• Journal of Veterinary Science
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• v.23 no.5
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• pp.32.1-32.11
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• 2022

Background: Classical swine fever (CSF) is a severe infectious disease of pigs that causes significant economic losses to the swine industry. Objectives: This study developed a solid-phase blocking enzyme-linked immunosorbent assay (spbELISA) method for the specific detection of antibodies against the CSF virus (CSFV) in porcine serum samples. Methods: A spbELISA method was developed based on the recombinant E2 expressed in Escherichia coli. The specificity of this established spbELISA method was evaluated using reference serum samples positive for antibodies against other common infectious diseases. The stability and sensitivity were evaluated using an accelerated thermostability test. Results: The spbELISA successfully detected the antibody levels in swine vaccinated with the C-strain of CSFV. In addition, the detection ability of spbELISA for CSFV antibodies was compared with that of other commercial ELISA kits and validated using an indirect immunofluorescence assay. The results suggested that the spbELISA provides an alternative, stable, and rapid serological detection method suitable for the large-scale screening of CSFV serum antibodies. Conclusions: The spbELISA has practical applications in assessing the vaccination status of large pig herds.

• Environmental Analysis Health and Toxicology
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• v.28
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• pp.16.1-16.8
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• 2013

Objectives Fish vitellogenin (VTG) is produced in the female liver during oogenesis through the estradiol cycle and produced in the male liver by endocrine disrupting chemicals (EDCs) such as alkylphenols. In this study, we propose that the VTG concentration in the pale chub could be detected using monoclonal antibodies and polyclonal antibodies against vitellin (Vn) in a VTG enzyme-linked immunosorbent assay (ELISA) system. Methods Monoclonal antibodies and polyclonal antibodies were produced using the Vn extracted from the matured ovum of the ovary. The VTG was extracted from the plasma of the male pale chub. The Vn and VTG were confirmed by measuring the molecular weight of their proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of the antibodies was checked through western blotting methods. The assay system was validated with respect to optimal assay concentrations, specificity, recovery, and intra- and inter-assay variations. Results The Vn consisted of two protein bands with apparent molecular weights of 64 and 37 kDa. The SDS-PAGE indicated protein weights of 146 and 77 kDa in the VTG. The assay range was 15.6 ng/mL to 2,000 ng/mL, and the value of the intra- and inter-assay variations were within 10.0% and 14.7%, respectively. The recovery rate was $99.5{\pm}5.5%$. Conclusions A sandwich ELISA was developed that could be used to qualify the VTG of pale chub in screening for EDCs. Pale chub is an ideal species for observing estrogen activity in the environment because of its extensive habitat and extensive food chain. The ELISA developed here would be more favorable than those for other species for determining the effect of long-term food chain accumulation of EDCs in aquatic environments.

• 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%)

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.571-578
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• 2007

A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The $IC_{50}$ value by IC-ELISA with scFv antibody was 4.8ng/ml, compared with 1.6ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.

• The Journal of the Korean Society for Microbiology
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• v.22 no.4
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• pp.377-392
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• 1987

Monoclonal antibody to the Escherichia coli heat-stable enterotoxin(ST) was produced to develop a rapid and convenient diagnostic method to the ST. The toxin was purified from culture supernatant of enterotoxigenic E. coli O148H28($ST^+/LT^+$) and conjugated to bovine serum albumin(BSA). The ST-BSA conjugate was used to immunize BALB/c mice and the immune spleen cells from these mice were fused with $P3{\times}63$ Ag8.V653 plasmacytoma cells. Hybridomas were screened by ELISA and positive hybridomas were cloned by limiting dilution. Finally, one stable clone (AS36) specific to ST was selected for further growth and characterization. Antibody titers of culture supernatant and ascitic fluid from BALB/c mice were 1:1,024 and 1:20,480 respectively in ELISA. The isotype and subclass of monoclonal antibody was IgG1 in sandwich ELISA. To test the neutralizing effect of monoclonal antibody on toxin activity of ST, mixture of ascitic fluid and ST was assayed by infant mouse assay and this monoclonel antibody was proved to be a neutralizing antibody. The titer of ascitic fluid which completely neutralized biological activity of 4 units of ST was 1:4. Purified ST was quantitatively measured by competitive ELISA and minimum amount of ST detectable by this assay was 250pg, which was an amount six-fold smaller than that detectable by infant mouse assay. Four reference strains of enterotoxigenic E. coli from WHO were detected by competitive ELISA and highly specific, sensitive and reproducible result was obtained.

