B형 간염 표면항원 검사시약 성능평가를 위한 B형 간염 표면항원 저역가 표준패널 확립

권소영,하건우,조연정,윤경원,최경영,주현아,오덕자,조남선,이주헌,유숙원 대한수혈학회 2009 大韓輸血學會誌 Vol.20 No.2

Background: A range of well characterized materials are needed for validating the performance of hepatitis B surface antigen (HBsAg) immunoassays. These materials are purchased currently from overseas manufacturers at a high cost and with limited quantity. This study was conducted to establish an HBsAg low titer performance panel for use as a national standard for validation of HBsAg immunoassays in Korea. Methods: 476 plasma units reactive on blood donor screening were collected HBsAg was tested using 3 enzyme immunoassays (EIA) and 1 chemiluminescence immunoassay (CIA). Units reactive on the CIA assay or on 2 or more immunoassays were subjected to hepatitis B virus (HBV) DNA quantification, HBV genotyping and subtyping. Units reactive on HBV DNA quantification were confirmed for HBsAg by neutralization. Candidates for the panel were subjected to a collaborative study performed at 7 laboratories using 7 immunoassays. Results: Eleven HBsAg positive units were selected for the low titer performance panel based on HBsAg immunoassay, HBV DNA quantification, HBV genotyping and subtyping results. The range of the HBsAg concentration of the panel members was 0.05∼1.28 IU/mL. Two HBsAg negative units were also included as negative controls. Conclusion: As a result of this study, a low titer performance panel [KFDA standard (08/028); HBsAg low titer performance panel (BTRL HBV/LP)] for validation of HBsAg immunoassays has been established as a Korean national standard. Use of this panel will improve performance assessment of HBsAg immunoassays. Because the performance of immunoassays cannot be assessed properly with a limited number of panels, continuous efforts are needed to develop a range of performance panels. 배경: HBsAg 검사시약의 성능평가와 품질관리를 위해서는 특성이 잘 규명된 다양한 종류의 검체들이 필요하다. 현재 이들 물질들은 고가의 가격과 제한된 양으로 외국으로부터 구입되고 있다. 본 연구는 HBsAg 검사시약의 성능평가를 위한 HBsAg 저역가 패널을 제조하여 이를 국가표준품으로 확립하고 하였다. 방법: 헌혈자 선별검사에서 양상결과를 보인 476단위의 혈장을 수집하였다. 이들 혈장에 대해 3종의 효소면역검사(enzyme immunoassay, EIA) 시약과 1종의 화학발광면역검사(chemiluminescence immunoassay, CIA) 시약을 사용하여 HBsAg을 검사하였다. CIA에서 양성결과를 보이거나 2종 이상의 면역혈청 검사시약에서 양성결과를 보인 경우 B형 간염바이러스(hepatitis B virus, HBV) DNA 정량검사, HBV 유전자형과 아형 검사를 시행하였다. HBV DNA 정량검사에서 DNA가 검출된 혈장에 대해서는 HBsAg neutralization을 통해 HBsAg 진양성 여부를 확인하였다. HBsAg 저역가 패널을 위한 후보물질들에 대해 7개 검사기관에서 7종의 검사시약을 사용하여 공동연구를 수행하였다. 결과: HBsAg 면역혈청검사, HBV DNA 정량검사, HBV 유전자형과 아형 검사결과를 근거로 11개의 HBsAg 진양성 혈액을 HBsAg 저역가 패널 구성원으로 선정하였다. 이들 검체의 HBsAg 농도는 0.05∼1.28 IU/mL의 범위 내에 있었다. HBsAg 음성 혈액 2단위는 음성대조물질로 패널에 포함되었다. 결론: 본 연구의 결과로 HBsAg 검사시약 성능평가를 위한 저역가 패널[KFDA standard (08/ 028); HBsAg low titer performance panel (BTRL HBV/LP)]이 국가표준품으로 확립되었다. 본 패널의 사용으로 HBsAg 검사시약의 성능평가가 개선될 것으로 기대된다. 검사시약의 성능을 정확하게 평가하기 위해서는 다양한 특성을 갖는 패널이 필요하기 때문에 보다 많은 수의 표준품이 지속적으로 개발되어야 할 것이다.

