Next Article in Journal
Cold Enrichment Methods for the Detection of Foodborne Yersiniosis: Friend or Foe?
Next Article in Special Issue
Phage ImmunoPrecipitation Sequencing (PhIP-Seq): The Promise of High Throughput Serology
Previous Article in Journal
Imaging Aspects of Hepatic Alveolar Echinococcosis: Retrospective Findings of a Surgical Center in Turkey
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pathogens
Volume 11
Issue 2
10.3390/pathogens11020277
pathogens-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparative Performance of Recombinant GRA6, GRA7, and GRA14 for the Serodetection of T. gondii Infection and Analysis of IgG Subclasses in Human Sera from the Philippines

by
Rochelle Haidee Ybañez
1,2 and
Yoshifumi Nishikawa
1,*
1
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
2
Institute of Molecular Parasitology and Protozoan Diseases, Main Campus and College of Veterinary Medicine, Barili Campus, Cebu Technological University, Cebu City 6000, Philippines
*
Author to whom correspondence should be addressed.
Pathogens 2022, 11(2), 277; https://doi.org/10.3390/pathogens11020277
Submission received: 28 January 2022 / Revised: 9 February 2022 / Accepted: 18 February 2022 / Published: 21 February 2022
(This article belongs to the Special Issue Sero-Epidemiology of Natural Infections and Vaccination)
Browse Figures
Review Reports Versions Notes

Abstract

:
Highly specific and sensitive diagnostic methods are vital for the effective control and treatment of toxoplasmosis. Routine diagnosis is primarily serological because T. gondii infections stimulate persistently high IgG antibody responses. The sensitivity and specificity of methods are crucial factors for the proper diagnosis of toxoplasmosis, primarily dependent on the antigens used in different assays. In the present study, we compared the serodiagnostic performances of three recombinant dense granule antigens, namely, the GRA6, GRA7, and GRA14, to detect IgG antibodies against T. gondii in human sera from the Philippines. Moreover, we evaluated the IgG1, IgG2, IgG3, and IgG4 responses against the different recombinant antigens, which has not been performed previously. Our results revealed that the TgGRA7 has consistently displayed superior diagnostic capability, while TgGRA6 can be a satisfactory alternative antigen among the GRA proteins. Furthermore, IgG1 is the predominant subclass stimulated by the different recombinant antigens. This study’s results provide options to researchers and manufacturers to choose recombinant antigens suitable for their purpose.

1. Introduction

Toxoplasmosis is known to infect nearly one-third of the world’s human population [1]. The accurate detection of T. gondii infection through highly specific and sensitive diagnostic methods is vital for the effective control and treatment of toxoplasmosis [2,3]. T. gondii infections in humans are characterized by persistent high IgG antibody titers [4,5]. Thus, routine diagnosis for toxoplasmosis is mainly through detecting specific T. gondii antibodies.
The sensitivity and specificity of methods are crucial factors for the proper diagnosis of toxoplasmosis to avoid false-positive and false-negative results. These factors are primarily dependent on the antigens used in different assays. The recombinant antigens have been considered alternative diagnostic markers to replace the native antigens, given the tedious standardization and high cost to produce crude T. gondii antigens. Aside from improving T. gondii diagnosis, differentiating the different phases of infection using recombinant antigens has also been widely recognized [6,7]. Among the recombinant antigens that showed outstanding serodiagnostic performance are the SAG1 [8,9,10] and SAG2 [11,12] of the surface antigen (SAG) family; the dense granule (GRA) proteins GRA1 [8], GRA2 [13,14], GRA 3 [9], GRA5 [15], GRA6 [16,17], GRA7 [10,18,19,20], and GRA8 [21,22]; the rhoptry proteins ROP1 [14,23], ROP2 [24], and ROP8 [25]; and MAG1 [26] and MIC2 [9].
Moreover, several studies have investigated the dynamics of human IgG subclasses in their varied responses to different infectious agents. The prevalent IgG responses to protein antigens are IgG1 and IgG3, while IgG2 is against carbohydrates. IgG1 and IgG3 are stimulated for viral antigens, while bacterial antigens primarily elicit the IgG2 subclass [27,28]. These IgG responses also vary depending on the host organism [29].
In the present study, we compared the serodiagnostic performances of three recombinant GRA antigens, namely, the GRA6, GRA7, and GRA14, to detect IgG antibodies against T. gondii in human sera from the Philippines. Furthermore, we evaluated the IgG1, IgG2, IgG3, and IgG4 responses against the recombinant antigens, which has not been conducted previously. Our findings confirmed the superior diagnostic potential of TgGRA7 and the suitability of the TgGRA6 as an alternative antigen for toxoplasmosis serodiagnosis. In addition, the first use of TgGRA14 as a diagnostic marker for human toxoplasmosis is documented in this study. Furthermore, IgG1 is the predominant subclass recognized by the different recombinant antigens.

2. Results

In the present study, we assessed the reactivity of IgG and IgG subclasses (IgG1, IgG2, IgG3, IgG4) in human sera from the Philippines against three recombinant antigens expressed in E. coli as GST-fused (TgGRA7 and TgGRA14) and His-tagged (TgGRA6) proteins through indirect ELISA. We also compared the performance of the recombinant antigens with Platelia IgG-ELISA (com-ELISA). The ELISAs using TgGRA6 (25 positives of 88 samples) and TgGRA7 (27 positives of 88 samples) obtained similar results as the com-ELISA (27 positives of 88 samples) for the detection of IgG antibodies (Table 1). The TgGRA14 showed the lowest IgG detection rate (11 positives of 88 samples) (Table 1). The TgGRA7-ELISA detection results were in perfect agreement with the com-ELISA. For the TgGRA6- and TgGRA14-ELISA, 2 and 16 human serum samples were found below the cut-off values, respectively. These samples were judged positive by com-ELISA (Figure 1). Similarly, the TgGRA14-ELISA showed the lowest sensitivity (40.7%), although with 100% specificity against the com-ELISA. Meanwhile, high sensitivity (92.6–100%), specificity (100%), and kappa values (0.945–1) in the ELISAs using TgGRA6 and TgGRA7 were found (Table 2).
The 27 serum samples that detected IgG antibodies using any of the three recombinant antigens were further evaluated for the reactivity of specific IgG subclasses by ELISA (Figure 2). TgGRA6 and TgGRA7 showed significant differences (p-value < 0.001) in the detection results of anti-Toxoplasma IgG subclasses from human sera (Table 3). Moreover, the detection rate of IgG1 in both antigens was significantly higher than IgG2, IgG3, and IgG4 (Table 3).

