• 대한바이러스학회지
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• 제28권1호
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• pp.31-38
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• 1998

In Korea, all domestic made test systems for detecting antibodies in HIV-1 contain the antigens from human immunodeficiency type 1 (HIV-1) subtype B. However, because HIV-1 subtype O is significantly different in amino acid sequences from all other subtypes of HIV-1, there has been a need for developing a test for detecting antibodies in subtype O. For this purpose, the entire nucleotide sequence corresponding to the extracellular domain of the transmembrane glycoprotein of HIV-1 subtype O was synthesized with consideration of Escherichia coli condon usage. Various regions of the extracellular domain were cloned into E. coli expression vectors and tested for levels of protein production. The nucleotide sequence, named ECTM, that can encode a 129 amino acid-long peptide, was found to be expressed at a high level in E. coli. The protein of approximately 17 kDa specifically reacted with sera from individuals infected with HIV-1 subtype O. The ECTM protein was purified to near homogeneity by the CM-T gel chromatography, using concentrated, denatured inclusion bodies. In Western blot analysis, the purified viral antigen reacted with sera from individuals infected with subtype O more efficiently than subtype B. The enzyme linked immunoabsorbent assay (ELISA) system was developed using the subtype O viral protein and compared with the commercially available kit lacking the antigens from subtype O. The ELISA kit containing the subtype O antigen ECTM alone efficiently reacted with sera from individuals infected with subtype O. The subtype O antigen-containing kit produced a positive absorbence even when sera were diluted 512-fold, suggesting a high sensitivity. The commercially available kit also reacted with subtype O sera, but produced a negative result at a dilution of 8-fold. Our results suggest that the currently available kit may not be able to efficiently detect subtype O sera and that the viral protein developed in this study may be added to the current system to maximize the detection of sera from individuals infected with subtype O.

• 농약과학회지
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• 제5권2호
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• pp.1-12
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• 2001

다클론항체를 이용하여 환경 시료중 유기인계 살충제 acephate를 분석할 수 있는 경합적 간접효소면역측정법(competitive indirect ELISA)을 개발하기 위하여 분석물과 화학구조가 유사하며 hexanoic acid를 지닌 3종의 hapten을 합성하였다. 이들 hapten을 active ester 방법으로 담체단백질인 keyhole limpet hemocyanin과 접합시켜 면역원으로 사용하있고, bovine serum albumin과 접합시켜 homologous 혹은 heterologous ELISA를 위한 코팅항원으로 사용하였다. 생산된 항체들과 코팅항원을 최종선발하여 acephate 잔류분석을 위한 ELISA를 위하여 최적화하였다. 단백질의 농도가 $1{\mu}g/mL$인 코팅항원 (hapten-3-BSA)과 다클론항체 #8377를 16000배로 희석한 heterologous ELISA에서 최적화된 acephate의 $IC_{50}$ 값은 110 ng/mL이었고, 분석범위와 최소검출한계는 각각 $10{\sim}1000$과 4 ng/mL 이었다. Acephate의 주분해산물이고 살충제로 사용되는 methamidophos을 위시한 몇몇 유기인계 살충제의 항체에 대한 교차반응은 모두 0.02%이하였다. 이와 같은 결과는 본 ELISA가 농산물 및 환경시료중의 acephate 잔류분석을 위하여 편리한 방법이 될 수 있음을 시사한다.

• Parasites, Hosts and Diseases
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• 제51권6호
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• pp.763-766
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• 2013

A synthetic peptide was prepared based on the antigenic region of Paragonimus westermani pre-procathepsin L, and its applicability for immunodiagnosis for human paragonimiasis (due to Paragonimus heterotremus) was tested using an ELISA to detect IgG4 antibodies in the sera of patients. Sera from other helminthiases, tuberculosis, and healthy volunteers were used as the references. This peptide-based assay system gave sensitivity, specificity, accuracy, and positive and negative predictive values of 100%, 94.6%, 96.2%, 100%, and 88.9%, respectively. Cross reactivity was frequently seen against the sera of fascioliasis (75%) and hookworm infections (50%). Since differential diagnosis between paragonimiasis and fascioliasis can be easily done by clinical presentation and fascioliasis serology, this cross reaction is not a serious problem. Sera from patients with other parasitoses (0-25%) rarely responded to this synthetic antigen. This synthetic peptide antigen seems to be useful for development of a standardized diagnostic system for paragonimiasis.

