Displaying publications 1 - 20 of 26 in total

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  1. Epitope Identification and Application for Diagnosis of Duck Tembusu Virus Infections in Ducks
    Li C, Liu J, Shaozhou W, Bai X, Zhang Q, Hua R, et al.
    Viruses, 2016 Nov 10;8(11).
    PMID: 27834908
    Duck Tembusu virus (DTMUV) causes substantial egg drop disease. DTMUV was first identified in China and rapidly spread to Malaysia and Thailand. The antigenicity of the DTMUV E protein has not yet been characterized. Here, we investigated antigenic sites on the E protein using the non-neutralizing monoclonal antibodies (mAbs) 1F3 and 1A5. Two minimal epitopes were mapped to (221)LD/NLPW(225) and (87)YAEYI(91) by using phage display and mutagenesis. DTMUV-positive duck sera reacted with the epitopes, thus indicating the importance of the minimal amino acids of the epitopes for antibody-epitope binding. The performance of the dot blotting assay with the corresponding positive sera indicated that YAEYI was DTMUV type-specific, whereas (221)LD/NLPW(225) was a cross-reactive epitope for West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) and corresponded to conserved and variable amino acid sequences among these strains. The structure model of the E protein revealed that YAEYI and LD/NLPW were located on domain (D) II, which confirmed that DII might contain a type-specific non-neutralizing epitope. The YAEYI epitope-based antigen demonstrated its diagnostic potential by reacting with high specificity to serum samples obtained from DTMUV-infected ducks. Based on these observations, a YAEYI-based serological test could be used for DTMUV surveillance and could differentiate DTMUV infections from JEV or WNV infections. These findings provide new insights into the organization of epitopes on flavivirus E proteins that might be valuable for the development of epitope-based serological diagnostic tests for DTMUV.
    Matched MeSH terms: Serologic Tests/methods*
  2. 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.
    Matched MeSH terms: Serologic Tests/methods
  3. Fulltext Optimization of the expression of surface antigen SAG1/2 of Toxoplasma gondii in the yeast Pichia pastoris
    Thiruvengadam G, Init I, Fong MY, Lau YL
    Trop Biomed, 2011 Dec;28(3):506-13.
    PMID: 22433878 MyJurnal
    Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.
    Matched MeSH terms: Serologic Tests/methods
  4. Fulltext A low molecular weight lipopolysaccharide antigen preparation reactive to acute leptospirosis heterologous sera
    Riazi M, Abdul Rani B, Fairuz A, Zainul FZ
    Trop Biomed, 2010 Aug;27(2):241-53.
    PMID: 20962722 MyJurnal
    There is a need for identification of new infection markers against common Leptospira isolates in Malaysia. To achieve this goal, seven-day-old cultures of Leptospira interrogans serogroup Icterohemorrhagiae (L44) and Leptospira interrogans serogroup Javanica (L55) were used for antigen preparation by sequential extraction method using 40 mM Tris, 8M Urea and 2M thiourea. Immunoblot analysis of the antigens were performed using serum samples from 46 local patients with confirmed acute leptospirosis, 28 patients with other infections and 14 healthy controls. The patients serum samples used in this study contained heterologous antibody against a number of different leptospira serovars. A strong IgM reactivity to a broad diffuse band of 10-15 kDa was observed. Combining results using L44 and L55 antigens showed sensitivity of 80.4% and specificity of 95.2% for detection of leptospirosis. Proteinase K and periodate treatment indicated that the band is likely to be lipopolysaccharide (LPS) in nature. This study showed that the 10-15 kDa antigen could potentially be useful for serodiagnosis of acute leptospirosis in Malaysia.
    Matched MeSH terms: Serologic Tests/methods
  5. Comparison of two IgG4 assay formats (ELISA and rapid dipstick test) for detection of brugian filariasis
    Noordin R, Shenoy RK, Rahman RA
    Southeast Asian J. Trop. Med. Public Health, 2003 Dec;34(4):768-70.
    PMID: 15115085
    Brugia malayi infection is endemic in several Asian countries. Filaria-specific IgG4 antibody detection based on BmR1 recombinant antigen has been shown to be sensitive and specific for the diagnosis of brugian filariasis. Two formats of the test has been reported ie indirect ELISA (BE) and rapid dipstick test (BR). Since different test formats use different amounts of sample and reagents which may affect its sensitivity and specificity, this study was performed to compare these two test formats in the detection of B. malayi. A total of 264 blinded serum samples from India and Malaysia were employed. Group 1 comprised 164 samples from actively infected individuals and group 2 comprised 100 samples from filaria non-endemic areas. Sensitivity was 96.3% (158/164) and 90.8% (149/164) for rapid test and ELISA respectively; chi-square p=0.00. Both test formats demonstrated 100% specificity. Therefore the rapid test format was equally specific but more sensitive than the ELISA format. The ELISA format would be able to demonstrate decline in IgG4 titer post-treatment while the rapid test would be very useful for screening and diagnosis in the field.
