• Title, Summary, Keyword: western blot

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Western blot analysis of stray cat sera against Toxoplasma gondii and the diagnostic availability of monoclonal antibodies in sandwich-ELISA

  • Sohn, Woon-Mok;Nam, Ho-Woo
    • The Korean Journal of Parasitology
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    • v.37 no.4
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    • pp.249-256
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    • 1999
  • A total of 198 sera from stray cats was assayed against Toxoplasma gondii antigen by western blot. Out of 198 sera assayed, 26 sera (13.1%) showed typical blot patterns against T gondii. When spotted by ELISA absorbance and indirect latex agglutination lest (ILAT) titer, all 26 cases were distributed over the cut-off value of ELISA whereas 24 cases (92.3%) were in the positive range of 1:32 or higher and 2 cases in negative range by ILAT. Among western blot negative 172 sera, 162 cases were negative in both ILAT and ELISA while 10 cases were reactive falsely such that three cases were ILAT positive with 1:32 titer and 9 cases were ELISA positive (2 cases overlapped). These 10 cases reacted peculiarly without typical binding pattern in Western blot. Sandwich-ELISA was performed with monoclonal antibodies (mAbs) of Tg563 (30 kDa, SAG 1), Tg505 (22 kDa, SAG2), Tg605 (43 kDa, SAG3), Tg556 (28 kDa, GRA2), Tg737 (32 kDa, GRA6). Tg695 (66 kDa, ROP2), Tg786 (42 kDa, ROP6), and Tg621 (32 kDa, anonymous but cytosolic) clone, respectively. All western blot-positive cases were in the positive range and negative cases in the negative range clearly. Among the 10 false reactive cases, 3 cases were in the positive range with one or more mAbs. All mAbs used in this study were confirmed to be specific to T. gondii infection as a standardized sandwich-ELISA to differentiate it from other pathogens.

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Imported Intraocular Gnathostomiasis with Subretinal Tracks Confirmed by Western Blot Assay

  • Yang, Ji-Ho;Kim, Moo-Sang;Kim, Eung-Suk;Na, Byoung-Kuk;Yu, Seung-Young;Kwak, Hyung-Woo
    • The Korean Journal of Parasitology
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    • v.50 no.1
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    • pp.73-78
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    • 2012
  • We report a case of intraocular gnathostomiasis diagnosed by western blot assay in a patient with subretinal tracks. A 15-year-old male patient complained of blurred vision in the right eye, lasting for 2 weeks. Eight months earlier, he had traveled to Vietnam for 1 week and ate raw wild boar meat and lobster. His best-corrected visual acuity was 20/20 in both eyes and anterior chamber examination revealed no abnormalities. Fundus examination showed subretinal tracks in the right eye. Fluorescein angiography and indocyanine green angiography showed linear hyperfluorescence of the subretinal lesion observed on fundus in the right eye. Ultrasound examination revealed no abnormalities. Blood tests indicated mild eosinophilia (7.5%), and there was no abnormality found by systemic examinations. Two years later, the patient visited our department again for ophthalmologic evaluation. Visual acuity remained 20/20 in both eyes and the subretinal tracks in the right eye had not changed since the previous examination. Serologic examination was performed to provide a more accurate diagnosis, and the patient's serum reacted strongly to the $Gnathostoma$ $nipponicum$ antigen by western blot assay, which led to a diagnosis of intraocular gnathostomiasis. This is the first reported case of intraocular gnathostomiasis with subretinal tracks confirmed serologically using western blot in Korea.

