• BMB Reports
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• 제34권1호
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• pp.47-50
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• 2001

A one-step, two-site enzyme immunoassay was developed for measuring human alpha-fetoprotein (AFP) in serum and amniotic fluid using monoclonal antibodies (McAb) by eliminating the high-dose hook effect. Three McAbs that recognize different epitopes were selected among 16 different clones on the basis of epitope mapping, two for immobilization and one for horseradish peroxidase conjugation. This one-step immunoassay system is more convenient and rapid compared to a conventional two-step sandwich immunoassay system. It did not exhibit the hook effect to around 2.7 mg/ml of AFP, which is probably one of the highest concentrations of AFP in the serum. The dose-response curve of the system was linear to 500 mg/ml of AFP and the system could differentiate as low as 1 mg/ml of AFP The intra- and inter-assay variations were in an acceptable range; 95~104% and 97~105% respectively Its correlation with other commercial systems was around 95%.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권2호
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• pp.112-117
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• 2004

The performance of an immuno-analytical system can be assessed in terms of its analytical sensitivity, i.e., the detection limit of an analyte, which is determined by the amount of analyte molecules bound to the capture antibody that has been immobilized onto a solid surface. To increase the number of the binding complexes, we have investigated a site-directed immobilization of an antibody that has the ability to resolve a current problem associated with a random arrangement of the insolubilized immunoglobulin. The binding molecules were chemically reduced to produce thiol groups that were limited at the hinge region, and then, the reduced products were coupled to biotin. This biotinylated antibody was bound to a streptavidin-coated surface via the streptavidin-biotin reaction. This method can control the orientation of the antibody molecules present on a solid surface and also can significantly reduce the possibility of steric hindrance in the antigen-antibody reactions. In a two-site immunoassay, the introduction of the site-directly immobilized antibody as the capture enhanced the sensitivity of analyte detection approximately 10 times compared to that of the antibody randomly coupled to biotin. Such a novel approach would offer a protocol of antibody immobilization in order for the possibility of constructing a high performance immunochip.

• Journal of Microbiology and Biotechnology
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• 제34권2호
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• pp.340-348
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• 2024

Salmonella, a major contributor to foodborne infections, typically causes self-limiting gastroenteritis. However, it is frequently invasive and disseminates across the intestinal epithelium, leading to deadly bacteremia. Although the genus is subdivided into >2,600 serotypes based on their antigenic determinants, only few serotypes are responsible for most human infections. In this study, a rapid dot-blot immunoassay was developed to diagnose multiple Salmonella enterica serotypes with high incidence rates in humans. The feasibility of 10 commercial antibodies (four polyclonal and six monoclonal antibodies) was tested using the 18 serotypes associated with 67.5% Salmonella infection cases in the United States of America (U.S.A) in 2016. Ab 3 (polyclonal; eight of 18 serotypes), Ab 8 (monoclonal; 13 of 18 serotypes), and Ab 9 (monoclonal; 10 of 18 serotypes) antibodies exhibited high detection rates in western blotting and combinations of two antibodies (Ab 3+8, Ab 3+9, and Ab 8+9) were applied to dot-blot assays. The combination of Ab 3+8 identified 15 of the tested 18 serotypes in 3 h, i.e., S. Enteritidis, S. Typhimurium, S. Javiana, S. I 4,[5],12:i:-, S. Infantis, S. Montevideo, S. Braenderup, S. Thompson, S. Saintpaul, S. Heidelberg, S. Oranienburg, S. Bareilly, S. Berta, S. Agona, and S. Anatum, which were responsible for 53.7% Salmonella infections in the U.S. in 2016. This cost-effective and rapid method can be utilized as an on-site colorimetric method for Salmonella detection.

• 한국가축번식학회지
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• 제9권2호
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• pp.133-139
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• 1985