    Matched MeSH terms: Serologic Tests/methods*
  6. A review of the serological results obtained in a routine diagnostic laboratory for Mycoplasma pneumoniae infections
    Tay ST, Cheong YM
    Malays J Pathol, 1995 Jun;17(1):35-7.
    PMID: 8907003
    The results of a Mycoplasma pneumoniae serology test performed routinely at the Bacteriology Division, Institute for Medical Research were reviewed. A total of 1402 patients were screened over a period of 4 years (January, 1990-December, 1993), of which 327 (23.3%) were seropositive. The seropositivity rates among Malays, Chinese and Indians were 25.2, 25.4 and 17.8% respectively. The male to female ratio was 1:1. The age specific rate was highest amongst patients of the 6-12 years (35.1%) followed by the 13-20 years age groups (35.0%). In general, infection with M. pneumoniae appears to be relatively common in this country.
    Matched MeSH terms: Serologic Tests/methods
  7. Production of Toxocara cati TES-120 Recombinant Antigen and Comparison with its T. canis Homolog for Serodiagnosis of Toxocariasis
    Zahabiun F, Sadjjadi SM, Yunus MH, Rahumatullah A, Moghaddam MH, Saidin S, et al.
    Am J Trop Med Hyg, 2015 Aug;93(2):319-25.
    PMID: 26033026 DOI: 10.4269/ajtmh.15-0190
    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.
    Matched MeSH terms: Serologic Tests/methods
  8. Lateral Flow Dipstick Test for Serodiagnosis of Strongyloidiasis
    Yunus MH, Arifin N, Balachandra D, Anuar NS, Noordin R
    Am J Trop Med Hyg, 2019 08;101(2):432-435.
    PMID: 31218996 DOI: 10.4269/ajtmh.19-0053
    The conventional method of detecting Strongyloides stercoralis in fecal samples has poor diagnostic sensitivity. Detection of Strongyloides-specific antibodies increases the sensitivity; however, most tests are ELISAs that use parasite extract which may cross-react with the sera of other helminth infections. To improve the serological diagnosis of strongyloidiasis, this study aimed at developing a sensitive and specific lateral flow rapid dipstick test. Two recombinant proteins, recombinant NIE (rNIE) and recombinant Ss1a (rSs1a), were used in preparing the dipstick, with gold-conjugated antihuman IgG4 as detector reagent. In parallel, the corresponding ELISA was performed. Both assays demonstrated diagnostic sensitivity of 91.3% (21/23) when tested with serum samples of patients with Strongyloides infection, and 100% specificity with 82 sera of asymptomatic (healthy) and those with other parasitic infections. The ELISA and dipstick test results were positively correlated to each other (r = 0.6114, P = 0.0019). The developed lateral flow dipstick test may improve the serodiagnosis of strongyloidiasis and merit further validation studies.
    Matched MeSH terms: Serologic Tests/methods*
  9. Evaluation of Toxoplasma gondii-recombinant dense granular protein (GRA2) for serodiagnosis by western blot
    Ching XT, Lau YL, Fong MY, Nissapatorn V
    Parasitol Res, 2013 Mar;112(3):1229-36.
    PMID: 23274488 DOI: 10.1007/s00436-012-3255-5
    Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the food industry. Commonly used serological tests involve preparation of whole Toxoplasma lysate antigens from tachyzoites which are costly and hazardous. An alternative method for better antigen production involving the prokaryotic expression system was therefore used in this study. Recombinant dense granular protein, GRA2, was successfully cloned, expressed, and purified in Escherichia coli, BL21 (DE3) pLysS. The potential of this purified antigen for diagnosis of human infections was evaluated through western blot analysis against 100 human serum samples. Results showed that the rGRA2 protein has 100 and 61.5 % sensitivity towards acute and chronic infection, respectively, in T. gondii-infected humans, indicating that this protein is useful in differentiating present and past infections. Therefore, it is suitable to be used as a sensitive and specific molecular marker for the serodiagnosis of Toxoplasma infection in both humans and animals.
