• Journal of Food Hygiene and Safety
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• v.6 no.3
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• pp.111-117
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• 1991

We examined to develop the enzyme linked immunosorbent assay (ELISA) for determination of zearalenone in animal feeds. Zearalenone was first converted to 6'-(carboxymethyl) zearalenone oxime(zearalenone oxime) to get a coupling site and then conjugated to bovine serum albumin(BSA) for use as immunogen and to horseradish peroxidase(HRP) for use as enzyme marker. Antibody against zearalenone was obtained after 11 weeks of immunization of rabbit with zearalenone oxime-BSA. Cross reactivity of the antibody with ${\alpha}-zearalanol,\;{\beta}-zearalenol,\;{\alpha}-zearalanol\;and\;{\beta}-zearalanol$ were 168, 46, 26 and 20% respectiviely. A simple procedure was devised for the screening of zearalenone in feeds using ELISA. Feeds samples(5g) were extracted by blending with 25 ml of methanol-phospate butTered saline-dimethylformate(70 : 29 : 1) and the extract was filtered and aqueous filterate analyzed. It took only 1 hours to do whole procedure for the analysis of zearalenone in feeds by the direct competitive ELISA, and detectable limit was 1-100 ppb. Using this procedure, only 4 of 24 feed samples showed positive results with 3.93-7.43 ppb levels.

• Biomaterials and Biomechanics in Bioengineering
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• v.2 no.1
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• pp.23-32
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• 2015

Facile immobilization of growth factors in hyaluronic acid (HA) hydrogels using dual enzymes is reported in the paper. The hydrogels were formed by using horseradish peroxidase (HRP) and hydrogen peroxide ($H_2O_2$) and transforming growth factor-${\beta}3$ (TGF-${\beta}3$) was covalently conjugated on the hydrogels in situ using tyrosinase (Ty) without any modifications. For the preparation of hydrogels, HA was grafted with poly(ethylene glycol) (PEG), which was modified with a tyrosine. The gelation times of the HA hydrogels were ranging from 415 to 17 s and the storage moduli was dependent on the concentration of $H_2O_2$ and Ty (470-1600 Pa). A native TGF-${\beta}3$ (200 ng/mL) was readily encapsulated in the HA hydrogels and 17% of the TGF-${\beta}3$ was released over 1 month at the Ty concentration of 0.5 KU/mL, while the release was faster when 0.3 KU/mL of Ty was used for the encapsulation (27%). It can be suggested that the growth factors resident in the hydrogels for a long period of time may lead cells proliferating and differentiating, whereas the growth factors that are initially released from the hydrogels can induce the ingrowth of cells into the matrices. Therefore, the dual enzymatic methods as facile gel forming and loading of various native growth factors or therapeutic proteins could be highly promising for tissue regenerative medicines.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.501-509
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• 2021

More than half the world's population is thought to be infected with Helicobacter pylori. Although the majority of infected people are asymptomatic, H. pylori infection may cause gastric ulcers and deadly gastric cancer. Owing to the difficulty and invasiveness of current routine culture and diagnostic methods, a highly sensitive and specific noninvasive assay for H. pylori is of interest. This study highlighted the design and performance of a colorimetric magneto loop-mediated isothermal amplification (CM-LAMP) assay to detect H. pylori in spiked saliva samples. LF primers were coated on magnetic nanoparticles by carbodiimide-induced immobilization and functionally used for solid-phase amplification. During the LAMP reaction at 66℃, biotin-tagged FIPs were incorporated into LAMP amplicons. The colorimetric signal developed after the addition of NeutrAvidin horseradish peroxidase conjugate (NA-HRP) and ABTS. None of the tested microorganisms, including closely related bacteria, was shown positive by the CM-LAMP assay except H. pylori isolates. This novel platform was highly specific and 100-fold more sensitive (40 CFU/ml or 0.2 CFU per reaction) than the PCR and conventional LAMP assays for the detection of H. pylori in spiked saliva. Our results demonstrated the feasibility of using this noninvasive molecular diagnostic test to detect H. pylori in saliva samples.

