• Bulletin of the Korean Chemical Society
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• v.18 no.1
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• pp.44-50
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• 1997

A method of dual-signal generation from two different enzymes was developed and utilized to simultaneously perform dual immunoassays in a single microwell. Two enzymes selected as tracers were horseradish peroxidase (HRP) and β-galactosidase (GAL). 3, 3', 5, 5'-Tetramethylbenzidine (TMB) and chlorophenolred-β-galactopyranoside (CPRG) as chromogenic substrates for the respective enzyme were used. Although the two enzymes showed their maximum activities at distinct pH conditions (pH 5.1 for HRP and 7.5 for GAL), the enzyme reactions were able to be concurrently carried out at pH 5.75 in a dual-substrate solution without signal loss. This performance was achieved by increasing TMB concentration two-fold, introducing potassium salt as activator of GAL reaction, and extending total reaction time 50%. The signal generation method was then used for dual-enzyme immunoassays to detect antibodies with co-immobilized Hepatitis C virus antigens (core and NS5) and a Hepatitis B virus antigen (PreS(2)) in a microwell. Dose-response curves of the assays revealed cooperativity between different antigen-antibody complex formation, which suggested that dual immunoassays can only be used for qualitative screening tests unless the antigens immobilized were spatially separated.

• Bulletin of the Korean Chemical Society
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• v.22 no.4
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• pp.417-420
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• 2001

• The Korean Journal of Nuclear Medicine
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• v.18 no.2
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• pp.61-69
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• 1984

• Bulletin of the Korean Chemical Society
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• v.18 no.5
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• pp.515-519
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• 1997

In a competitive enzyme immunoassay, the performance was tested with different analyte-enzyme conjugates (signal generators) in their binding constants to antibody. Analyte (progesterone)-enzyme (glucose oxidase; GO) conjugates were chemically synthesized and purified by using a gel column with an immobilized antibody to progesterone. In an elution range from the column, four peaks were detected by measuring total enzyme activities. Results from further analysis indicated that the first peak contained mainly unreacted GO while the next three peaks conjugated GO with progesterone. These three conjugate preparations were compared in dose-response curves along with the unpurified mixture. The purified conjugates showed higher detection capabilities than did the mixture. Especially, the preparation in the second peak next to the free GO peak improved the detection limit five times. This performance was comparable to that of a progesterone-horseradish peroxidase conjugate that has been identified to have one progesterone ligand.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.04a
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• pp.281-281
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• 1996

The immunoassay method is frequently used for the identification and quantitation of ultratrace amount of bioactive substances. Homogeneous liposome immunoassays, which can avoid the use of radioisotopes and separation steps, have recently been reported in many publications. Cytolysin-mediated liposome immunoassay using melittin ever been studied but showed limited applications. Here, we designed a homogeneous liposome immunoassay using Clostridium perfringens phospholipase C (PLC), an enzyme which catalyzes the hydrolysis of phosphatidylcholine in biological membranes, as a cytolysin.

• Korean Journal of Veterinary Research
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• v.31 no.2
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• pp.223-228
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• 1991

Most progesterone enzyme immunoassays(EIA) are used liquid-phase double-antibody or single-antibody seperation. These methods consume considerable time and reagents because of the requirements for several washing and centrifugation steps involving the reactants. Because of these several problems, we were prompted to develop an effective enzyme-linked immunosorbent assay(ELISA) system that would be equal or superior to RIA for assay of progesterone. The results were obtained as follows. 1. Cross reaction of the progesterone antiserum with other steroids determined was shown with progesterone(100%), $11{\alpha}$-deoxycorti-costerone(2.271%), but the other steroids were shown below 0.9%. 2. Standard curve for progesterone ELISA was shown available difference according to progesterone concentration from 0 to 1,000pg/ml. 3. The lower limit of sensitivity was 0.2pg/well 4. Progesterone concentration was 1.6ng/ml for before parturition, and that was below 0.5ng/ml for after parturition. This development enzyme-linked immunosorbent assay for progesterone can be detected pregnancy diagnosis in cattle, and also applicable 10 research on physiological function including such as reproductive disorders.