소아에서 4제요법 후 enzyme immunoassay에 의한 Helicobacter pylori 대변 항원 검출법의 유용성에 대한 연구

양혜란,서정기,Yang, Hye Ran,Seo, Jeong Kee 대한소아소화기영양학회 2004 Pediatric gastroenterology, hepatology & nutrition Vol.7 No.2

목적: H. pylori 대변항원(Helicobacter pylori stool antigen; HpSA) 검사는 H. pylori 감염 여부를 진단하는 데 이용되는 비침습적 검사이지만, 소아에서 제균요법 후 H. pylori 대변항원검사의 유용성에 대한 연구가 거의 이루어지지 못한 상태이다. 본 연구에서는 소아에서 H. pylori의 제균 여부를 확인하는 데 있어 enzyme immunoassay에 의한 HpSA 검사의 진단 정확도를 평가하고자 하였다. 연구방법: 2001년 1월부터 2003년 10월까지 서울대학교병원 소아과에서 146명의 소아(평균 연령 $9.3{\pm}4.3$세)를 대상으로 총 164회의 H. pylori 대변항원검사(Primier platinum HpSA)를 시행하였다. H. pylori 감염여부를 확인하기 위해 모든 환아에서 H. pylori 대변항원검사와 상부위장관내시경에 의한 위점막 생검을 병행하였다. H. pylori 감염이 확인되어 사제요법(omeprazole, amoxicillin, metronidazole, bismuth subcitrate)을 1주 동안 시행 받은 환아들에서는 치료 종결 후 최소 4주가 경과하였을 때 위내시경과 H. pylori 대변항원검사를 반복 실시하였다. H. pylori 대변항원검사는 OD값이 0.16 이상일 때 양성, 0.14~0.16 사이는 감염 미확정, 0.14 미만은 음성으로 판정하였다. 결과: 1) 제균 전 시행한 131회의 위내시경 조직검사 결과 28명이 H. pylori 양성이었고 나머지 103명은 H. pylori 음성이었다. 동시에 시행한 H. pylori 대변항원검사 결과에서 30명이 H. pylori 양성이었고, 101명이 음성으로 판정되었다. 따라서 제균요법 시행 전 H. pylori 대변항원검사의 민감도, 특이도, 양성 예측치, 음성 예측치는 각각 96.4%, 97.1%, 90% 그리고 99%이었다. 2) 제균요법 4주 후 33명의 환아에서 시행한 위내시경에 의한 조직검사에서 H. pylori는 24명에서 음성이었으나 9명은 여전히 양성이었다. 동시에 시행한 H. pylori 대변항원검사는 10명에서 양성, 23명에서 음성이었다. 따라서 제균요법 후 H. pylori 대변항원검사의 민감도, 특이도, 양성 예측치, 음성 예측치는 각각 88.9%, 91.7%, 80% 그리고 95.7%이었다. 결론: 소아에서 H. pylori 대변항원검사는 제균 후에도 높은 진단 정확도를 보였다. 따라서 H. pylori 대변항원검사는 소아에서 제균요법 후 균 박멸여부를 확인하는 데에도 매우 유용한 비침습적인 검사방법이라고 하겠다. Purpose: The Helicobacter pylori stool antigen (HpSA) enzyme immunoassay is a non-invasive test for the diagnosis and monitoring of H. pylori infection. But, there are few validation studies on the HpSA test after eradication in children. The aim of this study was to assess the diagnostic accuracy of HpSA enzyme immunoassay for the detection of H. pylori to confirm eradication in children. Methods: From January 2001 to October 2003, 164 tests were performed in 146 children aged 1 to 17.5 years (mean $9.3{\pm}4.3$ years). H. pylori infection was confirmed by endoscopy-based tests (rapid urease test, histology, and culture). All H. pylori infected children were treated with quadruple regimens (Omeprazole, amoxicillin, metronidazole and bismuth subcitrate for 7 days). Stool specimens were collected from all patients for the HpSA enzyme immunoassay (Primier platinum HpSA). The results of HpSA tests were interpreted as positive for $OD{\geq}0.160$, unresolved for $$0.140{\leq_-}OD$$<0.160, and negative for OD<0.140 at 450 nm on spectrophotometer. Results: 1) One hundred thirty-one HpSA tests were performed before treatment. The result of HpSA enzyme immunoassay showed three false positive cases and one false negative case. The sensitivity, specificity, positive predictive value, and negative predictive value of HpSA enzyme immunoassay before treatment were 96.4%, 97.1%, 90%, and 99%, respectively. 2) Thirty-three HpSA enzyme immunoassay were performed at least 4 weeks after eradication therapy. The results of HpSA enzyme immunoassay showed two false positive cases and one false negative case. The sensitivity, specificity, positive predictive value, and negative predictive value after treatment were 88.9%, 91.7%, 80%, and 95.7%, respectively. Conclusion: Diagnostic accuracy of the HpSA enzyme immunoassay after eradication therapy was as high as that of the HpSA test before eradication therapy. The HpSA enzyme immunoassay was found to be a useful non-invasive method to confirm H. pylori eradication in children.

