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Displaying publications 41 - 60 of 72 in total

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Eurycomanone suppresses expression of lung cancer cell tumor markers, prohibitin, annexin 1 and endoplasmic reticulum protein 28

Wong PF, Cheong WF, Shu MH, Teh CH, Chan KL, AbuBakar S

Phytomedicine, 2012 Jan 15;19(2):138-44.
PMID: 21903368 DOI: 10.1016/j.phymed.2011.07.001

Bioactive compounds from the medicinal plant, Eurycoma longifolia Jack have been shown to promote anti-proliferative effects on various cancer cell lines. Here we examined the effects of purified eurycomanone, a quassinoid found in Eurycoma longifolia Jack extract, on the expression of selected genes of the A549 lung cancer cells. Eurycomanone inhibited A549 lung cancer cell proliferation in a dose-dependent manner at concentrations ranging from 5 to 20 μg/ml. The concentration that inhibited 50% of cell growth (GI(50)) was 5.1 μg/ml. The anti-proliferative effects were not fully reversible following the removal of eurycomanone, in which 30% of cell inhibition still remained (p<0.0001, T-test). At 8 μg/ml (GI(70)), eurycomanone suppressed anchorage-independent growth of A549 cells by >25% (p<0.05, T-test, n=8) as determined using soft agar colony formation assay. Cisplatin, a chemotherapy drug used for the treatment of non small cell lung cancer on the other hand, inhibited A549 cells proliferation at concentrations ranging from 0.2 μg/ml to 15 μg/ml with a GI(50) of 0.58 μg/ml. The treatment with eurycomanone reduced the abundance expression of the lung cancer markers, heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1, p53 tumor suppressor protein and other cancer-associated genes including prohibitin (PHB), annexin 1 (ANX1) and endoplasmic reticulum protein 28 (ERp28) but not the house keeping genes. The mRNA expressions of all genes with the exception of PHB were significantly downregulated, 72 h after treatment (p<0.05, T-test, n=9). These findings suggest that eurycomanone at viable therapeutic concentrations of 5-20 μg/ml exhibited significant anti-proliferative and anti-clonogenic cell growth effects on A549 lung cancer cells. The treatment also resulted in suppression of the lung cancer cell tumor markers and several known cancer cell growth-associated genes.

Matched MeSH terms: Immunoblotting
Chemical-Induced Inhibition of Blue Light-Mediated Seedling Development Caused by Disruption of Upstream Signal Transduction Involving Cryptochromes in Arabidopsis thaliana

Ong WD, Okubo-Kurihara E, Kurihara Y, Shimada S, Makita Y, Kawashima M, et al.

Plant Cell Physiol, 2017 01 01;58(1):95-105.
PMID: 28011868 DOI: 10.1093/pcp/pcw181

Plants have a remarkable ability to perceive and respond to various wavelengths of light and initiate regulation of different cascades of light signaling and molecular components. While the perception of red light and the mechanisms of its signaling involving phytochromes are largely known, knowledge of the mechanisms of blue light signaling is still limited. Chemical genetics involves the use of diverse small active or synthetic molecules to evaluate biological processes. By combining chemicals and analyzing the effects they have on plant morphology, we identified a chemical, 3-bromo-7-nitroindazole (3B7N), that promotes hypocotyl elongation of wild-type Arabidopsis only under continuous blue light. Further evaluation with loss-of-function mutants confirmed that 3B7N inhibits photomorphogenesis through cryptochrome-mediated light signaling. Microarray analysis demonstrated that the effect of 3B7N treatment on gene expression in cry1cry2 is considerably smaller than that in the wild type, indicating that 3B7N specifically interrupts cryptochrome function in the control of seedling development in a light-dependent manner. We demonstrated that 3B7N directly binds to CRY1 protein using an in vitro binding assay. These results suggest that 3B7N is a novel chemical that directly inhibits plant cryptochrome function by physical binding. The application of 3B7N can be used on other plants to study further the blue light mechanism and the genetic control of cryptochromes in the growth and development of plant species.

