• Title, Summary, Keyword: immunoblotting analysis

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Analysis of Culture Filtrate Antigens of Aspergillus fumigatus Strains and of Antibody Responce in Patients with Aspergillosis by Immunoblotting (Immunoblotting 에 의한 Aspergillus fumigatus 균주(菌株)의 항원분석(抗原分析)과 이 균(菌)에 감염(感染)된 환자의 항체반응(抗體反應)에 관한 연구)

  • Kim, Sang-Jae;Kim, Sin-Ok;Hong, Young-Pyo
    • The Korean Journal of Mycology
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    • v.17 no.2
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    • pp.66-75
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    • 1989
  • Heterogeneity in antigenic composition of Aspergillus fumigatus isolates from clinical specimens and in antibody response of patients infected with this fungus was investigated by immunoblotting. A considerable quantitative and qualitative difference was found in composition of the culture filtrate antigens derived from a reference strain (ATCC 13073) and 8 clinical isolates of A. fumigatus on SDS-PAGE and immunoblots. The crude CF antigen of a strain AFG7 was selected to identify the serologically reactive and specific components by immunoblotting. Out of more than 36 components separated by electrophoresis, transblotted to nitrocellulose sheet, and reacted with sera that showed a positive reaction to A. fumigatus or other fungal antigens on immunodiffusion tests, merely four or so were found useful to serodiagnosis of aspergillosis. An antigen of 82KD was found most reactive and specific component so as to be contained in the standard preparation. Several other components, for example 11KD, 26KD, 30KD and 31KD, also possessed relatively high reactivity and specificity and seemed to be worth while purifying and characterizing. Antibody binding activity (reactivity) of the antigenic components was clearly shown on immunoblots because some were faintly stained with Coomassie blue but darkly stained on immunoblots, while some others behaved contrary to them. A number of components seemed to carry not only species specific but cross reactive antigenic determinants. Immunoblotting proved very useful to identify serologically reactive and specific components that should be present in the antigen to be employed to the serodiagnosis of aspergillosis.

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Comparison of Four Commercial ELISA Kits and In-House Immunoblotting for Diagnosis of Helicobacter pylori Infection

  • Jeong, Hoar Lim;Jung, Yang-Sook;Jun, Jin-Su;Yeom, Jung Sook;Park, Ji Sook;Seo, Ji-Hyun;Lim, Jae-Young;Park, Chan-Hoo;Woo, Hyang-Ok;Youn, Hee-Shang;Ko, Gyung-Hyuck;Baik, Seung-Chul;Lee, Woo-Kon;Cho, Myung-Je;Rhee, Kwang-Ho
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • v.15 no.2
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    • pp.85-90
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    • 2012
  • Purpose: Commercial enzyme-linked immunosorbent assay (ELISA) kits have been considered less reliable for children than for adults. The aim of this study was to compare four ELISA kits and in-house immunoblotting based on the analysis of anti-H. pylori-IgG antibody reactivity. Methods: A total of 399 serum samples were collected at the GNU Hospital during 1998-1999. All sera were tested using ELISA and immunoblotting. Statistically significant differences were determined by the $x^2$ test. Results: The overall seropositivity rates using GAP IgG, Genedia IgG, HM-CAP, Pyloriset EIA-G, and immunoblotting were 13.0%, 25.1%, 18.3%, 15.8%, and 62.9%, respectively. Immunoblotting showed a higher seropositivity rate than did all four ELISA kits in all age groups. Genedia IgG had the highest seropositivity among the ELISA kits. The seropositivity rate for children aged 13 to 18 months was lowest, and that of children aged 15 years was highest (90.0%). The seropositivity rate for children aged 7 months to 5 years was significantly lower than that for children aged 6 to 15 years among the four ELISA kits (p<0.0001) and immunoblotting (p=0.02). Conclusion: Immunoblotting is the most sensitive test for detection of anti-Helicobacter pylori IgG antibodies among the serological tests in this study. These results emphasize the need for standardization when commercial ELISA tests are used in different nations or in young age groups. Immunoblotting could be a suitable noninvasive assay for serodiagnosis and seroepidemiologic study of H. pylori infection in Korean children.