3. Discussion

Our results revealed that IgG antibodies reacted more strongly and frequently with TgGRA6 and TgGRA7 ELISAs than TgGRA14. Detection rates for both antigens were similar to results obtained using the com-ELISA. It shows the importance of recombinant antigens in the serological detection of T. gondii infections in humans, as isolating T. gondii is difficult [30]. Many recombinant antigens produced in E. coli have been examined and confirmed to be reliable markers for T. gondii infection diagnosis [8,10,11,24]. Regardless of the clinical presentations in the infected individuals, T. gondii infections elicit intense and often lasting humoral immune responses characterized by high antibody titers [4,5]. Thus, serological assays to detect specific antibodies are still the favored method for diagnosing acute or chronic infections of T. gondii [31].
In the present study, the high sensitivity and specificity of TgGRA6-ELISA and its perfect agreement with conventional ELISA test reveal that TgGRA6 is a promising alternative antigen for toxoplasmosis serodiagnosis in the Philippines. The usefulness of TgGRA6 as a serodiagnostic antigen for human infections was previously validated in several ELISAs in Poland [11], Germany [16], Iran [17], and France [32]. A rTgGRA6 demonstrated high reactivity with specific IgG-positive human sera, reaching a sensitivity of 96% in an IgG ELISA test [32]. An ELISA using a GST-tagged TgGRA6 obtained an 89% sensitivity and 99.6% specificity for detecting IgG [16]. The same study also reported that the recombinant antigen successfully differentiated acute from chronic infections, with a sensitivity of 86% [16]. Another ELISA utilizing a rTgGRA6 with His-tag domain was able to detect more recent toxoplasmosis (93.9%) than chronic cases (60.6%) [11]. Furthermore, an IgM-ELISA using TgGRA6 demonstrated a high sensitivity of 97.1% in discriminating acute from past Toxoplasma infections in pregnant women, which confirms a remarkable correlation with VIDAS Toxo IgM kit [17].
An immunochromatographic test using TgGRA7 revealed a 93.1–100% sensitivity and 100% specificity for detecting IgG and IgM and/or IgG antibodies [20]. This affirms TgGRA7’s outstanding antigenic potential vis-à-vis standard tests. The GRA proteins are among the antigens well recognized for their excellent diagnostic ability [14,18,22]. Their vast immunogenic properties [33,34] stimulate cell-mediated or antibody-dependent immunity [35]. Moreover, the TgGRA7 is an excellent serodiagnostic marker for human toxoplasmosis, with a sensitivity of 81–98.9% and specificity of 98-100% using ELISA [9,10,18,36]. Furthermore, this antigen is more correlated with acute toxoplasmosis, with reported sensitivity ranging from 75 to 100% in ELISA [8,10,18,24].
Although the TgGRA14-ELISA in our current study demonstrated high specificity, it had low sensitivity to detect IgG antibodies in human sera. The current findings corroborated with a previous study where a TgGRA14-ELISA revealed low reactivity using experimentally infected mice characterized by lower OD values compared to TgGRA7. It also registered a sensitivity of only 81.25% for detecting IgG and/or IgM antibodies to infection in field pig sera, which was lower than TgGRA7 (90.63%) [37].
Apart from the present study, TgGRA14 has not been utilized yet as an ELISA antigen for human toxoplasmosis serodiagnosis. The GRA14, along with other GRA proteins, is found on the parasitophorous vacuole (PV) membrane extensions linking neighboring PVs [38]. It is also expressed in all life stages of T. gondii, which contributes to its robust antigenic properties [39]. Previous studies using DNA immunizations with GRA14 enhanced cellular and humoral responses, leading to relative protection against toxoplasmosis in mice [40,41]. However, the sensitivities and specificities of serological assays using recombinant diagnostic antigens may vary. These differences may be due to variances in cloning approaches, protocols used during the purification of recombinant proteins, and standards used in the data analyses [6,42]. Moreover, recombinant antigens often lose their antigenicity due to incorrect folding and probable contamination with E. coli antigens during protein expression [43,44,45,46].
For the IgG subclasses, our results found the IgG1 to be the primary subclass response stimulated by all the examined recombinant antigens, similar to previous studies showing the predominance of IgG1 antibodies in the humoral immune response to T. gondii infections in humans [29,47,48,49]. IgG3 and IgG4 antibodies were also detected in this study, although less frequently than the IG1, while IgG2 antibody detection was zero to very minimal. These results were similar to the ELISA study of [48] using sera from patients clinically suspected of toxoplasmosis. Furthermore, lesser responses in the IgG2 and IgG3 subclasses were observed among human sera from individuals with acute and chronic T. gondii infections [29]. Contrastingly, an elevated IgG2 response in a standardized ELISA using a cyst antigen was noted in the CSF of patients with cerebral toxoplasmosis [49].
The current and previous studies may explain the preferential recognition of T. gondii antigens by lgG antibodies where lgG1; IgG3; and, to a lesser extent, IgG4 react with protein antigens [50], while IgG2 reacts to polysaccharide antigens [51,52]. The high IgG2 levels detected in [49] could be explained by the presence of polysaccharides in the T. gondii cysts used as antigen in the ELISA [52]. Large quantities of polysaccharides are produced and stored in the bradyzoite cytoplasm and cyst wall during cyst formation [53,54]. Moreover, the predominance of IgG1 at different infection stages in immunocompetent individuals is presumed to be related to a T-cell control of humoral response during toxoplasmosis [47]. Some studies also suggest that lgG1 and lgG3 stimulate phagocytosis of parasites by stimulating the binding of mononuclear cells to the parasite [55] and activating the complement system [56]. Furthermore, other studies proposed that IgG subclasses specific to T. gondii could be markers of clinical outcome of toxoplasmosis [57,58,59].
In an ELISA using tachyzoite antigens, elevated levels in IgG1, IgG2, IgG3, and/or IgG4 in mothers and/or newborns were associated with offspring clinical problems or vertical transmission [57]. The association between clinical signs of congenital toxoplasmosis and IgG subclasses using antirMIC3-ELISA revealed that the IgG2 or IgG4 reactivity was linked to the occurrence of retinochoroidal lesions and intracranial calcifications [58]. Meanwhile, detecting T. gondii-specific IgG4 antibodies in serum and/or CSF samples reinforced the diagnosis of cerebral toxoplasmosis in HIV-infected patients [59].

