• Food Science of Animal Resources
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• v.24 no.2
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• pp.176-181
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• 2004

The specificity of sandwich enzyme-linked immunosorbent assay (ELISA) to detect Salmonella in milk was determined in this study. The antibodies used in sandwich ELISA were egg yolk immunoglobulin G (IgY) obtained after immunization of hen with outer membrane protein (OMP) fraction from Salmonella typhimurium and rabbit IgG obtained after immunization of rabbit with the purified OMP with the molecular weight of 40,000. The immunoblot assay showed that the IgY reacted strongly with OMP with the molecular weight of 6,000 and the rabbit IgG reacted strongly with OMP with the molecular weights of 40,000, 35,000, and 6,000 from the bacteria including Salmonella which belongs to Enterobacteriaceae. The IgY and rabbit IgG also reacted with other proteins from Salmonella typhimurium in immunoblot assay. Competitive ELISA showed that IgY showed specifity to react with two strains of Salmonella typhimurium and Salmonella cholerasuis but not with Escherichia coli and Yersinia enterocolitica. Two strains of Salmonella typhimurium added to UHT milk showed the highest absorbance of all the bacteria used in the sandwich ELISA. Some strains of Salmonella cholerasuis showed higher absorbances than non-Salmonella bacteria.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.385-389
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• 2004

The antibody-mix sandwich enzyme-linked immunosorbent assay (Ab-mix sELlS A) system was developed in order to simultaneously detect the extracellular polysaccharide (FPS) of Aspergillus, Penicillium, or Fusarium species using one detection system. The detection limit and detection range of the Ab-mix sELISA towards EPS of Penicilliun citrinum were not changed, and those towards Fusarium moniliforme EPS were changed a little compared to that of individual sandwich ELISA [9, 10]. The fungal culture filtrates of Aspergillus and Penicillium species showed nearly similar reactivity towards Ab-mix sELISA as that of sELISA using the MAb lB8 alone [9]. Also, the fungal culture filtrates of Fusarium species showed nearly the same reactivity towards Ab-mix sELISA as that of sELISA using the MAb lB8 alone [10]. Thus, this ELISA system showed that the three genera of molds, Aspergillus, Penicillium, or Fusarium, which are three major important molds producing mycotoxins in food or agricultural commodities, could be detected at the same time, using one detection system.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2113-2120
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• 2018

Cross-reactive material 197 ($CRM_{197}$) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. $CRM_{197}$ has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect $CRM_{197}$ and $CRM_{197}$ conjugate vaccines, we generated a human anti-$CRM_{197}$ monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-$CRM_{197}$ polyclonal antibody. The affinity ($K_D$) of 3F9 for $CRM_{197}$ was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of $CRM_{197}$. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml $CRM_{197}$. In addition, the 3F9 antibody bound to the $CRM_{197}$-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of $CRM_{197}$ and $CRM_{197}$ conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against $CRM_{197}$ and to develop a sandwich ELISA for $CRM_{197}$ conjugate vaccines.

• Journal of the Korean Society of Food Science and Nutrition
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• v.30 no.4
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• pp.600-604
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• 2001

Immunoassay was used to study the detection method of irradiated shrimp. Sandwich ELISA was formatted with monoclonal antibody (Ab) (M-IgG) and polyclonal Abs (P-IgG) and polyclonal Abs (P-IgG) individually produced against brown shrimp tropomyosin (TPM) as an antigen. When M-IgG was used as a coating Ab to capture TPM, and P-IgG were used as reaction Ab against captured TPM could be detected in the range of 12.5 to 50 $\mu\textrm{g}$/mL. Detected concentrations of TPM from irradiated shrimp decreased dose-dependently, and the concentration of Ag by combination of irradiation with heating or freezing treatments also decreased. This results suggests the possibility for Sandwich ELISA, one of immunological analyses, to be applied for detecting irradiated shrimp.