• 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.

• 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.

• Journal of Conservation Science
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• v.35 no.4
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• pp.363-372
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• 2019

Animal glue has been used as a binder in Dancheong since the Joseon dynasty. Binders play an important role in determining the physical characteristics of a painting layer. The analysis of binders can be used to identify the materials and techniques used in traditional Dancheong. Binders can be investigated using physicochemical component analyses methods such as gas chromatography/mass spectrometry, pyrolysis-gas chromatography/mass spectrometry, and fourier transform infrared spectroscopy, but the detection characteristics vary depending on the degradation properties of the pigment and binder. Therefore, cross-validation using a combination of physicochemical analysis and enzyme immunoassay is used to increase the reliability of the results. In this study, we present an enzyme-linked immunosorbent assay (ELISA) as an example of an enzyme immunoassay as a method for analyzing animal glue, a traditional binder used in Korea. The applicability of ELISA was tested using commercial animal glue, in addition to animal glue produced using a variety of extraction conditions. The animal glue was analyzed in a Noerok-additionally coated-replica sample to evaluate the possibility of analyzing the animal glue in a paint layer mixed with pigment. Based on the results, we performed an assay on the use of animal glue in the Dancheong sample of the temples of the Joseon dynasty, that are estimated to have been built in the 17^th century.

• Biomedical Science Letters
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• v.7 no.3
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• pp.103-110
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• 2001

$\alpha$-Fetoprotein(AFP) is a good marker for the detection of several diseases such as hepatocellular carcinoma, gonadal germ cell tumor, gastric tumor, and Down's syndrome. In this study, we developed ELISA, using synthetic peptides corresponding to the epitopes of AFP. Five kinds of peptides were synthesized from AFP to produce antibodies in rats that recognize AFP in human plasma as well as amniotic fluid and do not cross-react with serum albumin. All five kinds of antibodies showed good reactivities with their peptide-keyhole limpet hemocyanin conjugates. Anti-synthetic peptide 1 (R-N-E-Y-G-I-A-S-I-L, 4-13) antibody, in particular, reacted well with AEP as well as synthetic peptide 1-KLH but not with human serum albumin. The binding affinity(Kd) was 2.7$\times$10$^{-9}$M for peptide 1 and 6.8$\times$10$^{-8}$M for AEP. The range for measurement of AFP was 10~1,000 ng/ml. The within-assay and between-assay coefficients of variance(CV) were 4.83% and 10.97%, respectively. In a sample of 31 sera and 33 amniotic fluids, there was a good correlation between AFP values determined in this assay and those in a commercial kit. These results indicate that the antibodies against synthetic peptides corresponding to the epitopes of AFP are highly specific to APP and synthetic peptide-based ELISA would be useful for the measurement of human AFP.

• Bulletin of the Korean Chemical Society
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• v.23 no.4
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• pp.610-612
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• 2002

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of organophosphorus insecticide cyanophos. An analogue (hapten) of cyanophos was synthesized and was coupled to BSA to produce polyclonal antibodi es from rabbits. The antisera were screened against another hapten coupled to ovalbumin (OVA). Using the sera of highest specificity, an antigen-coated ELISA was developed, which showed an I50 of 310 ng/mL with the detection limit of 20 ng/mL. The antibodies showed negligible cross-reactivities with other organophosphorus pesticides except for parathion-methyl, which makes the assay suitable for the selective detection of cyanophos.