Matched MeSH terms: Immunoblotting
Magnoflorine Enhances LPS-Activated Pro-Inflammatory Responses via MyD88-Dependent Pathways in U937 Macrophages

Haque MA, Jantan I, Harikrishnan H, Abdul Wahab SM

Planta Med, 2018 Nov;84(17):1255-1264.
PMID: 29906814 DOI: 10.1055/a-0637-9936

Magnoflorine, a major bioactive metabolite isolated from Tinospora crispa, has been reported for its diverse biochemical and pharmacological properties. However, there is little report on its underlying mechanisms of action on immune responses, particularly on macrophage activation. In this study, we aimed to investigate the effects of magnoflorine, isolated from T. crispa on the pro-inflammatory mediators generation induced by LPS and the concomitant NF-κB, MAPKs, and PI3K-Akt signaling pathways in U937 macrophages. Differentiated U937 macrophages were treated with magnoflorine and the release of pro-inflammatory mediators was evaluated through ELISA, while the relative mRNA expression of the respective mediators was quantified through qRT-PCR. Correspondingly, western blotting was executed to observe the modulatory effects of magnoflorine on the expression of various markers related to NF-κB, MAPK and PI3K-Akt signaling activation in LPS-primed U937 macrophages. Magnoflorine significantly enhanced the upregulation of TNF-α, IL-1β, and PGE2 production as well as COX-2 protein expression. Successively, magnoflorine prompted the mRNA transcription level of these pro-inflammatory mediators. Magnoflorine enhanced the NF-κB activation by prompting p65, IκBα, and IKKα/β phosphorylation as well as IκBα degradation. Besides, magnoflorine treatments concentration-dependently augmented the phosphorylation of JNK, ERK, and p38 MAPKs as well as Akt. The immunoaugmenting effects were further confirmed by investigating the effects of magnoflorine on specific inhibitors, where the treatment with specific inhibitors of NF-κB, MAPKs, and PI3K-Akt proficiently blocked the magnoflorine-triggered TNF-α release and COX-2 expression. Magnoflorine furthermore enhanced the MyD88 and TLR4 upregulation. The results suggest that magnoflorine has high potential on augmenting immune responses.

Matched MeSH terms: Immunoblotting
Fulltext Highly pathogenic avian influenza virus nucleoprotein interacts with TREX complex adaptor protein Aly/REF

Balasubramaniam VR, Hong Wai T, Ario Tejo B, Omar AR, Syed Hassan S

PLoS One, 2013;8(9):e72429.
PMID: 24073193 DOI: 10.1371/journal.pone.0072429

We constructed a novel chicken (Gallus gallus) lung cDNA library fused inside yeast acting domain vector (pGADT7). Using yeast two-hybrid screening with highly pathogenic avian influenza (HPAI) nucleoprotein (NP) from the strain (A/chicken/Malaysia/5858/2004(H5N1)) as bait, and the Gallus gallus lung cDNA library as prey, a novel interaction between the Gallus gallus cellular RNA export adaptor protein Aly/REF and the viral NP was identified. This interaction was confirmed and validated with mammalian two hybrid studies and co-immunoprecipitation assay. Cellular localization studies using confocal microscopy showed that NP and Aly/REF co-localize primarily in the nucleus. Further investigations by mammalian two hybrid studies into the binding of NP of other subtypes of influenza virus such as the swine A/New Jersey/1976/H1N1 and pandemic A/Malaysia/854/2009(H1N1) to human Aly/REF, also showed that the NP of these viruses interacts with human Aly/REF. Our findings are also supported by docking studies which showed tight and favorable binding between H5N1 NP and human Aly/REF, using crystal structures from Protein Data Bank. siRNA knockdown of Aly/REF had little effect on the export of HPAI NP and other viral RNA as it showed no significant reduction in virus titer. However, UAP56, another component of the TREX complex, which recruits Aly/REF to mRNA was found to interact even better with H5N1 NP through molecular docking studies. Both these proteins also co-localizes in the nucleus at early infection similar to Aly/REF. Intriguingly, knockdown of UAP56 in A549 infected cells shows significant reduction in viral titer (close to 10 fold reduction). Conclusively, our study have opened new avenues for research of other cellular RNA export adaptors crucial in aiding viral RNA export such as the SRSF3, 9G8 and ASF/SF2 that may play role in influenza virus RNA nucleocytoplasmic transport.

