• Molecules and Cells
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• v.23 no.3
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• pp.340-348
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• 2007

Although considerable effort has been devoted in the mass spectrometric analysis of phosphorylated peptides, successful identification of multi-phosphorylated peptides in enzymatically digested protein samples still remains challenging. The ionization behavior of multi-phosphorylated peptides appears to be somewhat different from that of mono- or di-phosphorylated peptides. In this study, we demonstrate increased sensitivity of detection of multi-phosphorylated peptides of beta casein without using phosphopeptide enrichment techniques. Proteinase K digestion alone increased the detection limit of beta casein multi-phosphorylated peptides in the LC-MS analysis almost 500 fold, compared to conventional trypsin digestion (~50 pmol). In order to understand this effect, various factors affecting the ionization of phosphopeptides were investigated. Unlike ionizations of phosphopeptides with minor modifications, those of multi-phosphorylated peptides appeared to be subject to effects such as selectively suppressed ionization by more ionizable peptides and decreased ionization efficiency by multi-phosphorylation. The enhanced detection limit of multi-phosphorylated peptides resulting from proteinase K digestion was validated using a complex protein sample, namely a lysate of HEK 293 cells. Compared to trypsin digestion, the numbers of phosphopeptides identified and modification sites per peptide were noticeably increased by proteinase K digestion. Non-specific proteases such as proteinase K and elastase have been used in the past to increase detection of phosphorylation sites but the effectiveness of proteinase K digestion for multi-phosphorylated peptides has not been reported.

• Food Science of Animal Resources
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• v.30 no.1
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• pp.36-41
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• 2010

The Hen's egg is widely used in many processed foods as an ingredient and is one of the most prevalent food allergens in children. To detect egg proteins in processed foods, we developed a competitive indirect enzyme-linked immunosorbent assay (ciELISA) using an anti-ovomucoid (OM) antibody, which was produced by immunization of rabbits with OM, the most heat-stable component of the egg proteins. The detection limit of this quantitative assay system was 30 ng/mL. Cross-reactivity of the anti-OM antibody toward OM, ovalbumin, skim milk, casein, whey protein isolate, and isolated soy protein was 100, 0.4, 0.2, 0.04, 0, and 0%, respectively. In the spike test of egg white powder in milk replacer, commercial sausage, and in-house sausage, the assay recoveries ($mean{\pm}SD$) were $129{\pm}13.7%$, $73.9{\pm}12.5%$, and $65.5{\pm}13.6%$, respectively. When egg white in a commercial crab meat analog and sausage was determined by ciELISA, the assay recovery was found to be 108% and 127%, respectively. The combined results of this study indicate that this novel ciELISA for OM detection could be applied for the quantification of hen's egg proteins in processed foods.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.367-371
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• 2011

Multiplex PCR assays were developed for the simultaneous detection of ten important Korean quarantine phytoplasmas. The species-specific primers were designed based on ribosomal protein, putative preprotein translocase Y, immunodominant protein, elongation factor TU, chaperonin protein and the 16S rRNA genes of 'Candidatus (Ca.) Phytoplasma' species. Three main primer sets were prepared from ten designed primer pairs to limit nonspecific amplification as much as possible. The multiplex PCR assay using the three primer sets successfully amplified the correct conserved genes for each 'Ca. Phytoplasma' species. In addition, ten important 'Ca. Phytoplasma' species could be easily determined by recognizing band patterns specific for each phytoplasma species from three primer sets. Moreover, a high sensitivity of multiplex PCR for each primer set was observed for samples containing a low DNA concentration (10 ng/${\mu}l$). This study provides the useful multiplex PCR assay as a convenient method to detect the presence of ten important quarantine phytoplasmas in Korea.

• Interdisciplinary Bio Central
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• v.4 no.2
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• pp.5.1-5.9
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• 2012

Small peptide tags such as c-myc, HA, or FLAG tag have facilitated efficient Western-blotting of proteins of interest especially when specific antibodies for the proteins are not available. However, the conventional Western-blotting requires the multi-steps process taking at least several hours up to two days. With examples of various applications, here we show a convenient and time-saving method for protein detection which employs a fluorescent chemical BDED and its binding peptide RC-tag. And we propose "Estern staining", as a standard term for protein detection method using fluorescent chemicals and their binding small peptide tags. Eastern staining may substitutes for the time-consuming "immuno-staining" in many versatile applications.