    Matched MeSH terms: Serologic Tests/methods
  10. Fulltext Design and evaluation of a recombinant multi-epitope antigen for serodiagnosis of Toxoplasma gondii infection in humans
    Hajissa K, Zakaria R, Suppian R, Mohamed Z
    Parasit Vectors, 2015;8:315.
    PMID: 26062975 DOI: 10.1186/s13071-015-0932-0
    Serological investigation remains the primary approach to achieve satisfactory results in Toxoplasma gondii identification. However, the accuracy of the native antigen used in the current diagnostic kits has proven to be insufficient as well as difficult to standardize, so significant efforts have been made to find alternative reagents as capture antigens. Consequently, multi-epitope peptides are promising diagnostic markers, with the potential for improving the accuracy of diagnostic kits. In this study, we described a simple, inexpensive and improved strategy to acquire such diagnostic markers. The study was aimed at producing novel synthetic protein consisting of multiple immunodominant epitopes of several T. gondii antigens.
    Matched MeSH terms: Serologic Tests/methods*
  11. Fulltext Multi-isotype antibody responses against the multimeric Salmonella Typhi recombinant hemolysin E antigen
    Ong EB, Ignatius J, Anthony AA, Aziah I, Ismail A, Lim TS
    Microbiol. Immunol., 2015 Jan;59(1):43-7.
    PMID: 25399538 DOI: 10.1111/1348-0421.12211
    The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.
    Matched MeSH terms: Serologic Tests/methods
  12. Transcriptome analysis of Burkholderia pseudomallei T6SS identifies Hcp1 as a potential serodiagnostic marker
    Chieng S, Mohamed R, Nathan S
    Microb Pathog, 2015 Feb;79:47-56.
    PMID: 25616255 DOI: 10.1016/j.micpath.2015.01.006
    Burkholderia pseudomallei, the causative agent of melioidosis, is able to survive extreme environments and utilizes various virulence factors for survival and pathogenicity. To compete and survive within these different ecological niches, B. pseudomallei has evolved specialized pathways, including the Type VI secretion systems (T6SSs), that have a role in pathogenesis as well as interbacterial interactions. We examined the expression profile of B. pseudomallei T6SS six gene clusters during infection of U937 macrophage cells. T6SS-5 was robustly transcribed while the other five clusters were not significantly regulated proposing the utility of T6SS-5 as a potential biomarker of exposure to B. pseudomallei. Transcription of T6SS regulators VirAG and BprB was also not significant during infection when compared to bacteria grown in culture. Guided by these findings, three highly expressed T6SS genes, tssJ-4, hcp1 and tssE-5, were expressed as recombinant proteins and screened against melioidosis patient sera by western analysis and ELISA. Only Hcp1 was reactive by both types of analysis. The recombinant Hcp1 protein was further evaluated against a cohort of melioidosis patients (n = 32) and non-melioidosis individuals (n = 20) sera and the data clearly indicates a higher sensitivity (93.7%) and specificity (100%) for Hcp1 compared to bacterial lysate. The detection of anti-Hcp1 antibodies in patients' sera indicating the presence of B. pseudomallei highlights the potential of Hcp1 to be further developed as a serodiagnostic marker for melioidosis.
    Matched MeSH terms: Serologic Tests/methods
  13. Fulltext Immunogenicity of bacterial-expressed recombinant Plasmodium knowlesi merozoite surface protein-142 (MSP-142)
    Cheong FW, Fong MY, Lau YL, Mahmud R
    Malar J, 2013;12:454.
    PMID: 24354660 DOI: 10.1186/1475-2875-12-454
    Plasmodium knowlesi is the fifth Plasmodium species that can infect humans. The Plasmodium merozoite surface protein-1(42) (MSP-1(42)) is a potential candidate for malaria vaccine. However, limited studies have focused on P. knowlesi MSP-1(42).
    Matched MeSH terms: Serologic Tests/methods
  14. Establishment of an immunofluorescence assay to detect IgM antibodies to Nipah virus using HeLa cells expressing recombinant nucleoprotein
    Kaku Y, Park ES, Noguchi A, Inoue S, Lunt R, Malbas FF, et al.
    J Virol Methods, 2019 07;269:83-87.