• Journal of Microbiology and Biotechnology
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• v.31 no.5
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• pp.705-709
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• 2021

Porphyromonas gingivalis (P. gingivalis) is a major bacterial pathogen that causes periodontitis, a chronic inflammatory disease of tissues around the teeth. Periodontitis is known to be related to other diseases, such as oral cancer, Alzheimer's disease, and rheumatism. Thus, a precise and sensitive test to detect P. gingivalis is necessary for the early diagnosis of periodontitis. The objective of this study was to optimize a rapid visual detection system for P. gingivalis. First, we performed a visual membrane immunoassay using 3,3',5,5'-tetramethylbenzidine (TMB; blue) and coating and detection antibodies that could bind to the host laboratory strain, ATCC 33277. Antibodies against the P. gingivalis surface adhesion molecules RgpB (arginine proteinase) and Kgp (lysine proteinase) were determined to be the most specific coating and detection antibodies, respectively. Using these two selected antibodies, the streptavidin-horseradish peroxidase (HRP) reaction was performed using a nitrocellulose membrane and visualized with a detection range of 10³-10⁵ bacterial cells/ml following incubation for 15 min. These selected conditions were applied to test other oral bacteria, and the results showed that P. gingivalis could be detected without cross-reactivity to other bacteria, including Streptococcus mutans and Escherichia fergusonii. Furthermore, three clinical strains of P. gingivalis, KCOM 2880, KCOM 2803, and KCOM 3190, were also recognized using this optimized enzyme immunoassay (EIA) system. To conclude, we established optimized conditions for P. gingivalis detection with specificity, accuracy, and sensitivity. These results could be utilized to manufacture economical and rapid detection kits for P. gingivalis.

• Korean Journal of Microbiology
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• v.52 no.1
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• pp.10-17
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• 2016

Enzyme immunoassay to analyze specific binding activity of antibody to antigen uses horseradish peroxidase (HRP) or alkaline phosphatase (AP). Chemical methods are usually used for coupling of these enzymes to antibody, which is complicated and random cross-linking process. As results, it causes decreases or loss of functional activity of either antibody or enzyme. In addition, most enzyme assays use secondary antibody to detect antigen binding activity of primary antibody. Enzymes coupled to secondary antibody provide a binding signal by substrate-based color development, suggesting secondary antibody is required in enzyme immunoassay. Additional incubation time for binding of secondary antibody should also be necessary. More importantly, non-specific binding activity caused by secondary antibody should also be eliminated. In this study, we cloned AP isolated from Escherichia coli (E. coli) chromosome by PCR and fused to) hAY4 single-chain variable domain fragment (ScFv) specific to death receptor (DR4) which is a receptor for tumor necrosis factor ${\alpha}$ related apoptosis induced ligand (TRAIL). hAY4 ScFv-AP expressed in E. coli showed 73.8 kDa as a monomer in SDS-PAGE. However, this fusion protein shown in size-exclusion chromatography (SEC) exhibited 147.6 kDa as a dimer confirming that natural dimerization of AP by non-covalent association induced ScFv-AP dimerization. In several immunoassay such as ELISA, Western blot and immunocytochemistry, it showed antigen binding activity by color development of substrates catalyzed by AP directly fused to primary hAY4 ScFv without secondary antibody. In summary, hAY4 ScFv-AP fusion protein was successfully purified as a soluble dimeric form in E. coli and showed antigen binding activity in several immunoassays without addition of secondary antibody which sometimes causes time-consuming, expensive and non-specific false binding.

• Korean Journal of Food Science and Technology
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• v.36 no.6
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• pp.959-967
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• 2004

To screen probiotic lactic acid bacteria with potent adhesive property on human colonic mucosa, colonic mucin-binding assay was introduced. This colonic mucin-binding assay actually measures the binding activity of surface lectin-like protein (SLP) on colonic mucin, and the optimal conditions were examined. The optimal pH for colonic mucin coating on plate wells was 4.8, and ${\times}24,000$ diluted solution of commercially available horseradish peroxidase (HRP) conjugated streptoavidin yielded good results, for rapid screening, $5.0\;{\mu}g/mL$ of biotinylated SLP from lactic acid bacteria was optimal, and optimal scintillation time of 3,3',5,5'-tetramethyl benzidine (TMB) was 10 min. These conditions were useful for both rapid selection and quantitative analysis of lactic acid bacteria that have high adhesion property to human intestinal tract. Among 50 strains of lactic acid bacteria, including 32 type culture strains and 18 isolated strains from infant feces, Lactobacillus species FSB-1 isolated from kimchi showed the highest binding activity to colonic mucin. From taxonomical viewpoints based on morphological study, physico-biochemical study, partial 16S rDNA seguencing, and phylogenetic analysis, L. species FSB-1 was identified as Lactobacillus brevis.