4. Materials and Methods

4.1. Human Serum Samples

In this study, we tested 88 human serum samples from the Philippines, which a licensed phlebotomist aseptically collected, separated by centrifugation, and stored at −20 °C until further use.

4.2. Production of GST-Fused Recombinant TgGRA7 and TgGRA14

All the DNA samples used as a template for the amplification of the coding regions of the target antigens were from the type I RH strain of T. gondii tachyzoites. Previously described protocols were adopted for the amplification and cloning of the TgGRA7 [20,37] and TgGRA14 [37] into the Escherichia coli expression vector pGEX-4T1 (G.E. Healthcare, Amersham, Buckinghamshire, UK). The recombinant proteins of TgGRA7 and TgGRA14 were eventually transformed in E. coli BL21 for protein expression.
The transformed plasmids were grown in L.B. media supplemented with 50 µg/mL of ampicillin at 37 °C until an optical density (O.D.) at 600 nm of 0.6 was reached. Protein production was induced by isopropyl-b-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The recombinant proteins of TgGRA7 (rTgGRA7) and TgGRA14 (rTgGRA14) were expressed as GST fusion proteins in the E. coli (Takara Bio, Inc., Shiga, Japan). According to the manufacturer’s instructions, the GST tags of the rTGRA7 and rTgGRA14 were removed with thrombin protease (G.E. Healthcare). SDS-PAGE results revealed that the rTgGRA7 and rTgGRA14 were 29 kDa and 30 kDa proteins, respectively. Protein concentrations were quantified using a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA) before storage at −30 °C until use.

4.3. Production of His-Tagged Recombinant TgGRA6

The PrimeScript™ II 1st strand cDNA Synthesis Kit (Takara Bio, Inc., Shiga, Japan) was used to obtain the cDNA template for the PCR amplification of the TgGRA6. The following primers containing BamHI and HindIII recognition sequences (bold): 5′-AAGGATCCATGGCAGCAGACAGCGGTGGT-3′ (forward) and 5′-CGAAGCTTTCTGTGGCGTTTCTGTGTTCG-3′ (reverse) were used to facilitate cloning. The target amplicon (345 bp) was digested using the same set of restriction enzymes and ligated into the pET30b vector in-frame of the His-tag domain at the C-terminal of the fusion protein. The correct sequence of the His-tagged protein was also confirmed via double-enzyme digestion and sequencing.
The recombinant vector was transformed in BL21 (DE3) E. coli and cultivated in L.B. media supplemented with 100 µg/mL of kanamycin at 37 °C until an optical density (O.D.) at 600 nm of 0.6 was reached. Induction with 1 mM IPTG was performed 16 h at 25 °C with vigorous shaking (160 rpm). The culture was centrifuged to harvest the cells. The pellet was resuspended in lysis buffer (100 mM NaH2PO4, 10 mM Tris–HCl, 8 M urea; pH 8.0) with lysozyme (1 mg/mL), mixed gently using a rotary shaker for 1 h, and disrupted by sonication (ten 10 s cycles at 55% power). Centrifugation at 10,000 rpm for 30 min at 4 °C was performed to remove insoluble debris. The supernatant was added into 50% Ni-NTA beads (Qiagen, Germantown, MD, USA) previously stabilized in lysis buffer and mixed via mild rotation at 4 °C for 1 h. The mixture was then loaded into the Ni-NTA poly-prep chromatography columns (Bio-Rad, Hercules, CA, USA). The unbound proteins were removed by washing buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea; pH 6.3), which was performed twice. The TgGRA6 protein was eluted 3–5 times from the column using elution buffer (100 mM NaH2PO4, 10 mM Tris-Cl, 8 M urea; pH 5.9). The recombinant His-tagged TgGRA6 (rTgGRA6) was a 13 kDa protein, as confirmed using SDS-PAGE analysis.
The eluted rTgGRA6 fractions were pooled and dialyzed in reducing urea concentrations (6 M, 4 M, 2 M, 1 M) for 6 h each. Final dialysis was against PBS, twice for 6 h each. A BCA protein assay kit (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA) was also used to determine the protein concentration before storage at −30 °C until further use.

4.4. Indirect ELISA Using a Commercial Kit

Performance of the ELISA using the Platelia Toxo IgG (Bio-Rad, Hercules, CA, USA) followed previously documented protocols [20]. Interpretation of results was according to the manufacturer’s instructions.

