• 대한바이러스학회지
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• 제30권1호
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• pp.61-70
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• 2000

Standard plaque assay using agarose-overlay has long been used for titration of many infectious virus particle. Plaque assay for the titration of varicella-zoster virus and its live vaccine requires three intermittent agarose overlay to visualize plaques. Overall procedure of the assay takes at least nine days from virus inoculation and microbe contamination including fungi is frequently accompanied during incubation period. We studied whether an immunofocus assay in conjunction with peroxidase-mediated immunohistochemical reaction may replace the standard plaque assay for the virus titration by comparing the two methods. A linear relationship was observed between number of foci and virus dilution. The number of foci in a given dilution of virus appeared a little higher than counted plaques formed in standard plaque assay. Independent titration results obtained from two assay methods for a given dilution of virus demonstrated a strong correlation ($r^2=0.99$). Foci of virus infected cells as revealed by the enzyme reaction could be counted either 4 days post-infection (p.i.) under low magnification (40X) microscopy, or 6 days p.i. by naked eye observation. Larger size of cell cuture plate, virus adsorption at $35^{\circ}C$, and 10% FBS in diluent appeared to be better conditions for the assay. Immunofocus assay will be an effective and dependable titration method for varicella-zoster virus and its live vaccine in place of the standard plaque assay in respect to accuracy, costs, and experimental convenience.

• 대한수의사회지
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• 제27권12호
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• pp.725-728
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• 1991

The END method for titration of hog cholera virus and its serum neutralizing antibody was improved using ST cells grown and kept in modified media. ST cells were grown in Eagles media containing 0.5$\%$ lactalbumin hydrolysate, 10$\%$

• 약학회지
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• 제49권3호
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• pp.217-224
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• 2005

Human Immunodeficiency Virus type 1 (HIV-l) based lentivirus vector has demonstrated great potential as gene therapy vectors mediating efficient gene delivery and long-term transgene expression in both dividing and nondividing cells. However, for clinical studies it must be confirmed that vector preparations are safe and not contaminated by replication competent lentivirus (RCL) related to the parental pathogenic virus, HIV-l. In this study, we would like to establish the method for titration and RCL detection of lentivirus vector. The titration was determined by vector expression containing the green fluorescent protein, GFP in transduced cells. The titer was $1{\times}10^7$ Transducing Unit/ml in the GFP expression assay and $8.9{\times}10^7$ molecules/ml in the real-time PCR. Also, for the detection of RCL, we have used a combination method of PCR and p24 antigen detection. First, PBS/psi and VSV-G region in the genomic DNA of transduced cells was detected by PCR assay. Second, transfer and expression of the HIV-1 gag gene was detected by p24 ELISA. In an attempt to amplify any RCL, the transduced cells were cultured for 3 weeks (amplification phase) and the supernatant of amplified transduced cell was used for the second transduction to determine whether a true RCL was present (indicator phase). Analysis of cells and supernatant at day 6 in indicator phase were negative for PBS/psi, VSV-G, and p24 antigen. These results suggest that they are not mobilized and therefore there are no RCL in amplification phase. Thus, real-time PCR is a reliable and sensitive method for titration and RCL detection of lentivirus vector.

• 한국동물위생학회지
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• 제41권4호
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• pp.263-269
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• 2018

Developing drugs targeting respiratory pathogen is essential to control respiratory diseases. Many experiments have been performed under in vivo situation. However, in vivo experiments have economical and ethical issues. The objective of this study was to determine the possibility of developing an ex vivo lung culture system with possible application for respiratory infection studies. After isolating lungs from naïve pigs, agarose-inflated lung tissues were prepared and sliced manually. These sliced lung tissues were then subsequently placed on 24-well plates. Eight different combinations of media were used to determine the optimum ex vivo lung culture condition. In addition, lung tissues were infected with porcine reproductive and respiratory syndrome (PRRS) virus at a titer of $1{\times}10^4\;TCID_{50}/mL$. Virus growth was confirmed by titration in MARC-145 cells at 2, 4, 6 days post infection (dpi). We found that ex vivo lung culture in physiological environment was not media specific based on histopathology and cytotoxicity. However, under virus-infected condition, thickened alveolar walls in the lung tissues and stable virus titers at 2, 4, 6 dpi were shown in F12K medium suggesting that it was useful for tissue maintenance and virus infection using PRRS virus infected lung tissues. The present study shows the possibility of using porcine ex vivo lung model for respiratory infection studies.

