Displaying publications 21 - 40 of 226 in total

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  1. Testosterone regulates levels of cystic fibrosis transmembrane regulator, adenylate cyclase, and cAMP in the seminal vesicles of orchidectomized rats
    Ramli NS, Giribabu N, Muniandy S, Salleh N
    Theriogenology, 2016 Jan 15;85(2):238-46.
    PMID: 26483308 DOI: 10.1016/j.theriogenology.2015.09.036
    Secretions of chloride (Cl(-))- and bicarbonate (HCO3(-))-rich fluid by the seminal vesicles could involve cystic fibrosis transmembrane regulator (CFTR), which activity can be stimulated by cAMP generated from the reaction involving adenylate cyclase (AC). In this study, we investigated levels of CFTR, AC, and cAMP in the seminal vesicles under testosterone influence. Orchidectomized adult male rats received 7-day treatment with 125 or 250 μg/kg/day of testosterone with or without flutamide or finasteride. At the end of the treatment, animals were sacrificed and seminal vesicles were harvested for analyses of CFTR and AC protein expression level by Western blotting. Distribution of CFTR and AC in seminal vesicles was observed by immunohistochemistry. Levels of cAMP and dihydrotestosterone in seminal vesicle homogenates were measured by ELISA. Cystic fibrosis transmembrane regulator, AC, and cAMP levels increased with increasing doses of testosterone (P 
    Matched MeSH terms: Blotting, Western
  2. Estrogen, progesterone, and genistein differentially regulate levels of expression of α-, β-, and γ-epithelial sodium channel (ENaC) and α-sodium potassium pump (Na⁺/K⁺-ATPase) in the uteri of sex steroid-deficient rats
    Chinigarzadeh A, Muniandy S, Salleh N
    Theriogenology, 2015 Oct 1;84(6):911-26.
    PMID: 26154487 DOI: 10.1016/j.theriogenology.2015.05.029
    Estrogen, progesterone, and genistein could induce changes in uterine fluid volume and Na(+) concentration. Progesterone upregulates expression of epithelial sodium channel (ENaC) and Na(+)/K(+)-ATPase which contributed toward these changes. However, effects of estrogen and genistein were unknown. This study therefore investigated changes in expression of these proteins in the uterus under estrogen, progesterone, and genistein influences to further understand mechanisms underlying sex steroids and phytoestrogen effects on uterine fluid Na(+) regulation. In this study, uteri of ovariectomized female rats receiving 7-day treatment with genistein (25, 50, and 100 mg/kg/day), estrogen (0.8 × 10(-4) mg/kg/day), or progesterone (4 mg/kg/day) were harvested, and expression levels of α-, β-, and γ-ENaC proteins and messenger RNAs (mRNAs) and α-Na(+)/K(+)-ATPase protein were determined by Western blotting (proteins) and real-time polymerase chain reaction (mRNA). Meanwhile, distribution of α-, β-, and γ-ENaC and α-Na(+)/K(+)-ATPase proteins in the uterus was identified by immunohistochemistry. Our findings indicated that expression of α-, β-, and γ-ENaC proteins and mRNAs and α-Na(+)/K(+)-ATPase protein were enhanced under progesterone influence. Lower expressions were noted under estrogen and genistein influences compared to progesterone. Under estrogen, progesterone, and genistein influences, α- and β-ENaC were distributed at apical membrane and γ-ENaC was distributed at apical and basolateral membranes of uterine luminal epithelia. Under progesterone influence, α-Na(+)/K(+)-ATPase was highly expressed at basolateral membrane. In conclusion, high expression of α-, β-, and γ-ENaC and α-Na(+)/K(+)-ATPase under progesterone influence would contribute toward increased uterine fluid Na(+) reabsorption, whereas lesser expression of these proteins under estrogen and genistein influences would contribute toward lower reabsorption of uterine fluid Na(+).
    Matched MeSH terms: Blotting, Western
  3. Reduced expression of prostacyclin synthase and nitric oxide synthase in subcutaneous arteries of type 2 diabetic patients
    Safiah Mokhtar S, M Vanhoutte P, W S Leung S, Imran Yusof M, Wan Sulaiman WA, Zaharil Mat Saad A, et al.
    Tohoku J. Exp. Med., 2013 11;231(3):217-22.