4.5. Indirect ELISA Using TgGRA6, TgGRA7, and TgGRA14

The rTgGRA6, rTgGRA7, and rTgGRA14 were diluted in 50 mM carbonate-bicarbonate buffer (pH 9.6) at a final concentration of 0.1 μM. Then, each well of the ELISA plate (Nunc, Roskilde, Denmark) was coated with 50 μL of the diluted antigen and incubated overnight at 4 °C. The succeeding steps were performed as previously described [20]. For the detection of IgG subclasses, the same procedure was followed as above, except for the fact that secondary antibodies used were horseradish peroxidase (HRP)-conjugated mouse anti-human IgG1, IgG2, IgG3, or IgG4 (Thermo Fisher Scientific, USA) diluted at 1:1000.
The cutoff points for all antigens were determined as the mean value at OD415nm plus three standard deviations of human sera (N = 10) previously confirmed negative using commercial ELISA (Platelia Toxo IgG; Bio-Rad, Hercules, CA, USA) and LAT (Toxocheck-MT; Eiken Chemical, Tokyo, Japan) kits.

4.6. Statistical Analyses

The ELISA results were encoded to Microsoft Excel using appropriate coding for statistical analyses. Descriptive statistics were employed. The statistical significance of the test results was evaluated using analysis of variance with post hoc analyses. p-values of less than 0.05 were considered significant. An online statistical tool was used to calculate the sensitivity, specificity, and kappa values with a 95% confidence interval (http://vassarstats.net/ accessed on 9 June 2021). The strength of agreement was graded with kappa values of fair (0.21 to 0.40), moderate (0.41 to 0.60), substantial (0.61 to 0.80), and very good (0.81 to 1.00). Graphs were constructed using GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA).

5. Conclusions

All three recombinant proteins developed were found to detect IgG antibodies in human sera from the Philippines, albeit with varied performances. The TgGRA7 has consistently displayed superior diagnostic capability, while TgGRA6 could be a suitable alternative antigen among the GRA proteins. Moreover, IgG1 is the predominant subclass stimulated by the different recombinant antigens. Therefore, this study’s results provide options to researchers and manufacturers to choose recombinant antigens suitable for their purpose. Furthermore, analysis of the IgG subclass antibody response to T. gondii could be a potential tool to understand better the pathogenesis, diagnosis, and probable approach for treating toxoplasmosis.

Author Contributions

Conceptualization, R.H.Y. and Y.N.; methodology, R.H.Y.; formal analysis, R.H.Y.; investigation, R.H.Y.; visualization, R.H.Y.; writing—original draft preparation, R.H.Y.; writing—review and editing, R.H.Y. and Y.N.; supervision, Y.N.; project administration, Y.N.; funding acquisition, Y.N. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported in part by the Research Program on Emerging and Re-Emerging Infectious Diseases (21fk0108137h (Y.N.)) from the Agency for Medical Research and Development (AMED), and KAKENHI Grants from the Japan Society for the Promotion of Science (20KK0152 (Y.N.)).

Institutional Review Board Statement

The whole study was conducted in accordance with the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of the University of the Visayas, Cebu (UV-IRB2017-91), and Obihiro University of Agriculture and Veterinary Medicine (2017-05).

Informed Consent Statement

Informed consent was obtained from all respondents involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Acknowledgments