One-Step Homogeneous Immunoassay for the Detection of Influenza Virus Using Switching Peptide and Graphene Quencher

김홍래,봉지홍,김태훈,신승식,강민정,심원보,이도영,손동희,변재철 한국바이오칩학회 2022 BioChip Journal Vol.16 No.3

One-step homogeneous immunoassay was developed for detecting influenza viruses A and B (Inf-A and Inf-B) using the switching peptide H2. As the fluorescence-labeled switching peptide dissociated from the binding pocket of detection antibodies, the fluorescence signal could be directly generated by the binding of Inf-A and Inf-B without washing (i.e., one-step immunoassay). For the one-step homogeneous immunoassay with detection antibodies in solution, graphene was labeled with the antibodies as a fluorescence quencher. To test the feasibility of the homogeneous one-step immunoassay, the stability of the antibody complex with the switching peptide was evaluated under different pH and salt conditions. The one-step homogeneous immunoassay with switching peptide was conducted using influenza virus antigens in phosphate-buffered saline and real samples with inactivated Inf-A and Inf-B spiked in serum. Finally, the one-step homogeneous immunoassay results were compared with those of commercially available lateral flow immunoassays.

Two Types of Immunoassay Based on Nile Blue Labeling Polydopamine Nanospheres

Lili Liu,Wenbo Lu,Chang Liu,Ying Wang,Jian Dong,Weiping Qian 성균관대학교(자연과학캠퍼스) 성균나노과학기술원 2017 NANO Vol.12 No.8

The sandwich-type immunoassays have been developed by using electrochemical and surface-enhanced Raman scattering (SERS) techniques for the detection of carcinoembryonic antigen (CEA). Nile blue as a kind of Raman dye has been decorated on nanospheres with polydopamine resin (PDR) via π-stacking interaction. The Nile blue displays the strong SERS signals as well as a characteristic electrochemical reduction peak at -0.33V (versus Ag/AgCl). The implementation of the PDR nanospheres mixing with Au nanoparticles (AuNPs/PDR) exhibits a better electrical conductivity and large SERS enhancement. The immunoassays based on Nile blue-labeled AuNPs/PDR nanospheres have been fabricated by using electrochemical and SERS techniques for the detection of CEA. The proposed immunoassay shows higher sensitivity, high selectivity, lower detection limit and long-term stability. The performances of the electrochemical immunoassay are better than that of SERS immunoassay. For the electrochemical immunoassay, the linear range occurs from 1 pg/mL to 100 ng/mL (R = 0.995) with a detection limit of 0.68 pg/mL and signal-to-noise ratio of 3 in the detection of CEA. The data for the analysis of human serum samples by using the electrochemical method are acceptably consistent with those obtained from the enzyme-linked immunosorbent assay (ELISA). The proposed immunoassay exhibits a promising potential for application in clinical diagnosis.

Immunoassay분과 외부 신빙도조사 결과보고(1998)

권오헌,임환섭,김현숙,김덕언,김영기,김진규,신섭,정영순,조한수 대한임상검사정도관리학회 1999 臨床檢査와 精度管理 Vol.21 No.1

Comparison of Serologic Response of Hospitalized COVID-19 Patients Using 8 Immunoassays

Sun Min Lee,In-Suk Kim,Seungjin Lim,Su Jin Lee,Won-Joo Kim,Kyung-Hwa Shin,Soo Young Moon,Chulhun L. Chang 대한의학회 2021 Journal of Korean medical science Vol.36 No.9

Background: In Korea, there were issues regarding the use of immunoassays for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies to detect infection. So, we compared antibody results of eight kinds of commercial immunoassays using clinical remnant specimens. Methods: We compared the results of several immunoassay kits tested on 40 serum samples from 15 confirmed patients and 86 remnant serum samples from clinical laboratory. Eight kinds of IVD kits—four enzyme-linked immunosorbent assay, two lateral flow rapid immunochromatographic assays, and two chemiluminescent immunoassays with one RUO kit were tested. Results: Among 40 serum samples from 15 coronavirus disease 2019 (COVID-19) patients, 35 yielded at least one positive result for detecting antibodies in the combined assessment. There were inconsistent results in 12 (28%) samples by single immunoassay. Forty samples collected in 2019 before the first COVID-19 Korean case showed negative results except for one equivocal result. Conclusion: The discrepant results obtained with different immunoassay kits in this study show that serological assessment of SARS-CoV-2 by a single immunoassay requires caution not only in detecting infection but also in assessing immunologic status.

