• 한국식품영양과학회지
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• 제30권4호
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• pp.600-604
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• 2001

감마선 조사된 새우의 신속한 판별을 위한 방법으로 갈색새우의 TPM을 항원으로 하여 개별적으로 생산된 M-IgG와 P-IgG를 이용한 Sandwich ELISA를 분석법으로 확립하였다. M-IgG를 항원 포획을 이한 coating 항체로 사용하고, P-IgG를 포획된 TPM에 대한 반응항체로 사용하였을 때, 12.5에서 50$\mu\textrm{g}$/mL의 농도 범위에서 TPM을 정량할 수 있었다. 감마선 조사된 새우의 TPM 농도는 선량에 의존하며 감소하였고, 감마선 조사와 가열 또는 냉동 등의 병용 처리에서도 선량에 의존하며 감소하였다. 이 결과는 면역분석기법의 하나인 Sandwich ELISA가 감마선 조사된 새우의 검지법으로 이용될 수 있다는 가능성을 나타내었다.

• 환경생물
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• 제19권4호
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• pp.302-312
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• 2001

Immunoglobulin G was purified by 40% $(NH_4)_2SO_4$ precipitation, DEAE-Sephadex, Sephadex G-150 column chromatographies from rabbit antiserum against V. vulnificus ATCC 27562 O antigen and used for immunological test for V. vulnificus isolates. The profiles of cell lysate total protein and outer membrane protein from the isolates were analyzed by SDS-PAGE and densitometry. The overall profiles in all isolates were similar. Distict protein band was observed in comparison with V. parahaemolyticus. Western Blotting with rabbit Immunoglobulin G against cell lysates and OMP of V. vulnificus isolates showed a strong antigenic response to antigen 66, 60, 54, 48, 33 and 26 kDa which were common to all strains examined. The 26 kDa antigen showed V. vulnificus specific antigen in comparison with Vibrio parahaemolyticus. A sandwich enzyme-linked immunosorbent assay was developed by using rat anti-V. vulnificus ATCC 27562 polyclonal antibodies as capture antibody, a purified rabbit IgG antibody as detector antibody, and goat anti-rabbit IgG-alkaline phosphatase conjugate as developer antibody. When four V. vulnificus isolates were tested, the reactivity showed from 50 to 70% by sandwich ELISA.

• Bulletin of the Korean Chemical Society
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• 제30권1호
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• pp.77-82
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• 2009

Thrombin activatable fibrinolysis inhibitor (TAFI) also known as plasma procarboxypeptidase B or U is a 60 kD glycoprotein, which is the major modulator of fibrinolysis in plasma. TAFI is a proenzyme, which is activated by proteolytic cleavage to an active carboxypeptidase B-like enzyme (TAFIa, 35.8 kD) by thrombin/thrombomodulin and plasmin. Modulation of fibrinolysis occurs when TAFIa enzymatically removes C-terminal lysine residues of partially degraded fibrin, thereby inhibiting the stimulation of tissue plasminogen activator (t-PA) modulated plasminogen activation. TAFIa undergoes a rapid conformational change at $37{^{\circ}C}$ to an inactive isoform called TAFIai. Potato tuber carboxypetidase inhibitor (PTCI) was shown to specifically bind to TAFIa as well as TAFIai. In this study, a novel immunoassay TAFIa/ai ELISA was used for quantitation of the two TAFI activation isoforms TAFIa and TAFIai. The ELISA utilizes PTCI as the capture agent and a double antibody sandwich technique for the detection. Low levels of TAFIa/ai antigen levels were detected in normal plasma and elevated levels were found in hemophilia A plasmas. TAFIa/ai antigen represents a novel marker to monitor fibrinolysis and TAFIa/ai ELISA may be a valuable assay for studying the role of TAFI in normal hemostasis and in pathological conditions.

