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Fulltext A Toxoplasma gondii 10 kDa in vitro excretory secretory antigen reactive with human IgM and IgA antibodies

Saadatnia G, Ghaffarifar F, Khalilpour A, Amerizadeh A, Rahmah N

Trop Biomed, 2011 Dec;28(3):606-14.
PMID: 22433890 MyJurnal

Toxoplasmosis can cause serious disease in immunocompromised patients and to congenitally infected foetuses. Appropriate laboratory investigations in potential cases of acute Toxoplasma infection are important. Excretory secretory antigen (ESA) is immunogenic during both human and experimental infections, therefore is considered as a good candidate for investigation into new infection markers. In this study, ESA was prepared from in vitro cultures of Toxoplasma gondii to identify T. gondii ESA antigenic component(s) that is/are most reactive with serum samples from probable acute cases of toxoplasmosis. Serum samples were obtained from several categories of individuals with the following Toxoplasma serology: Group I: IgM+ IgG+ (low IgG avidity) or IgM+ IgG- from sera of patients who had clinical query of toxoplasmosis (n=35). Group II: IgM- IgG+ (high IgG avidity) from chronically infected individuals (n=30). Group III: normal/healthy individuals with anti-Toxoplasma IgMIgG- (n=20). Group IV: individuals with other infections who had anti-Toxoplasma IgM- IgG- (n=10). The ESA was subjected to SDS-PAGE, followed by Western blot analysis using the above sera and probed with peroxidase conjugated anti-human IgM and IgA antibodies. The blots were then developed using chemiluminescence substrate. The selected antigenic band was excised from the gel after two dimensional electrophoresis and sent for mass spectrometry analysis using MALDI TOF-TOF. The most promising antigenic band was a 10 kDa protein which showed sensitivity of 80% in both IgM and IgA blots, and specificity of 96.7% with sera from other infections and healthy controls. The two best identifications for the 10 kDa band were ubiquitin (ribosomal protein CEP52 fusion protein) and polyubiquitin.
An assay for selection of sera with circulating Toxoplasma gondii antigens

Emelia O, Zeehaida M, Sulaiman O, Rohela M, Saadatnia G, Yeng C, et al.

J Immunoassay Immunochem, 2010;31(1):79-91.
PMID: 20391020 DOI: 10.1080/15321810903405134

We have developed an ELISA that employs monoclonal anti-Toxoplasma SAG1 (p30) as the capture antibody to detect T. gondii circulating antigens in patients' serum samples. Using serum spiked with Toxoplasma soluble and with SAG1 recombinant proteins, the detection limits were 31.25 ng/mL and 62.50 ng/mL, respectively. We obtained positive results in 28% (21/75) and 11% (23/206) of probable active and chronic toxoplasmosis serum samples, respectively. Western blot analysis on pooled antigen-positive serum samples showed antigenic bands of molecular weights 25 and 75 kDa from sera of probable active infection and five antigenic bands ranging in size from 26 to 33 kDa from chronic infection sera. This assay would be useful as an initial serum selection step in developing a Toxoplasma antigen detection test and for characterization studies.
Fulltext Optimization of Toxoplasma gondii cultivation in VERO cell line

Saadatnia G, Haj Ghani H, Khoo BY, Maimunah A, Rahmah N

Trop Biomed, 2010 Apr;27(1):125-30.
PMID: 20562822

In vitro culture of Toxoplasma gondii can provide tachyzoites which are active, viable and with desirable purity. Thus the aim of this study was to optimize the cell culture method for T. gondii propagation to obtain a consistent source of parasites with maximum yield and viability, but minimum host cell contamination for use in production of excretory-secretory antigen. Tachyzoites with seed counts of 1x10(6), 1x10(7) and 1x10(8) harvested from infected mice were added to VERO cells of different degrees of confluence, namely 50%, 85% and 100%, and examined periodically using an inverted microscope. When the maximum release of the tachyzoites was observed from the host cells, the culture supernatant was removed and the tachyzoites harvested. Using a Neubauer chamber, the percentages of viable tachyzoites and host cell contamination were determined using trypan blue stain. Parameters that gave the best yield and purity of viable tachyzoites were found to be as follows: VERO cells at 85% confluence in DMEM medium and inoculum comprising 1x10(7) tachyzoites. After about 3 days post infection, the tachyzoites multiplied 78x, with a yield of ~7.8x10(8) per flask, 99% viability and 3% host cell contamination. This study has successfully optimized the method of propagation of T. gondii tachyzoites in VERO cells which produce parasites with high yield, purity and viability.
Production of refolded Toxoplasma gondii recombinant SAG1-related sequence 3 (SRS3) and its use for serodiagnosis of human toxoplasmosis

Mirzadeh A, Saadatnia G, Golkar M, Babaie J, Noordin R

Protein Expr Purif, 2017 05;133:66-74.
PMID: 28263855 DOI: 10.1016/j.pep.2017.03.001

SAG1-related sequence 3 (SRS3) is one of the major Toxoplasma gondii tachyzoite surface antigens and has been shown to be potentially useful for the detection of toxoplasmosis. This protein is highly conformational due to the presence of six disulfide bonds. To achieve solubility and antigenicity, SRS3 depends on proper disulfide bond formation. The aim of this study was to over-express the SRS3 protein with correct folding for use in serodiagnosis of the disease. To achieve this, a truncated SRS3 fusion protein (rtSRS3) was produced, containing six histidyl residues at both terminals and purified by immobilized metal affinity chromatography. The refolding process was performed through three methods, namely dialysis in the presence of chemical additives along with reduced/oxidized glutathione and drop-wise dilution methods with reduced/oxidized glutathione or reduced DTT/oxidized glutathione. Ellman's assay and ELISA showed that the protein folding obtained by the dialysis method was the most favorable, probably due to the correct folding. Subsequently, serum samples from individuals with chronic infection (n = 76), probable acute infection (n = 14), and healthy controls (n = 81) were used to determine the usefulness of the refolded rtSRS3 for Toxoplasma serodiagnosis. The results of the developed IgG-ELISA showed a diagnostic specificity of 91% and a sensitivity of 82.89% and 100% for chronic and acute serum samples, respectively. In conclusion, correctly folded rtSRS3 has the potential to be used as a soluble antigen for the detection of human toxoplasmosis.
Antigenic proteins of Helicobacter pylori of potential diagnostic value

Khalilpour A, Santhanam A, Wei LC, Saadatnia G, Velusamy N, Osman S, et al.

Asian Pac J Cancer Prev, 2013;14(3):1635-42.
PMID: 23679248

Helicobacter pylori antigen was prepared from an isolate from a patient with a duodenal ulcer. Serum samples were obtained from culture-positive H. pylori infected patients with duodenal ulcers, gastric ulcers and gastritis (n=30). As controls, three kinds of sera without detectable H. pylori IgG antibodies were used: 30 from healthy individuals without history of gastric disorders, 30 from patients who were seen in the endoscopy clinic but were H. pylori culture negative and 30 from people with other diseases. OFF-GEL electrophoresis, SDS-PAGE and Western blots of individual serum samples were used to identify protein bands with good sensitivity and specificity when probed with the above sera and HRP-conjugated anti-human IgG. Four H. pylori protein bands showed good (≥ 70%) sensitivity and high specificity (98-100%) towards anti-Helicobacter IgG antibody in culture- positive patients sera and control sera, respectively. The identities of the antigenic proteins were elucidated by mass spectrometry. The relative molecular weights and the identities of the proteins, based on MALDI TOF/ TOF, were as follows: CagI (25 kDa), urease G accessory protein (25 kDa), UreB (63 kDa) and proline/pyrroline- 5-carboxylate dehydrogenase (118 KDa). These identified proteins, singly and/or in combinations, may be useful for diagnosis of H. pylori infection in patients.
Toxoplasma gondii excretory secretory antigenic proteins of diagnostic potential

Saadatnia G, Mohamed Z, Ghaffarifar F, Osman E, Moghadam ZK, Noordin R

APMIS, 2012 Jan;120(1):47-55.
PMID: 22151308 DOI: 10.1111/j.1600-0463.2011.02810.x