    PMID: 24225501
    Diabetic endothelial dysfunction is characterized by impaired endothelium-dependent relaxation. In this study, we measured the expression of endothelial nitric oxide synthase (eNOS), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), prostacyclin synthase (PGIS), and prostacyclin receptor (IP) in subcutaneous arteries of type-2 diabetic and non-diabetic patients. Subcutaneous arteries were dissected from tissues from seven diabetics (4 males and 3 females) and seven non-diabetics (5 males and 2 females) aged between 18 to 65 years, who underwent lower limb surgical procedures. Diabetics had higher fasting blood glucose compared to non-diabetics, but there were no differences in blood pressure, body mass index and age. Patients were excluded if they had uncontrolled hypertension, previous myocardial infarction, coronary heart disease, renal or hepatic failure and tumor. The relative expression levels of eNOS, COX-1, COX-2, PGIS and IP receptor were determined by Western blotting analysis, normalized with the β-actin level. Increased expression of COX-2 was observed in subcutaneous arteries of diabetics compared to non-diabetics, whereas the expression levels of eNOS and PGIS were significantly lower in diabetics. There were no significant differences in expression levels of COX-1 and IP receptor between the two groups. Immunohistochemical study of subcutaneous arteries showed that the intensities of eNOS and PGIS staining were lower in diabetics, with higher COX-2 staining. In conclusion, type-2 diabetes is associated with higher COX-2 expression, but lower eNOS and PGIS expression in subcutaneous arteries. These alterations may lead to impaired endothelium-dependent vasodilatation, and thus these proteins may be potential targets for protection against the microvascular complications of diabetes.
    Matched MeSH terms: Blotting, Western
  4. Cloning and expression of Toxoplasma gondii dense granule antigen 2 (GRA2) gene by Pichia pastoris
    Lau YL, Fong MY, Idris MM, Ching XT
    Southeast Asian J. Trop. Med. Public Health, 2012 Jan;43(1):10-6.
    PMID: 23082548
    Detection of Toxoplasma gondii infection is essential in pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several Toxoplasma antigens, including dense granule antigens (GRAs) has high potential as diagnostic reagents. In the present study, we produced GRA2 using Pichia pastoris system. RNA of T. gondii RH strain tachyzoite was used as a template to produce cDNA clones of full-length GRA2 via reverse transcriptase PCR. Amplicons were inserted into pPICZalpha A and the recombinant plasmid transformed into P. pastoris, X-33 strain. The expressed recombinant protein was identified by SDS-PAGE and Western blotting. A recombinant protein of -28 kDa was produced, which could be detected by toxoplasmosis positive human sera indicating that the recombinant protein retained its antigenicity. The present study indicates that P. pastoris-expressed GRA2 should be useful for detection of Toxoplasma infection.
    Matched MeSH terms: Blotting, Western
  5. Optimization for high-level expression in Pichia pastoris and purification of truncated and full length recombinant SAG2 of Toxoplasma gondii for diagnostic use
    Ling LY, Ithoi I, Yik FM
    Southeast Asian J. Trop. Med. Public Health, 2010 May;41(3):507-13.
    PMID: 20578535
    SAG2 is one of the major surface antigens of the intracellular protozoan parasite Toxoplasma gondii. In the present study, truncated recombinant SAG2(S) and full length recombinant SAG2(T) of T. gondii were optimally produced (approximately 15 mg/liter) in Pichia pastoris expression system using BMMY medium at pH 3, 25 degrees C in 0.5-1% methanol and a time-course of 1-2 days. The recombinant proteins were purified using a commercial gel filtration purification system obtaining approximately 33% recovery. The purified SAG2(S) and SAG2(T) showed molecular masses of 45 and 36 kDa by SDS-PAGE, respectively. The recombinant proteins were evaluated by Western blotting with patients' sera and demonstrated 90% sensitivity and 100% specificity for detection of toxoplasmosis. This study provided a means for large-scale expression and purification of SAG2, which should be useful for diagnosis of toxoplasmosis.
    Matched MeSH terms: Blotting, Western
  6. Differential expression of aldolase, alpha tubulin and thioredoxin peroxidase in peripheral blood mononuclear cells from dengue fever and dengue hemorrhagic fever patients
    Thayan R, Huat TL, See LL, Khairullah NS, Yusof R, Devi S
    Southeast Asian J. Trop. Med. Public Health, 2009 Jan;40(1):56-65.