Development of ELISA for brucella abortus RB51 I. Analysis on antigens of Brucella abortus RB51 by Westeren blot (부루세라 RB51의 ELISA 진단법개발 I. Westeren blot에 의한 Brucella abortus RB51균의 항원 분석)

  • Her, Moon;Cho, Dong-hee;Jung, Byeong-yeal;Cho, Seong-kun;Jung, Suk-chan;Kim, Ok-kyung
    • Korean Journal of Veterinary Research
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    • v.41 no.1
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    • pp.43-49
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    • 2001
  • As compared with reaction of antibody for sonicated antigen of Brucella abortus strain RB51 and 1119-3 by Western blot analysis, Brucella field positive sera was detected strong reaction at 40~80 kDa LPS of strain 1119-3, but detected very weak reaction at strain RB51 partly. Otherwise, as we analyzed major immunogen of RB51 by antisera bled periodically during 6 months after RB51 vaccination. we detected strong immunological reaction at 17, 18 and 8 kDa antigen of RB51. Especially, reaction of 8 kDa antigen by Western blot coincided with reaction of dot-blot assay in RB51-antibody detection method. We also compared with reaction of field sera by STAT(standard tube agglutination test), dot-blot assay and Western blot (reaction of 8 kDa antigen of strain RB51). 16 sera of 4~5 months after RB51 vaccination are all negative by STAT, and 12 field brucellosis positive serum are all positive, and also 12 of 16 sera vaccinated RB51 are positive by dot-blot assay and reaction of 8kDa antigen by Western blot. but 1 of 15 Brucellosis negative sera reacted nonspecifically dot-blot assay.

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Comparison of IHNV Detection Limits by IMS-RT-PCR, Western Blot and ELISA

  • Kim Soo-Jin;Lee Eun-Young;Oh Myung-Joo;Choi Tae-Jin
    • Fisheries and aquatic sciences
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    • v.4 no.1
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    • pp.32-38
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    • 2001
  • Several molecular biological techniques have been used to detect virus rapidly and accurately, but these methods have limitations in the early stage of viral infection with very low concentration of virus. We compared the detection limits of IMS-PCR, Western blot and ELISA with infectious hematopoietic necrosis virus OHNV). Four antibodies, rabbit anti-IHNV polyclonal antibody, anti-IHNV nucleocapsid protein monoclonal antibody, anti-IHNV nucleocapsid protein polyclonal antibody, and anti-IHNV glycoprotein polyclonal antibody, were tested to find out the most effective antibody for each method. The detection limit with IMS- PCR was $2\times10^6$ pfu when the viral RNA was extracted before RT-PCR. In the western blot with rabbit anti­IHNV polyclonal antibody one pfu of virus could be detected. In ELISA, 10 pfu of virus particles were detected with the same antibody.

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Seroprevalence of Sarcocystis falcatula in Two Islands of Malaysia using Recombinant Surface Antigen 4

  • Nadzirah, Tengku-Idris Tengku Idzzan;Yik, Fong Mun;Ling, Lau Yee
    • The Korean Journal of Parasitology
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    • v.58 no.1
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    • pp.1-5
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    • 2020
  • 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.

A study of osteonectin expression patterns in BAPN-induced cleft palate formed rats (BAPN으로 유도된 구개파열 백서의 osteonectin발현 양상에 대한 연구)

  • Tae, Ki-Chul;Kim, En-Chel;Lee, Sun-Kyeong
    • The korean journal of orthodontics
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    • v.30 no.4
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    • pp.433-440
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    • 2000
  • The purpose of this study was to investigate osteonectin expression patterns in cleft palate compare to normal palate rats. We used 4 pregnant rats, and beta-aminoproprionitrile was oral dose to rat according to 1g/kg body weight at gestation days 13 to induce cleft palate. Total 6 fetus was got with cleft formed, then 3 fetus was used for immunohistostain and 3 fetus was used for western blot analysis. Expression patterns of osteonectin in mesenchymal cells of cleft palate was more dilute to mesenchymal cells of normal palate with immnunohistostain, and width and length of band of maxilla in cleft palate was more thin than maxilla of normal palate with western blot study.