We thank the members of the Research Unit for Host Defense (National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine) for their technical support with the experiments.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Dubey, J.P. Toxoplasmosis—An overview. Southeast Asian J. Trop. Med. Public Health 1991, 22, 88–92. [Google Scholar] [PubMed]
  2. Liu, Q.; Wang, Z.D.; Huang, S.Y.; Zhu, X.Q. Diagnosis of toxoplasmosis and typing of Toxoplasma gondii. Parasit. Vectors 2015, 8, 292. [Google Scholar] [CrossRef] [Green Version]
  3. Pomares, C.; Montoya, J.G. Laboratory Diagnosis of congenital toxoplasmosis. J. Clin. Microbiol. 2016, 54, 2448–2454. [Google Scholar] [CrossRef] [Green Version]
  4. Parmley, S.F.; Sgarlato, G.D.; Mark, J.; Prince, J.B.; Remington, J.S. Expression, characterization, and serologic reactivity of recombinant surface antigen P22 of Toxoplasma gondii. J. Clin. Microbiol. 1992, 30, 1127–1133. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Dubey, J.P. The history of Toxoplasma gondii—The first 100 years. J. Eukaryot. Microbiol. 2008, 55, 467–475. [Google Scholar] [CrossRef]
  6. Holec-Gąsior, L. Toxoplasma gondii recombinant antigens as tools for serodiagnosis of human toxoplasmosis—The current status of studies. Clin. Vaccine Immunol. 2013, 20, 1343–1351. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Ybañez, R.H.D.; Ybanez, A.; Nishikawa, N. Review on the current trends of toxoplasmosis serodiagnosis in humans. Front. Cell. Infect. Microbiol. 2020, 10, 204. [Google Scholar] [CrossRef]
  8. Pietkiewicz, H.; Hiszczyńska-Sawicka, E.; Kur, J.; Petersen, E.; Nielsen, H.V.; Stankiewicz, M.; Andrzejewska, I.; Myjak, P. Usefulness of Toxoplasma gondii-specific recombinant antigens in serodiagnosis of human toxoplasmosis. J. Clin. Microbiol. 2004, 42, 1779–1781. [Google Scholar] [CrossRef] [Green Version]
  9. Beghetto, E.; Spadoni, A.; Bruno, L.; Buffolano, W.; Gargano, N. Chimeric antigens of Toxoplasma gondii: Toward standardization of toxoplasmosis serodiagnosis using recombinant products. J. Clin. Microbiol. 2006, 44, 2133–2140. [Google Scholar] [CrossRef] [Green Version]
  10. Kotresha, D.; Poonam, D.; Muhammad Hafiznur, Y.; Saadatnia, G.; Nurulhasanah, O.; Sabariah, O.; Tan, S.Y.; Izzati Zahidah, A.K.; Rahmah, N. Recombinant proteins from new constructs of SAG1 and GRA7 sequences and their usefulness to detect acute toxoplasmosis. Trop. Biomed. 2012, 29, 129–137. [Google Scholar]
  11. Hiszczyńska-Sawicka, E.; Kur, J.; Pietkiewicz, H.; Holec, L.; Gąsior, A.; Myjak, P. Efficient production of the Toxoplasma gondii GRA6, P35 and SAG2 recombinant antigens and their applications in the serodiagnosis of toxoplasmosis. Acta Parasitol. 2005, 50, 249–254. [Google Scholar]
  12. Lau, Y.L.; Fong, M.Y. Toxoplasma gondii: Serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris. Exp. Parasitol. 2008, 119, 373–378. [Google Scholar] [CrossRef] [PubMed]
  13. Golkar, M.; Rafati, S.; Abdel-Latif, M.S.; Brenier-Pinchart, M.P.; Fricker-Hidalgo, H.; Sima, B.K.; Babaie, J.; Pelloux, H.; Cesbron-Delauw, M.F.; Mercier, C. The dense granule protein GRA2, a new marker for the serodiagnosis of acute Toxoplasma infection: Comparison of sera collected in both France and Iran from pregnant women. Diagn. Microbiol. Infect. Dis. 2007, 58, 419–426. [Google Scholar] [CrossRef] [PubMed]
  14. Holec-Gąsior, L.; Kur, J.; Hiszczynska-Sawicka, E. GRA2 and ROP1 recombinant antigens as potential markers for detection of Toxoplasma gondii specific immunoglobulin G in human with acute toxoplasmosis. Clin. Vaccine Immunol. 2009, 16, 510–514. [Google Scholar] [CrossRef] [Green Version]
  15. Holec-Gąsior, L.; Kur, J. Toxoplasma gondii: Recombinant GRA5 antigen for detection of immunoglobulin G antibodies using enzyme-linked immunosorbent assay. Exp. Parasitol. 2010, 124, 272–279. [Google Scholar] [CrossRef] [PubMed]
  16. Redlich, A.; Müller, W.A. Serodiagnosis of acute toxoplasmosis using a recombinant form of the dense granule antigen GRA6 in an enzyme-linked immunosorbent assay. Parasitol. Res. 1998, 84, 700–706. [Google Scholar] [CrossRef]
  17. Golkar, M.; Azadmanesh, K.; Khalili, G.; Khoshkholgh-Sima, B.; Babaie, J.; Mercier, C.; Brenier-Pinchart, M.P.; Fricker-Hidalgo, H.; Pelloux, H.; Cesbron-Delauw, M.F. Serodiagnosis of recently acquired Toxoplasma gondii infection in pregnant women using enzyme-linked immunosorbent assays with a recombinant dense granule GRA6 protein. Diagn. Microbiol. Infect. Dis. 2008, 61, 31–39. [Google Scholar] [CrossRef]
  18. Jacobs, D.; Vercammen, M.; Saman, E. Evaluation of recombinant dense granule antigen 7 (GRA7) of Toxoplasma gondii for detection of immunoglobulin G antibodies and analysis of a major antigenic domain. Clin. Diagn. Lab. Immunol. 1999, 6, 24–29. [Google Scholar] [CrossRef] [Green Version]
  19. Selseleh, M.; Keshavarz, H.; Mohebali, M.; Shojaee, S.; Selseleh, M.; Eshragian, M.R.; Mansouri, F.; Modarressi, M.H. Production and evaluation of Toxoplasma gondii recombinant GRA7 for serodiagnosis of human infections. Korean J. Parasitol. 2012, 50, 233–238. [Google Scholar] [CrossRef] [PubMed]
