, were separated on a 10% SDS-PAGE gel with 20 μg/lane. Afterward, the proteins were transferred onto activated PVDF membranes and then blocked with 5% skim milk at RT for 2 h. Following three washes with PBS-T, 10⁶ T. gondii tachyzoites of mouse sera collected at 5 dpi (dilution 1:100 in 5% BSA) were used as the primary antibody, whereas normal mouse sera served as the control, and the membranes were incubated for 1 h at RT. The membrane was then washed again three times with PBS-T and incubated with goat anti-mouse IgM (heavy chain) secondary antibody HRP (Thermo Fisher 62-6820) and diluted 1:3000 in 5% BSA for 1 h at RT. Finally, the protein bands were visualized as described previously.

To generate complexes of Eu NP and antibody, PS-COOH Eu NP beads with a size of 0.2 μm (#FCEU002, Bangs Lab) were conjugated with either goat anti-mouse IgM antibody (ARG21517, Arigo Biolaboratories) or goat anti-human IgM antibody (K0211481, KOMABIOTECH). Initially, 20 μL of Eu NPs was added to 754 μL of 0.05 M MES buffer (pH 6.1, Sigma–Aldrich) and then agitated for 1 h at 25°C in the presence of 26 μL of 5 mM EDC (Thermo #22980) and 200 μL of 50 mM sulfo-NHS (Sigma #56485-1G) to activate the COOH groups present on the surface of Eu NPs. The surplus EDC and sulfo-NHS were removed by subjecting the mixture to centrifugation at 27,237×g for 10 min at 4°C. Subsequently, the activated Eu NPs were combined with 60 μL of 1 mg/mL antibody in 940 μL of 0.1 M sodium phosphate at pH 8. This mixture was allowed to react for 2 h at 25°C. Following centrifugation at 27,237×g for 10 min, the precipitated bioconjugates were collected, washed once, and resuspended in 400 μL of 2 mM borax pH 9.0 containing 0.1% BSA. Finally, the bioconjugates were stored in the dark at 4°C for subsequent use (Duong et al., 2022).

To establish immunochromatographic test strips, the NC membrane (10 μM) was coated with antigen or antibody using the BioDot Dispenser machine (BioDot, California, US). For standardizing the lateral flow reaction, the membrane was coated with 0.05 mg/mL of rabbit anti-Goat IgG (H + L) antibody (ARG21945, Arigo Biolaboratories) to serve as the control line (CL). The test line (TL) was coated with 0.3 mg/mL purified rAg T. gondii-GAP50 to detect IgM in the serum of patients with toxoplasmosis. The conjugate, sample, and absorbent pads were then attached to a backing card to complete the process.

The strip was used for FICT after drying at 30°C for 2 days. In summary, 6 μL of Eu NP conjugated either with anti-mouse IgM or anti-human IgM was placed onto the conjugate pad. Then, a mixture of either mouse or human serum in 75 μL of distilled water (DW) was thoroughly diluted into 125 μL of diluent buffer (0.1 M Tris pH 9, 0.1% gelatin, and 0.5% Tween 20) and then applied to the sample pad. After 15–20 min, a portable fluorescent strip reader (MEDISENSOR, Daegu, Korea) was used to interpret the results at excitation and emission wavelengths of 365 and 610 nm, respectively. The TL/CL ratio was used to calculate the quantitative diagnostic parameters of the FICT.

The cutoff value of the FICT was determined by calculating the mean of the normal sera (n = 10) plus three times the standard deviation (SD) using the TL/CL value. Subsequently, to establish the LOD (Armbruster and Pry, 2008) for the FICT test involving the coating rAg Tg-GAP50, standard T. gondii-infected human sera (code 13/132) were prepared by spiking 10 μL of normal sera, which were then subjected to the FICT.

All graphs were generated using GraphPad Prism (version 9.0, La Jolla, CA, USA) and presented as the mean and standard deviations (SD) of biological replicates. One-way and two-way analysis of variance (ANOVA) were used to analyze the ELISA and FICT.

To obtain highly pure protein samples, tachyzoites were mass-cultivated with APRE19 cells and subsequently purified to ensure no contamination, as shown in Supplementary Figures S1A,B.

