• Journal of Veterinary Science
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• 제23권5호
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• pp.32.1-32.11
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• 2022

Background: Classical swine fever (CSF) is a severe infectious disease of pigs that causes significant economic losses to the swine industry. Objectives: This study developed a solid-phase blocking enzyme-linked immunosorbent assay (spbELISA) method for the specific detection of antibodies against the CSF virus (CSFV) in porcine serum samples. Methods: A spbELISA method was developed based on the recombinant E2 expressed in Escherichia coli. The specificity of this established spbELISA method was evaluated using reference serum samples positive for antibodies against other common infectious diseases. The stability and sensitivity were evaluated using an accelerated thermostability test. Results: The spbELISA successfully detected the antibody levels in swine vaccinated with the C-strain of CSFV. In addition, the detection ability of spbELISA for CSFV antibodies was compared with that of other commercial ELISA kits and validated using an indirect immunofluorescence assay. The results suggested that the spbELISA provides an alternative, stable, and rapid serological detection method suitable for the large-scale screening of CSFV serum antibodies. Conclusions: The spbELISA has practical applications in assessing the vaccination status of large pig herds.

• BMB Reports
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• 제39권1호
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• pp.55-60
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• 2006

We describe a phage display strategy, based on the differential resistance of proteins to denaturant-induced unfolding, that can be used to select protein variants with improved conformational stability. To test the efficiency of this strategy, wild-type and two stable variants of ${\alpha}_1$-antitrypsin (${\alpha}_1AT$) were fused to the gene III protein of M13 phage. These phages were incubated in unfolding solution containing denaturant (urea or guanidinium chloride), and then subjected to an unfavorable refolding procedure (dialysis at $37^{\circ}C$). Once the ${\alpha}_1AT$ moiety of the fusion protein had unfolded in the unfolding solution, in which the denaturant concentration was higher than the unfolding transition midpoint ($C_m$) of the ${\alpha}_1AT$ variant, around 20% of the phage retained binding affinity to anti-${\alpha}_1AT$ antibody due to a low refolding efficiency. Moreover, this affinity reduced to less than 5% when 10 mg/mL skimmed milk (a misfolding-promoting additive) was included during the unfolding/refolding procedure. In contrast, most binding affinity (>95%) remained if the ${\alpha}_1AT$ variant was stable enough to resist unfolding. Because this selection procedure does not affect the infectivity of M13, the method is expected to be generally applicable to the high-throughput screening of stable protein variants, when activity-based screening is not possible.

• 대한의생명과학회지
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• 제21권1호
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• pp.9-14
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• 2015

Mouse hepatitis virus (MHV) is a major pathogen in laboratory mice that usually leads to fatal diseases, such as hepatitis, multiple sclerosis, encephalitis, and respiratory disease. MHV has a high infection rate, and it needs to be detected as soon as possible to prevent its spread to other facilities. However, MHV detection by enzyme-linked immunosorbent assay (ELISA) often gives false positives; thus, it is very important that the results are confirmed as true positives in the early infection stage or distinguished as false positives with more accurate, reliable methods. Under microbiological screening, MHV ELISA-positive mice were found in four GFP-tagging transgenic mice. To verify the detection of the MHV antigen directly, reverse transcription polymerase chain reaction (RT-PCR) was performed, and the mice were determined to be MHV negative. Additional serum antibody-based screening was conducted with three different ELISA kits, and multiplexed fluorometric immunoassay (MFIA) was performed to confirm their accuracy/sensitivity. In brief, the ELISA kit for A59 nucleocapsid protein (MHV-A59N) revealed MHV ELISA positivity, while other ELISA kits (MHV-S lysate and MHV-JHM lysate) demonstrated MHV negativity. In MFIA, only the test for the recombinant A59 nucleocapsid antigen was MHV positive, which was consistent with the ELISA results. These results suggest that the ELISA kit with the recombinant A59 nucleocapsid antigen might induce non-specific MHV ELISA positivity and that confirmation is therefore essential.

• 대한핵의학회지
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• 제19권1호
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• pp.113-117
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• 1985

The present study was undertaken to examine serum concentration of free $T_3,\;T_4$, in various functional states of the thyroid by Amerlex RIA kit method, which uses unidentified $T_3,\;T_4-labelled$ analogues, said to be unreactive with $T_3,\;T_4-binding$ proteins in serum, together with an antibody that binds both analogue and $T_3,\;T_4$. The test method has been compared with free $T_4$ index, which was product of total serum thyroxine and $T_3$ resin uptake ratio. Free $T_4$ value by this method was found to have a highly significant positive correlation with a free $T_4$ index (r; 0.957, p<0.005) and total $T_4$ (r:0.89, p<0.005) also, it was similar to other free $T_4$ measuring methods previously reported. So, it was showen to have the clinical utility as the screening test for thyroid function because it was a rapid; convinient, and reliable method for quantifying the free $T_4,\;T_3$ concentration in various thyroidal functional states.

