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.
Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory-secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥ 95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank's balanced salt solution (HBSS). Concurrently, the effect of adding L: -cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥ 95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P>0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥ 95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only one fold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.
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.
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.
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.
This study describes the properties of colloidal gold nanoparticles (AuNPs) with sizes of 20, 30 and 40 nm, which were synthesized using citrate reduction or seeding-growth methods. Likewise, the conjugation of these AuNPs to mouse anti-human IgG(4) (MαHIgG(4)) was evaluated for an immunochromatographic (ICG) strip test to detect brugian filariasis. The morphology of the AuNPs was studied based on the degree of ellipticity (G) of the transmission electron microscopy images. The AuNPs produced using the seeding-growth method showed lower ellipticity (G ≤ 1.11) as compared with the AuNPs synthesized using the citrate reduction method (G ≤ 1.18). Zetasizer analysis showed that the AuNPs that were synthesized using the seeding-growth method were almost monodispersed with a lower polydispersity index (PDI; PDI≤0.079), as compared with the AuNPs synthesized using the citrate reduction method (PDI≤0.177). UV-visible spectroscopic analysis showed a red-shift of the absorbance spectra after the reaction with MαHIgG(4), which indicated that the AuNPs were successfully conjugated. The optimum concentration of the BmR1 recombinant antigen that was immobilized on the surface of the ICG strip on the test line was 1.0 mg ml(-1). When used with the ICG test strip assay and brugian filariasis serum samples, the conjugated AuNPs-MαHIgG(4) synthesized using the seeding-growth method had faster detection times, as compared with the AuNPs synthesized using the citrate reduction method. The 30 nm AuNPs-MαHIgG(4), with an optical density of 4 from the seeding-growth method, demonstrated the best performance for labelling ICG strips because it displayed the best sensitivity and the highest specificity when tested with serum samples from brugian filariasis patients and controls.
Diagnosis of human toxocariasis, caused by Toxocara canis or Toxocara cati, normally relies on a combination of the presence of clinical signs and symptoms backed by positive serology. The use of Toxocara excretory-secretory antigen (TES) in ELISA assays increases the test specificity. However, in tropical countries where soil-transmitted helminths are endemic, cross-reactivity from antibodies to these intestinal parasites poses a significant limitation for Toxocara serodiagnosis. To increase the specificity of serodiagnosis, we compared the use of IgG-ELISA to the use of IgG4-ELISA using commercially manufactured TES-coated plates. The sensitivity of the IgG-ELISA was 97.1%, while that of the IgG4-ELISA was 45.7%; the specificities were 36.0 and 78.6%, respectively. The study shows that employing both assays can improve the serodiagnosis of toxocariasis. An IgG4 immunoassay would also be useful in the secondary screening of antigen clones in the effort to develop improved serological tests for toxocariasis.
BmR1 recombinant antigen has previously been shown to demonstrate high sensitivity and specificity in the serological diagnosis of brugian filariasis in humans. In this study, the pattern of recognition of antibody to BmR1 during Brugia malayi infection was investigated by employing Meriones unguiculatus as the experimental model. Thirty two gerbils were infected subcutaneously with 120 L(3); and two control groups each comprising 25 animals were employed. ELISA using BmR1 was used to detect filaria-specific IgG antibodies elicited by the gerbils; using sera collected from the day 1 until day 150 post-inoculation (p.i.). The results showed that BmR1 detected B. malayi infection in gerbils harboring adult worms irrespective of the presence of circulating microfilaria, and was exemplified by positive ELISA results in nine a microfilaraemic animals that harbored live adult worms. The initial time of the antibody recognition was at day 8 p.i. and the antibody titre showed some correlation with adult worm burden.
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.
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.
This study was performed to identify circulating Plasmodium falciparum proteins in patient serum, which may be useful as diagnostic markers. Depletion of highly abundant proteins from each pooled serum sample obtained from P. falciparum-infected patients and healthy individuals was performed using the Proteoseek Antibody-Based Albumin/IgG Removal Kit (Thermo Scientific, Rockford, IL). In analysis 1, the depleted serum was analyzed directly by NanoLC-MS/MS. In analysis 2, the depleted serum was separated by two-dimensional electrophoresis followed by western blot analysis. Subsequently, the selected band was analyzed by NanoLC-MS/MS. The result of analysis 1 revealed the presence of two mature erythrocyte surface antigen (MESA) proteins and chloroquine resistance transporter protein (PfCRT). In addition, analysis 2 revealed an antigenic 75-kDa band when the membrane was probed with purified IgG from the pooled serum obtained from P. falciparum-infected patients. MS/MS analysis of this protein band revealed fragments of P. falciparum MESA proteins. Thus, in this study, two different analyses revealed the presence of Plasmodium MESA protein in pooled serum from malaria patients; thus, this protein should be further investigated to determine its usefulness as a diagnostic marker.