    PMID: 30954461 DOI: 10.1016/j.jviromet.2019.03.009
    A novel indirect fluorescent antibody test (IFAT) for detection of IgM against Nipah virus (NiV) was developed using HeLa 229 cells expressing recombinant NiV nucleocapsid protein (NiV-N). The NiV IFAT was evaluated using three panels of sera: a) experimentally produced sera from NiV-N-immunized/pre-immunized macaques, b) post-infection human sera associated with a Nipah disease outbreak in the Philippines in 2014, and c) human sera from a non-exposed Malaysian population. Immunized macaque sera showed a characteristic granular staining pattern of the NiV-N expressed antigen in HeLa 229 cells, which was readily distinguished from negative-binding results of the pre-immunized macaque sera. The IgM antibody titers in sequential serum samples (n = 7) obtained from three Nipah patients correlated well with previously published results using conventional IgM capture ELISA and SNT serology. The 90 human serum samples from unexposed persons were unreactive by IFAT. The IFAT utilizing NiV-N-expressing HeLa 229 cells to detect IgM antibody in an early stage of NiV infection is an effective approach, which could be utilized readily in local laboratories to complement other capabilities in NiV-affected countries.
    Matched MeSH terms: Serologic Tests/methods
  15. Limited diagnostic value of two commercial rapid tests for acute leptospirosis detection in Malaysia
    Chang CH, Riazi M, Yunus MH, Osman S, Noordin R
    Diagn Microbiol Infect Dis, 2014 Dec;80(4):278-81.
    PMID: 25241641 DOI: 10.1016/j.diagmicrobio.2014.08.012
    This study evaluated 2 rapid leptospirosis serological tests, Leptorapide® (Linnodee, Northern Ireland) and VISITECT®-LEPTO (Omega Diagnostics, Scotland, UK), which are commonly used in Malaysia. A total of 183 samples comprised 113 sera from leptospirosis patients, and 70 sera from other infections and healthy controls were used. The leptospirosis sera were grouped into 2 serum panels, i.e., Group I (MAT+, PCR+) and Group II (MAT+). When inconclusive results were interpreted as positives, both tests showed lower diagnostic sensitivities (≤ 34%) with Group I sera, as compared to Group II sera (Leptorapide®, 93%; VISITECT®-LEPTO, 40%). When inconclusive results were interpreted as negatives, the 2 tests showed ~20% sensitivity with both serum panels. The diagnostic specificity of VISITECT®-LEPTO (94%) was superior to Leptorapide® (69%). Since both tests had misdiagnosed a large proportion of Group I patients and showed many inconclusive results among Group II patients, they have limited diagnostic value in detecting acute leptospirosis.
    Matched MeSH terms: Serologic Tests/methods*
  16. Expression of recombinant proteins of Orientia tsutsugamushi and their applications in the serodiagnosis of scrub typhus
    Tay ST, Rohani MY, Ho TM, Devi S
    Diagn Microbiol Infect Dis, 2002 Oct;44(2):137-42.
    PMID: 12458119
    In this study, recombinant proteins that encompassed the AD I-AD III regions of 56 kDa immunodominant gene of 2 Orientia tsutsugamushi (OT) serotypes; Gilliam and TA763 were expressed in Escherichia coli. Both recombinant proteins exhibited serologic cross-reactivity with the rabbit antisera against various OT serotypes, as evaluated by enzyme-linked immunosorbent assay (ELISA), but not against other rickettsial species, including Rickettsia typhi, R. prowazekii and TT118 SFG rickettsiae. The feasibility of using the recombinant proteins as a diagnostic reagent was further evaluated by ELISA using sera from blood donors and scrub typhus patients. The results suggested a higher affinity of the antihuman IgM than IgG with both recombinant proteins. The IgM ELISA findings were agreeable with the results of indirect immunoperoxidase (IIP) assay especially with sera of high antibody (1:1600). However, more than one antigen are probably needed for development of an effective assay for serodiagnosis of scrub typhus in endemic areas.
    Matched MeSH terms: Serologic Tests/methods
  17. Preliminary assessment of the diagnostic performances of a new rapid diagnostic test for the serodiagnosis of human cystic echinococcosis
    Vola A, Tamarozzi F, Noordin R, Yunus MH, Khanbabaie S, De Silvestri A, et al.
    Diagn Microbiol Infect Dis, 2018 Sep;92(1):31-33.
    PMID: 29776711 DOI: 10.1016/j.diagmicrobio.2018.04.007
    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.
    Matched MeSH terms: Serologic Tests/methods*
  18. Current status of diagnostic methods for henipavirus
    Tamin A, Rota PA
    Dev Biol (Basel), 2013;135:139-45.