• 한국동물위생학회지
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• 제36권3호
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• pp.181-186
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• 2013

This study was conducted to isolate the Actinobacillus pleuropneumoniae (APP) and to find out the distribution of 15 serovars mainly in southern Gyeonggi province, Korea. From July 2011 to Nov. 2012, a total of 2,204 slaughter pigs (110 herds) were inspected for evaluation of APP like pneumonic lesions. 48 (33.8%) APP strains were isolated from the 142 lungs and identified using PCR assays (cps, apx/omlA, biovar). Consequently, the serotype ratio were as in the following; type2 41.7% (n=20), type5 33.3% (n=16), type12 10.4% (n=5), type1 6.2% (n=3), type4 and 7 2.1% (n=1) and unknown 4.2% (n=2). Also serological test was implemented for 452 (83 herds) serum samples randomly collected from above slaughter pigs using commercial ELISA kits. The positive ratio of each serotype for tested pigs were 19.1% (77/404) on [2], 7.1% (32/452) on [3, 6, 8], 6.9% (28/404) on [5a, 5b], 6.2% (28/452) on [4, 7], 2.8% (9/320) on [12], 2.0% (9/452) on [1, 9, 11] and 0.0% (0/452) on [10]. And 49.3% (223/452) of pigs were positive on apxIV antibody. On the basis of latter screening test, the infected farm ratio accounted for 71.1% (59/83) and that was much higher than previously reported data.

• Journal of Periodontal and Implant Science
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• 제46권5호
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• pp.320-328
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• 2016

Purpose: Human saliva, as a vital part of the immune defense system, contains a number of distinct proteins and peptides. Recently human common salivary protein 1 (CSP1) has been identified as an abundant salivary protein and may play a role in promoting the binding of cariogenic bacteria to salivary pellicles. However, nothing else is known regarding the role of CSP1 in periodontology. The aim of this study was to quantify and compare CSP1 levels between healthy subjects and periodontal patients. Methods: This controlled clinical study was conducted in periodontally healthy individuals and patients with chronic periodontitis Chonbuk National University Hospital, with Institutional Review Board approval. Whole saliva samples were collected from 36 healthy subjects and 33 chronic periodontitis patients and analyzed. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immune blotting were conducted to ensure that anti-CSP1 monoclonal antibody (mAb) binds to CSP1 in human saliva. A sandwich enzyme-linked immunosorbent assay (ELISA) system was house-fabricated using mAb-hCSP1#14 and mAb-hCSP1#4 as a capture and a detector mAb, respectively. The CSP1 concentrations in saliva from 36 healthy subjects and 33 periodontal patients were quantified using the CSP1 sandwich ELISA system, and the results were analyzed using the Student's t-test. Results: Immunoblot analysis using mAb-hCSP1 as a probe confirmed that CSP1 in human saliva existed as a single band with a molecular weight of approximately 27-kDa. The quantification of CSP1 concentrations by CSP1 ELISA showed that the median values (25th to 75th percentiles) of periodontal patients and healthy subjects were 9,474 ng/mL (range, 8,434.10,139 ng/mL) and 8,598 ng/mL (range, 7,421.9,877 ng/mL), respectively. The Student's t-test indicated the presence of a statistically significant difference between the 2 groups (P=0.024). Conclusions: The presence of a significant difference in CSP1 levels between healthy subjects and periodontal patients suggests that CSP1 may be a potential biomarker for the detection or screening of periodontitis patients.