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Production of A Monoclonal Antibody (MAb) Against a Thermal Stable-Soluble Protein in Mackerel and Confirmation of the Properties for the MAb (고등어 어육 중 열안정성 단백질에 특이한 단클론성 항체 개발과 특성 확인)

  • Lee, Jeong-Eun;Kim, Jeong-Sook;Chung, Duck-Hwa;Shim, Won-Bo
    • Journal of Food Hygiene and Safety
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    • v.32 no.1
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    • pp.75-81
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    • 2017
  • For people who have a food allergy the only way to manage the allergy is to avoid the food allergen. The mackerel is one of the major food allergens, but no immunoassay for the rapid and simple detection of mackerel has been reported. The objectives of this study are to develop and characterize monoclonal antibodies (MAbs) specific to mackerel using thermal stable-soluble proteins (TSSP) as an immunogen and to characterize the MAbs by indirect enzyme-linked immunosorbent assay (iELISA). The mice immunized with mackerel TSSP and showing high titer were used for cell fusion and cloning. The characterization of MAbs produced from hybridoma cells obtained was confirmed by indirect ELISA and western blot. Four MAbs were confirmed to be specific to mackerel without cross-reaction to other marine products and livestock products in the both methods. The iELISA and western blot based on the MAbs can sensitively detect 1% mackerel protein in other marine products. These results support that immunochemical methods based on the MAb produced could be used as rapid means to detect low levels of mackerel and to identify mackerel adulterated in food.

Immunological Characteristics of the Vitellogenin Induced by $Estradiol-17\beta$ in Male Spotted Flounder, Verasper variegatus (범가자미, Verasper variegatus 수컷에서 $estradiol-17\beta$에 의해 유도된 vitellogenin의 면역학적 특성)

  • KIM Yoon;KIM Woo Jin;BAEK Hea Ja;KIM Kyung Kil;BANG In Chul;HAN Chang Hee
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.30 no.3
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    • pp.480-487
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    • 1997
  • Vitellogenin (Vg) was purified from plasma of $estradiol-17\beta(E_2)-treated$ spotted flounder, verasper variegatus, by preripitation with cold distilled water, followed by fractionation using a Sepharose CL-6B column chromatography. $E_2-induced$ protein was identified as Vg by SDS-PAGE and western blot analysis. The molecular weight of the purified Vg was estimated 175 kD as determined by SDS-PAGE. In order to measure the Vg level, monoclonal antibodies against Vg were produced by hybridoma technique. Purified Vg was immunized into Balb/c mice and then the spleen cells from mice were fused with NS-1 myeloma cells. The hybridoma cells were screened by enzyme-linked immunosorbent assay (WLISA) and subcloned by limiting dilution. The hybridoma clones which secreted antibodies highly reactive to the purified Vg were designated as 4D6. Its specificity was demonstrated by western blot from plasma of untreated, $E_2-treated$ male fish, and purified Vg.

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Comparative Study of Korean (Viscum album var. coloratum) and European Mistletoes (Viscum album)

  • Lyu, Su-Yun;Park, Sun-Myo;Choung, Bo-Yun;Park, Won-Bong
    • Archives of Pharmacal Research
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    • v.23 no.6
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    • pp.592-598
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    • 2000
  • A lectin (agglutinin, VCA) from Korean mistletoe (Viscum album L. coloratum) was isolated by affinity chromatograpy on a asialofetuin-Sepharose 4B. The molecular weights of A- and B-chains of VCA were different from those of VAAS. The VCA recognized the antibody of VAAs in the Western blot analysis and ELLA system. We also investigated the synergistic effects of the components in mistletoe by dividing the extract into different molecular weight fractions.

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Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite

  • Son, Eui-Sun;Kim, Tong-Soo;Nam, Ho-Woo
    • The Korean Journal of Parasitology
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    • v.39 no.2
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    • pp.171-176
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    • 2001
  • Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), meroxoite surface protein (MSP-1), apical merozoite antigen (AMA- 1), serine repeat antigen (SERA), and exported antigen (EXP- 1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG. The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination. When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera. In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively. We obtained higher detection rates when using S antigens (91.9%) rather than using each antigen solely (55.6 - 80%), a combination of 2 (76.3 - 87.5%), 3 (85.6 - 90.6%), or 4 antigens (89.4 - 91.3%). This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria.

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