Expression of major piroplasm protein(p33)of Theileria sergenti (Korean isolate) and its immunogenicity in guinea pigs

  • Kang, Seung-Won;Kweon, Chang-Hee;Choi, Eun-Jin;Yoon, Yong-Dhuk
    • The Korean Journal of Parasitology
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    • v.37 no.4
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    • pp.277-283
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    • 1999
  • To investigate the development of a subunit vaccine against theileriosis in cattle, the DNA fragments encoding piroplasm surface protein (p33) of Theileria sergenti of a Korean isolate were expressed in baculoviruses. The expressed p33 was characterized by indirect fluorescent antibody (IFA) and western blotting analysis. The expression of p33 was mainly detected on the surface of infected Sf21 cells by IFA. The immunoblotting analysis revealed the presence of a same molecular weight protein band of p33. The antigenicity of expressed polypeptide was further examined through the inoculation of a guinea pig. The sera of guinea pigs immunized with p33 expressed cell Iysate showed similar fluorescent antibody patterns and reacted with the same molecular weight protein of T. sergenti in immunoblotting analysis, thus indicating that this protein can be a promising candidate for a subunit vaccine in the future.

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Anti-tumor Promoting Activity of Some Malaysian Traditional Vegetable (Ulam) Extracts by Immunoblotting Analysis of Raji Cells

  • Ali, A.M.;Mooi, L.Y.;Yih, K. Yih;Norhanom, A.W.;Saleh, K. Mat;Lajis, N.H.;Yazid, A.M.;Ahmad, F.B.H.;Prasad, U.
    • Natural Product Sciences
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    • v.6 no.3
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    • pp.147-150
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    • 2000
  • The extracts of Carica papaya (flower), Barringtonia macrostachya (leaves), Coleus tuberosus (tuber), Mangifera indica (fruit skin) and Eugenia polyantha (leaves) showed strong in vitro anti-tumor promoting activity when assayed using Raji cells (Mooi et al., 1999). The antitumor promoting activity of the crude extracts was further analyzed by immunoblotting analysis of Raji cells carving Epstein-Barr virus genome. The expression of early antigens diffuse (EA-D) and early antigens restricted (EA-R) was determined by performing western blotting of treated Raji cells with human sera of nasopharyngeal carcinoma patients. All the plant extracts were shown to be able to suppress both EA-D and EA-R.

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Expression of Escherichia coli Heat-labile Enterotoxin B Subunit (LTB) in Saccharomyces cerevisiae

  • Rezaee Mohammad Ahangarzadeh;Rezaee Abbas;Moazzeni Seyed Mohammad;Salmanian Ali Hatef;Yasuda Yoko;Tochikubo Kunio;Pirayeh Shahin Najar;Arzanlou Mohsen
    • Journal of Microbiology
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    • v.43 no.4
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    • pp.354-360
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    • 2005
  • Heat-labile enterotoxin B subunit (LTB) of enterotoxigenic Escherichia coli (ETEC) is both a strong mucosal adjuvant and immunogen. It is a subunit vaccine candidate to be used against ETEC-induced diarrhea. It has already been expressed in several bacterial and plant systems. In order to construct yeast expressing vector for the LTB protein, the eltB gene encoding LTB was amplified from a human origin enterotoxigenic E. coli DNA by PCR. The expression plasmid pLTB83 was constructed by inserting the eltB gene into the pYES2 shuttle vector immediately downstream of the GAL1 promoter. The recombinant vector was transformed into S. cerevisiae and was then induced by galactose. The LTB protein was detected in the total soluble protein of the yeast by SDS-PAGE analysis. Quantitative ELISA showed that the maximum amount of LTB protein expressed in the yeast was approximately $1.9\%$ of the total soluble protein. Immunoblotting analysis showed the yeast-derived LTB protein was antigenically indistinguishable from bacterial LTB protein. Since the whole-recombinant yeast has been introduced as a new vaccine formulation the expression of LTB in S. cerevisiae can offer an inexpensive yet effective strategy to protect against ETEC, especially in developing countries where it is needed most.