Matched MeSH terms: Immunoblotting
Fulltext Scalable Production of Recombinant Membrane Active Peptides and Its Potential as a Complementary Adjunct to Conventional Chemotherapeutics

Rothan HA, Ambikabothy J, Abdulrahman AY, Bahrani H, Golpich M, Amini E, et al.

PLoS One, 2015;10(9):e0139248.
PMID: 26418816 DOI: 10.1371/journal.pone.0139248

The production of short anticancer peptides in recombinant form is an alternative method for costly chemical manufacturing. However, the limitations of host toxicity, bioactivity and column purification have impaired production in mass quantities. In this study, short cationic peptides were produced in aggregated inclusion bodies by double fusion with a central protein that has anti-cancer activity. The anticancer peptides Tachiplicin I (TACH) and Latarcin 1 (LATA) were fused with the N- and C-terminus of the MAP30 protein, respectively. We successfully produced the recombinant TACH-MAP30-LATA protein and MAP30 alone in E. coli that represented 59% and 68% of the inclusion bodies. The purified form of the inclusion bodies was prepared by eliminating host cell proteins through multiple washing steps and semi-solubilization in alkaline buffer. The purified active protein was recovered by inclusive solubilization at pH 12.5 in the presence of 2 M urea and refolded in alkaline buffer containing oxides and reduced glutathione. The peptide-fusion protein showed lower CC50 values against cancer cells (HepG2, 0.35±0.1 μM and MCF-7, 0.58±0.1 μM) compared with normal cells (WRL68, 1.83±0.2 μM and ARPE19, 2.5±0.1 μM) with outstanding activity compared with its individual components. The presence of the short peptides facilitated the entry of the peptide fusion protein into cancer cells (1.8 to 2.2-fold) compared with MAP30 alone through direct interaction with the cell membrane. The cancer chemotherapy agent doxorubicin showed higher efficiency and selectivity against cancer cells in combination with the peptide- fusion protein. This study provides new data on the mass production of short anticancer peptides as inclusion bodies in E. coli by fusion with a central protein that has similar activity. The product was biologically active against cancer cells compared with normal cells and enhanced the activity and selective delivery of an anticancer chemotherapy agent.

Matched MeSH terms: Immunoblotting
Fulltext Effects of Amyloid Precursor Protein Overexpression on NF-κB, Rho-GTPase and Pro-Apoptosis Bcl-2 Pathways in Neuronal Cells

Chan HH, Leong YQ, Voon SM, Pan ML, Leong CO, Lim CL, et al.

Rep Biochem Mol Biol, 2021 Jan;9(4):417-425.
PMID: 33969135 DOI: 10.52547/rbmb.9.4.417

Background: Alzheimer's disease (AD) is a neurodegenerative disorder that causes cognitive dysfunction. Previous studies have suggested that amyloid plaques, mainly comprising of amyloid-beta peptides, play a pivotal role in AD pathophysiology. This study focuses on the evaluation of the effects of amyloid precursor protein (APP) overexpression on NF-κB, Rho-GTPase and Bcl-2 mediated pro-apoptotic pathways in neuronal cells.
Methods: A lentiviral transduction system was used to generate SH-SY5Y cells overexpressing APP. Immunoblotting was conducted to determine expression levels of NF-κB, Rho-GTPase, and Bcl-2 family proteins in the APP overexpressed cells.
Results: In the NF-κB signaling pathway, APP-overexpressing SH-SY5Y cells showed that there was a reduction of p-NF-κB (p< 0.05) and IKKα. Subsequently, there was upregulation of protein expression of NF-Κb, IKKβ and IκBα. On the other hand, protein expression of RhoC (p< 0.05) and Rac1/2/3 was upregulated as compared to the control group. Meanwhile, a decrease in RhoA, Cdc42 (p< 0.05) and p-Rac1/cdc42 protein levels was observed in the APP-overexpressed group. Lastly, in the pro-apoptotic pathway, the expression of Bcl-2, Bid, Bok and Puma (p< 0.05) was up regulated in the APP-overexpressed group. Downregulation of Bad and Bim expression was observed in the APP-overexpressed as compared to the control group, and Bax expression remained unchanged in the APP-overexpressed group.
Conclusion: APP overexpression regulated signaling in the NF-κB, Rho-GTPase and Bcl-2 family pathways in neuronal cells, suggesting that these are involved in promoting neuronal survival and modulating synaptic plasticity in AD. However, further studies are essential to elucidate the APP-mediated mechanism of action.

