• The Journal of the Korean Society for Microbiology
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• v.22 no.4
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• pp.445-451
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• 1987

Coagglutination method is widely used for the diagnosis of Salmonella infection. This test, however, has a disadvantage of false positive reaction due to the coagglutination of staphylococci with non-specific immune complexes or anti-staphylococci antibody in serum. Salmonell O antigen was detected by enzyme immunoassay with protein A-bearing Staphylococcus aureus as in the solid phase. Horse radish peroxidase was labeled to IgG specific against Salmonella O antigen. This enzyme immunoassay was much more sensitive than conventional coagglutination method without false poitive agglutination. To improve the sensitivity for detection of Salmonella O antigen in samples, we tried to determine the optimal concentration of normal IgG that inhibits non-specific binding of horse radish peroxidase labeled IgG to staphylococci, and to establish the optimal condition of reaction between antigen-antibody complex and staphylococci. Non-specific binding of horse radish peroxidase labeled specific IgG to staphylococci was almost blocked when the enzyme labeled IgG was 500-fold diluted with phosphate buffered saline containing 2mg/ml of normal IgG. When staphylococci coated with antibody to Salmonella O antigen were mixed with antigen-antibody complex and then incubated for 1 hour at room temperature, the minimal detectable concentration of Salmonella O antigen was 1ng/ml. The sensitivity of enzyme immunoassay was 100-fold greater than a conventional coagglutination method. This enzyme immunoassay could be expected as an improved method for detection of other infectious agents.

• Journal of Powder Materials
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• v.16 no.3
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• pp.180-184
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• 2009

In this paper, the electrochemical non-enzyme immunosensor has been developed for the determination of salmonella antigen, using inverse voltammetry. For the estimation of salmonella antigen concentration, the $Fe_3O_4$ nanoparticles synthesized by microemulsion method were conjugated with salmonella antigen. Then, the immunocomplex between antibody immobilized on the transducer surface and antigen containing a magnetic nanoparticles was formed. From the linear relationship between the reduction peak current of Fe(III) and salmonella antigen concentration, it is suggested that the electrochemical non-enzyme biosensor is applicable to detect salmonella antigen in the concentration range of $10^1-10^5$ CFU/ml.

• Korean Journal of Veterinary Research
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• v.38 no.4
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• pp.793-802
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• 1998

Salmonellosis caused by a number of serotypes of Salmonella is an infectious, acute or chronic, zoonotic disease and characterized by enteritis and diarrhea, septicemia in animal. In these studies we investigated the prevalent serotypes of Salmonella causing animal salmonellosis in Korea and the 71 strains of Salmonella spp. were isolated from materials such as mesenteric lymph nodes, fecal samples from slaughtered animal. With the identification test results, the most prevalent serotypes were, in order, S stanley 31 strains (43.7%), S typhimurium 19 strains (26.8%) and S montevideo 11 strains (15.5%), respectively. And we could establish the method for detection of antibodies to broad variety of Salmonella serotypes. Lipopolysaccharide(LPS) antigen extracted from Salmonella was more sensitive and specific than outer membrane protein antigen from that for detection of Salmonella antibody by using an indirect ELISA. The optimal concentration of antigen was 100ng/ml of LPS, the dilutions of conjugate and serum were 1 : 1,000~2,000 and 1 : 200~400, respectively. The mix LPS-ELISA which was used by mixing LPS from S typhimurium (group B), S choleraesuis (group C) and S enteritidis (group D) were more rapid and effective than that used LPS from individual strain for detection of Salmonella serogroup O4, O7 and O9 antibody at the same time. We could obtain the high values of optical density ($0.73{\pm}0.32$) by mix LPS-ELISA on the farm which had occurred salmonellosis, but very low values of $0.17{\pm}0.06$ on the negative farm of salmonellosis. So, the mix LPS-ELISA may be used to monitor the serological surveillance for the presence of infection with a number of serotypes of Salmonella and would be useful for prevention and control of salmonellosis in animal.

• Proceedings of the Microbiological Society of Korea Conference
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• 2000.05a
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• pp.58-65
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• 2000

Diarrheal diseases are a major cause of both illness and death in developing countries and are caused by rotavirus, Shigella spp., Salmonella spp., enterotoxigenic Escherichia coli (ETEC), and Vibrio spp. In this study, for the development of vaccine against diarrheal diseases caused by Shigella sonei, Salmonella typhimurium, E. coli O157, and Vibrio cholerae, cloning and nucleotide sequence analysis of genes and characteristics of their gene products in E. coli were performed. For construction of attenuated strain of S. sonnei KNIH104 and Salmonella typhimurium KNIH100, the aroA genes were cloned, respectively. The recombinant plasmid $_pJP{\Delta}A45$ containing aroA deleted region and suicide vector $(_pJP5603)$ was constructed. The aroA gene deleted mutants were constructed using this recombinant plasmid. For cloning gene encoding antigenic region of E. coli O157 KNIH317, the O-antigen synthesis gene cluster and sit gene was cloned. The E. coli XL1-Blue cells harboring this recombinant plasmid showed cytotoxicity in Vero cells. The ctx gene was cloned for tile purpose of antigenic region against V. cholerae KNIH002. Sequence analysis confirmed that the virulence gene cassette was consisted of ace, zot, ctxA and ctxB genes.