  20. Ybañez, R.H.D.; Nishikawa, Y. Serological detection of T. gondii infection in humans using an immunochromatographic assay based on dense granule protein 7. Parasitol. Int. 2020, 76, 102089. [Google Scholar] [CrossRef] [PubMed]
  21. Suzuki, Y.; Ramirez, R.; Press, C.; Li, S.; Parmley, S.; Thulliez, P.; Remington, J.S. Detection of immunoglobulin M antibodies to P35 antigen of Toxoplasma gondii for serodiagnosis of recently acquired infection in pregnant women. J. Clin. Microbiol. 2000, 38, 3967–3970. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  22. Lu, B.; Wu, S.; Shi, Y.; Zhang, R.; Zou, L.; Gao, S.; Lin, M.; Zhou, Y. Toxoplasma gondii: Expression pattern and detection of infection using full-length recombinant P35 antigen. Exp. Parasitol. 2006, 113, 83–90. [Google Scholar] [CrossRef]
  23. Holec-Gąsior, L.; Drapała, D.; Lautenbach, D.; Kur, J. Toxoplasma gondii: Usefulness of ROP1 recombinant antigen in an immunoglobulin G avidity assay for diagnosis of acute toxoplasmosis in humans. Pol. J. Microbiol. 2010, 59, 307–310. [Google Scholar] [CrossRef] [PubMed]
  24. Nigro, M.; Gutierrez, A.; Hoffer, A.M.; Clemente, M.; Kaufer, F.; Carral, L.; Martin, V.; Guarnera, E.A.; Angel, S.O. Evaluation of Toxoplasma gondii recombinant proteins for the diagnosis of recently acquired toxoplasmosis by an immunoglobulin G analysis. Diagn. Microbiol. Infect. Dis. 2003, 47, 609–613. [Google Scholar] [CrossRef]
  25. Sonaimuthu, P.; Fong, M.Y.; Kalyanasundaram, R.; Mahmud, R.; Lau, Y.L. Serodiagnostic evaluation of Toxoplasma gondii recombinant rhoptry antigen 8 expressed in E. coli. Parasit. Vectors 2014, 7, 297. [Google Scholar] [CrossRef] [Green Version]
  26. Holec, L.; Hiszczynska-Sawicka, E.; Gąsior, A.; Brillowska-Dabrowska, A.; Kur, J. Use of MAG1 recombinant antigen for detection of Toxoplasma gondii infection in humans. Clin. Vaccine Immunol. 2007, 14, 220–225. [Google Scholar] [CrossRef] [Green Version]
  27. Wagner, D.K.; Nelson, D.L.; Walsh, E.E.; Reimer, C.B.; Henderson, F.W.; Murphy, B.R. Differential immunoglobulin G subclass antibody titers to respiratory syncytial virus F and G glycoproteins in adults. J. Clin. Microbiol. 1987, 25, 748–750. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  28. Sousa, A.O.; Henry, S.; Marója, F.M.; Lee, F.K.; Brum, L.; Singh, M.; Lagrange, P.H.; Aucouturier, P. IgG subclass distribution of antibody responses to protein and polysaccharide mycobacterial antigens in leprosy and tuberculosis patients. Clin. Exp. Immunol. 1998, 111, 48–55. [Google Scholar] [CrossRef] [PubMed]
  29. Huskinson, J.; Stepick-Biek, P.N.; Araujo, F.G.; Thulliez, P.; Suzuki, Y.; Remington, J.S. Toxoplasma antigens recognized by immunoglobulin G subclasses during acute and chronic infection. J. Clin. Microbiol. 1989, 27, 2031–2038. [Google Scholar] [CrossRef] [Green Version]
  30. Robert-Gangneux, F.; Dardé, M.L. Epidemiology of and diagnostic strategies for toxoplasmosis. Clin. Microbiol. Rev. 2012, 25, 264–296. [Google Scholar] [CrossRef] [Green Version]
  31. Candolfi, E.; Pastor, R.; Huber, R.; Filisetti, D.; Villard, O. IgG avidity assay firms up the diagnosis of acute toxoplasmosis on the first serum sample in immunocompetent pregnant women. Diagn. Microbiol. Infect. Dis. 2007, 53, 83–88. [Google Scholar] [CrossRef] [PubMed]
  32. Lecordier, L.; Fourmaux, M.P.; Mercier, C.; Dehecq, E.; Masy, E.; Cesbron-Delauw, M.F. Enzyme-linked immunosorbent assay using recombinant dense granule antigens GRA6 and GRA1 of Toxoplasma gondii for detection of immunoglobulin G antibodies. Clin. Diagn. Lab. Immunol. 2000, 7, 607–611. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Darcy, F.; Deslee, D.; Santoro, F.; Charif, F.; Auriault, C.; Decoster, A.; Duquesne, V.; Capron, A. Induction of a protective antibody-dependent response against toxoplasmosis by in vitro excreted/secreted antigens from tachyzoites of Toxoplasma gondii. Parasite Immunol. 1988, 10, 553–567. [Google Scholar] [CrossRef] [PubMed]
  34. Prigione, I.; Facchetti, P.; Lecordier, L.; Deslée, D.; Chiesa, S.; Cesbron-Delauw, M.F.; Pistoia, V. T cell clones raised from chronically infected healthy humans by stimulation with Toxoplasma gondii excretory-secretory antigens cross-react with live tachyzoites: Characterization of the fine antigenic specificity of the clones and implications for vaccine development. J. Immunol. 2000, 164, 3741–3748. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  35. Zenner, L.; Estaquier, J.; Darcy, F.; Maes, P.; Capron, A.; Cesbron-Delauw, M.F. Protective immunity in the rat model of congenital toxoplasmosis and the potential of excreted-secreted antigens as vaccine components. Parasit. Immunol. 1999, 21, 261–272. [Google Scholar] [CrossRef] [PubMed]
  36. Felgner, J.; Juarez, S.; Hung, C.; Liang, L.I.; Jain, A.; Döşkaya, M.; Felgner, P.L.; Caner, A.; Gürüz, Y.; Davies, D.H. Identification of Toxoplasma gondii antigens associated with different types of infection by serum antibody profiling. Parasitology 2015, 142, 827–838. [Google Scholar] [CrossRef] [Green Version]
  37. Terkawi, M.A.; Kameyama, K.; Rasul, N.H.; Xuan, X.; Nishikawa, Y. Development of an immunochromatographic assay based on dense granule protein 7 for serological detection of Toxoplasma gondii infection. Clin. Vaccine Immunol. 2013, 20, 596–601. [Google Scholar] [CrossRef] [Green Version]
