• Journal of Microbiology and Biotechnology
• /
• v.30 no.11
• /
• pp.1760-1768
• /
• 2020

Vibrio cholerae, cause of the life-threatening diarrheal disease cholera, can be divided into different serogroups based on the structure of its lipopolysaccharide (LPS), which consists of lipid-A, core-polysaccharide and O-antigen polysaccharide (O-PS). The O1 serogroup, the predominant cause of cholera, includes two major serotypes, Inaba and Ogawa. These serotypes are differentiated by the presence of a single 2-O-methyl group in the upstream terminal perosamine of the Ogawa O-PS, which is absent in the Inaba O-PS. To ensure the consistent quality and efficacy of the current cholera vaccines, accurate measurement and characterization of each of these two serotypes is highly important. In this study, we efficiently screened a phage-displayed human synthetic Fab library by bio-panning against Ogawa LPS and finally selected three unique mAbs (D9, E11, and F7) that specifically react with Ogawa LPS. The mAbs bound to Vibrio cholerae vaccine in a dose-dependent fashion. Sequence and structure analyses of antibody paratopes suggest that IgG D9 might have the same fine specificity as that of the murine mAbs, which were shown to bind to the upstream terminal perosamine of Ogawa O-PS, whereas IgGs F7 and E11 showed some different characteristics in the paratopes. To our knowledge, this study is the first to demonstrate the generation of Ogawa-specific mAbs using phage display technology. The mAbs will be useful for identification and quantification of Ogawa LPS in multivalent V. cholerae vaccines.

• Korean Journal of Veterinary Research
• /
• v.26 no.1
• /
• pp.69-77
• /
• 1986

The purpose of this study was the examination for presence of K99 antigen (K99), enterotoxigenicity, 0-groups, colicin and antibiotic susceptibility among E. coil isolated from calves and cows. A total of 49(18.7%) among 262 strains, isolated from 30(26.5%) out of 113 calves and cows, possesed K99, and thirty three of 49 $K99^+$ strains produced ST. Of the strains of diarrheal calf origin which less than 15 days old, a high correlation was observed between enterotoxigenic ability and K99: 92.3% of the $K99^+$ strains produced heat stable enterotoxin(ST). In O group typing of 33 $ST^+$ strains, they belonged to O20(48.4%), O8(9.1%), O9(6.1%), O139(6.1%), O149(6.1%), O101(3.0%), O115(3.0%), except six which were untypable or autoagglutinable. Of 262 E. coil isolates, 30 strains(13.3%) produced colicin and K99 were detected in 6 strains. One hundred eighty eight strains(71.8%) of 262 E. coil isolates were resistance to ampicillin, chloramphenicol, gentamicin, kanamycin, streptomycin, tetracycline, alone or in combination thereof. One hundred and fourteen(60.6%) out of 188 drug resistance strains carried R factor($R^+$) which were transferable to the recipients by conjugation. Sixty five $R^+$ strains(57.0%) carried thermo-sensitive R plasmid.

• Food Science of Animal Resources
• /
• v.31 no.2
• /
• pp.158-165
• /
• 2011

A sensitive reverse transcriptase-PCR (RT-PCR) method to detect viable Escherichia coli O157:H7 in milk was established. The primer sets were designed based on the nucleotide sequences of the rfbE (per) and wbdN genes in the O157 antigen gene cluster of E. coli O157:H7. RT-PCR using five different primer sets yielded DNA with sizes of 655, 518, 450, and 149-bp, respectively. All five of the E. coli O157:H7 strains were detected by RT-PCR, but 11 other bacterial species were not. The sensitivity of RT-PCR was improved by adding yeast tRNA as a carrier to the crude RNA extract. The RT-PCR amplifying the 149-bp DNA fragment was the most sensitive for detecting E. coli O157:H7 and the most refractory to the bactericidal treatments. Heat treatment at $65^{\circ}C$ for 30 min was the least inhibitory of all bactericidal treatments. Treatment with RNase A strongly inhibited the RT-PCR of heated milk but not unheated milk. This study described RT-PCR methods that are specific and sensitive with a detection limit of 10 E. coli O157:H7 cells, and showed that pre-treating milk samples with RNase A improved the specificity to detect viable bacteria by RT-PCR.

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

• Korean Journal of Veterinary Service
• /
• v.33 no.2
• /
• pp.135-141
• /
• 2010

Bovine brucellosis, an important zoonosis, is diagnosed with serological tests such as the RBT, TAT using inactivated whole bacterial cells or bacterial lipopolysaccharide (LPS) antigen in Korea. However, a strong cross-reaction between Brucella spp. and Yersinia enterocolitica O9 in these tests has seriously complicated the diagnosis of animal brucellosis because Brucella spp. shares common antigenic determinants with Y. enterocolitica O9 in the smooth LPS region. In this study, Brucella-field strains were isolated from Brucellapositive Hanwoo in Kimhae, Korea and outer membrane protein (omp) which has low cross-reaction with Y. enterocolitica O9 and high immunogenicity was extracted from the field strains Then we compared ELISA using the extract with RBT-TAT. Fifteen field strains were isolated from 47 supra-mammary-lymph nodes, which were collected from 18 farms. Isolation rate was 32%. Brucella-specific antigen was identified by performing SDS-PAGE or Western blotting on extracted omp with at 0.5% n-lauroylsarcosine One hundred and ninety-two serum-samples were used in the experiment: 142 negative and 50 positive samples verified by RBT-TAT. According to ELISA results, 127 samples were negative and 15 appeared positive among 142 negatives by RBT-TAT, while 42 samples were positive and 8 were negative among 50 positives by RBT-TAT. Therefore, it showed 89.4% of specificity and 84% of sensi-tivity. Through the current experiments, we could set up an ELISA based on the omp which has low cross-reaction and high immunogenicity and concluded that the omp could be a good material for accurate diagnosis of bovine brucellosis.