• Korean Journal of Food Science and Technology
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• v.35 no.3
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• pp.366-372
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• 2003

Enzyme-linked immunosorbent assay (ELISA) for assaying the 5-enolpyruvyshikimate-3-phosphate synthase from Agribacterium sp. CP4 (CP4 EPSPS) in genetically modified soybeans was developed. Polyclonal and monoclonal antibodies (Pab, Mab) specific to the CP4 EPSPS were produced. When using the Pab, the detection limit of sandwich ELISA toward CP4 EPSPS (0.03 ${\mu}g/mL$) was better than that of competitive indirect ELISA(ciELISA) (1 ${\mu}g/mL$). It was found that 2 of 3 monoclonal antibodies, Mab1 and Mab2, recognized the same antigenic determinant on CP4 EPSPS, but Mab3 recognized different antigenic determinant when competitive ELISA was performed using the Mabs. On the other hand, when the sensitivity of sandwich ELISA using combination of Pab and/or Mabs was determined, the sandiwich ELISA using Mab2 as a capture antibody and Pab-HRP as a secondary antibody showed the lowest detection limit of CP4 EPSPS (0.02 ${\mu}g/mL$). The sandwich ELISA developed in this study could be applied to detect glyphosate-tolerant soybeans.

• Korean Journal of Food Science and Technology
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• v.35 no.4
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• pp.556-560
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• 2003

It was determined whether the sandwich ELISA using specific anti-CP4 EPSPS polyclonal and monoclonal antibodies, developed in the previous study, could be applied to detect GM soybean or not. The soybeans (47 imported and 20 domestic soybeans) were analyzed by a sandwich ELISA. The results of imported soybeans were divided into two groups which were high contents $(39.1{\pm}13.5\;{\mu}g/g,\;n=33)$ and low contents of CP4 EPSPS $(2.6{\pm}1.2\;{\mu}g/g,\;n=14)$. The ratio of GM in imported soybeans was about 70.2%. One the other hand, the contents of CP4 EPSPS in domestic soybeans was very low $(0.9{\pm}0.5\;{\mu}g/g,\;n=20)$ which determined to be non-GM soybeans. In case of soybean sprouts, the contents of CP4 EPSPS in soybean sprouts were different between GM and non-GM soybean sprout. The CP4 EPSPS in cotyledon of GM soybeans sprout was higher than that in root hair. The contents of CP4 EPSPS in soybeans sprout of domestic soybeans were very low. Thus, it was possible to determine that the soybeans sprout was made of GM or non-GM soybeans. Also, PCR experiment showed that the sandwich ELISA was accurate to distinguish the soybeans to be GM or non-GM. These results showed the sandwich ELISA could determine the soybeans were GM or non-GM, rapidly and simply.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.764-772
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• 1999

To develop an enzyme-linked immunosorbent assay (ELISA) for detecting mold, we produced anti-mold polyclonal antibodies by immunizing extracellular polysaccharide (EPS) of Aspergillus flavus or Penicillium citrinum in rabbits subcutaneously. Using the purified antibody (Ab) and Ab-HRP conjugate, a sandwich ELISA for EPS was established. The standard curve of the ELISA showed the detection limit for P citrinum EPS to be $0.003{\;}\mu\textrm{g}/ml$. The cross-reactivities of the anti-P citrinum EPS Ab toward components of P citrinum such as EPS, liquid, and solid culture mycelium were 100, 10.5, and 0.58%, respectively, and those toward components of A. flavus such as EPS, liquid and solid culture mycelium, and spore were 300, 0.67, 0.29, and 0%, respectively. When the reactivities toward culture broths of 59 mold strains were tested by the sandwich ELISA, most of the Aspergillus (16 of 18) and Penicillium (14 of 16) strains along with one of the two Cladosporium strains gave positive signals in the culture broths even when diluted 1,000 fold, while the rest of species such as Fusarium, Absidia, Alternaria, and Candida gave negative signals. When the water extracts of 30 corn samples were analyzed by the sandwich ELISA, the EPS in the com could be detected in the concentration range of $0.1-1.6{\;}\mu\textrm{g}/g$.