• 생명과학회지
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• 제22권6호
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• pp.703-712
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• 2012

AAV는 매우 안전하고 효율적인 유전자전달방법으로 인정되어 왔다. 그러나, AAV가 가진 생물의약품으로서 단점은 효율적이고 재현성 높은 생산법이 취약하고, 또한 다양한 혈청형을 간단한 한 가지 공통적 방법으로 신뢰성 있게 정량하는 방법이 개발되어야 하는 것이다. 따라서, 본 연구에서는 AAV2와 아데노바이러스를 동시에 감염하는 종래의 생산법에 의한 효율성과 새로운 생산법, 즉 AAV2 감염 후 pHelper 플라스미드를 transfection 하는 방법을 통한 생산효율성을 비교하였고, HEK293과 293T를 생산세포주로 하여 시간에 따른 생산효율성도 분석하였다. 그 결과 AAV2와 pHelper DNA를 포함한 새로운 생산법은 기존의 방법에 비해 10배 이상 높은 생산효율성을 보였고, 293T에서 AAV2를 10 MOI로 감염한 후 5일째에 가장 높은 생산효율성을 보였는데, 생산세포 한 개 당 $1.61{\times}10^5$ virus genomes (v.g.)을 생산하는 결과였다. 따라서 이 생산조건을 다른 혈청형 생산에 적용한 결과, 모든 혈청형에서 생산세포주 한 개 당 $10^5$ v.g. 이상을 생산하는 효율성을 보였다. 한편, 다양한 AAV 혈청형을 한 가지 공통적인 방법으로 정량하기 위해 the universal PCR 프라이머를 제작하였고, 그것을 이용하여 신뢰성 높고 10개 분자까지도 증폭이 가능한 결과를 모든 혈청형에서 얻었다. 그러므로 이 한 쌍의 정량용 the universal 프라이머는 임상시험용 아데노바이러스벡터에 존재하는 AAV오염을 검출하는 것에도 사용 가능하다.

• Journal of Microbiology and Biotechnology
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• 제21권2호
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• pp.170-175
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• 2011

This study was carried out for the molecular level identification of recombinant protein vaccine efficacy, by oral feeding against white spot syndrome virus infection, with the comparison of viral mRNA transcriptional levels in shrimp cells. For the determination of WSSV dilution ratio for the vaccination experiment by oral feeding, in vivo virus titration was carried out using different virus dilutions of virus stock ($1{\times}10^2$, $2{\times}10^2$, and $1{\times}10^3$). Among the dilution ratios, $2{\times}10^2$ diluted WSSV stock was chosen as the optimal condition because this dilution showed 90% mortality at 10 days after virus injection. Recombinant viral proteins, rVP19 and rVP28, produced as protein vaccines were delivered in shrimps by oral feeding. The cumulative mortalities of the shrimps vaccinated with rVP19 and rVP28 at 21 days after the challenge with WSSV were 66.7% and 41.7%, respectively. This indicates that rVP28 showed a better protective effect against WSSV in shrimp than rVP19. Through the comparison of mRNA transcriptional levels of viral genes from collected shrimp organ samples, it was confirmed that viral gene transcriptions of vaccinated shrimps were delayed for 4~10 days compared with those of unvaccinated shrimps. Protection from WSSV infection in shrimp by the vaccination with recombinant viral proteins could be accomplished by the prevention of entry of WSSV due to the shrimp immune system activated by recombinant protein vaccines.

• 대한바이러스학회지
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• 제29권1호
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• pp.33-38
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• 1999

Retroviral vector provide a highly efficient method for gene transfer into eukaryotic cells. This vector system can be divided into two components; the retroviral vector itself and the retroviral packaging cell line. The key improvement in the design of these two components are, focused on two aspects; the reduction of helper virus production and high titer-virus. We used PA317 for retrovirus packaging cell line, for its high producibility of viral titer. To test the ability of the vectors to generate high titer-virus, we have chosen four different retroviral vectors; LN, LNSX, LNCX and LXSN. To test easily the viral titer, we have made recombinant construction with CD4 and CD8, checked their viral titer and stained their surface expression. LXSN which contain SV40 early promoter in front of neo gene showed best results in viral transient transfection assay, dot blot assay and surface expression. In addition, recombinant containing CD8 generally showed much higher viral titration and surface expression than CD4.

• 한국동물위생학회지
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• 제24권1호
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• pp.21-29
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• 2001