Matched MeSH terms: Immunoblotting
Evaluation of bovine antibody responses to haemorrhagic septicaemia vaccine

Johnson RB, Dawkins HJ, Spencer TL, Saharee AA, Bahaman AR, Ramdani, et al.

Res Vet Sci, 1989 Sep;47(2):277-9.
PMID: 2508206

ELISA and immunoblotting techniques were used to examine the humoral immune response to Pasteurella multocida, in bovine sera from Indonesia and Malaysia. Elevated levels of antibody to a crude lipopolysaccharide preparation were found in vaccinated animals. In addition to the response to lipopolysaccharide, antibodies from the vaccinated cattle strongly labelled five to six of the 40 protein bands in this organism.

Matched MeSH terms: Immunoblotting
Frequency and role of low molecular weight IgM in systemic lupus erythematosus. Study of patients from different ethnic origins

Roberts-Thomson PJ, Shepherd K, Bradley J, Boey ML

Rheumatol Int, 1990;10(3):95-8.
PMID: 2392640

Low molecular weight IgM (LMW IgM) is the monomeric subunit of the naturally occurring pentameric IgM. It is not seen in health but has been previously observed in systemic lupus erythematosus (SLE) particularly in those patients with active disease and may reflect an adverse prognostic finding. We have therefore studied the presence of LMW IgM in 33 Chinese or Malay SLE patients (Singapore) and 21 Caucasian patients (Adelaide). LMW IgM was measured using filtration chromatography or by a sensitive immunoblotting technique. LMW IgM was observed in all patients in the Adelaide group and in 32 patients in the Singapore group with slightly greater quantities being seen in the Adelaide group. LMW IgM constituted up to 15.3% of the total IgM and was frequently associated with the presence of other low molecular weight IgM oligomers. In both groups LMW IgM correlated significantly with the total IgM levels (P less than 0.01). In a more detailed study in the Singapore group LMW IgM also correlated significantly with the IgM anticardiolipin levels (P = 0.02) but not with IgG anticardiolipin or with IgG or IgM anti-DNA levels or with rheumatoid factor. Patients with more extensive organ involvement had higher levels of LMW IgM but not at a significant level. We conclude that circulating LMW IgM occurs almost universally in SLE, is closely related to the total IgM levels and appears independent of ethnic background. The significance of LMW IgM in this disorder is unclear.

Matched MeSH terms: Immunoblotting
Penyaringan domain ADP-ribosilasi gen toksin daripada Burkholderia pseudomallei virulen

Shahrul Hisham Zainal Ariffin, Rahmah Mohamed, Zulkeflie Zamrod, Mohammad Noor Embi

Sains Malaysiana, 2008;37.

Bahagian aktif bagi enzim toksin bakteria daripada Burkholderia pseudomallei, Pseudomonas aeruginosa dan difteria merupakan domain ADP-ribosilasi. Domain ini didapati terpelihara di antara ketiga-tiga mikroorganisme. Di dalam kajian ini, domain ADP-ribosilasi Burkholderia pseudomallei telah diamplifikasi daripada genom B. pseudomallei virulen dengan menggunakan pencetus-pencetus yang dibina berdasarkan kepada jujukan domain ADP ribosilasi Pseudomonas aeruginosa. Hasil DNA amplifikasi ditulenkan dan digunakan sebagai prob (HPCR2) untuk menyaring DNA selitan daripada B. pseudomallei yang diklonkan ke dalam vektor pengekspresan pSport-I. Objektif kajian ini adalah untuk menyaring lapan klon yang positif hasil daripada penyaringan awal melalui pendekatan immunoblot menggunakan antitoksin daripada arnab. Penyaringan ini juga melibatkan tiga klon yang tidak memberikan isyarat positif semasa penyaringan secara immunoblot. Keputusan menunjukkan hanya satu klon (L31) daripada lapan klon immunoblot positif mempunyai domain ADP-ribosilasi. Penjujukan DNA separa klon L31 secara manual melibatkan dua pencetus menghasilkan jujukan sepanjang 450pb. Analisis selanjutnya mendapati daripada enam kemungkinan translasi kepada polipeptida hanya satu polipeptida wujud yang tidak mempunyai sebarang kodon penamat pada jujukan kodonnya.

