• Journal of Food Hygiene and Safety
• /
• v.9 no.3
• /
• pp.123-131
• /
• 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%)

• Korean Journal of Veterinary Research
• /
• v.29 no.4
• /
• pp.503-515
• /
• 1989

Critical parameters affecting sensitivity and specificity of an enzyme-linked immunosorbent assay(ELISA) for detection of antibodies to avian infections bronchitis virus(IBV) were standardized. By adopting the optimized conditions an equation calculating ELISA antibody titers from the observations at single serum dilution was formulated. The purified antigen of IBV Mass-41 strain was dispensed into polystyrene microplate wells at a concentration of 300ng per well($100{\mu}l$) and the plates were coated by completey drying at $37^{\circ}C$. Diluted chicken serum and horseradish peroxidase conjugated goat anti-chicken IgG were added in order in $100{\mu}l$ volumes per well and allowed to react for 30 minutes each at room temperature. Just before use and after each reaction the plates were washed three times with distilled water. Finally o-phenylenediamine solution was added as an enzyme substrate. After incubation for another 15 minutes at room temperature absorbances were read at 492nm. Hyperimmune serum against Mass-41 strain was used as internal reference positive(IRP) serum. After repeated titration of IRP and negative serum, a constant titer of IRP was determined. Serum titrations were carried out for various sample sera together with IRP and negative sera and the observed titers of sample sera were corrected by reflecting the ratio between observed and constant titers of IRP serum. These corrected titers of the sample sera were plotted against sample/positive(S/P) OD ratios. All the OD's measured in the serum titrations were also corrected by substracting negative serum OD. The following equation was formulated from the above data; $Log_{10}$ ELISA titer=$5.568({\log}_{10}S/P)+4.161$ Thus it was possible to calculate ELISA titer by measuring absorbance at 1/400 single serum dilution. Titer measured by cross ELISA tests employing Mass-41 strain and three local IBV isolates were similar. These results suggest that the ELISA tests standardized in this study can be used for evaluating not only vaccinal immunity but also for infection status against fields IBV's.

• Korean Journal of Food Science and Technology
• /
• v.32 no.3
• /
• pp.538-543
• /
• 2000

This study was conducted to develop an enzyme-linked immunosorbent assay(ELISA) for the determination of cooked goat meat. Muscle proteins were extracted from goat meat by heating at $98^{\circ}C$ for 15 min. Major thermostable(TS) protein, whose size and pI are 36 and 38 kDa and 4.5 respectively, were purified by DEAE-Sephadex A-50 and Sephadex G-75 column chromatography. The TS protein was immunized into rabbits in order to produce goat specific antibodies. Competitive indirect ELISA(ciELISA) was established by using the anti-TS antibody. The antibody showed high reactivity toward the TS antigen and the boiled goat meat extract but it did not show any reactivities toward extracts of boiled chicken, pork, lamb, and beef. Thus, this ciELISA developed in this study could be applicable to identify goat species from cooked meat.

• Journal of fish pathology
• /
• v.9 no.2
• /
• pp.169-176
• /
• 1996

An indirect double antibody enzyme-linked immunosorbent assay (ELISA) was developed for rapid detection of a new virus isolated from abnormally swimming salmonid fish, RVS (Retrovirus of salmonid). Results using brain tissue homogenates, and infected cell cultures are described. The sensitivity of the methods is $10^{2.6}$ $TCID_{50}/100{\mu}l$ of the examined cell culture fluid. The specificity was confirmed by the ELISA inhibition test and virological examinations. Viral antigen could be detected in artificially infected fish tissue homogenates. The assay will allow the diagnosis of RVS-infected fish within a day.