• Journal of Microbiology and Biotechnology
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• v.13 no.5
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• pp.794-799
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• 2003

Enzyme-linked immunosorbent assay (ELISA) was developed for the rapid detection of Fusarium species, known as harmful fungi in food. One of the hybridoma cell lines (lB8) which produced a monoclonal antibody (Mab) specific to Fusarium extracellular polysaccharide (EPS) was screened and the Mab was produced and purified. A detection limit of the sandwich ELISA against F. moniliforme EPS was $0.001\;\mu\textrm{g}/ml$ in the standard curve. Among the 59 strains tested, most Fusarium species showed hight reactivity with Mab lB8, even when the culture broths were diluted 100,000 times. On the other hand, the other genera, except A. versicolor and Trichoderma viride, had no reactivity. When 1 to $100\;\mu\textrm{g}$ of F. moniliforme EPS was spiked into rice, potato, and mandarine orange, the average recoveries were 151%, 84%, and 94%, respectively, determined by sandwich ELISA. The correlation coefficients between the EPS levels determined by sandwich ELISA and the dry mycelial weight of the liquid culture of F. moniliforme, as well as between the EPS and colony forming unit in solid culture of potato, were 0.97 and 0.91, respectively.

• Parasites, Hosts and Diseases
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• v.37 no.4
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• pp.249-256
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• 1999

A total of 198 sera from stray cats was assayed against Toxoplasma gondii antigen by western blot. Out of 198 sera assayed, 26 sera (13.1％) showed typical blot patterns against T gondii. When spotted by ELISA absorbance and indirect latex agglutination lest (ILAT) titer, all 26 cases were distributed over the cut-off value of ELISA whereas 24 cases (92.3％) were in the positive range of 1:32 or higher and 2 cases in negative range by ILAT. Among western blot negative 172 sera, 162 cases were negative in both ILAT and ELISA while 10 cases were reactive falsely such that three cases were ILAT positive with 1:32 titer and 9 cases were ELISA positive (2 cases overlapped). These 10 cases reacted peculiarly without typical binding pattern in Western blot. Sandwich-ELISA was performed with monoclonal antibodies (mAbs) of Tg563 (30 kDa, SAG 1), Tg505 (22 kDa, SAG2), Tg605 (43 kDa, SAG3), Tg556 (28 kDa, GRA2), Tg737 (32 kDa, GRA6). Tg695 (66 kDa, ROP2), Tg786 (42 kDa, ROP6), and Tg621 (32 kDa, anonymous but cytosolic) clone, respectively. All western blot-positive cases were in the positive range and negative cases in the negative range clearly. Among the 10 false reactive cases, 3 cases were in the positive range with one or more mAbs. All mAbs used in this study were confirmed to be specific to T. gondii infection as a standardized sandwich-ELISA to differentiate it from other pathogens.

• Korean Journal of Veterinary Service
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• v.43 no.4
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• pp.237-244
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• 2020

Porcine transmissible gastroenteritis (TGE) has been a significant cause of economic losses in pig farming industry since 1950s. Although transmissible gastroenteritis virus (TGEV) has declined in recent years, it should not be excluded because of its characteristics; the frequency of gene mutation, the mortality in piglets, and the possibility for sudden incidence. Therefore, the herd-level monitoring of the virus is important to prevent further circulation of TGE. The aim of this study is to develop a large-scale sandwich enzyme-linked immunosorbent assay (ELISA) with high specificity to rapidly detect TGEV in feces by using monoclonal antibodies (Mabs). The TGEV specific Mabs were produced in hybridoma cells. Among the Mabs belonged to the IgG class developed by this study, the final selected 8H6, 1B7, 4G3, and 1F8 were identified to have the neutralization ability against TGEV. The sandwich ELISA was established using 8H6 as a reporter antibody and 1B7 and the reported 5C8 as a capture antibody. The developed sandwich ELISA was able to distinguish TGEV from other pathogenic diarrheal agents (porcine rotavirus, porcine reovirus, porcine epidemic diarrhea virus (PEDV), E. coli, and C. perfringens) in tissue culture as well as fecal samples. And the detection rate of TGEV in feces was 80% compared with RT-PCR. The results suggested that the developed sandwich ELISA may be useful in the herd-level monitoring for effective preventive measures due to the early diagnosis of TGEV using a large amount of samples.