• Pediatric Gastroenterology, Hepatology & Nutrition
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• 제15권2호
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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.

• 한국균학회지
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• 제17권2호
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• pp.66-75
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• 1989

임상가검물(臨床可檢物)로부터 분리(分離)된 Aspergillus fumigatus 균주(菌株)의 항원조성(抗原造成)과 이균에 감염(感染)된 환자의 항체반응(抗體反應)의 다양성을 Immunoblotting으로 관찰하였다. SDS-PAGE와 immunoblots상에 나타난 결과를 보면 비교분석된 배양려액항원(培養慮液抗原)의 조성에 있어서 균주간(菌株間)에 정성(定性) 및 정량적(定量的) 차이가 있음을 알수 있었다. 혈청학적(血淸學的) 반응력(反應力)과 특이성(特異性)이 비교적 높은 항원성분(抗原成分)을 동정(同定)하기 위해 그러한 항원들이 비교적 많이 함유된 AFG7을 선택하였다. 전기영동(電氣泳動)으로 분리하여 nitrocellulose paper로 전이흡착(轉移吸着)시켜 A. fumigatus나 기타 진균항원(眞菌抗原)에 양성침강항체(陽性沈降抗體)(면역확산시험(免疫擴散試驗)에서)를 가진 혈청과 반응시킨 결과 적어도 36개의 항원성분이 검출되었다. 그러나 혈청학적 진단가치가 있는 항원성분은 4개 정도에 불과했다. 특이성과 반응력이 가장 높아 표준항원(標準抗原)에 반드시 함유되어야 할 것으로 보는 항원성분은 분자량(分子量)이 82 KD 정도되는 항원이었고 그 외에 11 KD, 26 KD, 30 KD 및 31 KD 등도 우수한 항원성분으로서 앞으로 더 연구검토되어야 할 것으로 본다. 반응력이 큰 항원이면 Coomassie청(靑)에는 흐리게 염색되어도 immunoblots상에는 짙게 염색되는데 비해 반응력이 약한 항원은 그와 반대현상을 나타내어 immunoblotting 분석법이 항원의 반응력과 특이성 관찰에 매우 유용함을 알 수 있다. 많은 항원성분이 균종특이(菌種特異) 항원결정기(抗原決定基)와 함께 광범한 균군(菌群)에 분포하는 항원결정기(抗原決定基)도 가지고 있었다.

• 한국식품과학회지
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• 제36권3호
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• pp.369-374
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• 2004

유전자재조합 콩 Roundup Ready는 Agrobacterium sp. CP4에서 유래한 유전자를 함유하고 있으며 이 삽입 유전자에서 5-enolpyruvylshikimate-3-phosphate synthase(CP4EPSPS)가 발현된다. 본 연구실에서는 이 단백질을 유전자재조합 콩과 시중에서 구입한 두부 시료에서 CP4EPSPS 특이 단클론 및 다클론 항체를 이용하여 immunoblotting법으로 검사하였다. 분리 정제된 CP4EPSPS 재조합 단백질은 단클론 또는 다클론 항체를 이용할 때 각각 $0.006{\mu}g$ 또는 $0.0006{\mu}g$의 검증 한계치로 확인할 수 있었다. 유전자재조합 콩에 발현된 CP4EPSPS 검증 한계치는 단클론 또는 다클론 항체를 이용할 때 각각 $0.001{\mu}g$과 $0.0001{\mu}g$이었다. 다클론 항체를 이용하여 조사된 9종의 두부에서는 2종에서 양성결과를 확인하였다. 본 연구에서 생산된 단클론 및 다클론 항체를 이용하여 확립된 immunoblotting법은 콩 가공식품 중의 glyphosate 내성 유전자재조합 콩 검사에 적용될 수 있을 것이다.

• 대한수의학회지
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• 제32권2호
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• pp.195-205
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• 1992

The soluble antigen profiles and antigenic specificities of Leptospira interrogans serovars icterhaemorrhagiae, canicola, pomona and hardjo were examined by SDS-polyacrylamide gel electrophoresis, crossed immunoeletrophoresis and immunoblotting. The profiles of protein, glycoprotein and fraction containing N-acetylglucosamine of 4 serovars were compared. The protein profiles of 4 serovars were very similar except the range of 14,400 to 30,000 daltons. Molecular weight of glycoprotein of L, pomona was lower than other serovars. L canicola showed extra N-acetylglucosamine bands having molecular weight of 82,000 and 90,000 daltons. In crossed immunoelectrophoresis, a close antigenic relationship was found between L icterohaemorrhagiae and L canicola. In immunoblottings conducted with soluble antigens and rabbit antisera of 4 serovars, Leptospira interrogans serovars possessed cross-reactive antigens and serovar-specific antigens. The molecular weights of serovar-specific antigens were 45,000, 82,000 and 90,000, 31,000 and 24,000 daltons in L icterohaemorrhagiae, L canicola, L pomona, and L hardjo, respectively.

• Parasites, Hosts and Diseases
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• 제53권1호
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• pp.85-93
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• 2015

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.