• Microbiology and Biotechnology Letters
• /
• v.20 no.2
• /
• pp.225-232
• /
• 1992

In order to develop an enzyme-linked immunosorbent assay(ELISA) for detecting aflatoxin $B_1(AFB_1)$, we produced and purified antibodies, thereafter established and evaluated methods of direct and indirect competitive ELISA. Anti-AFB, antisera, produced by immunizing rabbits with $AFB_1$-1-(0-carboxymethy1)oxime-bovine serum albumin conjugate ($AFB_1$-BSA), were removed of anti-BSA antibodies by quantitative precipitation reaction and further purified by ammonium sulfate precipitation and DEAE-Sephadex A-50 ion exchange chromatography. Purified IgG fractions were used as anti-$AFB_1$ antibodies. The antibodies, whose titer was deterrnined extremely high above $2 \times 10^6$, showed low cross-reactivity of 3~34% against $AFB_1$ analogues such as G2, B2, and GI. From the standard curves of direct and indirect competitive ELISA for AFBI, the detection ranges were found 0.2~20 and 1~10, 000 ng/ml(ppb) respectively. In their sensitivity, stability, simplicity, and rapidity, the direct method was more suitable than the indirect method for practical use.

• Journal of Microbiology and Biotechnology
• /
• v.33 no.9
• /
• pp.1170-1178
• /
• 2023

Food allergy represents a severe problem for many societies, including sensitive populations, academies, health authorities, and the food industry. Peanut allergy occupies a special place in the food allergy spectrum. To prevent consumption by consumers suffering from a peanut allergy, a rapid and sensitive detection method is essential to identify unintended peanut adulteration in processed foods. In this study, we produced four monoclonal antibodies (MAbs; RO 3A1-12, PB 4C12-10, PB 5F9-23, and PB 6G4-30) specific to thermo-stable and soluble proteins (TSSPs) of peanut and developed an enzyme-linked immunosorbent assay (ELISA) based on the MAbs. Among them, PB 5F9-23 MAb was firmly bound to Ara h 1, and other MAbs strongly reacted to Ara h 3 in the Western blot analysis. An antibody cocktail solution of the MAbs was used to enhance the sensitivity of an indirect ELISA, and the limit of detection of the indirect ELISA based on the antibody cocktail solution was 1 ng/ml and improved compared to the indirect ELISA based on the single MAb (11 ng/ml). The cross-reaction analysis revealed the high specificity of developed MAbs to peanut TSSPs without cross-reaction to other food allergens, including nuts. Subsequently, analyzing processed foods by indirect ELISA, all foods labeled as containing peanuts in the product description were confirmed to be positive. The results indicate that the developed antibodies exhibit high specificity and sensitivity to peanuts and can be used as bio-receptors in immunoassays or biosensors to detect intentional or unintentional adulteration of peanuts in processed foods, particularly heat-processed foods.

• The Korean Journal of Food And Nutrition
• /
• v.19 no.3
• /
• pp.296-300
• /
• 2006

Immunoblotting and competitive indirect enzyme-liked immunosorbent assay(Ci-ELISA) was used for detection of ${\beta}$-lactoglobulin(BLG) in dairy products, such as milk, dried milk and fermented milk. In immunoblotting, human IgE weakly recognized proteins of fermented milk, but still responded to dried milk even though become weak. Rabbit polyclonal antibody to BLG, used as a model of antigen, and milk allergic patients' IgE was used in the ELISA. Reactivities of Abs were the highest in market milk. BLG in fermented milk was detected in a low content. This result indicates the fermented milk have the lowest BLG content and could be used as hypo-allergenic food for milk-allergic individual.

• Korean Journal of Veterinary Research
• /
• v.29 no.4
• /
• pp.517-523
• /
• 1989

This study was undertaken to establish reliable diagnostic-procedures for the microbiological monitoring of laboratory animals. Murine(mice and rats) antibodies against hemagglutinating virus of Japan(HVJ), mouse hepatitis virus(MHV) and Mycoplasma pulmonis(Mp) were detected sensitively and specifically in experimentally and naturally infected animals' sera by an indirect enzyme-linked immunosorbent assay(ELISA), using urease conjugated antimurine immunoglobulin. The sensitivity and specificity of the complement fixation test which has been apllied widely for serodiagnosis of HVJ, MHV and Mp infections were apparently lower than those of ELISA. From these results, the ELISA was found to be available for the serodiagnosis of HVJ, MHV and Mp infections in mice and rats.

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

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