• Journal of Microbiology and Biotechnology
• /
• v.12 no.5
• /
• pp.780-786
• /
• 2002

An immunosensor based on surface plasmon resonance (SPR) with a self-assembled protein G layer was developed for the detection of Escherichia coli O157:H7. A self-assembled protein C layer on a gold (Au) surface was fabricated by adsorbing the mixture of 11-mercaptoundecanoic acid (MUA) and hexanethiol at various molar ratios and by activating chemical binding between free amine (-$NH_2$) of protein G and 11-(MUA) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) in series. The formation of a self-assembled protein G layer on an Au substrate and the binding of the antibody and antigen in series were confirmed by SPR spectroscopy. The surface morphology analyses of the self-assembled protein G layer on the Au substrate, monoclonal antibody (Mab) against E. coli O157:H7 which was immobilized on protein G, and bound E. coli O157:H7 extracts on Immobilized Mab against E. coii O157:H7 were performed by atomic force microscopy (AFM). The detection limit of the SPR-based immunosensor for E. coli O157:H7 was found to be about $10^4$ cells/ml.

• Korean Journal of Veterinary Research
• /
• v.31 no.4
• /
• pp.491-500
• /
• 1991

This study was conducted to detect the serum antibodies in the experimentally toxoplasma infected dogs and street dogs by use the of an enzyme-linked immunosorbent assay (ELISA). And this test was performed on the polystylene microplate by coating with the tachyzoites soluble antigen of T gondii (RH strain), incubated with sera diluted then, added with HPO-conjugated rabbit anti-dog IgG and o-phenylenediamine used as a substrate. Tachyzoites of T gondii harvested from mouse peritoneal cavity were purified by 30, 40 and 50% Percoll density gradient centrifugation and used as the source of antigen. The results obtained were summarized as follows; 1. The highest ratio of positive to negative (P/N ratio) was obtained at the level of $l{\mu}g/ml$ protein concentration of antigen with the 1/4000 dilution of the conjugate measured by checker-board titration. It was regarded as the optimum concentration of the antigen and conjugate. 2. Cut-off value in this IgG ELISA was 0.375 that was determined by mean absorbance (at 492nm) of IFA negative serum added with the dauble value of the standard deviation $(mean{\pm}2S.D.)$. 3. Serum ELISA IgG antibodies to T gondii in the exyerimentally infected dogs were detected firstly at the Week 3 after inoculation and the highest titer was recognized at the Week 4, 5 and 6 after inoculation. 4. Stability of the antigen absorbed in the microplates that were preserved at $4^{\circ}C$ and $-25^{\circ}C$ separately were prolonged up to 3 weeks and 10 weeks at $4^{\circ}C$ and $-25^{\circ}C$, respectively. However the reproducibility was not reliable after the preservation of 4 weeks and longer. 5. Positive rate of the specific antibodies in 312 test sera was 28.5% and there was no significant differences between the male (27.8%) and female (29.5%), respectively. 6. The IgG ELISA was proved to be a specific procedure for the detection of antibodies to canine toxoplasma infection and also evaluated as a screening test for the large scale of test samples in laboratory.

• Biomedical Science Letters
• /
• v.28 no.2
• /
• pp.127-136
• /
• 2022

Uropathogenic Escherichia coli (UPEC) is main causative agent of urinary tract infections. They are classified based on various types of O antigen. UPEC strains commonly possess many genes encoding virulece-associated factors. E. coli strains are generally divided into four main phylogenetic groups. The virulence factor (VF) profiles of UPEC are related with their O-serogroups in each strains. A total of 681 strains of UPEC clinical isolates were collected from Korean healthcare facility (1989: 123 strains and 2010-2014: 558 strains). The UPEC clinical isolates were analyzed by polymerase chain reaction (PCR) methods. A total of 14 O-serotypes (O1, O2, O4, O6, O7, O8, O15, O16, O18, O21, O22, O25, O75 and O83), 6 virulence factors (papC, fimG/H, sfaD/E, hly1, cnf1 and usp) and phylogenetic groups were identified. The most prevalent O-serogroups were O6 (11.1%) in 1989 UPEC strains and O25 (21.0%) in 2010-2014 UPEC strains. The identified VFs, phylogenetic groups in 1989 UPEC strains and 2010-2014 UPEC strains were fimG/H and B2 group. In this study, O6 serotype was revealed the close relationships with VFs. Also, the distribution of prevalence O-serogroups of UPEC has been changed from O6 to O25 and virulence of UPEC strains was increased during past twenty-one years.

• 한국생물공학회:학술대회논문집
• /
• 2003.10a
• /
• pp.745-745
• /
• 2003

• Proceedings of the PSK Conference
• /
• 2000.10a
• /
• pp.181.2-182
• /
• 2000