  38. Rome, M.E.; Beck, J.R.; Turetzky, J.M.; Webster, P.; Bradley, P.J. Intervacuolar transport and unique topology of GRA14, a novel dense granule protein in Toxoplasma gondii. Infect. Immun. 2008, 76, 4865–4875. [Google Scholar] [CrossRef] [Green Version]
  39. Cesbron-Delauw, M.F. Dense granule organelles of Toxoplasma gondii: Their role in the host parasite relationship. Parasitol. Today 1994, 10, 293–296. [Google Scholar] [CrossRef]
  40. Ahmadpour, E.; Sarvi, S.; Hashemi Soteh, M.B.; Sharif, M.; Rahimi, M.T.; Valadan, R.; Tehrani, M.; Khalilian, A.; Montazeri, M.; Daryani, A. Evaluation of the immune response in BALB/c mice induced by a novel DNA vaccine expressing GRA14 against Toxoplasma gondii. Parasite Immunol. 2017, 39, e12419. [Google Scholar] [CrossRef]
  41. Pagheh, A.S.; Sarvi, S.; Gholami, S.; Asgarian-Omran, H.; Valadan, R.; Hassannia, H.; Ahmadpour, E.; Fasihi-Ramandie, M.; Dodangeh, S.; Hosseni-Khah, Z.; et al. Protective efficacy induced by DNA prime and recombinant protein boost vaccination with Toxoplasma gondii GRA14 in mice. Microb. Pathog. 2019, 134, 103601. [Google Scholar] [CrossRef]
  42. Zhang, K.; Lin, G.; Han, Y.; Li, J. Serological diagnosis of toxoplasmosis and standardization. Clin. Chim. Acta 2016, 461, 83–89. [Google Scholar] [CrossRef] [PubMed]
  43. Biemans, R.; Grégoire, D.; Haumont, M.; Bosseloir, A.; Garcia, L.; Jacquet, A.; Dubeaux, C.; Bollen, A. The conformation of purified Toxoplasma gondii SAG1 antigen, secreted from engineered Pichia pastoris, is adequate for serorecognition and cell proliferation. J. Biotechnol. 1998, 66, 137–146. [Google Scholar] [CrossRef]
  44. Zhou, H.; Gu, Q.; Zhao, Q.; Zhang, J.; Cong, H.; Li, Y.; He, S. Toxoplasma gondii: Expression and characterization of a recombinant protein containing SAG1 and GRA2 in Pichia pastoris. Parasitol. Res. 2007, 100, 829–835. [Google Scholar] [CrossRef] [PubMed]
  45. Lau, Y.L.; Thiruvengadam, G.; Lee, W.W.; Fong, M.F. Immunogenic characterization of the chimeric surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii expressed in the yeast Pichia pastoris. Parasitol. Res. 2011, 109, 871–878. [Google Scholar] [CrossRef]
  46. Thiruvengadam, G.; Init, I.; Fong, M.Y.; Lau, Y.L. Optimization of the expression of surface antigen SAG1/2 of Toxoplasma gondii in the yeast Pichia pastoris. Trop. Biomed. 2011, 28, 506–513. [Google Scholar]
  47. Derouin, F.; Sulcebe, G.; Ballet, J.J. Sequential determination of IgG subclasses and IgA specific antibodies in primary and reactivating toxoplasmosis. Biomed. Pharmacother. 1987, 41, 429–433. [Google Scholar]
  48. Ee, T.Y.; Singh, M.; Yap, E.H. The determination of anti-Toxoplasma gondii antibodies in different IgG subclasses of human sera by the enzyme-linked immunosorbent assay (ELISA). Southeast Asian J. Trop. Med. Public Health 1989, 20, 71–79. [Google Scholar]
  49. Nascimento, F.S.; Suzuki, L.A.; Branco, N.; Franco, R.M.B.; Andrade, P.D.; Costa, S.C.B.; Pedro, M.N.; Rossi, C.L. Toxoplasma-specific IgG subclass antibody response in cerebrospinal fluid samples from patience with cerebral toxoplasmosis. Rev. Inst. Med. Trop. Sao Paulo 2015, 57, 439–442. [Google Scholar] [CrossRef]
  50. Mineo, J.R.; Camargo, M.E.; Ferreira, A.W. Enzyme-linked immunosorbent assay for antibodies to Toxoplasma gondii polysaccharides in human toxoplasmosis. Infect. Immun. 1980, 27, 283–287. [Google Scholar] [CrossRef] [Green Version]
  51. Barrett, D.J.; Ayoub, E.M. IgG2 subclass restriction of antibody to pneumococcal polysaccharides. Clin. Exp. Immunol. 1986, 63, 127–134. [Google Scholar] [PubMed]
  52. Schroeder, H.W., Jr.; Cavacini, L. Structure and function of immunoglobulins. J. Allergy Clin. Immunol. 2010, 125, S41–S52. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  53. Coppin, A.; Dzierszinski, F.; Legrand, S.; Mortuaire, M.; Ferguson, D.; Tomavo, S. Developmentally regulated biosynthesis of carbohydrate and storage polysaccharide during differentiation and tissue cyst formation in Toxoplasma gondii. Biochimie 2003, 85, 353–361. [Google Scholar] [CrossRef]
  54. Sullivan, W.J., Jr.; Jeffers, V. Mechanisms of Toxoplasma gondii persistence and latency. FEMS Microbiol. Rev. 2012, 36, 717–733. [Google Scholar] [CrossRef] [Green Version]
  55. Alexander, M.D. Specificity of Fc receptors on human monocytes for lgG1 and lgG3. Int. Archs. Allergy Appl. Immun. 1980, 62, 99. [Google Scholar] [CrossRef]
  56. Schreiber, R.D.; Feldman, H.A. Identification of the activator system for antibody to Toxoplasma as the classical complement pathway. J. Infect. Dis. 1980, 141, 366–399. [Google Scholar] [CrossRef]
  57. Cañedo-Solares, I.; de la Luz Galván-Ramírez, M.; Luna-Pastén, H.; Rodríguez Pérez, L.R.; Ortiz-Alegría, L.B.; Rico-Torres, C.P.; Vela-Amieva, M.; Pérez-Andrade, M.; Figueroa-Damián, R.; Correa, D. Congenital toxoplasmosis: Specific IgG subclasses in mother/newborn pairs. Pediatr. Infect. Dis. J. 2008, 27, 469–474. [Google Scholar] [CrossRef] [Green Version]