To compare of serum antibody titers using ELISA and HI, serum samples were collected from 100 breeders and their progeny 550 broilers. The breeders and broilers were vaccinated with infectious bronchitis(IB)- and Newcastle disease(ND)-viruses according to general vaccination program. The antibodies in serum samples against IB and ND viruses were detected by enzyme-linked immunosorbent assay(ELISA) using commercial ELISA kit and hemagglutination inhibition(HI) test. Geometric mean titer(GMT) of ELISA and In titers were monitored from 1-day-old to 35-day-old broilers and compared to those of breeder chickens. The antibody titers of breeders vaccinated with ]B virus showed 47,800, ELISA and 7.2, HI, respectively. Progeny chicks, 1-day-old, vaccinated with IBV showed high antibody titers than those of breed chickens. Those chicks were maintained protective antibody levels until 11-day-old. From 14-day-old, the antibody level decreased below protective levels. In ND, breeders serum antibody titers ELISA and Eiu were 30,200 GMT and 8.7 HI titer, respectively. On 1-day-old chicks, antibody levels was decreased to half in ELISA(16,270) compared with those of breeders, but In titers was 7.4. Progeny broilers, protective antibody level was maintained until 14- day-old by ELISA, but at 11-day-old by HI titers. After then, ND antibody titer was continuously decreased underdefense level. These result indicated that the ELISA method be more sensitive than HI titration to detect serum antibody level for IBV and NDV.

• 대한바이러스학회지
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• 제27권1호
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• pp.19-27
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• 1997

본 연구에서는 영장류에서 감염성 면역결핍 증상을 일으키는 type D simian retrovirus (SRV)를 검출하기 위해 SRV env 유전자의 특정 지역을 선정하여 증폭, 검색하는 중합효소 연쇄반응 (PCR)법을 개발하였다. 증폭반응의 대상부위인 SRV env 유전자의 3' 후반부는 서로 다른 3 종류의 SRV subtype 1, 2, 그리고 subtype 3에 걸쳐 높은 보존율을 보이고 있다. 반응 결과, 1차 PCR 만으로 3 종류의 SRV subtypes를 동시에 검출, 증폭하였으며 SRV와 더불어 영장류에서 감염성 면역결핍을 유도하는 주요 바이러스 simian immunodeficiency virus 또는 simian T-Iymphotropic virus type 1에 감염된 영장류의 peripheral blood mononuclear cells (PBMCs)을 비교, 완전한 증폭 특이성이 확인되었고 교차반응으로 인한 위양성반응은 발견되지 않았다. 한편, 위 증폭반응의 검출 민감도 측정을 위해 준정량적 적정 PCR을 수행하였으며 SRV 게놈과 증폭 대상 부위의 분자량을 기준으로 한 본 PCR법의 검출 한계는 단 한번의 증폭과 간단한 ethidium bromide 염색만으로 적어도 $5-7{\times}10^4$ 개의 PBMCs 중 한 개의 감염세포를 검출할 수 있는 것으로 확인되었다. 본 연구에서 확인된 신속성과 반응 특이성, 그리고 높은 민감도의 본 PCR 법은 현재 유일한 AIDS 연구모델인 영장류의 SIV 감염 연구 전후에 반드시 필요한 효과적인 SRV 감염검색에서 ELISA법의 위양성에 의한 약점을 보완하고 보다 높은 검출 민감도를 확보함으로써 연구모델 실험의 오류를 최소화하는 데 중요한 역할을 하게 될 것이다.

• 대한수의학회지
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• 제29권4호
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• pp.503-515
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• 1989

Critical parameters affecting sensitivity and specificity of an enzyme-linked immunosorbent assay(ELISA) for detection of antibodies to avian infections bronchitis virus(IBV) were standardized. By adopting the optimized conditions an equation calculating ELISA antibody titers from the observations at single serum dilution was formulated. The purified antigen of IBV Mass-41 strain was dispensed into polystyrene microplate wells at a concentration of 300ng per well($100{\mu}l$) and the plates were coated by completey drying at $37^{\circ}C$. Diluted chicken serum and horseradish peroxidase conjugated goat anti-chicken IgG were added in order in $100{\mu}l$ volumes per well and allowed to react for 30 minutes each at room temperature. Just before use and after each reaction the plates were washed three times with distilled water. Finally o-phenylenediamine solution was added as an enzyme substrate. After incubation for another 15 minutes at room temperature absorbances were read at 492nm. Hyperimmune serum against Mass-41 strain was used as internal reference positive(IRP) serum. After repeated titration of IRP and negative serum, a constant titer of IRP was determined. Serum titrations were carried out for various sample sera together with IRP and negative sera and the observed titers of sample sera were corrected by reflecting the ratio between observed and constant titers of IRP serum. These corrected titers of the sample sera were plotted against sample/positive(S/P) OD ratios. All the OD's measured in the serum titrations were also corrected by substracting negative serum OD. The following equation was formulated from the above data; $Log_{10}$ ELISA titer=$5.568({\log}_{10}S/P)+4.161$ Thus it was possible to calculate ELISA titer by measuring absorbance at 1/400 single serum dilution. Titer measured by cross ELISA tests employing Mass-41 strain and three local IBV isolates were similar. These results suggest that the ELISA tests standardized in this study can be used for evaluating not only vaccinal immunity but also for infection status against fields IBV's.