• 한국동물위생학회지
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• 제36권2호
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• pp.105-110
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• 2013

Bovine viral diarrhea virus (BVDV) is very important disease in domestic and wild ruminants and has a world wide distribution. Cattle persistently infected with BVDV (BVDV-PI) are the primary reservoir for BVDV infection in Korean native cattle herds. The prevalence of cattle persistently infected with BVDV (BVD-PI) was determined using 4,260 heads from 29 Korean native cattle farms at 8 districts from 2011 to 2012. The sera and ear nothches were collected for each sample. We surveyed BVD-PI cattle using antibody ELISA and antigen capture ELISA for detection of antibody and antigen respectively. Three thousand seventy-six cattle (72.2%) were positive for BVDV antibody and a total of 27 BVD-PI cattle were found in 12 farms. 11 cattle (40.7%) out of the total 27 BVDV-PI cattle were six months old or under. The positive rate of BVDV antibody (83.2%) from 12 farms with BVD-PI cattle was higher than the positive rate of BVDV antibody (63.6%) from 17 farms without BVD-PI cattle.

• 대한바이러스학회지
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• 제30권2호
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• pp.125-130
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• 2000

Infection with human cytomegalovirus (HCMV) is of considerable clinical relevance after placental transmission and in immunosuppressed patients such as transplant recipients or patients with AIDS. The rapid detection method of HCMV has been required to overcome the time-consuming methods such as classical plaque assay or other immunological methods. This study was performed to establish the in situ ELISA, in which human lung fibroblasts infected with HCMV were fixed and used directly as antigen in 96 well culture plate. Expressed HCMV antigens were detected with HCMV-specific monoclonal antibodies. This method could detect HCMV dose-dependently upto $3{\times}10^2\;pfu/ml$. Antiviral activity of ganciclovir could be assayed within the known range of effective dose. This result showed that HCMV could be quantitated by in situ ELISA. The chemical, which was selected on the basis of component analysis in natural product, was tested to have the anti-HCMV activity by in situ ELISA, and three among five samples were found to have anti-HCMV activity with the dose-dependent manner. Conclusively in situ ELISA could be useful method for quantitation of HCMV and screening antiviral activity of samples to HCMV.

• 한국식품과학회지
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• 제32권3호
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• pp.538-543
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• 2000

가열 처리된 우육, 돈육, 계육, 양육과 염소육과의 종판별을 위한 효소면역측정법(ELISA)을 확립하기 위하여 염소육을 $98^{\circ}C$에서 15분간 열처리해서 추출액을 분리하였다. 열에 안정한 주요 단백질(TS)은 분자량이 36과 38kDa이었고 두 분자량대의 단백질의 pI값은 4.5로 확인되었으며 면역원으로 사용하기 위해 이온교환 (DEAE-Sephadex A-50)과 겔 여과(Sephadex G-75) 크로마토그래피로 정제하였다. 분리, 정제된 TS단백질을 토끼에 면역하여 염소육에 대해 특이성을 갖는 항체를 생산하고 항TS 항체를 이용하여 간접경합 ELISA를 확립하였다. 항TS 항체는 가열 처리된 계육, 우육, 돈육, 양육과는 반응성이 전혀 없었고 동일하게 처리된 염소육과 지표단백질인 TS에만 반응성을 보였다. 따라서, 본 연구에서 확립한 간접경합 효소면역측정법은 가열 처리된 육류 중 염소육 판별에 웅용 가능함을 시사하였다.