• 대한수혈학회지
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• 제29권3호
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• pp.320-327
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• 2018

72세 남자 환자가 전신 무력감을 호소하면서 본원 혈액내과 외래로 내원하였다. 환자는 내원 2년 전 온난자가항체에 의한 자가면역용혈빈혈로 진단받고 치료 중이었다. 내원 당시 환자의 혈색소 수치는 6.3 g/dL로, 빈혈을 교정하기 위해 수혈이 의뢰되었다. 환자의 혈액형은 A형, RhD 양성이었고, 비예기항체 검사에서 범응집 소견 및 자가대조검사에서 양성 소견을 보이고 과거 교차시험에서 최소반응강도를 보이는 적혈구 3단위를 수혈받은 기왕력이 있어, ABO 동형의 적혈구와 교차시험을 하여 최소반응강도를 보이는 적혈구 1단위를 출고하였다. 환자는 수혈을 받은 후 별다른 증상 없이 귀가하였으나, 귀가 후 약 5시간 후부터 발생한 발열, 오한, 호흡곤란, 복통, 혈뇨를 주소로 수혈 다음 날 본원 응급실로 내원하였다. Polyethylene glycol을 이용한 자가흡착검사 후 획득한 상층액을 이용하여 시행한 비예기항체검사에서 항-$Fy^a$가 동정되어, 자가 항체에 의해 가려져 수혈 전에 검출하지 못한 항-$Fy^a$에 의한 급성용혈성수혈반응으로 진단하였다. 본 증례를 통해 자가항체가 동정되는 환자에 대해서 반드시 공존하는 동종항체 확인에 대한 고려가 필요하다는 것을 인지하게 되었고, 이런 자가항체를 제거하여 검사하기 위해 자가흡착검사 방법에 대해 좀 더 익숙해질 필요가 있다는 경험을 하였다.

• Pediatric Infection and Vaccine
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• 제10권2호
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• pp.200-207
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• 2003

목 적 : 말라리아는 국내에서 1970년대 말에 소멸된 것으로 알려져 있었으나, 1993년 비무장지대에 근무하던 군인에서 첫 환자가 발생한 이후, 지속적으로 증가되어 2000년에는 4,142명, 2001년에는 2,556명, 2002년에는 1,797명의 말라리아 환자가 보고되었다. 1999년부터는 민간인의 비율이 점차 증가되고 있고, 토착형 말라리아 환자가 늘어남에 따라 국내에 존재하는 매개숙주(중국 얼룩날개 모기)에 의해 주민간에 전파될 가능성도 높아진다. 저자들은 말초혈액 도말 검사 및 말라리아 항체 검사에서 말라리아로 확진된 소아 환자 13명을 조사하여 역학적 분석 및 임상적 특성을 알아보고자 하였다. 방 법 : 2000년 1월부터 2003년 8월까지 일산백병원에 내원한 소아 환자 중 말초혈액 도말 검사 및 말라리아 항체 검사로 확진된 15세 이하의 소아 환자 13명에 대하여 의무기록을 후향적으로 조사하였다. 결 과 : 13례 모두 토착형 말라리아 감염이었고, 원인 원충은 삼일열 말라리아(Plasmodium vivax)였다. 13례 중 남아와 여아의 비율은 9 : 4이었고, 발병 당시 평균 연령은 $9.5{\pm}3.6$세였다. 지역별로는 13례 중 9례가 일산 시내였으며, 11례의 발병 시기가 6~8월로 여름에 주로 발생했다. 13례 중 2례만이 3일 주기의 발작적인 발열을 보였고 나머지 11례는 지속적인 발열 및 불규칙한 발열 양상을 보였다. 혈소판 감소증은 가장 두드러진 소견으로 13례 중 12례에서 동반되었으며, 3례에서는 범혈구 감소증을 보였다. 신생아를 제외한 12례에서 hydroxychloroquine과 primaquine을 투약했고, 이후 시행한 혈액 도말 검사상 더 이상의 원충은 발견되지 않았고 혈액학적 이상 소견 역시 호전되었다. 결 론 : 저자들이 경험한 소아 말라리아 환아는 모두 Plasmodium vivax에 의해 발병되고 일부 환아는 유행외 지역에서 발병된 것으로 보아 이 원충에 의한 토착화의 확산을 추정할 수 있었다. 또한 전형적 삼일열 말라리아의 발열 양상이 없다하여도 혈소판 감소증 소견이 동반된 발열 환아에서 이 질환을 의심하여야 하며 말라리아 항체 검사가 선별 검사로 유용한 것으로 추정된다.

