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

• 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.

• Asian-Australasian Journal of Animal Sciences
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• v.18 no.10
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• pp.1498-1504
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• 2005

Competitive direct ELISA was developed to detect gentamicin residues. Mice immunized with gentamicin-keyhole limpet hemocyanin (KLH) conjugate developed good antiserum titers, which gradually increased with booster injections, indicating immunization was successfully processed. Monoclonal antibody against gentamicin was prepared using hybridoma cells cloned by limit dilution of fused cells. IgG was purified from ascites fluid of hybridoma cell-injected mice through ammonium sulfate precipitation and Sephadex G-25 gel filtration. After the gel filtration, fractions of high antibody titer were further purified through affinity chromatography on protein A/G column. Monoclonal antibody against gentamicin was confirmed as IgG1, which has kappa light chain. Cross-reactivities ($CR_{50}$) of gentamicin monoclonal antibody to other aminoglycosides (kanamycin, neomycin, and streptomycin) were less than 0.005%, indicating the monoclonal antibody was highly specific for gentamicin. Standard curve constructed through competitive direct ELISA showed measurement range (from 80 to 20% of B/$B_0$ ratio) of gentamicin was between 1 and 40 ng/ml, and 50% of B/$B_0$ ratio was about 4 ng/ml. The gentamicin concentration rapidly increased to 1,300 ng/ml after the intramuscular administration up to 2 h, then sharply decreased to less than 300 ng/ml after 4 h of withdrawal, during which the elimination half-life ($t_{1/2}$) of gentamicin in the rabbit plasma was estimated to be 1.8 h. Competitive direct ELISA method developed in this study using the prepared monoclonal antibody is highly sensitive for gentamicin, and could be useful for detecting gentamicin residues in plasma of live animals.

• 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 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%)

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.161-167
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• 2010

Early diagnoses of pregnancy for animal such as swine and bovine is extremely important to increase income of a farmhouse and for the management of farm. For the development of immunoasaay system of pregnancy in swine, we report a competitive heterologous enzyme linked immunosorbent assay (ELISA) for the direct measurement of oestrone sulfate (E1S) in diluted urine using anti-E1G (glucuronide) monoclonal antibody which cross react with ElS. The principle of assay was based on the typical solid-phase competitive ELISA methods using E1G-HRP (horseradish peroxidase) as a tracer and E1S for standard. The method had a reasonable sensitivity for the detection of E1S with 0.15 ng/ml as a detection limit. The intra-assay and inter-assay precisions were raging coefficient of from 8.50~9.67% and 8.50~9.87%, respectively, which were quite acceptable. In a field trial with a group 37 sows (18 non-pregnancy and 19 pregnancy sows) after day 29~30 post service, the concentration of E1S were determined to be below 30 ng/ml in all non-pregnancy group and over 48 ng/ml in pregnancy group except one sample. The method described here, heterologous ELISA for the measurement of E1S in urine is good enough for monitoring the early pregnancy test of swine.

• 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.

• Journal of Microbiology and Biotechnology
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• v.23 no.1
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• pp.69-75
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• 2013

Enrofloxacin is a fluoroquinolone antibiotic approved for the treatment of infections in animals. Because of the side effects to consumers of animal products, the maximum residue limits (MRLs) of enrofloxacin in animal tissues for consumption are regulated. In this study, a monoclonal antibody (mAb) against enrofloxacin was prepared and characterized for the development of a direct competitive enzyme-linked immunosorbent assay (ELISA). The obtained mAb, Enro44, was highly specific for enrofloxacin and had a 50% inhibition concentration ($IC_{50}$) of 1.99 ng/ml in a competitive ELISA, and the limit of detection (LOD) was 0.50 ng/ml. The cross-reactivity of the mAb with other quinolones and fluoroquinolones was lower than 0.01%. The subclass of the mAb Enro44 was identified as IgG1. The antigen (Ag)-captured direct competitive ELISA using the mAb Enro44 was tested on different spiked samples, including chicken muscle, cattle milk, and cattle urine, and the assay demonstrated recoveries of 82-112%, 80-125%, and 78-124%, respectively. Furthermore, the quantitation of enrofloxacin obtained from the ELISA and from high-performance liquid chromatography (HPLC) was in good agreement, with the linear regression coefficient between 0.933 and 1.056. The cDNAs encoding a heavy-chain Fd fragment (VH and CH1) and a light chain of the mAb Enro44 were cloned and sequenced. Taken together, the results obtained reveal a potential use of this mAb in an ELISA for the detection of enrofloxacin in food samples. The information of amino acid sequence of this mAb will be useful for further modification and production of the mAb in a bioreactor.

• Applied Biological Chemistry
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• v.36 no.1
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• pp.7-10
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• 1993

ELISA method was applied for the screening of zearalenone producing strains. The developed ELISA was as follow: $125\;{\mu}l$ of diluted solution (1 : 500) of antibody was added to each microtiter well and incubated overnight at $40^{\circ}C$. For direct competitive ELISA, samples and zearalenone-peroxidase conjugate were mixed in a 1 : 1 ratio, and a $100\;{\mu}l$ of aliquot was then added to antisera-coated wells. Plates were incubated for 30 minutes at $37^{\circ}C$, and wells washed 6 times, and $100\;{\mu}l$ of ABTS substrates was added. Plates were incubated for antother 15 minutes at $37^{\circ}C$, and $100\;{\mu}l$ of stopping reagent was added to the wells and absorbance was recorded at 410nm on ELISA Reader. Among 19 strains showed zearalenone-producing ability by ELISA, 3 strains (R-5, C-46, S-134) produced more than 50 ng/ml of zearalenone.

• Toxicological Research
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• v.27 no.2
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• pp.125-131
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• 2011

Through the present study, we produced a monoclonal antibody against aflatoxin B1 (AFB1) using AFB1-carboxymethoxylamine BSA conjugates. One clone showing high binding ability was selected and it was applied to develop a direct competitive ELISA system. The epitope densities of AFB1-CMO against BSA and KLH were about 1 : 6 and 1 : 545, respectively. The monoclonal antibody (mAb) from cloned hybridoma cell was the IgG1 subclass with ${\lambda}$-type light chains. The $IC_{50}s$ of the monoclonal antibody developed for AFB1, AFB2, AFG1 and AFG2 were 4.36, 7.22, 6.61 and 29.41 ng/ml, respectively, based on the AFB1-KLH coated ELISA system and 15.28, 26.62, 32.75 and 56.67 ng/ml, respectively, based on the mAb coated ELISA. Cross-relativities of mAb to AFB1 for AFB2, AFG1 and AFG2 were 60.47, 65.97 and 14.83% in the AFB1-KLH coated ELISA, and 59.41, 46.66 and 26.97% in the mAb coated ELISA, respectively. Quantitative calculations for AFB1 from the AFB1-Ab ELISA and AFB1-Ag ELISA ranged from 0.25 to 25 ng/ml ($R^2$ > 0.99) and from 1 to 100 ng/ml ($R^2$ > 0.99), respectively. The intra- and inter-assay precision CVs were < 10% in both ELISA assay, representing good reproducibility of developed assay. Recoveries ranged from 79.18 to 91.27%, CVs ranged from 3.21 to 7.97% after spiking AFB1 at concentrations ranging from 5 to 50 ng/ml and following by extraction with 70% methanol solution in the Ab-coated ELISA. In conclusion, we produced a group specific mAb against aflatoxins and developed two direct competitive ELISAs for the detection of AFB1 in feeds based on a monoclonal antibody developed.