  58. de Souza-e-Silva, C.H.; Vasconcelos-Santos, D.V.; de Andrade, G.Q.; Carellos, E.V.; de Castro Romanelli, R.M.; de Resende, L.M.; Januário, J.N.; Carneiro, M.; de Aguiar Vasconcelos Carneiro, A.C.; de Almeida Vitor, R.W. Association between IgG subclasses against Toxoplasma gondii and clinical signs in newborns with congenital toxoplasmosis. Pediatr. Infect. Dis. J. 2013, 32, 13–16. [Google Scholar] [CrossRef]
  59. Meira, C.S.; Vidal, J.E.; Costa-Silva, T.A.; Motoie, G.; Gava, R.; Hiramoto, R.M.; Pereira-Chioccola, V.L. IgG4 specific to Toxoplasma gondii excretory/secretory antigens in serum and/or cerebrospinal fluid support the cerebral toxoplasmosis diagnosis in HIV-infected patients. J. Immunol. Methods 2013, 395, 21–28. [Google Scholar] [CrossRef]
Pathogens 11 00277 g001 550
Figure 1. Detection of IgG antibodies against TgGRA6, TgGRA7, and TgGRA14 by indirect ELISA. Each symbol represents the mean values of the duplicate wells for each serum sample. The round and square symbols represent the negative (N) and positive (P) samples determined by Platelia IgG-ELISA, respectively. Antibody titers in OD (optical density at 415 nm) are plotted on the y-axis. Horizontal broken lines are the cut-off values for each recombinant antigen: 0.1506 (TgGRA6), 0.2205 (TgGRA7), and 0.0800 (TgGRA14).
Figure 1. Detection of IgG antibodies against TgGRA6, TgGRA7, and TgGRA14 by indirect ELISA. Each symbol represents the mean values of the duplicate wells for each serum sample. The round and square symbols represent the negative (N) and positive (P) samples determined by Platelia IgG-ELISA, respectively. Antibody titers in OD (optical density at 415 nm) are plotted on the y-axis. Horizontal broken lines are the cut-off values for each recombinant antigen: 0.1506 (TgGRA6), 0.2205 (TgGRA7), and 0.0800 (TgGRA14).
Pathogens 11 00277 g001
Pathogens 11 00277 g002 550
Figure 2. IgG subclass responses to TgGRA6, TgGRA7, and TgGRA14 by indirect ELISA (N = 27). The scatter plot shows mean OD values at 415 nm. Horizontal broken lines are the cut-off values for each recombinant antigen: TgGRA6 (IgG1, 0.0533; IgG2, 0.062; IgG3, 0.1517; IgG4, 0.0325), TgGRA7 (IgG1, 0.2162; IgG2, 0.1419; IgG3, 0.3246; IgG4, 0.0772), and TgGRA14 (IgG1, 0.0583; IgG2, 0.0275; IgG3, 0.0064; IgG4, 0.0261).
Figure 2. IgG subclass responses to TgGRA6, TgGRA7, and TgGRA14 by indirect ELISA (N = 27). The scatter plot shows mean OD values at 415 nm. Horizontal broken lines are the cut-off values for each recombinant antigen: TgGRA6 (IgG1, 0.0533; IgG2, 0.062; IgG3, 0.1517; IgG4, 0.0325), TgGRA7 (IgG1, 0.2162; IgG2, 0.1419; IgG3, 0.3246; IgG4, 0.0772), and TgGRA14 (IgG1, 0.0583; IgG2, 0.0275; IgG3, 0.0064; IgG4, 0.0261).
Pathogens 11 00277 g002
Table
Table 1. Comparison of IgG detection by ELISA using recombinant TgGRA6, TgGRA7, and TgGRA14 in human sera with commercial ELISA as reference test (N = 88).
Table 1. Comparison of IgG detection by ELISA using recombinant TgGRA6, TgGRA7, and TgGRA14 in human sera with commercial ELISA as reference test (N = 88).
com-ELISA aTgGRA6TgGRA7TgGRA14
(−)(+)(−)(+)(−)(+)
Negative (−)610610610
Positive (+)2250271611
Total632561277711
a Commercial ELISA: Platelia™ Toxo IgM/IgG (Bio-Rad, Hercules, CA, USA).
Table
Table 2. Sensitivity and specificity of different ELISAs using recombinant proteins to detect specific T. gondii IgG antibodies using a commercial ELISA as a reference test.
Table 2. Sensitivity and specificity of different ELISAs using recombinant proteins to detect specific T. gondii IgG antibodies using a commercial ELISA as a reference test.
ParametersTgGRA6TgGRA7 TgGRA14
Sensitivity (%)92.610040.7
Specificity (%)100100100
Kappa value0.94510.488
Table
Table 3. Detection of anti-Toxoplasma IgG subclasses from human sera using TgRA6, TgGRA14, and TgGRA7 (N = 27).
Table 3. Detection of anti-Toxoplasma IgG subclasses from human sera using TgRA6, TgGRA14, and TgGRA7 (N = 27).
Recombinant AntigensIgG1IgG2IgG3IgG4p-Value
No. (%)No. (%)No. (%)No. (%)
TgGRA614 (51.90)0 (0)2 (7.40)6 (22.22)< 0.001 *,a,b,c
TgGRA714 (51.85)1 (3.70)6 (22.22)6 (22.22)< 0.001 *,d,e,f
TgGRA144 (14.81)0 (0)1 (3.70)5 (18.52)0.057
* p < 0.05 is considered statistically significant (ANOVA); a TgGRA6-IgG1 vs. TgGRA6-IgG2; b TgGRA6-IgG1 vs. TgGRA6-IgG3; c TgGRA6-IgG1 vs. TgGRA6-IgG4; d TgGRA7-IgG1 vs. TgGRA7-IgG2; e TgGRA7-IgG1 vs. TgGRA7-IgG3; f TgGRA7-IgG1 vs. TgGRA7-IgG4
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Ybañez, R.H.; Nishikawa, Y. Comparative Performance of Recombinant GRA6, GRA7, and GRA14 for the Serodetection of T. gondii Infection and Analysis of IgG Subclasses in Human Sera from the Philippines. Pathogens 2022, 11, 277. https://doi.org/10.3390/pathogens11020277

AMA Style

Ybañez RH, Nishikawa Y. Comparative Performance of Recombinant GRA6, GRA7, and GRA14 for the Serodetection of T. gondii Infection and Analysis of IgG Subclasses in Human Sera from the Philippines. Pathogens. 2022; 11(2):277. https://doi.org/10.3390/pathogens11020277

Chicago/Turabian Style

Ybañez, Rochelle Haidee, and Yoshifumi Nishikawa. 2022. "Comparative Performance of Recombinant GRA6, GRA7, and GRA14 for the Serodetection of T. gondii Infection and Analysis of IgG Subclasses in Human Sera from the Philippines" Pathogens 11, no. 2: 277. https://doi.org/10.3390/pathogens11020277

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop