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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.
The design of target specific antibodies (scFv) by applying de novo workflow: Case study on BmR1 antigen from Brugia malayi

Khor BY, Lim TS, Noordin R, Choong YS

J Mol Graph Model, 2017 09;76:543-550.
PMID: 28811153 DOI: 10.1016/j.jmgm.2017.07.004

De novo approach was applied to design single chain fragment variable (scFv) for BmR1, a recombinant antigen from Bm17DIII gene which is the primary antigen used for the detection of anti-BmR1 IgG4 antibodies in the diagnostic of lymphatic filariasis. Three epitopes of the BmR1 was previously predicted form an ab initio derived three-dimensional structure. A collection of energetically favourable conformations was generated via hot-spot-centric approach. This resulted in a set of three different scFv scaffolds used to compute the high shape complementary conformations via dock-and-design approach with the predicted epitopes of BmR1. A total of 4227 scFv designs were generated where 200 scFv designs produced binding energies of less than -20 R.E.U with shape complementarity higher than 0.5. We further selected the design with at least one hydrogen bond and one salt bridge with the epitope, thus resulted in a total of 10, 1 and 19 sFv designs for epitope 1, 2 and 3, respectively. The results thus showed that de novo design can be an alternative approach to yield high affinity in silico scFv designs as a starting point for antibody or specific binder discovery processes.
Fulltext Serodiagnosis and early detection of Strongyloides stercoralis infection

Arifin N, Hanafiah KM, Ahmad H, Noordin R

J Microbiol Immunol Infect, 2019 Jun;52(3):371-378.
PMID: 30482708 DOI: 10.1016/j.jmii.2018.10.001

Strongyloidiasis is a major neglected tropical disease with the potential of causing lifelong infection and mortality. One of the ways for effective control of this disease is developing improved diagnostics, particularly using serological approaches. A serological test can achieve high diagnostic sensitivity and specificity, has the potential for point-of-care translation, and can be used as a screening tool for early detection. More research is needed to find clinically important antibody biomarkers for early disease detection, mapping, and epidemiological surveillance. This article summarizes human strongyloidiasis and the available diagnostic tools for the disease, focusing on describing the current antibody assays for strongyloidiasis. Finally, prospects of developing a more effective serodiagnostic tool for strongyloidiasis are discussed.
Seroprevalence of Hydatid Disease Among Migrant Workers in Malaysia

Khan AH, Khanbabaie S, Yunus MH, Mohd Zain SN, Mohd Baharudeen Z, Sahimin N, et al.

J Immigr Minor Health, 2020 Oct;22(5):1105-1108.
PMID: 32445161 DOI: 10.1007/s10903-020-01029-y

Hydatid disease is not endemic in Malaysia; however, its migrant workers originate from neighboring countries where the disease is prevalent. Thus, this study was aimed at investigating the seroprevalence of hydatid disease among the workers. A total of 479 migrant workers were screened for hydatid disease. The sociodemographic information was collected, and serum samples were tested with a rapid dipstick test for hydatid disease called Hyd Rapid™. The present study showed that 13.6% of the migrant workers were found to be seropositive for hydatid disease. The highest seroprevalence was seen among Indian workers (29.41%), followed by Myanmarese (21.43%), Bangladeshis (14.92%), Nepalese (10.68%), and Indonesian (10.66%). This is the first study that highlights the likely presence of hydatid disease among the migrant workers in Malaysia, which may be of interest to the health authorities.
Fulltext Development and evaluation of a sensitive and specific assay for diagnosis of human toxocariasis by use of three recombinant antigens (TES-26, TES-30USM, and TES-120)

Mohamad S, Azmi NC, Noordin R

J Clin Microbiol, 2009 Jun;47(6):1712-7.
PMID: 19369434 DOI: 10.1128/JCM.00001-09

Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.
Fulltext Evaluation of the Diagnostic Performance of Recombinant Antigen B1 for Detection of Cystic Echinococcosis Using Lateral Flow Dipstick Test

Noordin R, Khanbabaie S, Hafiznur Yunus M, Marti H, Nickel B, Fasihi Harandi M, et al.

Iran J Parasitol, 2020 10 22;15(3):290-298.
PMID: 33082792 DOI: 10.18502/ijpa.v15i3.4191

Background: Human echinococcosis is a neglected zoonotic disease distributed worldwide. It comprises cystic and alveolar forms, the former being the more prevalent disease. Imaging techniques are the first choice for diagnosis of cystic echinococcosis and serology is used as an additional diagnostic technique in doubtful cases or as the sole test in low-resource settings. Rapid diagnostic tests are useful and convenient for immunodiagnosis of cystic echinococcosis in endemic areas, where medical facilities often struggle with limited resources.
Methods: Recently, we have developed Hyd Rapid™, an IgG4 lateral flow dipstick test using recombinant antigen B1 for detection of cystic echinococcosis. This study was performed between 2016 until 2018 at the Institute for Research in Molecular Medicine, Universiti Sains Malaysia. The diagnostic performance of Hyd Rapid™ was tested in-house and at two international laboratories in Switzerland and Iran.
Results: The overall diagnostic sensitivity for detection of cystic and alveolar echinococcosis was 95% (56/59). Meanwhile, the diagnostic specificity, with and without exclusion of cysticercosis and fascioliasis, was 100% (n=48) and 88% (63/72), respectively.
Conclusion: Hyd Rapid™ detected cystic echinococcosis as well as probable cases of alveolar echinococcosis. Therefore, Hyd Rapid™ showed good potential as a serological tool for echinococcosis, and merits further evaluation.
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 Antibody-Based Protective Immunity against Helminth Infections: Antibody Phage Display Derived Antibodies against BmR1 Antigen

Rahumatullah A, Abdul Karim IZ, Noordin R, Lim TS

Int J Mol Sci, 2017 Nov 22;18(11).
PMID: 29165352 DOI: 10.3390/ijms18112376

Helminth parasite infections are significantly impacting global health, with more than two billion infections worldwide with a high morbidity rate. The complex life cycle of the nematodes has made host immune response studies against these parasites extremely difficult. In this study, we utilized two phage antibody libraries; the immune and naïve library were used to identify single chain fragment variable (scFv) clones against a specific filarial antigen (BmR1). The V-gene analysis of isolated scFv clones will help shed light on preferential VDJ gene segment usage against the filarial BmR1 antigen in healthy and infected states. The immune library showed the usage of both lambda and kappa light chains. However, the naïve library showed preferential use of the lambda family with different amino acid distributions. The binding characteristics of the scFv clones identified from this work were analyzed by immunoassay and immunoaffinity pull down of BmR1. The work highlights the antibody gene usage pattern of a naïve and immune antibody library against the same antigen as well as the robust nature of the enriched antibodies for downstream applications.
Fulltext The structure and dynamics of BmR1 protein from Brugia malayi: in silico approaches

Khor BY, Tye GJ, Lim TS, Noordin R, Choong YS

Int J Mol Sci, 2014 Jun 19;15(6):11082-99.
PMID: 24950179 DOI: 10.3390/ijms150611082

Brugia malayi is a filarial nematode, which causes lymphatic filariasis in humans. In 1995, the disease has been identified by the World Health Organization (WHO) as one of the second leading causes of permanent and long-term disability and thus it is targeted for elimination by year 2020. Therefore, accurate filariasis diagnosis is important for management and elimination programs. A recombinant antigen (BmR1) from the Bm17DIII gene product was used for antibody-based filariasis diagnosis in "Brugia Rapid". However, the structure and dynamics of BmR1 protein is yet to be elucidated. Here we study the three dimensional structure and dynamics of BmR1 protein using comparative modeling, threading and ab initio protein structure prediction. The best predicted structure obtained via an ab initio method (Rosetta) was further refined and minimized. A total of 5 ns molecular dynamics simulation were performed to investigate the packing of the protein. Here we also identified three epitopes as potential antibody binding sites from the molecular dynamics average structure. The structure and epitopes obtained from this study can be used to design a binder specific against BmR1, thus aiding future development of antigen-based filariasis diagnostics to complement the current diagnostics.
Selection of ssDNA aptamers and construction of aptameric electrochemical biosensor for the detection of Giardia intestinalis trophozoite protein

Alhindawi M, Rhouati A, Noordin R, Cialla-May D, Popp J, Zourob M

Int J Biol Macromol, 2024 Apr 11;267(Pt 2):131509.
PMID: 38608978 DOI: 10.1016/j.ijbiomac.2024.131509

Giardia intestinalis is one of the most widespread intestinal parasites and is considered a major cause of epidemic or sporadic diarrhea worldwide. In this study, we aimed to develop a rapid aptameric diagnostic technique for G. intestinalis infection. First, the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) process generated DNA aptamers specific to a recombinant protein of the parasite's trophozoite. Ten selection rounds were performed; each round, the DNA library was incubated with the target protein conjugated to Sepharose beads. Then, the unbound sequences were removed by washing and the specific sequences were eluted and amplified by Polymerase Chain Reaction (PCR). Two aptamers were selected, and the dissociation constants (Kd), were determined as 2.45 and 16.95 nM, showed their high affinity for the G. intestinalis trophozoite protein. Subsequently, the aptamer sequence T1, which exhibited better affinity, was employed to develop a label-free electrochemical biosensor. A thiolated aptamer was covalently immobilized onto a gold screen-printed electrode (SPGE), and the binding of the targeted protein was monitored using square wave voltammetry (SWV). The developed aptasensor enabled accurate detection of the G. intestinalis recombinant protein within the range of 0.1 pg/mL to 100 ng/mL, with an excellent sensitivity (LOD of 0.35 pg/mL). Moreover, selectivity studies showed a negligible cross-reactivity toward other proteins such as bovine serum albumin, globulin, and G. intestinalis cyst protein.
Purification of recombinant antigen BmSXP used in panLF rapid kit for lymphatic filariasis

Khoo TK, Noordin R, Santhanam A

Indian J Exp Biol, 2012 Apr;50(4):256-64.
PMID: 22611913

A rapid antibody detection test is very useful for the detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. panLF Rapid kit is suitable for this purpose since it can detect all species of lymphatic filaria. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant B. malayi antigens, BmR1 and BmSXP. There is an increase demand for the test due to its attributes of being rapid, sensitive and specific results, as well as its field-applicability. The main aim of this paper is to obtain high recovery and purity of recombinant antigen BmSXP via a modified method of immobilized metal affinity chromatography (IMAC). The highest product yield of 11.82 mg/g dry cell weight (DCW) was obtained when IMAC was performed using the optimized protocol of 10 mM imidazole concentration in lysis buffer, 30 mM imidazole concentration in wash buffer, and 10 column volume wash buffer containing 300 mM salt concentration. This gave a 54% protein recovery improvement over the manufacturer's protocol which recorded a product yield of only 7.68 mg/g DCW. The recovered BmSXP recombinant antigen showed good western blot reactivity, high sensitivity (31/32, 97%) and specificity (32/32, 100%) in ELISA, thus attesting to its good purity and quality.
Association of CTLA4 gene polymorphisms with lymphatic filariasis in an East Malaysian population

Idris ZM, Miswan N, Muhi J, Mohd TA, Kun JF, Noordin R

Hum Immunol, 2011 Jul;72(7):607-12.
PMID: 21513760 DOI: 10.1016/j.humimm.2011.03.017

Lymphatic filariasis (LF) is a parasitic disease caused by threadlike worms of the Brugia and Wuchereria species that live in the human lymphatic system. Regulatory T cells (Tregs) may play a key role in the pathogenesis of LF, and cytotoxic T-lymphocyte antigen-4 (CTLA4) expressed by Tregs is a potential candidate gene because it modulates T-cell activation. A case-control study was performed to establish a potential association of 5 CTLA4 gene promoter single nucleotide polymorphisms (SNPs; rs733618, rs11571316, rs5742909, rs231775, and rs16840252) with the occurrence of LF in an East Malaysian population (320 LF-infected individuals and 150 healthy controls). Polymorphisms were evaluated using TaqMan real-time polymerase chain reaction followed by direct sequencing. LF carriers of the rs733618 AG genotypes (p = 0.02) and those with combined minor allele G carriers (AG + GG; p = 0.01) exhibited a significantly decreased risk for LF. Among the asymptomatic amicrofilaremic cases, positive associations were reported for all genotypes and variants of rs733618 with odds ratios (ORs) ranging from 0.27 to 0.45. In the asymptomatic microfilaremic cases, marker rs231775 exhibited a significant decreased risk, with ORs ranging from 0.50 to 0.57. The study has identified SNPs in the CTLA4 promoter gene that may be functionally linked with susceptibility to LF.
Fulltext A pilot study on cytotoxic T lymphocyte-4 gene polymorphisms in urinary schistosomiasis

Idris ZM, Yazdanbakhsh M, Adegnika AA, Lell B, Issifou S, Noordin R

Genet Test Mol Biomarkers, 2012 Jun;16(6):488-92.
PMID: 22288822 DOI: 10.1089/gtmb.2011.0209

Urinary schistosomiasis is caused by the digenetic trematode Schistosoma haematobium, characterized by accumulation of eggs in the genitourinary tract. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) can play an important role in parasitic infection due to its major role as a negative regulator of T-cell activation and proliferation. This study was performed in patients with schistosomiasis and healthy controls to analyze the allele and genotype frequencies of four CTLA-4 gene polymorphisms. The CTLA-4 gene was amplified using Taqman real-time polymerase chain reaction, and allele and genotypes of 49 patients with schistosomiasis were analyzed using allelic discrimination analysis followed by subsequent direct sequencing. The results were compared with healthy control subjects. The frequencies of CTLA-4 rs733618 A allele at position -1722 (p=0.001), rs11571316 C allele at position -1577 (p<0.001), and rs231775 A allele at position +49 (p=0.002) in the patient group were significantly higher than the control group. The rs733618 AA genotype (p=0.001), rs11571316 CC genotype (p<0.001), and rs231775 AA genotype (p=0.007) were also significantly overrepresented. Meanwhile, rs733618 AG genotype (p=0.001), rs11571316 CT genotype (p=0.02), and rs231775 GG genotype (p=0.029) were significantly decreased in the patients with schistosomiasis, as compared with the controls. No significant difference was observed in both allele and genotype of rs16841252. The results of this study suggest that the rs733618, rs11571316, and rs231775 polymorphisms in the CTLA-4 gene may influence susceptibility to schistosomiasis infection in the Gabonese children.
Application of real-time polymerase chain reaction in detection of Entamoeba histolytica in pus aspirates of liver abscess patients

Othman N, Mohamed Z, Verweij JJ, Huat LB, Olivos-García A, Yeng C, et al.

Foodborne Pathog Dis, 2010 Jun;7(6):637-41.
PMID: 20132028 DOI: 10.1089/fpd.2009.0427

Entamoeba histolytica is the second major cause of liver abscess disease in humans, particularly in developing countries. Recently, DNA molecular-based methods have been employed to enhance the detection of E. histolytica in either pus or stool specimens. In this study, the results of real-time polymerase chain reaction (PCR) to detect E. histolytica DNA in pus from liver abscess cases were compared with those of indirect hemagglutination assay on the corresponding serum samples. Bacterial cultures were also performed on the pus samples for the diagnosis of pyogenic liver abscess. The real-time PCR detected E. histolytica DNA in 23 of 30 (76.7%) pus samples, when compared with 14 of 30 (46.7%) serum samples in which anti-Entamoeba antibodies were detected by indirect hemagglutination assay and 4 of 30 (13.3%) pus samples that showed bacterial infection by culture. The use of real-time PCR is a promising detection method for diagnosis and epidemiology assessment of amoebic liver abscess.
Pan LF-ELISA using BmR1 and BmSXP recombinant antigens for detection of lymphatic filariasis

Abdul Rahman R, Hwen-Yee C, Noordin R

Filaria journal, 2007;6:10.
PMID: 17961264

Anti-filarial IgG4 antibody has been shown to be a good marker for detection of lymphatic filaria infection. Previous studies demonstrated that anti-filarial IgG4 assay using BmR1 recombinant antigen was highly specific and sensitive for detection of brugian filariasis. For bancroftian filariasis, an equivalent assay employing recombinant antigen expressed from the ORF of SXP1 gene has been reported. In order to detect infections by all species of lymphatic filarial, BmR1 and BmSXP recombinant antigens were employed in the development of a pan LF-ELISA.
Multicentre evaluations of two new rapid IgG4 tests (WB rapid and panLF rapid) for detection of lymphatic filariasis

Noordin R, Itoh M, Kimura E, Abdul Rahman R, Ravindran B, Mahmud R, et al.

Filaria journal, 2007;6:9.
PMID: 17961262

In the global effort to eliminate lymphatic filariasis (LF), rapid field-applicable tests are useful tools that will allow on-site testing to be performed in remote places and the results to be obtained rapidly. Exclusive reliance on the few existing tests may jeopardize the progress of the LF elimination program, thus the introduction of other rapid tests would be useful to address this issue. Two new rapid immunochromatographic IgG4 cassette tests have been produced, namely WB rapid and panLF rapid, for detection of bancroftian filariasis and all three species of lymphatic filaria respectively. WB rapid was developed using BmSXP recombinant antigen, while PanLF rapid was developed using BmR1 and BmSXP recombinant antigens. A total of 165 WB rapid and 276 panLF rapid tests respectively were evaluated at USM and the rest were couriered to another university in Malaysia (98 WB rapid, 129 panLF rapid) and to universities in Indonesia (56 WB rapid, 62 panLF rapid), Japan (152 of each test) and India (18 of each test) where each of the tests underwent independent evaluations in a blinded manner. The average sensitivities of WB rapid and panLF rapid were found to be 97.6% (94%-100%) and 96.5% (94%-100%) respectively; while their average specificities were both 99.6% (99%-100%). Thus this study demonstrated that both the IgG4 rapid tests were highly sensitive and specific, and would be useful additional tests to facilitate the global drive to eliminate this disease.
Homologs of the Brugia malayi diagnostic antigen BmR1 are present in other filarial parasites but induce different humoral immune responses

Noordin R, Aziz RA, Ravindran B

Filaria journal, 2004 Dec 31;3(1):10.
PMID: 15627400

BACKGROUND: The recombinant antigen BmR1 has been extensively employed in both ELISA and immunochromatographic rapid dipstick (Brugia Rapid) formats for the specific and sensitive detection of IgG4 antibodies against the lymphatic filarial parasites Brugia malayi and Brugia timori. In sera of individuals infected with Wuchereria bancrofti the IgG4 reactivity to BmR1 is variable, and cross-reactivity of sera from individuals infected with Onchocerca volvulus or Loa loa was observed only in single cases. In order to characterize the homologs of the BmR1 antigen in W. bancrofti (Wb-BmR1), O. volvulus (Ov-BmR1) and L. loa (Ll-BmR1) the cDNA sequences were identified, the protein expressed and the antibody reactivity of patients' sera was studied. METHODS: PCR methodology was used to identify the cDNA sequences from cDNA libraries and/or genomic DNA of W. bancrofti, O. volvulus and L. loa. The clones obtained were sequenced and compared to the cDNA sequence of BmR1. Ov-BmR1 and Ll-BmR1 were expressed in E. coli and tested using an IgG4-ELISA with 262 serum samples from individuals with or without B. malayi, W. bancrofti, O. volvulus and L. loa infections or various other parasitic infections. BmR1, Ov-BmR1 and Ll-BmR1 were also tested for reactivity with the other three IgG subclasses in patients' sera. RESULTS: Wb-BmR1 was found to be identical to BmR1. Ov-BmR1 and Ll-BmR1 were found to be identical to each other and share 99.7% homology with BmR1. The pattern of IgG4 recognition of all serum samples to BmR1, Ov-BmR1 and Ll-BmR1 were identical. This included weak IgG4 reactivities demonstrated by L. loa- and O. volvulus-infected patients tested with Ov-BmR1 and Ll-BmR1 (or BmR1). With respect to reactivity to other IgG subclasses, sera from O. volvulus- and L. loa-infected patients showed positive reactions (when tested with BmR1, Ov-BmR1 or Ll-BmR1 antigens) only with IgG1. No reactivity was observed with IgG2 or with IgG3. Similarly, ELISAs to detect reactivity to other anti-filarial IgG subclasses antibodies showed that sera from individuals infected with B. malayi or W. bancrofti (active infections as well as patients with chronic disease) were positive with BmR1 only for IgG1 and were negative when tested with IgG2 and with IgG3 subclasses. CONCLUSIONS: This study demonstrates that homologs of the BmR1 antigen are present in W. bancrofti, O. volvulus and L. loa and that these antigens are highly conserved. Recognition of this antigen by patients' sera is similar with regard to IgG1, IgG2 and IgG3, but different for IgG4 antibodies. We conclude that the BmR1 antigen is suitable for detection of IgG4 antibodies in brugian filariasis. However, its homologs are not suitable for IgG4-based diagnosis of other filarial infections.
Comparison of IgG4 assays using whole parasite extract and BmR1 recombinant antigen in determining antibody prevalence in brugian filariasis

Noordin R, Wahyuni S, Mangali A, Huat LB, Yazdanbakhsh M, Sartono E

Filaria journal, 2004 Aug 12;3(1):8.
PMID: 15307892

BACKGROUND: Brugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi. METHODS: Serum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid trade mark ]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test. RESULTS: There were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children. CONCLUSIONS: Results and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study.
Entamoeba histolytica antigenic protein detected in pus aspirates from patients with amoebic liver abscess

Othman N, Mohamed Z, Yahya MM, Leow VM, Lim BH, Noordin R

Exp Parasitol, 2013 Aug;134(4):504-10.
PMID: 23680184 DOI: 10.1016/j.exppara.2013.05.001

Entamoeba histolytica is a causative agent of amoebic liver abscess (ALA) and is endemic in many underdeveloped countries. We investigated antigenic E. histolytica proteins in liver abscess aspirates using proteomics approach. Pus samples were first tested by real-time PCR to confirm the presence of E. histolytica DNA and the corresponding serum samples tested for E. histolytica-specific IgG by a commercial ELISA. Proteins were extracted from three and one pool(s) of pus samples from ALA and PLA (pyogenic liver abscess) patients respectively, followed by analysis using isoelectric focussing, SDS-PAGE and Western blot. Unpurified pooled serum samples from infected hamsters and pooled human amoebic-specific IgG were used as primary antibodies. The antigenic protein band was excised from the gel, digested and analysed by MALDI-TOF/TOF and LC-MS/MS. The results using both primary antibodies showed an antigenic protein band of ∼14kDa. Based on the mass spectrum analysis, putative tyrosine kinase is the most probable identification of the antigenic band.
Fulltext Triplex PCR using new primers for the detection of Toxoplasma gondii

Rahumatullah A, Khoo BY, Noordin R

Exp Parasitol, 2012 Jun;131(2):231-8.
PMID: 22561042 DOI: 10.1016/j.exppara.2012.04.009

Molecular methods are used increasingly for the detection of Toxoplasma gondii infection. This study developed a rapid, sensitive, and specific conventional triplex PCR for the detection of the B1 gene and ITS1 region of T. gondii using newly designed primers and an internal control based on the Vibrio cholerae HemM gene. The annealing temperature and concentrations of the primers, MgCl(2), and dNTPs were optimized. Two sets of primers (set 1 and 2) were tested, which contained different segments of the T. gondii B1 gene, 529 repeat region and ITS1 region. A series of sensitivity tests were performed using parasite DNA, whole parasites, and spiked human body fluids. Specificity tests were performed using DNA from common protozoa and bacteria. The newly developed assay based on set 2 primers was found to be specific and sensitive. The test was capable of detecting as little as 10 pg T. gondii DNA, 10(4) tachyzoites in spiked body fluids, and T. gondii DNA in the organ tissues of experimentally infected mice. The assay developed in this study will be useful for the laboratory detection of T. gondii infection.

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