Matched MeSH terms: Immunoblotting
Fulltext Characterization of major allergens of local mud crab (Scylla serrata)

Rosmilah Misnan, Nurul Izzah Abdul Rahman, Zailatul Hani Mohd Yadzir, Noormalin Abdullah, Mohd Faizal Bakhtiar, Shahnaz Murad

Scientific Research Journal, 2015;12(1):1-7.
MyJurnal

Crab meat is widely consumed in several countries around the world. However, when consumed, crab meat are frequent cause of allergic reactions throughout the world. Scylla serrata is among the most common mud crab in Malaysia. In a previous study two major allergens of mud crab at 36 and 41 kDa was identified. Thus, the aim of this study is to further identify these major allergens by a proteomic approach. Protein extract was prepared and resolved by 2-dimensional electrophoresis (2-DE). Immunoblotting was then performed using reactive sera from patients with crab allergy. Major allergenic spots were then excised from the 2-DE gel and analysed by mass spectrometry. The 2-DE profile of the extract revealed approximately >100 protein spots between pH of 4.00 to 8.00. Mass spectrometry analysis has identified the 36 and 41 kDa proteins as tropomyosin and arginine kinase, respectively. Our findings indicated that tropomyosin and arginine kinase play a major role in allergic reaction to mud crab meat among local patients with crab meat allergy, and should be included in diagnostics and therapeutic strategies of this allergy.

Matched MeSH terms: Immunoblotting
Fulltext IgE-binding residues analysis of the house dust mite allergen Der p 23

Pang SL, Matta SA, Sio YY, Ng YT, Say YH, Ng CL, et al.

Sci Rep, 2021 01 13;11(1):921.
PMID: 33441720 DOI: 10.1038/s41598-020-79820-y

House dust mites (HDMs) are one of the major causes of allergies in the world. The group 23 allergen, Der p 23, from Dermatophagoides pteronyssinus, is a major allergen amongst HDM-sensitized individuals. This study aims to determine the specific immunoglobulin E (sIgE) binding frequency and IgE-binding residues of recombinant Der p 23 (rDer p 23) allergen amongst a cohort of consecutive atopic individuals in a tropical region. We performed site-directed mutagenesis and carried out immuno-dot blot assays using 65 atopic sera. The immuno-dot blot assays results indicated that the two residues K44 and E46 which are located at the N-terminal region are the major IgE-binding residues. The rDerp-23 sIgE titers are strongly correlated to the number of IgE-binding residues for rDer p 23 (P

Matched MeSH terms: Immunoblotting
Effectiveness of a dot-blot immunoassay of anti-Rickettsia tsutsugamushi antibodies for serologic analysis of scrub typhus

Weddle JR, Chan TC, Thompson K, Paxton H, Kelly DJ, Dasch G, et al.

Am J Trop Med Hyg, 1995 Jul;53(1):43-6.
PMID: 7625532

We compared a commercially available dot-blot immunoassay system with the indirect immunofluorescence assay (IFA) in tests of known negative and known positive sera from scrub typhus cases. Using a panel of 100 sera from patients with various rickettsial and nonrickettsial infections, we observed that the IFA was 99% specific and the dipstick assay was 98% specific. In tests of 91 sera (30 negative and 61 positive for scrub typhus antibodies) from a study of febrile patients in Malaysia, using the standard of an IFA titer < 1:64 as negative, an IFA titer > 1:128 as positive, and an IFA titer = 1:64 as either positive or negative (supported by clinical records), dipsticks were 83% specific and 90% sensitive. The quantitative correlation of the dipsticks to IFA titers was confirmed by significant differences in geometric means of inverse IFA titers corresponding to the number of positive dipstick spots (no dots = 8.5, one dot = 43.3, two dots = 206.7, and three dots = 676.9). The assay would enable physicians and public health workers who deal with patients to quickly diagnose and appropriately treat most cases of the disease, especially in areas of high prevalence where the proportion of false-positive results to true-positive results would be low.

