• Title/Summary/Keyword: Polyclonal antibody

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Double-Enhancement Strategy: A Practical Approach to a Femto-Molar Level Detection of Prostate Specific $Antigen-{\alpha}_1-Antichymotrypsin$ (PSA/ACT Complex) for SPR Immunosensing

  • Cao, Cuong;Sim, Sang-Jun
    • Journal of Microbiology and Biotechnology
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    • v.17 no.6
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    • pp.1031-1035
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    • 2007
  • Prostate specific $antigen-{\alpha}_1-antichymotrypsin$ was detected by a double-enhancement strategy involving the exploitation of both colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation. The AuNPs were synthesized and conjugated with horse-radish peroxidase-PSA polyclonal antibody by physisorption. Using the protein-colloid for SPR-based detection of the PSPJACT complex showed their enhancement as being consistent with other previous studies with regard to AuNPs enhancement, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the signal. The limit of detection was found at as low as 0.027 ng/ml of the PSA/ACT complex (or 300 fM), which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

Immunoblotting Reactivity of Vitellogenin Antibodies against Native Vitellogenins and a Vitellogenin Protein Fragment Produced in E. coli

  • Ryu, Hyung-Seok;Jang, In-Young;Kim, Woo-Yeon
    • Journal of Applied Biological Chemistry
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    • v.52 no.4
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    • pp.170-173
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    • 2009
  • Vitellogenin (Vtg) is found in the serum of both female and male fish that have been exposed to environmental endocrine disrupters or estrogen hormones, and is used as a biomarker for such contamination. In our current study antibodies raised against the purified flatfish Vtg were tested for their reactivity on immunoblots to flatfish and carp Vtg, and also to a Vtg protein fragment produced in E. coli. Polyclonal antibodies raised against purified flatfish Vtg reacted well with Vtg in the serum of flatfish and carp induced with $17{\beta}$-estradiol, but not with the Vtg protein fragment produced in E. coli.

Development of Quantitative Vitellogenin ELISAs for Goldfish(Carassius auratus) used in Endocrine Disruptor Screening (내분비 장애물질 측정을 위한 붕어 비탈로제닌 정량 분석 ELISA 시스템 개발)

  • Li Chun-Ri;Kim Kwang-Tae;Kim Andre;Chung Kyu-Hyuck;Kim Dong-Kyoo;Kang Shin-Won;Park Jang-Su
    • Environmental Analysis Health and Toxicology
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    • v.19 no.4
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    • pp.353-357
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    • 2004
  • 난황단백질인 비탈로제닌을 성숙한 암컷 붕어 혈청으로부터 음이온 교환 크로마토그래피를 이용하여 정제 하였다. 정제한 비탈로제닌을 BALB/c mice를 이용하여 폴리크로날 항체를 생산하였고 이를 protein A column을 사용하여 정제하였다. 또한 이렇게 정제된 폴리크로날 항체를 이용한 붕어 비탈로제닌 측정용 효소면역측정법을 개발하였으며 그 측정 범위는 2∼l,000ng/mL이고 recovery 변동 범위는 88∼112%였다. 또한 이 효소면역측정법을 평가하기 위해 성숙한 수컷 붕어를 1,000ng/L ethinylestradiol(EE$_2$) 에 4주 동안 노출시켜 유도되어지는 비텔로제닌을 측정하였다. 그 결과 성숙한 수컷 붕어의 경우 비탈로제닌이 3주 만에 암컷 붕어의 평균수치만큼 유도됨을 알 수 있었다.

Demonstration of Newcastle Disease Virus Antigens in Paraffin Embedded Tissues of Experimentally Infected Chickens Using Peroxidase-antiperoxidase(PAP) Technique (Peroxidase-antiperoxidaes법을 이용한 실험감염 계의 조직내 뉴캣슬병 바이러스 항원동정)

  • 노환국;신종백;임기재;김병지
    • Korean Journal of Veterinary Service
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    • v.15 no.2
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    • pp.184-194
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    • 1992
  • This study was done to identify Newcastle disease virus(NDV) antigens in paraffin sections of various organs from experimentally NDV-infected chicken using peroxidase-antiperoxidase(PAP) technique. Sections were Incubated with rabbit anti-NDV polyclonal as first antibody, followed by incubation with goat anti-rabbit IgG conjugate and peroxidase anti-peroxidase ( PAP ). Positive reactions were often detected in the epithelim of trachea and in the lymphocyte of spleen at 24 hours after virus inoculation. The viral antigen was localized mainly in the cytoplasm of infected cells. The method approved to be highly specific for the indetification of NDV and allowed a precise localization of the viral antigens in infected cells.