Infection with Toxoplasma gondii is widespread and important in humans, especially pregnant women and immunosuppressed patients. A panel of tests is usually required for diagnosis toxoplasmosis. Excretory secretory antigen (ESA) is highly immunogenic, and thus it is a good candidate for investigation into new infection markers. ESA was prepared from tachyzoites of RH strain of T. gondii by mice intraperitoneal infection. Sera were obtained from several categories of individuals who differed in their status of anti-Toxoplasma IgM, IgG and IgG avidity antibodies. The ESA was subjected to SDS-PAGE, two-dimensional gel electrophoresis and Western blot analysis. Antigenic bands of approximate molecular weights of 12, 20 and 30 kDa, when probed with anti-human IgM-HRP and IgA-HRP, showed good potential as infection markers. The highest sensitivity of the bands was 98.7% with combination of IgM and IgA blots with sera of patients with anti-Toxoplasma IgM+ IgG+. The specificities were 84% and 70% with sera from other infections and healthy controls in IgM blots and IgA blots respectively. By mass spectrometry, the 12 kDa protein was identified as thioredoxin. The two top proteins identified for 20 kDa molecule were microneme protein 10 and dense granule protein 7; whereas that for 30 kDa were phosphoglycerate mutase 1 and phosphoglycerate mutase.
Fulltext Recombinant proteins from new constructs of SAG1 and GRA7 sequences and their usefulness to detect acute toxoplasmosis

Kotresha D, Poonam D, Muhammad Hafiznur Y, Saadatnia G, Nurulhasanah O, Sabariah O, et al.

Trop Biomed, 2012 Mar;29(1):129-37.
PMID: 22543613 MyJurnal

In this study we have cloned unreported gene fragments of Toxoplasma gondii GRA7 and SAG1 and expressed the corresponding recombinant proteins, followed by evaluation of their usefulness for the serological diagnosis of toxoplasmosis. Both recombinant proteins were expressed efficiently in insoluble form, purified by single step Ni-NTA affinity chromatography and their antigenicity to detect toxoplasma specific IgG antibodies were determined by immunoblotting. A total of 60 serum samples from three groups of individuals based on their anti-toxoplasma antibody profiles were tested, namely (I) IgM+, IgG+ (n=20), (II) IgM-, IgG+ (n=20) and (III) IgM-, IgG- (n=20). Both recombinant proteins exhibited high sensitivity (100%) with sera from Group I. rGRA7 and rSAG1 reacted 40% and 80% respectively with Group II sera. The specificity of the recombinant proteins based on reactivities with Group III sera were 100% and 80% with rGRA7 and rSAG1 respectively. Thus rGRA7 was found to be better at discriminating probable acute from chronic phases of toxoplasmosis, and it also showed higher specificity.
Fulltext Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples

Berizi M, Babaie J, Fard-Esfahani P, Enshaeieh M, Noordin R, Saadatnia G, et al.

Iran J Parasitol, 2022 1 28;16(4):621-630.
PMID: 35082891 DOI: 10.18502/ijpa.v16i4.7875

Background: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples.
Methods: This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016-2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction).
Results: The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant.
Conclusion: We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.
Fulltext Theoretical investigation on structural, functional and epitope of a 12 kDa excretory-secretory protein from Toxoplasma gondii

Tommy YB, Lim TS, Noordin R, Saadatnia G, Choong YS

BMC Struct Biol, 2012 Nov 27;12:30.
PMID: 23181504 DOI: 10.1186/1472-6807-12-30

BACKGROUND: Toxoplasma gondii is an intracellular coccidian parasite that causes toxoplasmosis. It was estimated that more than one third of the world population is infected by T. gondii, and the disease is critical in fetuses and immunosuppressed patients. Thus, early detection is crucial for disease diagnosis and therapy. However, the current available toxoplasmosis diagnostic tests vary in their accuracy and the better ones are costly.
RESULTS: An earlier published work discovered a highly antigenic 12 kDa excretory-secretory (ES) protein of T. gondii which may potentially be used for the development of an antigen detection test for toxoplasmosis. However, the three-dimensional structure of the protein is unknown. Since epitope identification is important prior to designing of a specific antibody for an antigen-detection based diagnostic test, the structural elucidation of this protein is essential. In this study, we constructed a three dimensional model of the 12 kDa ES protein. The built structure possesses a thioredoxin backbone which consists of four α-helices flanking five β-strands at the center. Three potential epitopes (6-8 residues) which can be combined into one "single" epitope have been identified from the built structure as the most potential antibody binding site.
CONCLUSION: Together with specific antibody design, this work could contribute towards future development of an antigen detection test for toxoplasmosis.
Fulltext IgG4 detection of Echinococcus granulosus paramyosin is a useful diagnostic test for human hydatidosis