Comparison of Six Automated Immunoassays With Isotope-Diluted Liquid Chromatography–Tandem Mass Spectrometry for Total Thyroxine Measurement

Songlin Yu,Weiyan Zhou,Xinqi Cheng,Qinghui Meng,Honglei Li,Li’an Hou,Jun Lu,Shaowei Xie,Qian Cheng,Chuanbao Zhang,Ling Qiu 대한진단검사의학회 2019 Annals of Laboratory Medicine Vol.39 No.4

Background: Accurate serum total thyroxine (TT4) measurement is important for thyroid disorder diagnosis and management. We compared the performance of six automated immunoassays with that of isotope-diluted liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) as the reference method. We also evaluated the correlation of thyroid stimulating hormone (TSH) with TT4 measured by ID-LC-MS/MS and immunoassays. Methods: Serum was collected from 156 patients between October 2015 and January 2016. TT4 was measured by immunoassays from Abbott (Architect), Siemens (ADVIA Centaur XP), Roche (E601), Beckman-Coulter (Dxi800), Autobio (Autolumo A2000), and Mindray (CL-1000i), and by ID-LC-MS/MS. Results were analyzed using Passing–Bablok regression and Bland–Altman plots. Minimum requirements based on biological variation were as follows: a mean bias of ≤4.5% and total imprecision (CV) of ≤3.7%. Results: All immunoassays showed a correlation >0.945 with ID-LC-MS/MS; however, the slope of the Passing–Bablok regression line varied from 0.886 (Mindray) to 1.23 (Siemens) and the intercept from -12.8 (Siemens) to 4.61 (Mindray). Only Autobio, Beckman-Coulter, and Roche included the value of one in the 95% confidence interval for slope. The mean bias ranged from -10.8% (Abbott) to 9.0% (Siemens), with the lowest value noted for Roche (3.5%) and the highest for Abbott (-10.8%). Only Abbott and Roche showed within-run and total CV ≤3.7%. Conclusions: Though all immunoassays correlated strongly with ID-LC-MS/MS, most did not meet the minimum clinical requirement. Laboratories and immunoassay manufacturers must be aware of these limitations.

Magnetic-bead-based immunoassay using E. coli cells with autodisplayed Z-domains

Yoo, G.,Bong, J.H.,Park, M.,Kang, M.J.,Jose, J.,Pyun, J.C. IPC Science and Technology Press ; Elsevier Scienc 2013 Enzyme and microbial technology Vol.53 No.2

Escherichia coli cells with autodisplayed Z-domains could increase the sensitivity of immunoassays by immobilizing antibodies in a controlled orientation. In the work presented here, E. coli cells with autodisplayed Z-domains were immobilized to magnetic beads for subsequent immunoassay. In comparing conventional immunoassay using the E. coli cells with autodisplayed Z-domains, the magnetic-bead-based immunoassay improved immunoassay efficiency by minimizing the loss of E. coli cells during repeated centrifugation steps during washing. For the immobilization of E. coli cells to magnetic beads, the magnetic beads were modified with poly-l-lysine to bind to negatively charged E. coli cells. During the surface modification process, physical parameters such as the surface charge and size of the magnetic beads were analyzed to confirm the formation of E. coli-magnetic bead complexes. To test the feasibility of the magnetic-bead-based immunoassay, horseradish peroxidase (HRP) was used as a model analyte, and a biomarker for inflammatory diseases, C-reactive protein (CRP), was used for a demonstration of an application in medical diagnosis.

Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay

Seo, Hyo-Deok,Lee, Joong-jae,Kim, Yu Jung,Hantschel, Oliver,Lee, Seung-Goo,Kim, Hak-Sung Elsevier 2017 Analytica chimica acta Vol.950 No.-

<P><B>Abstract</B></P> <P>Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL<SUP>−1</SUP> for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A human TNF-α (hTNF-α)-specific repebody was selected using a phage display. </LI> <LI> A monomeric alkaline phosphatase (mAP) was genetically fused to the repebody. </LI> <LI> mAP-fused repebody enabled detection of hTNF-α with high sensitivity and accuracy. </LI> <LI> mAP-fused repebody can be widely used as a new immuno-reagent in immunoassays. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Recent Developments in Innovative Magnetic Nanoparticles-Based Immunoassays: From Improvement of Conventional Immunoassays to Diagnosis of COVID-19

하연정,김이중 한국바이오칩학회 2022 BioChip Journal Vol.16 No.4

During the ongoing COVID-19 pandemic, the development of point-of-care (POC) detection with high sensitivity and rapid detection time is urgently needed to prevent transmission of infectious diseases. Magnetic nanoparticles (MNPs) have been considered attractive materials for enhancing sensitivity and reducing the detection time of conventional immunoassays due to their unique properties including magnetic behavior, high surface area, excellent stability, and easy biocompatibility. In addition, detecting target analytes through color development is necessary for user-friendly POC detection. In this review, recent advances in different types of MNPs-based immunoassays such as improvement of the conventional enzyme-linked immunosorbent assay (ELISA), immunoassays based on the peroxidase-like activity of MNPs and based on the dually labeled MNPs, filtration method, and lateral-flow immunoassay are described and we analyze the advantages and strategies of each method. Furthermore, immunoassays incorporating MNPs for COVID-19 diagnosis through color development are also introduced, demonstrating that MNPs can become common tools for on-site diagnosis.