Matched MeSH terms: Immunoblotting/methods*
Immunochemical characterization of edible bird's nest allergens

Goh DL, Chua KY, Chew FT, Liang RC, Seow TK, Ou KL, et al.

J Allergy Clin Immunol, 2001 Jun;107(6):1082-7.
PMID: 11398089

BACKGROUND: We have previously described anaphylaxis induced by edible bird's nest (BN) and demonstrated that this condition is IgE mediated.
OBJECTIVES: This study aimed at describing the immunochemical properties of the BN allergens. Comparative studies between 3 commercially available sources (according to the country of origin) of BN were also made.
METHODS: Crude extracts of commercially available processed BN from Sarawak (Malaysia), Thailand, and Indonesia and fresh unprocessed BN from the caves of Sarawak were obtained by means of aqueous extraction. Specific IgE toward these sources were determined by using fluorescence allergosorbent tests (FASTs). Cross-reactivity studies between the 3 sources of commercially available processed BN were carried out by means of FAST inhibition. Immunochemical characterization by means of IgE immunoblot, periodate treatment, and heat stability studies were carried out on fresh unprocessed BN from Sarawak.
RESULTS: Serum from allergic patients showed differences in IgE binding to the 3 sources of commercially available BN, with the highest levels of specific IgE recorded with the Sarawak source (P

Matched MeSH terms: Immunoblotting
Cloning, expression, and characterization of recombinant Hev b 3, a Hevea brasiliensis protein associated with latex allergy in patients with spina bifida

Wagner B, Krebitz M, Buck D, Niggemann B, Yeang HY, Han KH, et al.

J Allergy Clin Immunol, 1999 Nov;104(5):1084-92.
PMID: 10550757

BACKGROUND: Two natural rubber latex proteins, Hev b 1 and Hev b 3, have been described in spina bifida (SB)-associated latex allergy.
OBJECTIVE: The aim of this study was to clone and express Hev b 3 and to obtain the immunologic active and soluble recombinant allergen for diagnosis of SB-associated latex allergy.
METHODS: A complementary DNA (cDNA) coding for Hev b 3 was amplified from RNA of fresh latex collected from Malaysian rubber trees (Hevea brasiliensis). PCR primers were designed according to sequences of internal peptide fragments of natural (n) Hev b 3. The 5'-end sequence was obtained by specific amplification of cDNA ends. The recombinant (r) Hev b 3 was produced in Escherichia coli as a 6xHis tagged protein. Immunoblotting and inhibition assays were performed to characterize the recombinant allergen.
RESULTS: An Hev b 3 cDNA clone of 922 bp encoding a protein of 204 amino acid residues corresponding to a molecular weight of 22.3 kd was obtained. In immunoblots 29/35, latex-allergic patients with SB revealed IgE binding to rHev b 3, as did 4 of 15 of the latex-sensitized group. The presence of all IgE epitopes on rHev b 3 was shown by its ability to abolish all IgE binding to nHev b 3. Hev b 3 is related to Hev b 1 by a sequence identity of 47%. Cross-reactivity between these 2 latex allergens was illustrated by the large extent of inhibition of IgE binding to nHev b 1 by rHev b 3.
CONCLUSION: rHev b 3 constitutes a suitable in vitro reagent for the diagnosis of latex allergy in patients with SB. The determination of the full sequence of Hev b 3 and the production of the recombinant allergen will allow the epitope mapping and improve diagnostic reagents for latex allergy.