• Journal of Microbiology and Biotechnology
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• v.17 no.6
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• pp.1031-1035
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• 2007

Prostate specific $antigen-{\alpha}_1-antichymotrypsin$ was detected by a double-enhancement strategy involving the exploitation of both colloidal gold nanoparticles (AuNPs) and precipitation of an insoluble product formed by HRP-biocatalyzed oxidation. The AuNPs were synthesized and conjugated with horse-radish peroxidase-PSA polyclonal antibody by physisorption. Using the protein-colloid for SPR-based detection of the PSPJACT complex showed their enhancement as being consistent with other previous studies with regard to AuNPs enhancement, while the enzyme precipitation using DAB substrate was applied for the first time and greatly amplified the signal. The limit of detection was found at as low as 0.027 ng/ml of the PSA/ACT complex (or 300 fM), which is much higher than that of previous reports. This study indicates another way to enhance SPR measurement, and it is generally applicable to other SPR-based immunoassays.

• BMB Reports
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• v.30 no.5
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• pp.326-331
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• 1997

To construct a competitive ELISA standard curve for the detection of glucose-6-phosphate debydrogenase (G6PD), we used highly purified native G6PD (nG6PD) as both immobilized and soluble antigens and anti-G6PD serum raised against nG6PD as antibody. The polystyrene cuvettes coated with nG6PD were challenged with a mixture of a limiting amount of anti-G6PD serum and various doses of nG6PD as competitors followed by incubation with alkaline phosphatase-anti-IgG conjugate. The competitive ELISA did not exhibit the typical sigmoidal dose-response curve characteristic of competition immunoassays under the optimal concentrations of antigen and antibody. The soluble nG6PD used as competitor failed to effectively inhibit the binding of antibodies to the immobilized nG6PD. The addition of NADP, a cofactor of G6PD enzyme, to coating buffer used for immobilizing nG6PD to the cuvettes and PBS-Tween-BSA buffer for diluting competitors did not improve the inhibition of antibody binding to immobilized nG6PD by soluble n/G6PD. The addition of BSA to coating buffer did not increase inhibition, either. Surprisingly, when partially active G6PD (paG6PD), obtained by repeated freeze-thawing, was used as competitor, the antibody binding to either immobilized nG6PD or immobilized paG6PD was inhibited 49-58%. We conclude that an effective competitive ELISA system with nG6PD enzyme and anti-G6PD serum for the detection of G6PD may not be established due to the poor inhibition of antibody binding to immobilized nG6PD by soluble nG6PD under the present assay conditions and that the inhibition may be improved by using an inactivated enzyme as competitor regardless of the type of immobilized antigen used. These results imply that the immobilized nG6PD may undergo denaturation upon binding to the polystyrene cuvettes and that our anti-G6PD serum may recognize denatured enzyme better than active enzyme.

• Korean Journal of Veterinary Research
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• v.31 no.2
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• pp.217-222
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• 1991

Most progesterone enzyme immunoassays(EIA) are used liquid phase double-antibody separation. These methods consume considerable time and reagents because of the requirements for several washing and centrifugation steps involving the reactants. Because of there several problems, we were prompted to develop an effective EIA system by the use of higher titer of progesterone antiserum free of anti-bovine serum albumin antibodies (anti-BSA). The results obtained were as follows. 1. The antibody of progesterone antiserum was high as $1.5{\times}10^5$. 2. Percent activity bound of progesterone antiserum was about 77 at a dilution to $5{\times}10^3$ times. 3. Progesterone antiserum was contained a large amount of anti-BSA antibodies. 4. The anti-BSA was completely absorbed by using of polymerised BSA. 5. The molecular weight of albumin polymer (polymerised BSA) obtained by using 2.5% glut. araldehyde was $5{\times}10^5$.

• Bulletin of the Korean Chemical Society
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• v.34 no.3
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• pp.922-926
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• 2013

3,3',4,4'-Tetrachlorobiphenyl (IUPAC PCB77) is one of seven indicative polychlorinated biphenyls (PCBs) in the surface sediments. The current study presents a novel polyclonal antibody for the determination of the PCB77 using indirect competitive enzyme-linked immunosorbent assay. Under optimum conditions, PCB77 was determined within the concentration range of 0.01-100 ${\mu}g\;L^{-1}$, with a detection limit of 0.057 ${\mu}g\;L^{-1}$. The assays were tested for their cross-reactivity profiles using 3 selected congeners and 4 Aroclor products. The assays were highly specific for coplanar PCB congeners, but less specific for Aroclor1248. The spiked recoveries from five sediment samples were 86%-114% for PCB77 from ELISA, which were satisfactory. The current study demonstrated that the developed antiserum and immunoassay procedure can be used to detect PCB77 in environmental samples. The results of the sediment analysis were confirmed by conventional GC/ECD.