• The Journal of the Korean Society for Microbiology
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• v.20 no.1
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• pp.91-102
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• 1985

The advantages of enzyme-linked immunosorbent assay(ELISA) are its senstivity and simplicity in detecting IgG, IgM and IgA antibody. To apply ELISA to diagnosis of typhoid fever, antigen such as lipopolysaccharide of Salmonella typhi or killed whole cell must be coated on solid phase. It is easy to coat lipopolysaccharide on ELISA plate but troublesome to purify it. As it is easy to obtain the killed whole cells, the development of the appropriate method by which those antigens of S. typhi are optimally coated on solid phase is needed. To establish the appropriate method, carbonate buffer, methanol or poly-L-lysine was applied as binding substance on polystyrene or polyvinylchloride plate as solid phase when the killed whole cell antigens of S. typhi varided as follows: $10^6$, $10^7$, $10^8$ and $10^9\;cell/ml$. The criteria of the optimal method were determined as follows: 1. The optical density of positive sera is above 1.0(0.6 in IgM) at 1:10 serum dilution and is 0.3(0.2 in IgM) higher than that of negative sera: 2. The O.D. of sera is flat or lowering according to serum dilution: 3. It must be that the O.D. of negative sera is lower than 0.2 at the point of serum dilution where the O.D. of positive sera is higher than 1.0(0.5 in IgM). The results obtained were summarized as follows: 1. The methods which fitted the above criteria were to use poly-L-lysine as binding substance, polyvinylchloride plate as solid phase and $10^7\;cell/ml$ as antigen concentration of S. typhi(poly-L-lysine/polyvinylchloride/$10^7$) and poly-L-lysine/polyvinylchloride/$10^8$ in detecting IgG antibody, methanol/polystyrene/$10^9$, poly-L-lysine/polyvinylchloride/$10^8$ and poly-L-lysine/polyvinylchloride/$10^9$ in IgM and carbonate buffer/polystyrene/$10^8$, carbonate buffer/polystyrene/$10^9$, methanol/polystyrene/$10^8$, methanol/polyvinylchloride/$10^8$, methanol/polyvinylchloride/$10^9$, poly-L-lysine/polyvinylchloride/$10^8$ and poly-L-lysine/polyvinylchloride/$10^9$ in IgA. 2. The coaling method using poly-L-lysine, polyvinylchloride plate and $10^8\;cell/ml$ was best to assay IgG, IgM and IgA antibody all in one. By this method, to assay the each immunoglobulin calss with an appropriate fixed serum dilution, 1:320 dilution was best.

• Food Science of Animal Resources
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• v.32 no.6
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• pp.706-712
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• 2012

The present study was aimed to produce a chicken polyclonal antibody against Cronobacter muytjensii and to develop an immunoassay for its detection. Purification of anti-C. muytjensii IgY from egg yolk was accomplished using various methods such as water dilution and salt precipitation. As a result, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced two bands around 30 and 66 kDa, corresponding to a light and a heavy chain, respectively. Indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was performed to determine the effectiveness of the chicken IgY against C. muytjensii. The optimum conditions for detecting C. muytjensii by indirect ELISA and checkerboard titration of the antigen revealed an optimum average absorbance at the concentration of 18 ${\mu}g/mL$, having ca. $10^8$ coated cells per well. The anti-C. muytjensii IgY antibody had high specificity for C. muytjensii and low cross-reactivity with other tested pathogens. In this assay, no cross-reactivity was observed with the other genera of pathogenic bacteria including Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, Bacillus cereus, Enterobacter aerogenes, Salmonella Enteritidis and Listeria monocytogenes. In addition, detection of C. muytjensii in infant formula powder showed a low matrix effect on the detection curve of IC-ELISA for C. muytjensii, with similar detection limit of $10^5$ CFU/mL as shown in standard curve. These findings demonstrate that the developed method is able to detect C. muytjensii in infant formula powder. Due to the stable antibody supply without sacrificing animals, this IgY can have wide applications for the rapid and accurate detection of C. muytjensii in dairy foods samples.

• Journal of Food Hygiene and Safety
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• v.18 no.3
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• pp.118-124
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• 2003

For the rapid detection of various pathogenic microorganisms from food sample, various kinds of kits have been developed and commercially available in the markets. With the advantages of speed, accuracy and easiness, the market of these kits has gradually increased for the QC and QA field of food company as well as testing facilities or laboratories. In this study, the characteristics such as the detection limit and the sensitivity of immunochromatographic type of rapid detection kit (Donga Co, Korea, D-kit) for E. coli 0157:H7 developed by monoclonal antibody were examined and also the possibility of application of the kit to food samples was evaluated. The reference kits used for comparison study were Reveal E. coli 0157:H7 (Neogen Co., USA, R-kit) and VIP EHEC kit (Biocontrol Inc., USA, V-kit) occupying major market share. In the detection limit test with the E. coli 0157:H7 reference, both R-kit and D-kit showed a distinct positive reaction in $10^4$/ml and weak positive reaction in $10^3$/ml, whereas V-kit showed a same reaction in 105/ml. Also, it was identified that the culture treated with heat showed more sensitivity than no heat treated culture. The sensitivity test was conducted against 22 isolates of E. coli 0157:H7, 7 strains of non-O157:H7 verotoxin-producing E. coli, 40 strains of E. coli with different O and H antigen type, and 38 strains of non-E. coli Enterobacteriaceae, and all of the test strains except three were showed exactly three were showed exactly the same reaction against three kinds of the tested kits. All the three kinds of kits showed a positive reaction against E. coli O157:H19, E. coli O148:H18 and Salmonella galinarium. We suppose that there might be a similarity in serological property between these three strains and O157:H7. From the test results, it can be concluded that there is (was) no difference between the D-kit developed in this study and R-kit or V-kit based on the detection limit and sensitivity.