    PMID: 19323035
    We determined the differential expression levels of proteins in peripheral blood mononuclear cells of patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). Proteins were subjected to two-dimensional electrophoresis, mass spectrometry and Western blot analysis. We identified 8 proteins that were 2-fold or more up-regulated in patients compared to healthy control, three of which, aldolase, thioredoxin peroxidase and alpha tubulin, were related to dengue infection. Both thioredoxin peroxidase and alpha tubulin were over-expressed 4.9 and 3.3 times respectively in DHF compared to DF patients while aldolase was up-regulated 2.2 times in DF compared to DHF patients. Alpha tubulin and thioredoxin peroxidase have the potential to be utilized as biomarkers for DHF.
    Matched MeSH terms: Blotting, Western
  7. Cloning and expression of Vibrio cholerae virulence gene, accessory cholera enterotoxin (ace)
    Somarny WM, Mariana NS, Rozita R, Raha AR
    Southeast Asian J. Trop. Med. Public Health, 2004 Dec;35(4):856-62.
    PMID: 15916081
    The cholera enterotoxin (CT) has been considered a major virulence factor of Vibrio cholerae. The accessory cholera enterotoxin (ace) gene is the third gene of V. cholerae virulence cassette. The gene coding for the Ace toxin was amplified from V. cholerae isolates producing a single band of 314 bp. The presence of ace gene was confirmed by hybridization as well as by sequencing. The gene was successfully expressed in Escherichia coli (LMG194) using expression, pBAD/Thio-TOPO vector. Optimal conditions for expression included choice of host strain, temperature used for culturing, and concentration of antibiotic and arabinose inducer. The Ace protein was obtained from the cell supernatant as a fusion protein with a molecular mass 34 kDa which was detected using an anti V5-HRP epitope tagged antibody.
    Matched MeSH terms: Blotting, Western
  8. Borrelia burgdorferi (strain B. afzelii) antibodies among Malaysian blood donors and patients
    Tay ST, Kamalanathan M, Rohani MY
    Southeast Asian J. Trop. Med. Public Health, 2002 Dec;33(4):787-93.
    PMID: 12757227
    In this study, the presence of IgG and IgM antibodies against Borrelia burgdorferi (strain B. afzelii) among Malaysian blood donors and patients admitted to hospital with various infectious diseases was determined. Sera were screened using enzyme-linked immunosorbent assay (ELISA); positive sera were then subjected to Western blot testing. All but one of the blood donors were negative for borrelial antibodies. Of 121 patients' sera, IgM antibodies were detected in 24 (19.8%) and IgG antibodies were detected in 5 (4.1%) sera. Only one of two patients with skin manifestations suggestive of Lyme disease had IgM antibody against B. afzelii. Of 30 patients with exposure to tick typhus, 4 (13.3%) were IgM positive and 1 (3.3%) was IgG positive. Based on the detection of antigenic bands by Western blot, 6 patients' sera showed positive reactions. Antigenic bands of p39, p41 and p59/62 kDa were the commonest findings of Western blotting. This study provides serological evidence of B. afzelii infections in Malaysia; further investigation is needed to correlate serological and clinical findings.
    Matched MeSH terms: Blotting, Western
  9. Fulltext Antibodies to somatic L3 antigen not protective against Brugia malayi infection
    Noordin R, Abdullah KA, Azahri NA, Ramachandran CP
    Southeast Asian J. Trop. Med. Public Health, 1999 Dec;30(4):678-81.
    PMID: 10928359
    Western blot analysis of infective larvae (L3) antigen of Brugia malayi were performed on 200 sera from six groups of individuals: 36 samples from B. malayi microfilaremic individuals; 10 samples from individuals with elephantiasis; 50 and 20 samples from amicrofilaremic individuals in a B. malayi endemic area with no anti-filarial IgG4 antibodies (towards microfilaria and adult worm antigens) and samples with high titres of the anti-filarial IgG4 antibodies respectively; 50 samples from non-endemic normals and 34 samples from geohelminth-infected individuals. After protein transfer, PVDF membrane strips were successively incubated with blocking solution, human sera, monoclonal anti-human IgG4 antibody-HRP and developed with luminol chemiluminescence substrate. 28/36 (78%), 1/10 (10%) and 16/20(80%) of sera from individuals with microfilariae, elephantiasis and amicrofilaremic individuals with high titers of anti-filarial IgG4 antibodies respectively recognized L3 antigenic epitopes; the dominant and consistent antigenic bands were of approximately MW 43 kDa, 14 kDa, 15 kDa and 59 kDa. The rest of the sera were unreactive. This study showed that microfilaremics may or may not mount a notable antibody response to somatic L3 antigens, thus lending evidence that antibody response to this antigen is not protective against establishment of Brugia malayi infection.
    Matched MeSH terms: Blotting, Western
  10. Fulltext Toxoplasmosis in HIV/AIDS patients in Malaysia
    Nissapatorn V, Lee CK, Cho SM, Rohela M, Anuar AK, Quek KF, et al.
    Southeast Asian J. Trop. Med. Public Health, 2003;34 Suppl 2:80-5.
    PMID: 19238664
    Three hundred and one sera of HIV/AIDS patients were tested for anti-Toxoplasma IgG antibody by ELISA technique. The seroprevalence of toxoplasmosis was 41.2% (95% CI: 35.5-46.9) in HIV/AIDS patients. The seroprevalence was significantly higher in the Malay (57.9%) than the Chinese (38.7%), followed by the Indian patients (29.6%) (p<0.05). No possible risk factor, such as contact with cats, consumption of uncooked meat, and history of blood transfusions was found to have any significant association with the presence of anti-Toxoplasma antibody in the study sample (p>0.05). Multivariate analysis was employed to find any association between Toxoplasma seroprevalence and a single subject having single or multiple risk factors. It was found that the association was not statistically significant (p>0.05). Among the HIV/AIDS study samples, 124 (41.2%) samples were found to have positive anti-Toxoplasma antibody, the association between the presence of anti-Toxoplasma antibody and CD4 cell count was determined but no statistically significant association was found (p>0.05). During the study period, only one case of active CNS toxoplasmosis was registered and the diagnostic criteria included: clinical presentations, CT scan finding, serological evidence of anti-Toxoplasma IgG antibody, and respose to anti-Toxoplasma therapy.
    Matched MeSH terms: Blotting, Western
  11. Fulltext Serological evaluation of malaria patients in Thailand: antibody response against electrophoresed antigenic polypeptides of Plasmodium falciparum
    Kano S, Onda T, Matsumoto Y, Buchachart K, Krudsood S, Looareesuwan S, et al.
    Southeast Asian J. Trop. Med. Public Health, 1998 Jun;29(2):341-3.
    PMID: 9886125
    It was reported that a 47kDa antigenic polypeptide of Plasmodium falciparum had been strongly presented by the sera from 1) imported Japanese malaria patients with severe symptoms and 2) symptomatic and parasitemic inhabitants in endemic areas in the Sudan, Malaysia and the Philippines. In the present study, we observed the reactivity of the sera from falciparum malaria patients who had been hospitalized in the Bangkok Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, and compared the antibody response against the 47kDa antigenic polypeptide according to the severity of the patients. It was observed that antibodies to this molecule were more commonly shared in sera from severer patients, although the IFAT titers against the whole P. falciparum parasite antigen were lower in the group, which suggested that this antibody against the 47kDa molecule was playing a specific role at a severe stage of the infection. Determination of the immunological features of the antigenic molecules of parasites by this type of sero-epidemiological study will provide a new assay system for evaluation of immune status of individuals in different severity and suggest a way of vaccine development.
    Matched MeSH terms: Blotting, Western
  12. HTLV-I antibody study in normal individuals and unselected hospital patients in Malaysia
    Yap SF, Peh SC, Chan L, Wong HC, How VJ, Looi LM
    Southeast Asian J. Trop. Med. Public Health, 1992 Mar;23(1):26-9.
    PMID: 1355930
    A serological investigation for human T cell leukemia virus I (HTLV-I) infection was carried out at the University Hospital, Kuala Lumpur. A total of 626 sera from a non-patient population and 1,038 sera from unselected in-patients were screened for HTLV-I antibodies using an enzyme-linked immunosorbent assay (ELISA). 27/1664 (1.6%) were found to be reactive. However, on Western blotting, only 2 sera were confirmed positive, both showing reactions for the major core (p19 and p24) and the envelope (gp46) proteins. Both of the serum samples were from unselected hospital patients. Most of the remaining sera which were reactive on screening showed indeterminate results on Western blotting. These were further tested by radioimmunoprecipitation assay (RIPA) and none of these sera gave a positive reaction. Therefore, only 2/1038 (0.19%) unselected patients could be confirmed to have antibodies to HTLV-I. None of the normal individuals screened showed a positive Western blot result. Our data indicate that HTLV-I infection is present in our population, but at a low prevalence rate.