Toxocariasis is a cosmopolitan zoonotic disease caused by the infective larvae of Toxocara canis and T. cati. Diagnosis in humans is usually based on clinical symptoms and serology. Immunoglobulin G (IgG)-enzyme-linked immunosorbent assay kits using T. canis excretory-secretory (TES) larval antigens are commonly used for serodiagnosis. Differences in the antigens of the two Toxocara species may influence the diagnostic sensitivity of the test. In this study, T. cati recombinant TES-120 (rTES-120) was cloned, expressed, and compared with its T. canis homolog in an IgG4-western blot. The diagnostic sensitivity and specificity of T. cati rTES-120 were 70% (33/47) and 100% (39/39), respectively. T. canis rTES-120 showed 57.4% sensitivity and 94.4% specificity. When the results of assays using rTES-120 of both species were considered, the diagnostic sensitivity was 76%. This study shows that using antigens from both Toxocara species may improve the serodiagnosis of toxocariasis.
Rapid diagnostic tests for cystic echinococcosis (CE) are convenient to support ultrasound diagnosis in uncertain cases, especially in resource-limited settings. We found comparable diagnostic performances of the experimental Hyd Rapid Test and the commercial VIRapid HYDATIDOSIS Test, used in our diagnostic laboratory, using samples from well-characterized hepatic CE cases.
Entamoeba histolytica infection remains a public health concern in developing countries. Early diagnosis of amoebiasis can avoid disease complications, thus this study was aimed at developing a test that can rapidly detect the parasite antigens in stool samples. Rabbits were individually immunized with recombinant pyruvate phosphate dikinase (rPPDK) and E. histolytica excretory-secretory antigens to produce polyclonal antibodies. A rapid dipstick test was produced using anti-rPPDK PAb lined on the dipstick as capture reagent and anti-EhESA PAb conjugated to colloidal gold as the detector reagent. Using E. histolytica-spiked in stool sample of a healthy individual, the detection limit of the dipstick test was found to be 1000 cells ml-1. Meanwhile when rPPDK was spiked in the stool sample, the minimum concentration detected by the dipstick test was 0.1 μg ml-1. The performances of the dipstick, commercial Techlab E. histolytica II enzyme-linked immunosorbent assays (ELISA) and real-time PCR were compared using 70 stool samples from patients infected with Entamoeba species (n = 45) and other intestinal pathogens (n = 25). When compared to real-time PCR, the diagnostic sensitivity of the dipstick for detection of E. histolytica was 65.4% (n = 17/26); while the diagnostic specificity when tested with stool samples containing other intestinal pathogens was 92% (23/25). In contrast, Techlab E. histolytica II ELISA detected 19.2% (5/26) of the E. histolytica-positive samples as compared to real-time PCR. The lateral flow dipstick test produced in this study enabled rapid detection of E. histolytica, thus it showed good potential to be further developed into a diagnostic tool for intestinal amoebiasis.
At the end phase of the Global Programme to Eliminate Lymphatic Filariasis, antibody testing may have a role in decision-making for bancroftian filariasis-endemic areas. This study evaluated the diagnostic performance of BLF Rapid™, a prototype immunochromatographic IgG4-based test using BmSXP recombinant protein, for detection of bancroftian filariasis. The test was evaluated using 258 serum samples, comprising 96 samples tested at Universiti Sains Malaysia (in-house) and 162 samples tested independently at three international laboratories in the USA and India, and two laboratories in Malaysia. The independent testing involved 99 samples from Wuchereria bancrofti microfilaria or antigen positive individuals and 63 samples from people who were healthy or had other infections. The in-house evaluation showed 100% diagnostic sensitivity and specificity. The independent evaluations showed a diagnostic sensitivity of 84-100% and 100% specificity (excluding non-lymphatic filarial infections). BLF Rapid has potential as a surveillance diagnostic tool to make "Transmission Assessment Survey"-stopping decisions and conduct post-elimination surveillance.
