• Bulletin of the Korean Chemical Society
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• v.17 no.11
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• pp.1018-1022
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• 1996

Adenosine deaminase (ADA) has been utilized as the label in devising a potentiometric homogeneous assay for riboflavin and riboflavin binding protein (RBP). The proposed homogeneous assay method employs an ADA-biotin conjugate as the signal generator and an avidin-riboflavin conjugate as the signal modulator in the solution phase. The catalytic activity of the ADA-biotin conjugate is inhibited in the presence of an excess amount of the avidin-riboflavin conjugate, and the observed inhibition is reversed in an amount proportional to the concentration of RBP added. When the analyte riboflavin is added to this mixture of ADA-biotin, avidin-riboflavin and RBP, the activity of the enzyme conjugate is re-inhibited in an amount proportional to the concentration of riboflavin. Since the enzyme label used in this system is ADA, an ammonia-producing enzyme, a potentiometric rather than photometric detection scheme is used to monitor the enzymatic activity in the assay.

• Korean Journal of Veterinary Research
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• v.29 no.1
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• pp.21-25
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• 1989

This experiment was carried out to determine the progesterone concentration of bovine plasma by liquid phase double antibody enzyme immunoassay. The optimum conditions of assay-system, enzyme conjugate and gelatin were invested. The sensitivity, recovery rate and reproducibility by this assay were also analyzed. The results obtained were as follows: 1. The suitable supplementation level of gelatin was 0.2%. As the gelatin level increased to 1%, coefficient variation of interassay was shown to be irregular. 2. The optimum dilution rate of enzyme conjugate was 30 times. Enzyme activity was greatly fluctuated depending on the minor condition of enzyme conjugate technique. Therefore, it was considered to be checked when determined. 3. The sensitivity of the assay was 12 pg/tube. 4. Recovery rate was decreased when the amount of sample was too little or too much, but the recovery rate was high as 97.8% when the amount of sample between 50 and $200{\mu}l$. 5. Mean intra-assay and inter-assay coefficient of variation was 4.5% and 5.9%, respectively. By using liquid phase double antibody enzyme immunoassay, progesterone in plasma can be detected, and also this assay system is applicable to study on physiological function of progesterone and to diagnosis of reproductive disorders.

• 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.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.612-619
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• 2004

A competitive direct enzyme-linked immunosorbent assay (cdELISA) was developed for selective and rapid detection of a glycopeptide antibiotic, teicoplanin (TP). TP was conjugated to bovine serum albumin (BSA) for use as an immunogen. Repeated subcutaneous injections of 0.5 mg of the conjugate was effective in generating specific polyclonal antibody (PAb) toward TP in rabbits, as determined by cdELISA. TP-horseradish peroxidase conjugate (TP-HRP) was used as an enzyme marker. The cdELISA was developed based on a competition reaction between TP-BSA PAb and TP-HRP conjugate. The TP-BSA PAb was highly sensitive (detection limit, 0.3 ng/ml and specific toward teicoplanin, showing no cross-reactivity to other glycopeptide antibiotics including vancomycin. There were good correlations ($r^2$=0.84 and 0.76, respectively) between cdELISA and microbiological assay, and high-performance liquid chromatography. The cdELISA system developed in this work is expected to be useful not only for selective and rapid monitoring of TP but also for study of TP pharmacokinetics.

• YAKHAK HOEJI
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• v.45 no.3
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• pp.302-309
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• 2001

Wheat germ agglutinin (WGA) pectin, which binds to human melanoma cell line, was conjugated with Praecoxin A using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a cross-linking agent. Physical mixture (PM) of WGA and Praecoxin A was also prepared by using a non-specific binding property of Praecoxin A to WGA. The WGA:Praecoxin A ratio in the conjugate and PM was approximately 1:18 and 1:20, respectively. The results of hemagglutination assay and enzyme-linked lectin assay indicated that the conjugate and PM maintained the lectin-like properties of the WGA. The binding ratio of conjugate was about 70% during 4-24 hr, but the most of Praecoxin A was released within 24 hr in the case of PM. These results lead to the conclusion that the conjugate is potentially useful for the formulation of injection that requires targeting for melanoma as well as sustained release at the site.