Analysis of Antibodies Against Lyme Disease Agent, Borrelia burgdorferi, in Sera from Patients with Unknown Fever (열성질환 환자에서 라임병균 Borrelia burgdorferi 감염증 진단을 위한 혈청학적인 분석)

  • 김영미;김종배
    • Biomedical Science Letters
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    • v.3 no.2
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    • pp.95-105
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    • 1997
  • Currently, the laboratory diagnosis for lyme disease have been performed with the detection of antibodies against Borrelia burgdorferi. However there might be some difficulties in the interpretation of obtained results due to the usage of foreign isolates as an antigen in the test. Therefore the optimization of serological tests with Korean isolates of B. burgdorferi as an antigen would be needed to establish the standardized diagnostic method for the detection of antibodies against B. burgdorferi infection. In this study, the optimization of ELISA was investigated with experimentally challenged rabbit sera and sonicated B. burgdorferi antigens of Korean isolates. Of 217 human patient's sera with unknown fever, the mean seropositivities in ELISA, done under the optimized conditions obtained in this study, were found to be about 8% against B. burgdorferi sensu late, showing the highest seropositivity of 14.3% against B. afzelii. In immunoblotting assay with ELISA-positive human sera, the major reactive bands were 41kDa (flagellin) which might be the indication of early infection, and 27kDa, 31kDa (OspA), 34kDa (OspB) which are the characteristics of late infection. Obtained results in this study might strongly indicate the possibility of B. burgdorferi infections in Korea.

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Serological Studies on the Specific Antibodies Against P-pili of Uropathogenic Escherichia coli (요로 감염환자에서 혈청학적 방법을 이용한 P-pili특이혈중 항체의 조사)

  • 이원용;김종배
    • Biomedical Science Letters
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    • v.2 no.1
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    • pp.31-40
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    • 1996
  • Escherichia coli is one of the most common etiological agents in urinary tract infection. An important virulence factor is the adhesive capacity of E. coli to uroepithelial cell, mediated by bacterial fimbriae. The Adhesion property has been regarded as an important virulence determinant in urinary tract infections. A total of 60 patients, who were diagnosed microbiologically as urinary tract infections, were examined by immunoblotting and enzyme-linked immunosorbent assay(ELISA). Uropathogenic E. coli with recombinant plasmid were positive for mannose resistant hemagglutination (MRHA). For identification of p-fimbriae subtype in uropathogenic E. coli, In the immunoblot analysis, specific bands in the range of p-fimbriae molecular weight of 17KD-22KD were identified. For the distribution of p-fimbriae subtype in the patient sera, 34/60(56.7%) were positive for $F7_1$, 28/60(46.7%) were positive for $F7_2$, and 30/60(50%) were positive for F13 with immunoblotting method. similar trends were observed in the enzyme-linked immunosorbent assay. Relatively good specificity(92.6%) and sensitivity(90%) were found in the ELISA test system using mixed antigens of purified $F7_1$, $F7_2$, and F13 p-fimbriae, and 60 sera from patients with urinary tract infections. In conclusion The serological tests were convenient method in diagnosis of urinary tract infections. among those ELISA could be recommended in diagnosis of urinary tract infections.

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Comparison of Antigenic Spots between Escherichia coli O157:H7 Strains by 2-Dimensional Gel Electrophoresis (이차원전기영동을 이용한 Escherichia coli O157:H7 균주간 항원 Spot의 비교)

  • Ahn, Yeong-Chang;Shin, Gee-Wook;Shin, Yong-Seung;Lee, Eung-Goo;Lee, Hyoung-Jun;Park, Mi-Rim;Kim, Young-Rim;Jung, Tae-Sung;Kim, Gon-Sup;Kim, Yong-Hwan
    • Korean Journal of Veterinary Research
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    • v.42 no.2
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    • pp.231-239
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    • 2002
  • Proteomics is an emerging powerful tool in studying protein expression and function. At present study, proteomics was employed to evaluate the antigenicity among Escherichia coli O157:H7 strains using 2-dimensional gel electrophoresis (2-DE) and immunoblotting, SDS-PAGE and immunblotting analysis revealed no big differences among E coli O157:H7 strains. 2-DE analysis, however, revealed common antigens as well as specific antigens. The immunoblotting analysis revealed 20 common antigenic spots among E coli O157:H7 strains. In addition, there were 3 and 13 spots as common antigens between ATCC 43894 and KSC 109, and between ATCC 43894 and ACH 5, respectively. Antigenic spots specific for individual strain were also identified as 15, 8 and 22 for ATCC 43894, ACH 5 and KSC 109, respectively. The common antigens would be useful by employing either vaccine development or diagnosis marker, or both, whereas the specific antigens of individual strains would be applicable for epidemiological study. This study suggest that proteome analysis, representative as 2-DE, is valuable tool in exploring the E. coli antigenicity.