• Archives of Pharmacal Research
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• 제22권3호
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• pp.262-266
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• 1999

Dopamine transporter (DAT) plays the most important role in terminating the actions of dopamines released into the synaptic cleft. DAT is also the target of various psychotropic drugs such as cocaine and amphetamine. In this study were prepared DAT-specific antibodies using the 2nd extracellular loop of rat DAT as an antigen. The 2nd extracellular loop of the rat DAT was expressed in bacterial as a fusion protein with glutathione-S-transferase, and injected ito rabbits to raise antibodies. Produced antibodies clearly recognized the rat DAT in ELISA, immunoblotting, and immumoprecipitation. As expected from the high sequence homology between the rat and human DAT, the antibodies raised for the rat DAT cross-reacted with the human DAT in the immunoblotting. Considering the specificity for DAT with wide range of applications such as ELISA, immunoblotting, and immunoprecipitation, these antibodies would be valuable tool for understanding the pharmacological actions of dopamine transporter and drug addition.

• 대한수의학회지
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• 제42권1호
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• pp.81-88
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• 2002

The purpose of this study was to conduct diagnosis of bovine paratuberculosis in Kangwon area. Blood samples were collected from 2,261 dairy cows of 162 herds, and the ELISA and immunoblotting using recombinant 34KDa protein of M. paratuberculosis were conducted. The feces collected from dairy cows were cultured on HEY medium with mycobactin-J and PCR was conducted with washing solution of medium 4 weeks after culture. The ELISA had sensitivity of 83.3% and specificity of 96.7%. And the immunoblotting had sensitivity of 83.3% and specificity of 100%. Of the 2,261 dairy cows, 371 cows(16.4%) were positive in ELISA and 75 cows(3.3%) were positive in immunoblotting. And of the 162 herds, 109 herds(67.3%) had an apparent paratuberculosis prevalence by ELISA and 40 herds(24.7%) by immunoblotting. The geographic distribution of herds with paratuberculosis was not uniform. Of the 241 feces samples including 110 feces from ELISA positive cow, 9 feces were positive in culture and PCR. PCR was able to detect the growth of M. paratuberculosis as early as 4 weeks of culture.

• 한국식품영양학회지
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• 제19권3호
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• pp.296-300
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• 2006

시유, 분유, 발효유에 존재하는 우유알레르겐인 ${\beta}$-Lactoglobulin (BLG)를 측정하기 위해 면역 blotting 및 간접결합면역 분석법(Competitive-indirect Enzyme linked Immunosorbent Assay; Ci-ELISA)으로 분석하였다. Immunoblotting 결과, 발효유에서는 우유 알레르기 환자의 IgE와의 반응이 나타나지 않았고 분유 및 시유에서는 약하지만 반응이 나타났다. 다클론 항체 및 우유 알레르기 환자의 IgE로 이들의 BLG 함량을 정량한 결과, BLG 함량은 시유가 가장 높았으며 발효유는 매우 낮았다. 이상의 결과에서 발효유에서는 가장 낮은 BLG 함량이 측정되어 우유 알레르기 환자에 대한 저알레르기원성 식품으로 사용될 수 있을 것으로 사료된다.

• 대한의생명과학회지
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• 제18권2호
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• pp.96-103
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• 2012

Kv 4.2 ion channel protein has an ability to open at subthreshold membrane potentials and to recover quickly from inactivation. That is very important for neuronal signal transmission in vertebrate brain. In order to purify Kv 4.2 protein, the novel purification methods were experimented. The purification procedure utilized chromatography on DE-52 ion exchange column and affinity chromatography on a WGA-Sepharose 4B, and Kv 4.2 affinity column chromatography. It was found that 0.5% (wt./vol.) Triton X-100 detergent in lysis buffer worked well for Kv 4.2 protein solubilization from rat brain membrane. Protein quantitative determination was conducted by BCA method at 562 nm for each purification step to avoid determination interference of protein at 280 nm by detergent. The confirmation of Kv 4.2 existence and amount is performed using by SDS-PAGE/immunoblotting or 96-well dot blotting. The Kv 4.2 without interacting protein that contains carbohydrate, was purified from novel biochemical 3-steps purification method for further research.

• Journal of Oral Medicine and Pain
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• 제24권3호
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• pp.261-267
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• 1999

The present investigation was carried out to identify salivary components of mucosal pellicle and to explore the difference of mucosal pellicle components according to the location of oral mucosa. By using antisera and immunoblotting, high-(MG1) and low-(MG2) molecular-mass salivary mucins, amylase, IgA, proline-rich proteins(PRPs) were detected in mucosal pellicle in vivo. In addition, the data indicated that mucins, IgA and proline-rich proteins could be cleaved into lower-molecular-mass products, whereas the IgA, proline-rich proteins could also be cross-linked into higher-molecular-mass complexes. Mucosal pellicles from buccal, labial and palatal mucosa showed similar pattern in immunoblotting experiments using anti-MG2 and anti-PRPs antisera. The data from this study suggest that during mucosal pellicle formation multiple components of saliva adsorb to oral mucosal epithelial cell surfaces, and selected components can be proteolytically cleaved into smaller fragments and/or cross-linked into higher-molecular products.