Matched MeSH terms: Immunoblotting
Detection and precipitation of hepatitis B core antigen using a fusion bacteriophage

Hasmoni SS, Yusoff K, Tan WS

J Gen Appl Microbiol, 2005 Apr;51(2):125-31.
PMID: 15942873

The nucleocapsids of hepatitis B virus (HBV) are made of 180 or 240 subunits of core proteins or known as core antigens (HBcAg). A fusion bacteriophage bearing the WSFFSNI sequence that interacts tightly to HBcAg was employed as a diagnostic reagent for the detection of the antigen using the phage-enzyme-linked immunosorbent (phage-ELISA), dot blot and immunoprecipitation assays. The results from phage-ELISA and dot blot assay showed that as low as 10 ng of HBcAg can be detected optimally by 1.0x10(12) pfu/ml fusion M13 bacteriophage. The sensitivity of the dot blot assay corresponds with that of the phage-ELISA. HBcAg in HBV positive serum samples can also be detected using the fusion phage via the phage-ELISA and phage-dot blot assay. The phage cross-linked to cyanogen bromide (CNBr) activated agarose can also be used to precipitate HBcAg in bacterial lysate. The optimum amount of phage needed for cross-linking to 1 g of agarose is about 7.0x10(6) pfu/ml which could also precipitate purified and unpurified HBcAg in bacterial lysate. This study demonstrates the potential of fusion bacteriophage bearing the sequence WSFFSNI as a diagnostic reagent and a ligand for the detection and purification of HBcAg respectively.

Matched MeSH terms: Immunoblotting
Fulltext Identification of major and minor allergens of black tiger prawn (Penaeus monodon) and king prawn (Penaeus latisulcatus)

Sahabudin S, Misnan R, Yadzir ZH, Mohamad J, Abdullah N, Bakhtiar F, et al.

Malays J Med Sci, 2011 Jul;18(3):27-32.
PMID: 22135598 MyJurnal

BACKGROUND: Prawns and shrimp are a frequent cause of seafood allergy mediated by IgE antibodies. Penaeus monodon and Penaeus latisulcatus, commonly known as black tiger prawn and king prawn, respectively, are among the most frequently consumed prawns in Malaysia. The aim of this study was to identify the IgE-binding proteins of these 2 prawn species.
METHODS: Raw and boiled prawn extracts were prepared and then resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). IgE-immunoblotting was then performed using sera from patients with positive skin prick tests to the raw prawn extracts.
RESULTS: SDS-PAGE analysis of the raw extracts of both prawn species revealed 23 protein bands; the boiled extracts yielded fewer protein bands. The bands in the range of 40 to 100 kDa were sensitive to heat and therefore were not found in the boiled extracts. Immunoblot of raw extracts of black tiger prawns and king prawns yielded 14 and 11 IgE-binding proteins, respectively, with molecular weights of between 15 and 200 kDa. Proteins at 36, 42, and 49 kDa were detected as the major allergens in both species of prawns. A protein of 75 kDa was also identified as a major allergen in black tiger prawns. Other potential allergens were also observed at various molecular masses.
CONCLUSION: Proteins of 36, 42, and 49 kDa were identified as the major allergens of both species of prawns. The 36 and 42 kDa proteins are hypothesised to be tropomyosin and arginine kinase, respectively. A high molecular weight protein of 75 kDa was found to be an additional major allergen in black tiger prawns.
KEYWORDS: Penaeus; allergens; allergy and clinical immunology; hypersensitivity; immunoblotting; tropomyosin

Matched MeSH terms: Immunoblotting
Fulltext Antibody responses of dengue fever patients to dengue 2 (New Guinea C strain) viral proteins

AbuBakar S, Azmi A, Mohamed-Saad N, Shafee N, Chee HY

Malays J Pathol, 1997 Jun;19(1):41-51.
PMID: 10879241

The present study was undertaken to investigate the antibody responses of dengue fever (DF) patients to specific dengue virus proteins. Partially purified dengue 2 New Guinea C (NGC) strain virus was used as antigen. Under the present experimental protocols, it was observed that almost all DF patients' sera had detectable presence of antibodies which recognize the dengue 2 envelope (E) protein. The convalescent-phase sera especially had significant detectable IgG, IgM and IgE against the protein. In addition, IgGs specific against the NS1 dimer and PrM were also detected. Antibody against the core (C) protein, however, was not detectable in any of the DF patients' sera. The substantial presence of IgG against the PrM in the convalescent-phase sera, and the presence of IgE specific for the E, reflect the potential importance of these antibody responses in the pathogenesis of dengue.