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Immunohistochemical identification of newcastle disease virus with indirect immunoperoxidase technique (Indirect Immunoperoxidase 법을 이용한 조직내 뉴켓슬병 바이러스 항원동정)

  • Nho, Whan-goog;Sur, Jung-hyang;Kim, Soon-bok
    • Korean Journal of Veterinary Research
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    • v.30 no.3
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    • pp.309-315
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    • 1990
  • The present experiment was done to identify newcastle disease virus(NDV) antigens in frozen sections of various oragns from experimentally NDV-infected with indirect immunoperoxidase method. Section were incubated with rabbit anti-NDV polyclonal as first antibody, followed by incubation with goat anti-rabbit or protein A peroxidase conjugate. Positive reactions were often detected in the epithelium of trachea and in the lymphocyte of spleen at 24 hours after virus inoculation. the viral antigen was localized mainly in the cytoplasm of infected cells. The method approved to be highly specific for the identification of NDV and allowed a precise localization of the viral antigens in infected cells.

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Mutational Analysis of Cucumber Mosaic Virus Movement Protein Gene

  • You, Jin-Sam;Baik, Hyung-Suk;Paek, Kyung-Hee
    • BMB Reports
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    • v.32 no.1
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    • pp.82-85
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    • 1999
  • The movement protein of cucumber mosaic virus (CMV) is required for cell-to-cell movement of viral RNA. The movement of viral RNA occurs through the plant intercellular connection, the plasmodesmata. The viral movement protein was known to be multi-functional. In this work, a series of deletion mutants of CMV movement protein gene were created to identify the functional domains. The mutated movement proteins were produced as inclusion body in E. coli, and purified and renatured. A polyclonal antibody was raised against the CMV-Kor strain (Korean isolate) movement protein expressed in E. coli. The ability of the truncated proteins to bind to ssRNA was assayed by UV cross-linking and gel retardation analyses. The results indicate that the domain between amino acids 118 and 160 of CMV movement protein is essential for ssRNA binding.

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Immunolocalization of the 150 kDa protein in cyst fluid of Taenia solium metacestodes

  • Yang, Hyun-Jong;Chung, Young-Bae
    • Parasites, Hosts and Diseases
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    • v.42 no.2
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    • pp.81-84
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    • 2004
  • The 150 kDa protein of cyst fluid (CF) of Taenia solium metacestodes was purified by ammonium sulfate fractionation and Superose 6 HR gel filtration chromatography. The purified protein consisted of three subunits (15, 10 and 7 kDa proteins), which were analyzed with the use of a 7.5-15% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SOS-PAGE). Immunofluorescence study was carried out by using immunize specific polyclonal antibody. Positive reactions were noticed at bladder walls, calcareous corpuscles, granules of cyst fluid and some host tissue surrounding the bladder wall of the metacestodes. These results suggest that the 150 kDa protein was secreted into host tissues, inducing immune responses in the host, and it may play important roles in the cellular physiology of the parasites.

Purification and Characterization of an Antifungal Peptide from the Seeds of Phytolacca americana (미국자리공 종실로부터 항균성 펩타이드의 분리 및 특성 연구)

  • 손대영;신봉정;윤대진;성기영;정영륜
    • Korean Journal Plant Pathology
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    • v.14 no.3
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    • pp.203-208
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    • 1998
  • We isolated and characterized an antifungal peptide from the seeds of Phytolacca americana. Growth inhibition assay with Botrytis cinerea was used to screen inhibitory proteins from 60 different plant species. A 4 kDa antifungal peptide (Pa-AFP) inhibitory to hyphal growth of B. cinerea was found in the seeds of P. americana. The peptide Pa-AFP was purified to homogeneity by chromatographies of Sephadex G-50, DEAE-Sepharose, Sephacryl S-300, and C18 reverse-phase HPLC. Western blot analysis showed that a polyclonal antibody raised against the purified peptide cross-reacted with a 4 kDa protein in seeds but not in root and leaf tissues of P. americana. Pa-AFP inhibited the hyphal growth of Botrytis cinerea, Rihzoctonia solani, Fusarium oxysporum, and Magnaporthe grisea. Pa-AFP exhibited growth inhibition of Saccharomyces cerevisiae strain BWG7a, which was sensitive to osmotin.

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Identification and Characterization of Bull Sperm Hyaluronidase Hyal5

  • Kim, Ekyune;Chang, Kyu-Tae
    • Reproductive and Developmental Biology
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    • v.35 no.4
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    • pp.491-498
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    • 2011
  • During mammalian fertilization, germ cell-specific hyaluronidases, such as sperm adhesion molecule 1 (SPAM1) and hyaluronoglucosaminidase 5 (Hyal5), are important for the dispersal of the cumulus mass. In this study, we demonstrated that bull Hyal5 is a single copy gene on chromosome 4 that is expressed specifically in the testis. In addition, we expressed recombinant bull SPAM1 and Hyal5 in human embryonic kidney 293T cells and showed that these enzymes possessed hyaluronidase activity. We also demonstrated that a polyclonal antibody against bull sperm hyaluronidase inhibits sperm-egg interactions in an in vitro fertilization (IVF) assay. Our results suggested that bull Hyal5 may have a critical role in bull fertilization.