Pure and fresh tachyzoites of the T. gondii RH strain were i.p. injected into the mouse belly, and blood was collected 2–10 days post-infection (Figure 1A). The RH strain of T. gondii was highly virulent in mice, resulting in the mortality of all mouse groups infected with tachyzoites ranging from 10¹ to 10⁶ 12 days post-infection (Figures 1C,D).

During this study, mice inoculated with 10⁵ and 10⁶ tachyzoites were survived for only 7 dpi and lost 20% of their body weight before death. However, these two groups showed a strong IgM immunological response to the tachyzoite TLA base-indirect ELISA response at day 5 post-infection (Figure 1B).

The group of mice that received 10⁴ to 10¹ tachyzoites persisted longer, until 9, 10, 11, and 12 dpi, and experienced an approximately 10–15% decrease in body weight before eventually succumbing to the infection; however, the IgM responses were observed to be comparatively lower when mice were infected with higher tachyzoite doses (Figure 1).

To analyze the proteomics of T. gondii at tachyzoite stages, a comparative assessment was conducted between tachyzoite TLA and uninfected cells using 2DE gel, separating the proteins based on their isoelectric points (pIs) and molecular weights.

Using the data obtained from the 2DE of the IPG strip with a pH range of 3–10, distinct differences were observed in the tachyzoite sample (Figure 2A) compared with uninfected cells (Supplementary Figure S2). This indicated major variations in protein expression and composition primarily in the pH range of 5–8 area.

To specifically target the antigen identified by IgM antibodies, a group of mice infected with 10⁵ and 10⁶ tachyzoites, known to exhibit an intense IgM immune response, was selected for Western blotting analysis of tachyzoite TLA in 2DE. Compared with the sera from normal mice, the sera of T. gondii-infected mice exhibited extensive reactivity of IgM antibodies toward protein spots within the 40–55 kDa range at pH 5–8 (Figures 2B,C).

To investigate these proteins more thoroughly, Western blotting analysis of tachyzoite TLA in 2DE probed with mouse serum was conducted using IPG strips with a narrow pH range of 5–8, maintaining that the spots within the 40–55 kDa range at approximately pH 7 were consistently visible (Figures 2D–F).

The same spot pattern was consistently observed in the Western blotting assessment of the TLA of tachyzoites in 2DE probed with serum from mice via IPG strips at pH 3–10 in triplicate (Supplementary Figure S3) and pH 5–8 (Supplementary Figure S4).

To get insights into the composition and identification and characterization of molecules based on their mass-to-charge ratio through MS analysis, the spots in question were revealed to be T. gondii EF1γ (44 kDa at pI 5.97), PGKI (44.6 kDa at pI 6.57), and GAP50 (46.6 kDa at pI 6.46), with high percentages of peptide definition rates (Table 1).

In particular, EF1γ had coverage of 74.9, and 64% of the identified peptides corresponded to this protein, while PGKI and GAP50 exhibited coverage of 63.3 and 49%, respectively, of these sequences, and 22% of the recorded peptides were similar to them (Table 1).

The sequences of T. gondii EF1γ, PGKI, and GAP50 were evaluated using bioinformatics to predict linear epitopes using three different prediction programs: (ABCpred (threshold: 0.8), BCPreds (specificity: 75%), and [IEDB]-BepiPred (threshold 0.5) (Supplementary Figure S4). A total of 19 (threshold: 0.8), 8 (specificity: 75%), and 12 (threshold: 0.5) linear epitopes were predicted via ABCpred, BCPreds server 1.0, and IEDB-BepiPred, respectively, for EF1γ. PGKI was found to possess 17, 4, and 14 linear epitopes, respectively, and GAP50 comprised 15, 7, and 11 linear epitopes, respectively (Supplementary Figure S5).

In the case of EF1γ, the predictions from the three programs indicated the presence of GK (L1) at positions 165–166, AAAKKET (L2) at positions 205–211, and DVASAKD (L3) at positions 362–368. For PGKI, D (L1) at position 69 and G (L2) at position 137 were consistent across all three prediction programs. Similarly, GAP50 exhibited common epitopes, including SRVAGDSGR (L1) at positions 14–22, SGH (L2) at positions 203–205, SGASKGD (L3) at positions 262–268, and QPLKN at positions 362–366 (Table 2). These predicted common linear epitope sequences of EF1γ, PGKI, and GAP50 were analyzed and presented as 3D modeling (Figure 3).