    Matched MeSH terms: Blotting, Western/standards
  13. Cloning and expression of malaria and tuberculosis epitopes in mycobacterium bovis bacille calmette-guérin
    Suppian R, Zainuddin ZF, Norazmi MN
    Malays J Med Sci, 2006 Jan;13(1):13-20.
    PMID: 22589585
    Mycobacterium bovis bacille Calmette-Guèrin (BCG) represents one of the most promising live vectors for the delivery of foreign antigens to the immune system. A recombinant BCG containing a synthetic gene coding for the malarial epitopes namely, the fragment 2 of region II of EBA-175 (F2R(II)EBA) and the repeat sequence of the circumsporozoite protein NANP generated in favour of mycobacterium codon usage using assembly PCR was constructed. Two T-cell epitopes of the 6-kDa M. tuberculosis early-secreted antigenic target (ESAT-6) antigen were also clone in the same construct. Expression of the synthetic gene was driven by the heat shock protein 65 (hsp65) promoter from M. tuberculosis and the signal peptide from the MPT63 antigen of M. tuberculosis. Expression of the composite epitopes was detected by Western blotting of the cell extract and culture supernatant of the recombinant clones using a specific rabbit polyclonal antibody against F2R(II)EBA. This study demonstrates the possibility of cloning and expressing immunogenic epitopes from causative agents of two important diseases: malaria and tuberculosis (TB) in a single recombinant BCG construct.
    Matched MeSH terms: Blotting, Western
  14. Fulltext Seroprevalence of Sarcocystis falcatula in Two Islands of Malaysia using Recombinant Surface Antigen 4
    Nadzirah TTI, Yik FM, Ling LY
    Korean J Parasitol, 2020 Feb;58(1):1-5.
    PMID: 32145721 DOI: 10.3347/kjp.2020.58.1.1
    Sarcocystosis was diagnosed worldwide by serodiagnostic tests utilising the whole parasite, for which the protozoa were maintained in vitro are more costly. In this study, antigenicity of Sarcocystis falcatula recombinant protein (rSfSAG4) was investigated towards the local communities of Pangkor and Tioman Islands and its seroprevalence was surveyed in these islands. A total of 348 human sera were tested using rSfSAG4 by Western blot and ELISA. High prevalence of sarcocystosis was observed in Tioman Island (80.6%) than in Pangkor Island (50.0%) by Western blot. In ELISA, the seroprevalence observed in Tioman Island was 45.9%, whereas in Pangkor Island 63.0%. In other parasitic infections, the prevalence was 34.0% by Western blot and 46.0% by ELISA. In healthy control group, 7% by Western blot and 8% by ELISA showed positivity to rSfSAG4. It is suggested SfSAG4 is a candidate antigen to measure seroprevalence of sarcocystosis.
    Matched MeSH terms: Blotting, Western
  15. Effect of antisense oligodeoxynucleotides for ICAM-1 on renal ischaemia-reperfusion injury in the anaesthetised rat
    Kiew LV, Munavvar AS, Law CH, Azizan AN, Nazarina AR, Sidik K, et al.
    J Physiol, 2004 Jun 15;557(Pt 3):981-9.
    PMID: 15047774
    An antisense oligodeoxynucleotide (As-ODN) to the 3' untranslated region of the mRNA sequence expressing the intracellular adhesion molecule-1 (ICAM-1) was employed to determine ICAM-1's role in renal ischaemia-reperfusion injury in the rat. Wistar-Kyoto rats receiving i.v. either lipofectin-As-ODN (As-ODN group), lipofectin-reverse ODN (Rv-ODN group) or lipofectin (ischaemia control group) 8 h prior to study were anaesthetized and subjected to 30 min of renal artery occlusion. Renal haemodynamic and excretory parameters were monitored before and after renal ischaemia. On termination of the study renal tissue was subjected to histological and Western blot analysis. Renal blood flow decreased in the 3 h post-ischaemia period in the ischaemia control and Rv-ODN groups, but was maintained in the As-ODN group. Glomerular filtration rate was depressed initially but gradually increased to 10% above basal levels in the ischaemia control and Rv-ODN groups, but was below basal levels (20%) in the As-ODN group. There was a three- to fourfold increase in sodium and water excretion following ischaemia in the ischaemia control and reverse-ODN groups but not in the As-ODN treated group. The As-ODN ameliorated the histological evidence of ischaemic damage and reduced ICAM-1 protein levels to a greater extent in the medulla than cortex. These observations suggested that in the post-ischaemic period afferent and efferent arteriolar tone was increased with a loss of reabsorptive capacity which was in part due to ICAM-1. The possibility arises that the action of ICAM-1 at vascular and tubular sites in the deeper regions of the kidney contributes to the ischaemia-reperfusion injury.