An immunoassay may be defined as an analytical procedure involving the competitive reaction between a limiting concentration of specific antibody and two populations of antigen, one of which is labelled or immobillized. The advent of immunoassay has revolutionised our knowledge of reproductive physiology and the practice of veterinary and clinical medicine. Radioimmunoassay (RIA) was the first of these methods to be developed, which meausred the analyte with good sensitivity, accuracy and precision (1,2). The essential components of RIA are:-(i) a limited concentration of antibodies, (ii) a reference preparation, and (iii) an antigen labelled with a radioisotope (usually tritium or iodine-125). Most procedures invelove isolating the antibody-bound fraction and measuring the amount of labelled antigen. Good facilities are available for scintilltion counting, data reduction nd statistical analysis. RIA is undergoing refinement through:-(i) the introduction of new techniques to separate the antibody-bound and free fractions which minimize the misclassification of labelled antigen into these compartments, and the amount of non-specfic binding. (3), (ii) the development of non-extration for the measurement of haptens (4), (iii) the determination of a, pp.rent free (i.e. non-protein bound) analytes (5), and (iv) the use of monoclonal antibodies(6). In 1968, Miles and Hales introduced in important new type of immunoassay which they termed immunora-diometric assay (IRMA) based on t도 use of isotopically labelled specific antibodies(7) in a move from limited to excess reagent systems. The concept of two-site IRMAs (with a capture antibody on a solid-phase, and a second labelled antibody to a different antigenic determinant of the analyte) has enabled the development of more sensitive and less-time consuming methods for the measurement of protein hormones ovar wide concentration of analyte (8). The increasing use of isotopic methos for diverse a, pp.ications has exposed several problems. For example, the radioactive half-life and radiolysis of the labelled reagent limits assay sensitivity and imposes a time limit on the usefulness of a kit. In addition, the potential health hazards associated with the use and disposal of radioactive cmpounds and the solvents and photofluors necessary for liquid scientillation counting are incompatable with the development of extra-laboratory tests. To date, the most practical alternative labels to radioisotopes, for the measurement of analytes in a concentration > 1 ng/ml, are erythrocytes, polystyrene particiles, gold sols, dyes and enzymes or cofactors with a visual or colorimetric end-point(9). Increased sensitivity to<1 pg/ml may be obtained with fluorescent and chemiluminescent labels, or enzymes with a fluorometric, chemiluminometric or bioluminometric end-point. The sensitivity of any immunoassay or immunometric assay depends on the affinity of the antibody-antigen reaction, the specific activity of the label, the precision with which the reagents are manipulated and the nonspecific background signal (10). The sensitivity of a limited reagent system for the measurement of haptens or proteins is mainly dependent upon the affinity of the antibodies and the smalleest amount of reagent that may be manipulated. Consequently, it is difficult in practice to improve on the sensitivity obtained with iodine-125 as the label. Conversely, with excess reagent systems for the measurement of proteins it is theoretically possible to increase assay sensitivity at least 1000 fold with alternative luminescent labels. To date, a 10-fold improvement has been achieved, and attempts are being made to reduce the influence of other variables on the specific signal from the immunoreaction.

• 한국가축번식학회지
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• 제12권1호
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• pp.51-62
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• 1988

hCG로 면역화된 생쥐의 비장세포와 골수종양세포(SP 2/0 Ag14)를 융합하여 hCG에 대한 단일클론항체를 생산하는 잡종세포(hybridoma)를 얻었다. 생산된 면역글로부린 type과 titer 그리고 면역분석법 이용시 감도 등을 조사함으로써 그 특성을 조사하였다. 배지나 복수내에 존재하는 항체를 순수분리 정제하기 위하여 gel-filtration, DEAE-ion exchange chromatography, 그리고 affinity chromatography 원리에 의한 방법 등을 사용하여 전기영동(SDS-PAGE)으로 순수도를 조사함으로 상호 비교하였다. 또한 hCG의 정량을 위하여 항체를 plastic tube에 피복시킨 것과 화학발광체로 표지된 항체를 이용한 소위 two-site immunochemiluminometric assay(ICMA)를 개발하여 생산된 항체의 이용가능성을 제시하였다.

• Journal of Microbiology and Biotechnology
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• 제19권1호
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• pp.83-92
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• 2009

Individual immunochromatographic assays (ICG) for ochratoxin A (OTA) and zearalenone (ZEA) were optimized and used in the development of a one-step simultaneous immunochromatographic assay (OS-ICG) for the rapid multianalysis of two mycotoxins in corn samples. The nitrocellulose membrane of the OS-ICG was treated with OTA-bovine serum albumin (BSA), ZEA-ovalbumin (OVA), and anti-mouse IgG in the OTA test, ZEA test, and control zones, respectively. Monoclonal antibody-gold conjugates (OTA3 MAb-gold and ZEA2C5 MAb-gold) were sprayed onto the conjugate pad. The visual detection limits were 2.5 and 5 ng/ml for OTA and ZEA, respectively, and the results were obtained within 15 min after starting the analysis. An efficient, simple, and rapid extraction method using 30% MeOH/PBS was established and validated by analyzing the corn samples spiked with OTA/ZEA mixtures (0/0, 5/10, 10/20, and $20/30\;{\mu}g/kg$). The cut-off values of the OS-ICG for the spiked corn were 5 and $10\;{\mu}g/kg$ for OTA and ZEA, respectively. Natural corn samples were analyzed by OS-ICG, direct competitive enzyme-linked immunosorbent assay (DC-ELISA), and HPLC. Results of the OS-ICG were in good agreement with those obtained by DC-ELISA and HPLC. The developed OS-ICG offers a rapid, easy-to-use, and portable analytical system and can be used as a convenient qualitative tool for the on-site simultaneous determination of OTA and ZEA in cereals, food, and agricultural products in one analytical cycle.