• Korean Chemical Engineering Research
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• 제49권4호
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• pp.387-392
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• 2011

생촉매인 효소의 기질선택성은 다양한 분야에서 유용하게 이용되고 있다. 그 중에서도 효소결합면역흡착검사(enzyme-linked immunosorbent assay, ELISA)는 항원항체의 결합을 항체와 공유결합된 효소의 반응으로 나타냄으로써 다양한 항원들의 진단을 가능케 했다. 하지만 기존의 효소결합면역흡착검사는 하나의 항체당 하나의 효소가 결합된 형태이기 때문에 감도(sensitivity)의 증가 폭에 그 한계가 있으며, 이를 극복하기 위한 방안으로 하나의 항체당 결합된 효소의 수를 증가시킴으로써 혁신적인 감도의 향상을 가져오는 연구가 진행되었다. 최근 나노바이오촉매(nanobiocatalyst, NBC) 접근방식을 이용한 효소활성의 안정화는 효소결합면역흡착검사의 감도 향상뿐만 아니라 그 성능의 안정성을 확보할 수 있는 연구결과로 이어지고 있다. 본 총설에서는 일반적인 효소결합면역흡착검사의 기본적인 원리와 감도향상을 위한 연구, 그리고 성능안정성(performance stability)을 향상시키기 위한 나노바이오촉매-결합면역흡착검사(Nanobiocatalyst-Linked Immunosorbent Assay, NBC-LISA)에 대하여 살펴보고자 한다.

• The Plant Pathology Journal
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• 제26권1호
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• pp.25-31
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• 2010

Papaya ringspot virus (PRSV) causes the most widespread and devastating disease in papaya. Isolates of PRSV originating from different geographical regions in south India were collected and maintained on natural host papaya. The entire coat protein (CP) gene of Papaya ringspot virus-P biotype (PRSV-P) was amplified by RTPCR. The amplicon was inserted into pGEM-T vector, sequenced and sub cloned into a bacterial expression vector pRSET-A using a directional cloning strategy. The PRSV coat protein was over-expressed as a fusion protein in Escherichia coli. SDS-PAGE gel revealed that CP expressed as a ~40 kDa protein. The recombinant coat protein (rCP) fused with 6x His-tag was purified from E.coli using Ni-NTA resin. The antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified PRSV. The purified rCP was used as an antigen to produce high titer PRSV specific polyclonal antiserum. The resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay and compared its sensitivity levels with ELISA based assays for detection of PRSV isolates. IC-RT-PCR was shown to be the most sensitive test followed by dot-blot immunobinding assay (DBIA) and plate trapped ELISA.

• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.268-273
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• 2015

The fluorescent-antibody-to-membrane-antigen (FAMA) test is regarded as the "gold standard" to detect protective antibodies to varicella-zoster virus (VZV) because of its high sensitivity and specificity. Because the classic FAMA test uses an infectious virus for detection of antibodies to VZV, it is labor-intensive, and also requires special equipment for handling the virus. For this reason, we attempted to develop a simple and safe FAMA assay. Because VZV glycoprotein E (gE) is one of the major VZV glycoproteins, we used the gE protein for the FAMA test (gE FAMA). Here, we demonstrate that overexpression of gE in HEK293T cells can be used to measure antibodies in human serum, and that gE FAMA titers are closely correlated with gpEIA ELISA data. These results indicate that our gE FAMA test has the potential to measure antibodies to VZV.

• BMB Reports
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• 제30권6호
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• pp.403-409
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• 1997

Studies were undertaken to develop a competitive radioimmunoassay (RIA) and indirect antigen capture enzyme-linked immunosorbent assay (ELISA) for the determination of 5'-deoxy-5'-methylthioadenosine (MTA), which is formed from decarboxylated S-adenosylmethionine by spermidine and spermine synthase. Specific antiserum against MTA was raised in rabbits by immunization with MTA-BSA which was prepared by coupling BSA to oxidized MTA with periodate. Since MTA is oxidized easily to the sulfoxide, the sulfhydryl reagent, DTT. was added to the immunogen. For RIA, immunocomplexes were separated from free MTA by using ammonium sulfate precipitation. The antiserum showed almost no cross-reactivity with a variety of other nucleotides and riboses. But, the level of cross-reactivity of 5'-isobutylthioadenosine (SIBA) was high. These results showed the importance of hydrophobicity adjacent to the 5'-OH for determining antigenicity. The lower limit of detection by this assay was 100 fmol of MTA per tube. Using this assay. MTA levels were more easily and precisely determined in biological samples when compared with HPLC analysis. The RIA procedure is less time consuming. More than 24 analyses can be carried out in 2 h and required only a very small amount of sample ($20{\mu}l$ serum). In ELISA, biotin conjugated MTA-BSA was used as the labelled MTA. The sensitivity limit of this assay was lower than 100 pmol.