• IMMUNE NETWORK
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• 제6권1호
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• pp.43-51
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• 2006

Background: The DR-$70^{TM}$ immunoassay is a newly developed cancer diagnostic test which quantifies the serum fibrin degradation products (FDP), produced during fibrinolysis, by antibody reaction. The purpose of this study was to evaluate the potential of DR-$70^{TM}$ Immunoassay in screening malignant tumor. Methods: Sample subjects were 4,169 adults, both male and female, who visited the health promotion center of a general hospital from March 2004 to April 2005 and underwent the DR-$70^{TM}$ immunoassay test and other tests for cancer diagnosis. The patient group was defined as 42 adults out of the sample subjects who were newly diagnosed with cancer during the same time period when the DR-$70^{TM}$ immunoassay test was performed. Final confirmation of a malignant tumor was made by pathological analysis. Results: The mean DR-$70^{TM}$ level was $0.83{\pm}0.65{\mu}g/ml$ (range: 0.00 (0.0001)${\sim}7.42{\mu}g/ml)$ in the control group (n=4,127) as opposed to $2.70{\pm}2.33{\mu}g/ml$ (range: $0.12{\sim}9.30{\mu}g/ml)$ in the cancer group (n=42), and statistical significance was established (p<0.0001, Student t-test). When categorized by the type of malignant tumor, all cancer patients with the exception of the subgroups of colon and rectal cancer showed significantly higher mean DR-$70^{TM}$ levels compared with the control group (p<0.0001, Kruscal-Wallis test). The receiver operating characteristic (ROC) curve analysis revealed ${\geq}1.091{\mu}g/ml$ as the best cut-off value. Using this cut-off value, the DR-$70^{TM}$ immunoassay produced a sensitivity of 71.4%, a specificity of 70.1%, a positive predictability of 69.4%, and a negative predictability of 69.2% (1). Conclusion: A significant increase in the mean DR-$70^{TM}$ value was observed in the cancer group (thyroidal, gastric, breast, hepatic and ovarian) com pared with the control group. In particular, the specificity and sensitivity of the DR-$70^{TM}$ immunoassay was relatively high in the subgroups of breast, gastric, and thyroidal cancer patients. There is need for further studies on a large number of malignant tumor patients to see how the DR-$70^{TM}$ level might be changed according to the differentiation grade and postoperative prognosis of the malignant tumor.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2013년도 제45회 하계 정기학술대회 초록집
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• pp.273-273
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• 2013

Recently, Point-of-care (POC) testing microdevices enable to do the patient monitoring, drug screening, pathogen detection in the outside of hospital. Immunochromatographic strip (ICS) is one of the diagnostic technologies which are widely applied to POC detection. Relatively low cost, simplicity to use, easy interpretations of the diagnostic results and high stability under any circumstances are representative advantages of POC diagnosis. It would provide colorimetric results more conveniently, if the genetic analysis microsystem incorporates the ICS as a detector part. In this work, we develop a reverse transcriptase-polymerase chain reaction (RT-PCR) microfluidic device integrated with a ROSGENE strip for colorimetric influenza H1N1 virus detection. The integrated RT-PCR- ROSGENE device is consist of four functional units which are a pneumatic micropump for sample loading, 2 ${\mu}L$ volume RT-PCR chamber for target gene amplification, a resistance temperature detector (RTD) electrode for temperature control, and a ROSGENE strip for target gene detection. The device was fabricated by combining four layers: First wafer is for RTD microfabrication, the second wafer is for PCR chamber at the bottom and micropump channel on the top, the third is the monolithic PDMS, and the fourth is the manifold for micropump operation. The RT-PCR was performed with subtype specific forward and reverse primers which were labeled with Texas-red, serving as a fluorescent hapten. A biotin-dUTP was used to insert biotin moieties in the PCR amplicons, during the RT-PCR. The RT-PCR amplicons were loaded in the sample application area, and they were conjugated with Au NP-labeled hapten-antibody. The test band embedded with streptavidins captures the biotin labeled amplicons and we can see violet colorimetric signals if the target gene was amplified with the control line. The off-chip RT-PCR amplicons of the influenza H1N1 virus were analyzed with a ROSGENE strip in comparison with an agarose gel electrophoresis. The intensities of test line was proportional to the template quantity and the detection sensitivity of the strip was better than that of the agarose gel. The test band of the ROSGENE strip could be observed with only 10 copies of a RNA template by the naked eyes. For the on-chip RT-PCR-ROSGENE experiments, a RT-PCR cocktail was injected into the chamber from the inlet reservoir to the waste outlet by the micro-pump actuation. After filling without bubbles inside the chamber, a RT-PCR thermal cycling was executed for 2 hours with all the microvalves closed to isolate the PCR chamber. After thermal cycling, the RT-PCR product was delivered to the attached ROSGENE strip through the outlet reservoir. After dropping 40 ${\mu}L$ of an eluant buffer at the end of the strip, the violet test line was detected as a H1N1 virus indicator, while the negative experiment only revealed a control line and while the positive experiment a control and a test line was appeared.