Matched MeSH terms: Immunoblotting
Fulltext Hepatitis C: an update

Merican MI

Med J Malaysia, 1992 Sep;47(3):158-69.
PMID: 1283440

The identification of the Hepatitis C virus using molecular cloning techniques, besides making the term Non-A Non-B Hepatitis obsolete, enables the development of specific assays for the detection of antibodies in HCV-infected individuals, thus making it possible to obtain sero-epidemiological data of the disease. The carriage of Hepatitis C antibody varies worldwide. The disease is most prevalent in intravenous drug abusers or haemophiliacs. Parenteral transmission is the most important route of transmission. Sexual, intra-familial and perinatal transmissions are uncommon. About 40% could be community-acquired (sporadic). Diagnostic tests include enzyme-linked immunosorbant (ELISA) anti-HCV assay, recombinant immunoblot assay, HCV-RNA by polymerase chain reaction and HCV-Ag. More than 50% of acute cases becomes chronic and runs a benign and indolent course. About 20% progress to cirrhosis and some of these develop hepatocellular carcinoma. Several published trials have consistently shown that treatment with interferon in some patients is useful. There is however a relapse rate of 50%. Further trials with interferon and other anti-viral agents like ribavirin are awaited for more effective treatment.

Matched MeSH terms: Immunoblotting
Identification of tropomyosin as major allergen of white squid (Loligo edulis) by two-dimensional immunoblotting and mass spectrometry

Yadzir ZH, Misnan R, Murad S

Southeast Asian J. Trop. Med. Public Health, 2012 Jan;43(1):185-91.
PMID: 23082569

IgE-mediated allergic reaction to squid is one of the most frequent molluscan shellfish allergies. Previously, we have detected a 36 kDa protein as the major allergen of Loligo edulis (white squid) by immunoblotting using sera from patients with squid allergy. The aim of this present study was to further identify this major allergen using a proteomics approach. The major allergen was identified by a combination of two-dimensional electrophoresis (2-DE), immunoblotting, mass spectrometry and bioinformatics tools. The 2-DE gel fractionated the cooked white squid proteins to more than 50 different protein spots between 10 to 38 kDa and isoelectric point (pI) from 3.0 to 10.0. A highly reactive protein spot of a molecular mass of 36 kDa and pI of 4.55 was observed in all of the patients' serum samples tested. Mass spectrometry analysis led to identification of this allergen as tropomyosin. This finding can contribute to advancement in component-based diagnosis, management of squid allergic patients, to the development of immunotherapy and to the standardization of allergenic test products as tools in molecular allergology.

Matched MeSH terms: Immunoblotting
Antigenic types of Orientia tsutsugamushi in Malaysia

Tay ST, Rohani MY, Ho TM, Devi S

Southeast Asian J. Trop. Med. Public Health, 2002 Sep;33(3):557-64.
PMID: 12693591

The seroprevalence of various Orientia tsutsugamushi (OT) strains among Malaysian patients with suspected scrub typhus infections was determined using an indirect immunoperoxidase (IIP) assay. IgG against a single OT strain were detected in six sera (3 Karp, 1 Gilliam and 2 TC586), whereas IgM antibodies against a single OT strain (Gilliam) were noted in 3 sera (Gilliam). IgG reactive to all OT strains were present in 33 (47.1%) of the 70 sera and IgM reactive to all OT strains were present in 22 (78.6%) of the 28 sera. The fact that most sera were reactive to multiple OT strains suggests that group-specific antigens are involved in scrub typhus infections, whereas very few were due to strain-specific epitopes present on these strains. Peak IgG and IgM titers were noted more frequently against Gilliam, Karp, and TA763 strains: this suggests that these strains may be the commonest infecting strains among Malaysian patients. Two predominant OT polypeptides consistently reacted with patients' sera were the 70 kDa and 56 kDa proteins.

Matched MeSH terms: Immunoblotting

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