In addition, Figure 3, Tables 2, 3 highlighted the specific peptides associated with T-cell epitope processing, and MHC binding of T. gondii EF1γ, PGKI, and GAP50, respectively, is reliable between two out of three predictor systems.

The linear epitopes of GAP50 were mostly located on the outer surface, covering 5.57% of the sequence (see Table 2), representing the highest coverage compared with other types in the 3D model. In contrast, the linear epitopes of EF1γ and PGKI exhibited lower coverage, with 4.06 and 0.48% of the sequence, respectively. Regarding MHC-binding peptides, EF1γ, PGKI, and GAP50 exhibited total coverage percentages of 16.19, 21.35, and 20.61% of their respective sequences (Table 3). Notably, those of GAP50 were mainly exposed on the surface, while those of PGKI were hidden rather than surface-exposed. As a result, GAP50 seems to efficiently present strong epitopes and MHC-binding peptides on the surface compared with the others (Figure 3).

The full-length coding sequence of the EF1γ, PGKI, and GAP50 genes was cloned into expression vector pET21b (+) to produce a final construct. The purified recombinant antigens, fused with 6× His-tag, formed proteins of 46, 48.5, and 50 kDa, respectively, for EF1γ (Figures 4B,B1), PGKI (Figures 4C,C1), and GAP50 (Figures 4D,D1), which were confirmed by the presence of an intense band of approximately 40–55 kDa on SDS-PAGE and Western blotting probed with the anti-6× Hig-Tag antibody. Circular dichroism (CD) spectroscopy revealed that three recombinant proteins have α-helical structures (Supplementary Figure S6).

To verify the detectability of Toxoplasma IgM antibodies against recombinant antigens, Western blotting assays were performed using normal mouse sera and T. gondii-infected mouse sera as primary antibodies. The recombinant antigens EF1γ, PGKI, and GAP50 were compared with BSA, T. gondii tachyzoite TLA, and uninfected cells (Figures 4A,E,I). Figures 4B,F,J show that EF1γ was not recognized by IgM of T. gondii-infected mouse sera, whereas PGKI (Figures 4C,G,K) and GAP50 (Figures 4D,H,L) were extensively detected (Supplementary Figure S7).

Subsequently, PGKI and GAP50 were subjected to ELISA using mouse and human serum samples. The cross-reactivity with P. vivax and regular human serum was difficult to eliminate when PGKI was used as antigen coating (Supplementary Figure S8).

To further investigate the capacity of strip testing to detect antibodies against IgM in serum samples, purified rAg GAP50 was coated at a variety of concentrations (0.1, 0.3, 0.5, and 1 mg/mL) as a single TL on NC membranes. An immunochromatographic test strip (Figure 5A) utilizing Eu NP-conjugated anti-mouse IgM (diluted 80-fold) (Figure 5B) and/or anti-human IgM (diluted 320-fold) (Figure 5C) was used to detect mouse and human sera, respectively. With GAP50 coated at 0.3 mg/mL on the NC membrane, it was possible to reduce the non-specific fluorescence intensity when examining cross-interactions involving P. vivax, P. yoelii, or typical serum (raw data are shown in Supplementary Figure S9).

Furthermore, an FICT included the test line coated with 0.3 mg/mL GAP50 and the control line coated with 0.05 mg/mL rabbit anti-goat IgG (anti-gIgG) to quantify the conjugate required in each reaction.

The application of conjugated anti-mouse IgM (diluted 80-fold) (Figures 6A,C,E) and/or anti-human IgM (diluted 20-fold) (Figures 6B,D,F) efficiently minimized non-specific binding at the TL associated with Plasmodium or normal serum while maintaining an appropriate TL/CL ratio (raw data are shown in Supplementary Figure S10).