    Matched MeSH terms: Blotting, Western
  16. Isolation and characterization of the early nodule-specific protein homologue (Hev b 13), an allergenic lipolytic esterase from Hevea brasiliensis latex
    Arif SA, Hamilton RG, Yusof F, Chew NP, Loke YH, Nimkar S, et al.
    J Biol Chem, 2004 Jun 04;279(23):23933-41.
    PMID: 15024009
    Recurring reports of a highly allergenic 42-46-kDa protein in Hevea brasiliensis latex appeared to have been resolved with the discovery of a 43-kDa allergenic latex protein that was a homologue to patatin. However, the low to moderate prevalence of sensitization to the protein, designated Hev b 7, among latex-allergic patients could not adequately explain the frequent observations of the 42-46-kDa allergen. This led to the hypothesis that another, more allergenic protein of a similar molecular mass existed in Hevea latex. We report the isolation and purification of a 42.98-kDa latex glycoprotein showing homology to the early nodule-specific protein (ENSP) of the legumes Medicago sativa, Medicago truncatula, and Glycine max. The protein is allergenic, being recognized by immunoglobulin E (IgE) in sera from latex-allergic patients. The IgE epitope resides on the carbohydrate moiety of the protein, and the presence of a similar carbohydrate component on potato tuber patatin enables the latter to inhibit IgE binding to the ENSP homologue. The cDNA encoding the ENSP homologue was isolated by reverse transcription-PCR and cloned. The protein predicted from the cDNA sequence has 391 amino acids, the first 26 of which constitute a putative signal peptide. The deduced molecular mass of the mature protein is 40.40 kDa, while its isoelectric point is estimated at 5.0. The discrepancy between the predicted and observed molecular mass might be due to glycosylation, for which three N-sites on the protein are predicted. The purified protein showed lipase and esterase activities and may be involved in plant defense.
    Matched MeSH terms: Blotting, Western
  17. Diagnosis of natural rubber latex allergy: multicenter latex skin testing efficacy study. Multicenter Latex Skin Testing Study Task Force
    Hamilton RG, Adkinson NF
    J Allergy Clin Immunol, 1998 Sep;102(3):482-90.
    PMID: 9768592
    BACKGROUND: No characterized diagnostic natural rubber latex skin testing material is licensed for use in the United States.

    OBJECTIVE: We have conducted a multicenter clinical skin testing study to document the safety and diagnostic sensitivity and specificity of a candidate Hevea brasiliensis nonammoniated latex (NAL) extract. These data are intended to support the licensing of this reagent for the diagnosis of latex allergy in high-risk populations.

    METHODS: Three hundred twenty-four subjects (304 adults and 20 children) were classified by their clinical history as having latex allergy (LA group, 124 adults and 10 children) or having no latex allergy (NLA group, 180 adults and 10 children). All subjects provided blood samples and then received sequential puncture skin tests (PSTs) at 1, 100, or 1000 microg/mL protein with a bifurcated needle and NAL (Greer Laboratories) from Malaysian Hevea brasiliensis (clone 600) sap. A 2-stage glove provocation test was used to clarify latex allergy status of individuals with positive history/negative PST result and negative history/positive PST result mismatches.

