• Journal of Physiology & Pathology in Korean Medicine
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• v.18 no.3
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• pp.874-879
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• 2004

Alakaline phosphatase (ALP)- or horseradish peroxidase (HRP)-antibody conjugate was used frequently on the immunological detection methods such as enzyme-linked immunosobent assay (ELISA), immunobolt, immunohistochemistry. The classical enzyme-antibody coupling method by one-step (direction) injection of glutaraldehyde bring into being disadvantage such as low sensitivity of antigen detection because of homopolymers. This study was modified with the dialysis glutaraldehyde method to provide simple coupling through E-amino residues present in most protein. The dialysis glutaraldehyde coupling effects were better than the classical one-step glutaraldehyde injection in antigen detection of ELISA and immunobolt. Optimal dose of the dialysis glutaraldehyde solution was 0.10-0.25 %. This results suggest that the dialysis glutaraldehyde coupling method can readily applied to antigen detection of in vitro and in vivo.

• Analytical Science and Technology
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• v.23 no.6
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• pp.537-543
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• 2010

Dopamine (DA) is an important neurotransmitter molecule of catecholamines. Its deficiency could lead to brain disorder such as Parkinson's disease and schizophrenia. Therefore, it is necessary to establish a suitable analytical technique with sensitivity and simplicity. A competitive enzyme-linked immunosorbent assay for DA has been optimized and characterized. Assay sensitivity is controlled by two factors in competitive immunoassay. One is a nature and concentration of competitor, and the other is those of binder, antibody. Thus, optimization was performed: BSA-DA conjugate and antibody-avidin conjugate were prepared by dual heterobifunctional coupling method using SATA and SMCC. Assay condition was optimized with $6.66\;{\mu}gmL^{-1}$ of BSA-DA and $4.17{\times}10^{-10}\;M$ of antibody-avidin conjugate. A dose-response curve was constructed, and a limit of detection and a dynamic range for DA were accomplished to $2.3{\times}10^{-2}\;{\mu}g\;mL^{-1}$ and four orders of magnitude ($1.0{\times}10^{-7}\;M$ to $1.0{\times}10^{-3}\;M$), respectively. Calibration curve was constructed on dynamic range and least-squares regression of this data gave the following relationship: absorbance = -0.1098 log[DA]+0.0353 ($R^2$ = 0.9956).

• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1689-1695
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• 2010

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the identification, cloning, and characterization of panK-sp from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acids with a calculated molecular mass of 36.8 kDa and high homology with PanK from S. avermitilis and S. coelicolor A3(2). To elucidate the putative function of PanK-sp, it was cloned into pET32a(+) to construct pPKSP32, and the PanK-sp was then expressed in E. coli BL21(DE3) as a His-tag fusion protein and purified by immobilized metal affinity chromatography. The enzyme assay of PanK-sp was carried out as a coupling assay. The gradual decrease in NADH concentration with time clearly indicated the phosphorylating activity of PanK-sp. Furthermore, the ca. 1.4-fold increase of DXR and the ca. 1.5-fold increase of actinorhodin by in vivo overexpression of panK-sp, constructed in pIBR25 under the control of a strong $ermE^*$ promoter, established its positive role in secondary metabolite production from S. peucetius and S. coelicolor, respectively.

• BMB Reports
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• v.30 no.6
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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.

• YAKHAK HOEJI
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• v.43 no.3
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• pp.334-341
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• 1999

Thyroid peroxidase is a biochemical target protein for the antithyroid drugs. Ethanol extracts from one hundred and thirty seven natural products were screened for the inhibition of thyroid peroxidase activity. Thyroid peroxidase was purified from porcine thyroids, and the inhibition of peroxidase activity was evaluated using guaiacol oxidation (C-C coupling) assay. Twenty one natural products expressed a remarkable inhibition (>50%) of peroxidase activity at $330{\mu\textrm{g}}$ solid weight/m. The 50% inhibitory concentration ($IC_{50}$) of 70% ethanol extract from six potent natural products ranged from 3.1 to $31.2{\;}{\mu\textrm{g}}$ solid weight/m, in contrast to the range ($0.33~0.54{\;}{\mu\textrm{g}}/ml$) of $IC_{50}$ values fro catechin and epigallocatechin gallate as positive controls. Noteworthy, the extract of Camellia taliensis showed irreversible inhibition of the enzyme. It is suggested that extract from some natural products such as Camellia taliensis, Rheum undulatum or Euphorbia perinensis, exhibiting a potent inhibition of peroxidase activity, may be developed as sources of potent antithyroid agents.