Supplementary Figure S11 reveals that the ideal volume of human sera per reaction for FICT was 2 μL when various quantities (1, 2, 4, 8, 10, and 15 μL) of sera were compared. In our system, the utilization of 1 μL sera (diluted 1:200) in 200 μL per reaction was insufficient to distinguish IgM in a standard T. gondii patient serum 13/132 from other samples, while an increase in 15 μL of sera (diluted 1:13.3) was unable to eliminate the non-specific reaction when measuring the TL area. The greatest fluorescent signal at the test line (TL) was noted when using 8 μL of sera (diluted 1:25). However, this also led to an overall increase in the intensity at the control line (CL), resulting in a less impressive TL/CL ratio compared with the sample from patients infected with P. vivax. Non-specific signals appeared at the test line (TL) when 4 μL of serum (diluted 1:50) was tested with P. vivax patient sera. The TL/CL ratio of standard T. gondii patient serum 13/132 was three and five times higher than that of P. vivax and normal serum, when 10 μL of sera were used per reaction. However, there were big differences in TL density or TL/CL ratios when only 2 μL of the sample was used. (Supplementary Figure S11). It aids in cost reduction and facilitates efficient human serum screening for various diseases and evaluation of diverse experimental conditions, a particularly crucial consideration when using rodent models to align with ethical and resource considerations.

To establish the FICT threshold value, 2 μL of sera per strip reaction was applied. For mouse sera, the cutoff value was determined by calculating the mean of normal sera (n = 10) plus three times the standard deviation (SD) based on the TL/CL ratio (Figure 7A and Supplementary Figure S12). These data provide support for the tachyzoite TLA-based ELISA, indicating that sera at 2 dpi from mice infected with T. gondii ranging from 10¹ to 10⁶ exhibited fluorescence levels similar to the background signal. However, on days 5 and 7 pi, sera from mice infected with T. gondii ranging from 10³ to 10⁶ showed significantly higher detectability. In contrast, sera from T. gondii-infected mice at densities of 10¹ and 10² only produced positive results on day 10 pi (Figures 7B–G and Supplementary Figure S13).

We conclude that the FICT approach using GAP50 was more sensitive than TLA-based ELISA for detecting IgM levels in sera from rodents infected with T. gondii at 5 dpi (10³, 10⁴) and 7 dpi (10³).

In the context of FICT for human sera, the highest ideal volume of sera per reaction (10 μL of each sera per strip) (Supplementary Figure S11) was applied to determine the cutoff value. It similarly determined as 677.28 by calculating the mean of human seronegative sera (n = 10) plus three times the standard deviation (SD) based on the TL/CL ratio (Figure 8A and Supplementary Figure S14). Data demonstrate that the standard human T. gondii sera (code 13/132) (n = 1) significantly differ from that of seronegative sera and human cross-reaction controls (n = 20).

To establish the LOD for the GAP50-based FICT to human serum, a series of standard human T. gondii sera (code 13/132) with varying quantities (10, 8, 4, 2, and 1 μL equivalent to 3.2, 2.6, 1.3, 0.65, and 0.325 IU, respectively) were tested. In conclusion, positive results were observed up to 2 μL, which corresponded to 0.65 IU of anti-T. gondii antibodies (Figure 8B and Supplementary Figure S15).

Additionally, the OnSite commercial kit (REF: R0234CL) for IgM and IgG serum was employed to compare the results in parallel. According to the instructions of the kit, standard T. gondii 13/132 sera were tested in triplicate, with 10 μL utilized for each test, corresponding to 3.2 IU/strip. The outcome was visually inspected under the naked eye, with a faintly visible IgM-positive band (Supplementary Figure S16A). To assess the limit of detection (LOD) in parallel with FICT, the sera were similarly prepared by spiking them into 10 μL of normal sera and then analyzed in both the commercial strip and GAP50-FICT simultaneously. At each point of the LOD test, triplicated strips were evaluated. Inconsistent results were observed among them, while at least one strip exhibited a faint IgM band, not all strips showed the same result. This variation poses challenges in accurately determining the LOD when testing T. gondii 13/132 by OnSite commercial kit (Supplementary Figure S16A). In addition, the OnSite commercial kits were evaluated by seronegative sera and human cross-reaction controls (Supplementary Figures S16B–D). The specificity was 96.77% (n = 30) with one Dengue sample showing cross-reactivity. However, as only one positive T. gondii sample was available, the sensitivity could not be determined.

These findings highlight the distinct immunoreactivity of GAP50, emphasizing its potential as a specific diagnostic biomarker to enhance the sensitivity of the FICT in detecting IgM in rodent samples. Additionally, novel GAP50 emerges as an attractive candidate antigen for integration in POCT for the IgM detection of T. gondii infections in patient samples.