• Applied Microscopy
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• v.38 no.4
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• pp.375-382
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• 2008

In order to investigate shape of synaptic vesicles of the tooth pulp afferent boutons and their presynaptic endings (p-endings), and the neuroactive substance of the p-endings in the trigeminal nucleus principalis, rat incisor tooth pulp afferents were labeled by the horseradish peroxidase (HRP) and quantitative ultrastructural analysis and postembedding immunogold labeling were performed. Labeled tooth pulp afferent boutons contained clear, spherical synaptic vesicles (diameter: $45{\sim}55\;nm$) and occasionally dense core vesicles(diameter: $80{\sim}120\;nm$). They formed symmetrical synapses with unlabeled axon terminals (p-endings) containing pleomorphic synaptic vesicles. The ratio of short to long diameter (form factor) of synaptic vesicles of pulp afferent boutons was 0.6 to 0.99, whereas that of p-endings was 0.25 to 0.99. In addition, most of the p-endings showed GABA-like immunoreactivity. These results indicate that the shape of synaptic vesicles is quite different between the tooth pulp afferent boutons and p-endings, and the p-endings may contain GABA as a neuroactive substance in the trigeminal nucleus principalis.

• 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.

• Development and Reproduction
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• v.4 no.1
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• pp.115-123
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• 2000

A few enzymeimmunoassay (EfA) for testosterone (T) have been reported but was not suitable for all biological samples. The present study was designed to develop a rapid, ultrasensitive EIA and to apply this technique for study the physiological changes of T in biological samples. Saliva samples were collected at 06:00～09:00 hour during one menstrual cycle from 18 normally menstruating women and on 09:00～10:00 hour from 20 normal men. The present study shows an established EIA for testosterone, using horseradish peroxidase (HRP), which was covalently bonded to testosterone-3-carboxymethyloxime (T-3-CMO). One batch of T-antisera was also covalently linked to microcrystalline cellulose particles by a mixed anhydride method in order to facilitate separation of bound and free steroids. The established EIA was validated in terms of sensitivity, accuracy, specificity, precisions etc., comparing with conventional radioimmunoassay. The sensitivity of the established EIA was less than 25 pg/tube. The correlation coefficients between the expected T-values and observed T-values measured by EIA or RIA were r=0.985 and r=0.941 respectively. The cross reactivity of antiserum in EIA was a little higher than that of RIA, especially by 5 ${\alpha}$-DHT. The intra- and inter-assay precisions of the present EIA were similar to those of RIA. The present study also demonstrates that the normal T-values in saliva of Korean male ＆ female samples are 265.65${\pm}$15.80 pmol／l and 109.74${\pm}$ 12.01 pmol／l, respectively. The present EIA seems to be established and suitable for use in the endocrinological studies. The advantages of this EIA system also might make the present T-EIA an ideal procedure for use in a routine assay of ordinary laboratory with a conventional spectrophotometer.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.15 no.4
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• pp.281-301
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• 1993

It was revealed that the morphology and projection pattern of terminal arbors from single primary afferent are different among distinct fiber types, functional types and the different subdivision of trigeminal sensory nucleus complex(TSNC). But it was not identified the ultrastructural morphology and synaptic connections of terminal arbors from each primary afferent within TSNC. So we employed the intra-axonal horseradish peroxidase(HRP) injection technique to define the terminal arbors of primary afferent fiber from slowly adapting mechanoreceptors in the periodontal ligament of the cat, and examined 66 labeled terminal arbors within the rostrodorsomedial part(Vo.r) of the trigeminal nucleus oralis, electromicroscopically with 90nm serial sections. All the boutons labelled with HRP contained clear, spherical and uniform sized synaptic vesicles(diameter : $47.66{\pm}3.58nm$ ). Most of the labelled boutons were boutons en passant type and they were connected by unmyelinated axonal strand. In which neurofilament and microtubule was not developed but occasionally contained synaptic vesicle in contrast to the myelinated axon. The size of the labelled bouton was relatively small(long diameter : $1.46{\pm}0.24{\mu}m$, short diameter $0.85{\pm}0.26{\mu}m$, average diameter $1.15{\pm}0.24{\mu}m$) and the shape of which varied from dome to elongated shape, but scalloped glomerulus shape was not developed. Each primary ending in Vo.r made synapse with one or two neuronal propiles(average : $1.11{\pm}0.31$), of which, 89.4% of labelled boutons made synapse with only one neuronal pro pile, the remainder, 10.6% of labelled boutons, made synapse with two neuronal propile. So characteristically they made very simple synapse. Most of labelled boutons(80.03%) made asymmetrical synapse only with dendritic shaft or spine, and 6.1% of labelled boutons received symmetrical synapse from pleomorphic vesicle containing axonal ending(p-ending). So presynaptic inhibiton was relatively scarce. Synaptic triad, in which a p-ending is presynaptic both pre-and post-synaptic element of the axo-dendritic contact from the labelled primary ending was not observed.