    PMID: 23689891 DOI: 10.1159/000189236
    Hendra virus (HeV) and Nipah virus (NiV) are the causative agents of emerging transboundary animal disease in pigs and horses. They also cause fatal disease in humans. NiV has a case fatality rate of 40 - 100%. In the initial NiV outbreak in Malaysia in 1999, about 1.1 million pigs had to be culled. The economic impact was estimated to be approximately US$450 million. Worldwide, HeV has caused more than 60 deaths in horses with 7 human cases and 4 deaths. Since the initial outbreak, HeV spillovers from Pteropus bats to horses and humans continue. This article presents a brief review on the currently available diagnostic methods for henipavirus infections, including advances achieved since the initial outbreak, and a gap analysis of areas needing improvement.
    Matched MeSH terms: Serologic Tests/methods
  19. Fulltext Production and characterization of a polyclonal antibody of anti-rLipL21-IgG against leptospira for early detection of acute leptospirosis
    Seenichamy A, Bahaman AR, Mutalib AR, Khairani-Bejo S
    Biomed Res Int, 2014;2014:592858.
    PMID: 24860824 DOI: 10.1155/2014/592858
    Leptospirosis is one of the zoonotic diseases in animals and humans throughout the world. LipL21 is one of the important surface-exposed lipoproteins in leptospires and the most effective cross protective immunogenic antigen. It is widely considered as a diagnostic marker for leptospirosis. In this study, we evaluated the serodiagnostic potential of LipL21 protein of Leptospira interrogans serovar Pomona. We have successfully amplified, cloned, and expressed LipL21 in E. coli and evaluated its specificity by immunoblotting. Purified recombinant LipL21 (rLipL21) was inoculated into rabbits for the production of polyclonal antibody. Characterization of the purified IgG antibody against rLipL21 was performed by cross reactivity assay. Only sera from leptospirosis patients and rabbit hyperimmune sera recognized rLipL21 while the nonleptospirosis control sera showed no reaction in immunoblotting. We confirmed that anti-rLipL21-IgG antibody cross reacted with and detected only pathogenic leptospiral species and it did not react with nonpathogenic leptospires and other bacterial species. Results observed showed that anti-rLipL21-IgG antibody has high specificity and sensitivity to leptospires. The findings indicated that the antibody could be used in a diagnostic assay for detection of leptospires or their proteins in the early phase of infection.
    Matched MeSH terms: Serologic Tests/methods
  20. Fulltext Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess
    Saidin S, Yunus MH, Zakaria ND, Razak KA, Huat LB, Othman N, et al.
    BMC Infect Dis, 2014 Apr 04;14:182.
    PMID: 24708664 DOI: 10.1186/1471-2334-14-182
    BACKGROUND: Amoebic liver abscess (ALA) is the most common clinical manifestation of extraintestinal amoebiasis especially in developing countries, causing up to 100 000 fatal cases annually. Accurate and early diagnosis is important to prevent the disease complications, however its diagnosis still poses many challenges due to the limitations of the available detection tools. Pyruvate phosphate dikinase (PPDK), an excretory-secretory protein of E. histolytica, has been reported as a potential diagnostic marker for ALA, hence it may be exploited in the development of a new test for ALA.

    METHODS: Recombinant PPDK (rPPDK) was expressed, purified and evaluated by Western blot. In parallel, recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (Gal/GalNAc lectin) was produced and tested similarly. The protein identity was confirmed by analysis using MALDI-TOF/TOF. A lateral flow dipstick (LFD) test using rPPDK was subsequently developed (rPPDK-LFD) and evaluated for serodiagnosis of ALA.

    RESULTS: rPPDK was expressed as soluble protein after 4 hours of induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C. Purification using nickel-nitrilotriacetic acid (Ni-NTA) resin yielded 1.5 mg of rPPDK from 1 L of culture with estimated molecular mass of 98 kDa on SDS-PAGE. Western blots using sera from patients with ALA, healthy individuals and other diseases probed with anti-human IgG4-HRP showed the highest sensitivity (93.3%) and specificity (100%); as compared to blots using IgG and IgG1 as secondary antibodies. Moreover, rPPDK showed better specificity when compared to rGal/GalNAc lectin. In the development of the LFD test, the optimum amount of rPPDK was 0.625 μg per dipstick and the optimum working concentration of colloidal gold conjugated anti-human IgG4 was optical density (OD) 5 (1.7 μg of anti-human IgG4). Evaluation of rPPDK-LFD using ALA patients and controls serum samples showed 87% diagnostic sensitivity and 100% specificity.

    CONCLUSION: The developed rPPDK-LFD showed good potential for rapid diagnosis of ALA, and merit further multicentre validation using larger number of serum samples.

    Matched MeSH terms: Serologic Tests/methods*
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