• Journal of Food Hygiene and Safety
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• v.6 no.3
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• pp.111-117
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• 1991

We examined to develop the enzyme linked immunosorbent assay (ELISA) for determination of zearalenone in animal feeds. Zearalenone was first converted to 6'-(carboxymethyl) zearalenone oxime(zearalenone oxime) to get a coupling site and then conjugated to bovine serum albumin(BSA) for use as immunogen and to horseradish peroxidase(HRP) for use as enzyme marker. Antibody against zearalenone was obtained after 11 weeks of immunization of rabbit with zearalenone oxime-BSA. Cross reactivity of the antibody with ${\alpha}-zearalanol,\;{\beta}-zearalenol,\;{\alpha}-zearalanol\;and\;{\beta}-zearalanol$ were 168, 46, 26 and 20% respectiviely. A simple procedure was devised for the screening of zearalenone in feeds using ELISA. Feeds samples(5g) were extracted by blending with 25 ml of methanol-phospate butTered saline-dimethylformate(70 : 29 : 1) and the extract was filtered and aqueous filterate analyzed. It took only 1 hours to do whole procedure for the analysis of zearalenone in feeds by the direct competitive ELISA, and detectable limit was 1-100 ppb. Using this procedure, only 4 of 24 feed samples showed positive results with 3.93-7.43 ppb levels.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.231-234
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• 2001

We performed a basic experiment for rapid. on-line, real-time measurement of HBsAg by using a BIACORE biosensor, a chip-based sensor utilizing surface plasmon resonance technology to quantify the recognition and interaction of biomolecules. We immobilized an a -HBsAg antibody on a CM5 chip surface which was activated by N-hydroxysuccinimide for amine coupling with HBsAg, and measured the mass increase from the coupling. This study showed the potential of this biosensor-based method as a rapid, multi-sample, on-line assay. Once properly validated, it can serve as a more powerful method for HBsAg quantification.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.66-66
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• 2003

In skeletal muscle cells, depolarization of the transverse tubules (T-tubules) results in Ca$\^$2+/ release from the sarcoplasmic reticulum (SR), leading to elevated cytoplasmic Ca$\^$2+/ and muscle contraction. This process has been known as excitation-contraction coupling (E-C coupling). Several proteins, such as the ryanodine receptor (RyR), triadin, junctin, and calsequestrin (CSQ), have been identified to be involved in the Ca$\^$2＋/ release process. However, the molecular interactions between the SR proteins have not been resolved. In the present study, the mechanisms of interaction between RyRl and triadin have been studied by in vitro protein binding and $\^$45/Ca$\^$2+/ overlay assays. Our data demonstrate that the intraluminal loop II of RyR1 binds to triadin in Ca$\^$2+/-independent manner. Moreover, we could not find any Ca$\^$2+/ binding sites in the loop II region. GST-pull down assay revealed that a KEKE motif of triadin, which was previously identified as a CSQ binding site (Kobayasi et al.,2000 JBC) was also a binding site for RyR1. Our results suggest that the intraluminal loop II of RyR could participate in the RyR-mediated Ca$\^$2+/ release process by offering a direct binding site to luminal triadin.

• Journal of Food Hygiene and Safety
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• v.9 no.3
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• pp.123-131
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• 1994

An enzyme-linked immunosorbent assay(ELISA) was developed for the detection of residual gentamicin(GM) in edible animal products. The immunogen(GM-KLH conjugate) and coating antigen(GM-BSA conjugate) were prepared by coupling GM sulfate to keyhole limpet hemocyanin(KLH) and bovine serum albumin(BSA) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, respectively. Polyclonal antibody to GM was produced in rabbits(New Zealand White, female) by using the immunogen and the antibody titer was measured by indirect ELISA. A competitive ELISA was developed using GM-bovine serum albumin conjugate as a coating antigen, GM(as standards or sample), polyclonal antibody to GM, secondary antibody conjugated with horseradish peroxidase as an enzyme, and H2O2 and o-phenylenediamine dihydrochloride as a substrate and a chromophore, respectively. The detection limit of GM was 10 ng/ml and the standard curve of GM(n=26) was linear up to 10 $\mu\textrm{g}$/ml in this competitive ELISA system. There were no cross-reactivities of the partially purified antibody between GM and the various antibiotice such as amikacin, benzyl-penicillin, chloramphenicol, erythromycin, furazlidone, kanamycin, neomycin, oleandomycin, streptomycin, sulfathiazole and thiamphenicol(CR50<0.05%)

• KSBB Journal
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• v.17 no.1
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• pp.20-25
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• 2002

We performed a basic experiment for rapid, on-line, real-time measurement of HBsAg by using a surface plasmon resonance biosensor to quantify the recognition and interaction of biomolecules. We immobilized the anti-HBsAg polyclonal antibody to the dextran layer on a CM5 chip surface which was pre-activated by N-hydroxysuccinimide for amine coupling. The binding of the HBsAg to the immobilized antibody was measured by the mass increase detected by the change in the SPR signal. The binding characteristics between HBsAg and its antibody followed typical monolayer adsorption isotherm. When the entire immobilized antibody was interacted, there was no additional, non-specific binding observed, which suggested the biointeraction was very specific as expected and independent of the ligand density. No significant steric hindrance was observed at 17.6 nm/$mm^2$ immobilization density. The relationship between the HBsAg concentration in the sample solution and the antigen bound to the chip surface was linear up to ca. $40\mu\textrm{g}$/mL, which is much wider than that of the ELISA method. It appeared the antigen-antibody binding was increased as the immobilized ligand density increased, but verification is warranted. This study showed the potential of this biosensor-based method as a rapid, simple, multi-sample, on-line assay. Once properly validated, it can serve as a more powerful method for HBsAg quantification replacing the current ELISA method.