• Journal of Microbiology
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• 제35권2호
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• pp.87-91
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• 1997

Monoclonal antibodies were prepared in order to an assay method for Vibrio vulnificus. Sixteen mouse ybridoma cell lines were established by immunization of whole cell antigen to BALB/c mice, fusion with SP2/O myeloma cells, and cloning. Most of them secreted IgM.lambda. antibodies. A sandwich enzyme-linked immunosorbent assay was developed with rabbit anti-V. vulnificus polyclonal antibodies as capture antibody, an IgM monoclonal antibody as detector antibody, and goat anti-mouse IgM-alkaline phosphatase conjugate as developer antibody. The range of detection was 10$\^$4/ to 10 V. vulnificus cells per microplate well. When four related Vibrio species were tested for cross-reactions, V. parahaemolyticus showed 3.5% reactively and V. carchariae, V. fluvialis, and V. furnisii showed negligibal (<1%) cross-reactivity.

• 대한수의학회지
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• 제43권1호
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• pp.129-137
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• 2003

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failures in sows and respiratory problems in piglets. The nucleocapsid(N) protein, encoded by the open reading frame 7 (ORF7) gene, is known to be the most abundant and antigenic protein in PRRS virus. Therefore, it was suggested that the N protein could be a suitable candidate for the detection of PRRS virus-specific antibodies and diagnosis of PRRS. In the present study, the ORF7 gene encoding the N protein was cloned and expressed as a fusion protein with the glutathione S-transferase (GST) in Escherichia coli. The resulting GST-N recombinant protein was used as an antigen for an indirect sandwich enzyme-linked immunosorbent assay (i-ELISA). Expressed GST-N recombinant protein was migrated at 41 kDa and reacted with ORF7-specific monoclonal antibody by Western blotting. In order to increase the specificity of the ELISA for the detection of PRRS virus-specific antibodes, an i-ELISA was developed using an anti-GST antibody as a capture antibody. The sensitivity and specificity of developed i-ELISA were 92% and 96%, respectively. Based on these results, it was suggested that the i-ELISA is a simple and rapid test for screening a large number of swine sera for the anti-PRRS virus antibodies.

• 대한수의학회지
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• 제44권4호
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• pp.625-635
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• 2004

Trichnuris suis does not excrete eggs during larval stage as well as in particular adult stage, It is impossible to diagnose by use of fecal examination method in those periods. Therefore, serological diagnostic method can be very useful for those stages. In order to produce monoclonal antibody, specific somatic and secretory-excretory (SE) antigens of T. suis were identified and analyzed by SDS-PAGE and Western blot. Monoclonal antibody-producing hybridoma cells were cloned, which were made of popliteal lymph node of BALB/c mice immunized with a 45 kDa somatic antigen of T. suis. Five clones (1B9, 2C4, n2C5, 2D7 and 2D8) showing strong responses to T. suis antigens were selected and the isotype identified. All monoclonal antibodies were IgG1 isotype and the light chains were k chain. Established monoclonal antibodies reacted specifically to somatic and SE antigens of T. suis and did not cross-reacted to antigens of ascaris suum, trichuris vulpis, or Trichinella spiralis. The sensitivity of somatic and SE antigens against these monoclonal antibodies were significant (p<0.01) associated with those of positive and negative sera.

• 센서학회지
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• 제23권4호
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• pp.278-283
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• 2014

In this study, we propose an immunosensor using zinc oxide nanorods (NRs) inside PDMS channel for detecting the influenza A virus subtype H7N9. ZnO with high isoelectric point (IEP, ~9.5) makes it suitable for immobilizing proteins with low IEP. In this proposed H7N9 immunosensor structure ZnO NRs were grown on the PDMS channel inner surface to immobilize H7N9 capture antibody. A sandwich enzyme-linked immunosorbent assay (ELISA) method with was used 3,3',5,5' tetramethylbenzidine (TMB) for detecting H7N9 influenza virus. The immunosensor was evaluated by amperometry at various H7N9 influenza antigen concentrations (1 pg/ml - 1 ng/ml). The redox peak voltage and current were measured by amperometry with ZnO NWs and without ZnO NWs inside PDMS channel. The measurement results of the H7N9 immunosensor showed that oxidation peak current of TMB at 0.25 V logarithmically increased from 2.3 to 3.8 uA as the H7N9 influenza antigen concentration changed from 1 pg/ml to 1 ng/ml. And then we demonstrated that ZnO NRs inside PDMS channel can improve the sensitivity of immunosensor to compare non-ZnO NRs inside PDMS channel.