Inhibition of cell growth and induction of apoptosis by bilobalide in FaDu human pharyngeal squamous cell carcinoma

  • Jeong, Kyung In;Kim, Su-Gwan;Go, Dae-San;Kim, Do Kyungm
    • International Journal of Oral Biology
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    • v.45 no.1
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    • pp.8-14
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    • 2020
  • Bilobalide isolated from the leaves of Ginkgo biloba has several pharmacological activities such as neuroprotective, anti-inflammatory, and anticonvulsant. However, the effect of bilobalide on cancer has not been clearly established. The main purpose of this study was to investigate the effect of bilobalide on cell growth and apoptosis induction in FaDu human pharyngeal squamous cell carcinoma. This was examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, nuclear 4′,6-diamidino-2-phenylindole dihydrochloride staining, DNA fragmentation analysis, and immunoblotting. Bilobalide inhibited the growth of FaDu cells in dose- and time-dependent manners. Treatment with bilobalide resulted in nuclear condensation and DNA fragmentation in FaDu cells. Furthermore, it promoted the proteolytic cleavage of procaspase-3/-7/-8/-9 with increase in the amount of cleaved caspase-3/-7/-8/-9. Bilobalide-induced apoptosis in FaDu cells was mediated by the expression of Fas and the activation of caspase-8, caspase-3, and poly (ADP-ribose) polymerase. Immunoblotting revealed that the antiapoptotic mitochondrial protein Bcl-2 was downregulated, but the proapoptotic protein Bax was upregulated by bilobalide in FaDu cells. Bilobalide significantly increased Bax/Bcl-2 ratio. These results suggest that bilobalide inhibits cell proliferation and induces apoptosis in FaDu human pharyngeal squamous cell carcinoma via both the death receptor-mediated extrinsic apoptotic pathway and the mitochondrial-mediated intrinsic apoptotic pathway.

Characterization of Carp (Cyprinus carpio L.) Immunoglobulin Structure

  • Choi, Sang-Hoon;Park, Kwan-Ha;Yoon, Jong-Man
    • Asian-Australasian Journal of Animal Sciences
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    • v.15 no.2
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    • pp.290-296
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    • 2002
  • Serum immunoglobulins (Igs) from Israeli carp were purified using affinity chromatography. Fish were immunized with purified mouse IgG, and the specific fish antibodies were purified from the immune serum on a mouse IgG-immobilized agarose gel. Rabbit anti-Israeli carp Igs (R $\alpha$ I. carp Igs) antibodies were produced following hyperimmunization with mouse IgG specific carp antibodies. SDS-PAGE analysis under reducing condition showed that Israeli carp Igs were composed of two $\mu$-like heavy chains with about 82 and 50 kd, respectively, and one light chain with about 25 kd. On immunoblotting analysis, however, R $\alpha$ I. carp Igs failed to react with the light chain. When both protein A and protein G-purified normal carp Ig were compared with mouse IgG-specific Israeli carp Ig, no significant structural differences among them were observed. To investigate if there is any homology between other fish Ig molecules, cross-reactivity of R $\alpha$ I. carp Igs against Ig molecules from 6 different fish sera and mouse control serum was checked on immunoblotting analysis. As a result, R $\alpha$ I. carp Igs responded to Israeli carp, common carp, and tilapia Ig molecules. In flow cytometry study, however, R $\alpha$ I. carp Igs appeared to recognize 42.0%, 35.8% and <5% of Israeli carp, common carp and tilapia $Ig^+$ head kidney cells, respectively. The result suggests the heterogeneity between receptor Igs on B-like lymphocytes and soluble Igs in serum. It is crucial to obtain pure fish Igs to produce reagent antibodies as tools for the study on their specific immune responses.