Moghadam ZK, Ghaffarifar F, Khalilpour A, Abdul Aziz F, Saadatnia G, Noordin R

Clin Vaccine Immunol, 2013 Apr;20(4):501-5.
PMID: 23365208 DOI: 10.1128/CVI.00019-13

Hydatidosis is a public health problem in many parts of the world, and improvement in diagnosis of the disease is still being pursued. Protoscoleces of Echinococcus granulosus were isolated from hydatid cysts collected from naturally infected sheep slaughtered in abattoirs in Iran. Sonicated extract of protoscolex was subjected to two-dimensional gel electrophoresis and Western blot analysis. Primary antibodies were from serum samples from 130 hydatidosis patients, 38 individuals infected with other parasitic infections, and 30 healthy people, whereas peroxidase (HRP)-conjugated anti-human IgG and IgG4 were used as secondary antibodies. The recombinant form of the identified protein was produced and tested for its sensitivity and specificity for the detection of human hydatidosis. An antigenic band of ∼60 kDa was found to be sensitive (82%) and specific (100%) for the detection of hydatidosis when probed with anti-human IgG4-HRP, while the sensitivity and specificity were 33 and 100%, respectively, with anti-human IgG-HRP. By mass spectrometry, the band was identified as protoscolex tegument paramyosin. The sensitivity and specificity of full-length paramyosin-recombinant protein in IgG4 blots were found to be 86 and 98%, respectively. In conclusion, IgG4 detection of Echinococcus granulosus paramyosin was found to be useful for the diagnosis of human hydatidosis.
Identification of Toxoplasma gondii in-vivo induced antigens by cDNA library immunoscreening with chronic toxoplasmosis sera

Amerizadeh A, Idris ZM, Khoo BY, Kotresha D, Yunus MH, Karim IZ, et al.

Microb Pathog, 2013 Jan;54:60-6.
PMID: 23044055 DOI: 10.1016/j.micpath.2012.09.006

Toxoplasmosis is an infection caused by the parasite Toxoplasma gondii. Chronically-infected individuals with a compromised immune system are at risk for reactivation of the disease. In-vivo induced antigen technology (IVIAT) is a promising method for the identification of antigens expressed in-vivo. The aim of the present study was to apply IVIAT to identify antigens which are expressed in-vivo during T. gondii infection using sera from individuals with chronic toxoplasmosis. Forty serum samples were pooled, pre-adsorped against three different preparations of antigens, from each in-vitro grown T. gondii and Escherichia coli XLBlue MRF', and then used to screen a T. gondii cDNA expression library. Sequencing of DNA inserts from positive clones showed eight open reading frames with high homology to T. gondii genes. Expression analysis using quantitative real-time PCR showed that SAG1-related sequence 3 (SRS3) and two hypothetical genes were up-regulated in-vivo relative to their expression levels in-vitro. These three proteins also showed high sensitivity and specificity when tested with individual serum samples. Five other proteins namely M16 domain peptidase, microneme protein, elongation factor 1-alpha, pre-mRNA-splicing factor and small nuclear ribonucleoprotein F had lower RNA expression in-vivo as compared to in-vitro. SRS3 and the two hypothetical proteins warrant further investigation into their roles in the pathogenesis of toxoplasmosis.
IgG avidity Western blot using Toxoplasma gondii rGRA-7 cloned from nucleotides 39-711 for serodiagnosis of acute toxoplasmosis

Deshpande PS, Kotresha D, Noordin R, Yunus MH, Saadatnia G, Golkar M, et al.

Rev Inst Med Trop Sao Paulo, 2013 4 9;55(2):79-83.
PMID: 23563759

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

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