    RESULTS: Twenty-four subjects (15%) originally designated as having LA on the basis of their initial clinical history were reclassified to the NLA group on the basis of a negative glove provocation test result. Of the 134 subjects with LA, 54 (40%) were highly sensitive to latex, with a positive PST result at 1 microg/mL NAL. The Greer NAL reagent produced a positive PST rate (sensitivity) of 95% and 99% in subjects with LA at 100 microg/mL and 1 mg/mL, respectively. The negative PST rate (specificity) in 190 subjects with a negative history with the NAL extract at 100 microg/mL and 1 mg/mL, was 100% and 96%, respectively. Immediately after the PST, mild systemic reactions (mainly pruritus) were recorded in 16.1 % of the adults in the LA group and 4.4% of the adults in the NLA group. No reactions required treatment with epinephrine. Only mild delayed reactions were observed in 9.6% (LA group) and 2.8% (NLA group) of subjects 24 to 48 hours after PST. Mean wheal and erythema diameters measured in the 10 children in the LA group with spina bifida at 100 microg/mL and 1 mg/mL were similar to those observed in the adults in the LA group, suggesting that children are not at increased risk for systemic reactions compared with adults.

    CONCLUSIONS: A suggestive clinical history is necessary but not sufficient for a definitive diagnosis of IgE-dependent latex allergy. These data support the safety and diagnostic efficacy of the Greer NAL, skin test reagent at 100 micro/mL and 1 mg/mL for confirmatory PSTs.

    Matched MeSH terms: Blotting, Western
  18. Fulltext Evaluation of recombinant Plasmodium knowlesi merozoite surface protein-1(33) for detection of human malaria
    Cheong FW, Lau YL, Fong MY, Mahmud R
    Am J Trop Med Hyg, 2013 May;88(5):835-40.
    PMID: 23509118 DOI: 10.4269/ajtmh.12-0250
    Plasmodium knowlesi is now known as the fifth Plasmodium species that can cause human malaria. The Plasmodium merozoite surface protein (MSP) has been reported to be potential target for vaccination and diagnosis of malaria. MSP-1(33) has been shown to be immunogenic and its T cell epitopes could mediate cellular immune protection. However, limited studies have focused on P. knowlesi MSP-133. In this study, an approximately 28-kDa recombinant P. knowlesi MSP-1(33) (pkMSP-1(33)) was expressed by using an Escherichia coli system. The purified pkMSP-1(33) reacted with serum samples of patients infected with P. knowlesi (31 of 31, 100%) and non-P. knowlesi malaria (27 of 28, 96.43%) by Western blotting. The pkMSP-1(33) also reacted with P. knowlesi (25 of 31, 80.65%) and non-P. knowlesi malaria sera (20 of 28, 71.43%) in an enzyme-linked immunosorbent assay (ELISA). Most of the non-malarial infection (49 of 52 in by Western blotting and 46 of 52 in the ELISA) and healthy donor serum samples (65 of 65 by Western blotting and ELISA) did not react with recombinant pkMSP-1(33).
    Matched MeSH terms: Blotting, Western
  19. Fulltext Evaluation of Pichia pastoris-expressed recombinant rhoptry protein 2 of Toxoplasma gondii for its application in diagnosis of toxoplasmosis
    Chang PY, Fong MY, Nissapatorn V, Lau YL
    Am J Trop Med Hyg, 2011 Sep;85(3):485-9.
    PMID: 21896809 DOI: 10.4269/ajtmh.2011.11-0351
    Rhoptry protein 2 (ROP2) of Toxoplasma gondii is a rhoptry-secreted protein that plays a critical role in parasitophorous vacuole membrane formation during invasion. In previous studies, ROP2 has been shown to be efficient in triggering humoral and cell-mediated responses. High immunogenicity of ROP2 makes it a potential candidate for diagnosis and vaccination against toxoplasmosis. In this study, the ROP2 gene was cloned into pPICZα A expression vector and extracellularly expressed in the yeast Pichia pastoris, which has numerous advantages over other expression systems for eukaryotic proteins expression. The effectiveness of the secreted recombinant ROP2 as a diagnosis agent was assessed by Western Blot with 200 human serum samples. Recombinant ROP2 reacted with toxoplasmosis-positive human serum samples and yielded an overall sensitivity of 90% and specificity of 95%. However, recombinant ROP2 is a better marker for detection of IgG (91.7%) rather than IgM (80%).
    Matched MeSH terms: Blotting, Western/methods
  20. Mature Erythrocyte Surface Antigen Protein Identified in the Serum of Plasmodium falciparum-Infected Patients
    Zainudin NS, Othman N, Muhi J, Abdu Sani AA, Noordin R
    Am J Trop Med Hyg, 2015 Dec;93(6):1268-73.
    PMID: 26392156 DOI: 10.4269/ajtmh.15-0333
    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.
    Matched MeSH terms: Blotting, Western
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