• 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%)

• Analytical Science and Technology
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• v.13 no.5
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• pp.624-629
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• 2000

A simple and rapid homogeneous enzyme-linked binding assay for mistletoe lectin I(ML I) was developed using a coupled enzyme system of malate dehydrogenase (MDH) and D-galactose. A highly substituted MDH-galactose conjugate was prepared by employing an isothiocyanate method for formation of thiourea bond. In the presence of ML I, ML I inhibits the activity of the conjugate based on the ML I/D-galactose specific interaction. Thus, the concentration of ML I can be related to the homogeneous inhibition of the MDH-galactose conjugate. Using this method. ML I can be measured at the level of microgram per milliliter within 10 minutes.

• Korean Journal of Environmental Agriculture
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• v.11 no.1
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• pp.59-66
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• 1992

An enzyme immunoassay(EIA) was developed for the analysis of insecticide endosulfan and its degradation products. The sensitivity and specificity of the antibody produced were examined. Optimal conditions in the ELISA system for residue analysis were also discussed. A mixed suspension of endosulfan-hemocyanin conjugate(ES-KLH) 1.1 mg/ml and Freund's adjuvant was injected subcutaneously to white rabbits and then collected antisera were tested for titers by indirect ELISA(1/24,000). Because of difficulties in the synthesis of endosulfan peroxidase conjugate, amine derivative of endosulfan-diol was synthesized and it showed 40% of conjugate yields(2mg/ml of conjugate). the highest sensitivity obtained enzyme-conjugate was a concentration of 200ng/ml. The detection limit of endosulfan in ELISA system was 5 ppb on the standard curve. In application of ELISA for residue analysis, the recoveries were really 100% both in the spiked soil and apple sample regardless of endosulfan concentration treated. On the other hand, chlorinated hydrocarbons of similar structure with endosulfan showed low cross-reactivity$(2.2%{\sim}29.2%).$

• Microbiology and Biotechnology Letters
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• v.10 no.3
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• pp.205-209
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• 1982

The enzyme-linked immunosorbent assay using the so-called "double-sandwich"technique was applied to determine Clostridium botulinum type F toxin. Polystyrene tubes were coated with horse anti-type F toxin serum and then toxin sample was added. The tubes were subsequently treated with rabbit anti-type F toxin IgG and sheep anti-rabbit serum IgG-horseradish peroxidase conjugate. By this technique, about 10 mouse intraperitoneal 50% lethal doses (ip LD/50/) of type F toxin could be detected. Low back-ground reading was achieved with the use of phosphate-buffered saline containing 0.05% Tween 20 and 1% bovine serum albumin as diluents of rabbit IgG and conjugate. Addition of EDTA in the diluents of toxin increased ELISA extinction value significantly. No cross-reaction was observed with botulinum type A and B toxin, but type E toxin gave sleight cross-reaction.

• Archives of Pharmacal Research
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• v.12 no.3
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• pp.186-190
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• 1989

The release rates of methotrexate (MTX) from MTX-human serum albumin (HSA) conjugate, and 5-fluorouracil (5-FU) from 5-FU acetic acid (AA)-HSA conjugate were determined after incubation of the conjugates in various conditions. The concentrations of 5-FU released from the conjugate increased monoexponentially, however those of MTX increased biexponentially in all studies. It indicated that there are two distinct types of MTX-HSA linkage, weakly and tightly bound linkages. The release rates of 5-FU were lower than those of MTX in all studies indicating that the bond of 5-FU-AA-HSA conjugate is very stable, which is supported by the higher value of activation energy (39. 9 vs 10. 7 Kcal/mole) using Arrhenius equation. The release rates of MTX and 5 -FU from the conjugates increased with incubation temperatures. Proteolytic enzyme and liver homogenates accelerated significantly the release rates of MTX and 5-FU. Approximately 1.30 and 22.0% of MTX were released after 12 hours of incubation in the absence and presence of protease, respectively. The corresponding values for 5-FU were released after 12 hours of incubation with rat liver homogenates which were diluted 6 times with phosphate buffer of pH 6.0. The MTX-HSA and 5-FU-AA-HSA conjugates were very stable in rat plasma.