Entamoeba histolytica is a protozoan parasite that causes amebiasis and poses a significant health risk for populations in endemic areas. The molecular mechanisms involved in the pathogenesis and regulation of the parasite are not well characterized. We aimed to identify and quantify the differentially abundant membrane proteins by comparing the membrane proteins of virulent and avirulent variants of E. histolytica HM-1:IMSS, and to investigate the potential associations among the differentially abundant membrane proteins. We performed quantitative proteomics analysis using isobaric tags for relative and absolute quantitation labeling, in combination with two mass spectrometry instruments, that is, nano-liquid chromatography (nanoLC)-matrix-assisted laser desorption/ionization-mass spectrometry/mass spectrometry and nanoLC-electrospray ionization tandem mass spectrometry. Overall, 37 membrane proteins were found to be differentially abundant, whereby 19 and 18 membrane proteins of the virulent variant of E. histolytica increased and decreased in abundance, respectively. Proteins that were differentially abundant include Rho family GTPase, calreticulin, a 70-kDa heat shock protein, and hypothetical proteins. Analysis by Protein ANalysis THrough Evolutionary Relationships database revealed that the differentially abundant membrane proteins were mainly involved in catalytic activities (29.7%) and metabolic processes (32.4%). Differentially abundant membrane proteins that were found to be involved mainly in the catalytic activities and the metabolic processes were highlighted together with their putative roles in relation to the virulence. Further investigations should be performed to elucidate the roles of these proteins in E. histolytica pathogenesis.
Entamoeba histolytica membrane proteins are important players toward the pathogenesis of amoebiasis, but the roles of most of the proteins are not fully understood. Since efficient protein extraction method is crucial for a successful MS analysis, three extractions methods are evaluated for the use in studying the membrane proteome of E. histolytica: Two commercial kits (ProteoExtract from Calbiochem and ProteoPrep from Sigma), and a conventional laboratory method. The results show that ProteoExtract and the conventional method gave higher protein yields compared to ProteoPrep. LC-ESI-MS/MS identifies 456, 482, and 551 membrane fraction proteins extracted using ProteoExtract, ProteoPrep, and a conventional method, respectively. In silico analysis predicts 108 (21%), 235 (45%), and 177 (34%) membrane proteins from the extracts of ProteoExtract, ProteoPrep, and the conventional method, respectively. Furthermore, analysis of the cytosolic and membrane fractions shows the highest selectivity of the membrane proteins using the ProteoPrep extraction kit. Overall, this study reports 828 E. histolytica membrane fraction proteins that include 249 predicted membrane proteins. The data are available via ProteomeXchange with identifier PXD010171.
Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 109 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis.
Lateral flow assays (LFAs) are the mainstay of rapid point-of-care diagnostics, with the potential to enable early case management and transform the epidemiology of infectious disease. However, most LFAs only detect single biomarkers. Recognizing the complex nature of human disease, overlapping symptoms and states of co-infections, there is increasing demand for multiplexed systems that can detect multiple biomarkers simultaneously. Due to innate limitations in the design of traditional membrane-based LFAs, multiplexing is arguably limited to a small number of biomarkers. Here, we summarize the need for multiplexed LFA, key technical and operational challenges for multiplexing, inherent in the design and production of multiplexed LFAs, as well as emerging enabling technologies that may be able to address these challenges. We further identify important areas for research in efforts towards developing multiplexed LFAs for more impactful diagnosis of infectious diseases.
This study described the isolation of the coding region of human topoisomerase I (TopoI) from MDA-MB-231 and the expression of multiple copy recombinant genes in four Pichia pastoris strains. First, polymerase chain reaction (PCR)-amplification of the enzyme coding region was performed. The PCR fragment was cloned into pPICZ-α-A vector and sequenced. It was then transformed into X33, GS115, SMD1168H and KM71H strains of Pichia. PCR-screening for positive clones was performed, and estimation of multiple copy integrants in each Pichia strain was carried out using agar plates containing increasing concentrations of Zeocin(®). The selected clones of multiple copy recombinant genes were then induced for TopoI expression in shaker flasks. GS115 and SMD1168 were found to be better Pichia strains to accommodate the recombinant gene for the expression of TopoI extracellularly. However, the DNA relaxation activity revealed that only the target enzyme in the culture supernatants of GS115-pPICZ-α-A-TopoI exhibited consistent enzyme activity over the cultivation time-points. Active enzyme activity was inhibited by Camptothecin. The enzyme produced can be used for in-house gel-based DNA relaxation assay development in performing high throughput screening for target-specific growth inhibitors that display similar effect as the TopoI inhibitors. These inhibitors may contribute to the improvement of the treatment of cancer patients.