• Korean Journal of Food Science and Technology
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• v.31 no.1
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• pp.1-6
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• 1999

A piezoelectric (PZ) biosensor system detecting Salmonella spp. was developed. The system consisted of an oscillator, a frequency counter and an antibody-immobilized quartz crystal. An anti-Salmonella antibody was immobilized on one gold. surface of the quartz crystal with protein A. Salmonella detection was made by measuring resonant frequency shift owing to a mass change by specific binding of microbial cells to the gold surface of the PZ crystal. The PZ antibody sensor was operated optimally at 0.1M phosphate buffer, pH 7.2 and $35^{\circ}C$. The sensor was quite specific to Salmonella spp. The obtained frequency shift was correlated with the Salmonella concentration in the range of $10^5{\sim}10^6\;CFU/mL$. The frequency shift increased further by addition of polystyrene beads. The Salmonella detection which is indicated by a steady-state microbial adsorption to the quartz crystal was accomplished within 50min.

• KSBB Journal
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• v.22 no.6
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• pp.443-446
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• 2007

A prognostic indicator of coronary heart disease, C-reactive protein, was tried to be determined by a batch-type quartz crystal microbalance immunosensor. The sensor was operated by direct-binding mode and the optimum concentration for the corresponding antibody for immobilization was $50{\mu}g/ml$. The reaction buffer for the system was 0.1 M sodium phosphate (pH 7.0) and system operation was performed in the order of baseline stabilization, analyte addition and measurement, and regeneration of the sensor chip with 10 mM NaOH. When plotted in double-logarithmic scale, the sensor showed a linear detection range of 0.27-106.00 nM for rat C-reactive protein with the limit of detection of 0.53 nM. It also showed a good reusability.

• Korean Journal of Veterinary Research
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• v.43 no.1
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• pp.129-137
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• 2003

Porcine reproductive and respiratory syndrome (PRRS) is characterized by reproductive failures in sows and respiratory problems in piglets. The nucleocapsid(N) protein, encoded by the open reading frame 7 (ORF7) gene, is known to be the most abundant and antigenic protein in PRRS virus. Therefore, it was suggested that the N protein could be a suitable candidate for the detection of PRRS virus-specific antibodies and diagnosis of PRRS. In the present study, the ORF7 gene encoding the N protein was cloned and expressed as a fusion protein with the glutathione S-transferase (GST) in Escherichia coli. The resulting GST-N recombinant protein was used as an antigen for an indirect sandwich enzyme-linked immunosorbent assay (i-ELISA). Expressed GST-N recombinant protein was migrated at 41 kDa and reacted with ORF7-specific monoclonal antibody by Western blotting. In order to increase the specificity of the ELISA for the detection of PRRS virus-specific antibodes, an i-ELISA was developed using an anti-GST antibody as a capture antibody. The sensitivity and specificity of developed i-ELISA were 92% and 96%, respectively. Based on these results, it was suggested that the i-ELISA is a simple and rapid test for screening a large number of swine sera for the anti-PRRS virus antibodies.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.355-356
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• 2000

Three 'stress probe' plasmids were constructed and characterized which utilize a green fluorescent protein (CFP) as a non-invasive reporter to elucidate Escherichia coli cellular stress responses in quiescent or 'resting' cells. Facile detection of cellular stress levels was achieved by fusion of three heat shock stress protein promoter elements, those of the heat shock transcription factor ${\sigma}^{32}$, pretense subunit ClpB, and chaperone DnaK, to the reporter gene $gfp_{uv}$. When perturbed by chemical or physical stress (such as heat shock, nutrient (amino acid) limitation, addition of IPTG, acetic acid, ethanol, phenol, antifoam, and salt (osmotic shock), the E. coli cells produced GFPuv which was easily detected from within the cells as emitted green fluorescence. A temporal and amplitudinal mapping of these responses was performed, demonstrating regions where quantitative delineation of cell stress was afforded.

• Fisheries and Aquatic Sciences
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• v.4 no.1
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• pp.32-38
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• 2001

Several molecular biological techniques have been used to detect virus rapidly and accurately, but these methods have limitations in the early stage of viral infection with very low concentration of virus. We compared the detection limits of IMS-PCR, Western blot and ELISA with infectious hematopoietic necrosis virus OHNV). Four antibodies, rabbit anti-IHNV polyclonal antibody, anti-IHNV nucleocapsid protein monoclonal antibody, anti-IHNV nucleocapsid protein polyclonal antibody, and anti-IHNV glycoprotein polyclonal antibody, were tested to find out the most effective antibody for each method. The detection limit with IMS- PCR was $2\times10^6$ pfu when the viral RNA was extracted before RT-PCR. In the western blot with rabbit anti­IHNV polyclonal antibody one pfu of virus could be detected. In ELISA, 10 pfu of virus particles were detected with the same antibody.

• Journal of Food Hygiene and Safety
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• v.10 no.4
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• pp.213-217
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• 1995

A screening method has been developed for detecting sulfamethazine(SMZ) contamination of meat or feeds by using horseradish peroxidase (HRP) labeled protein A (Prot AHRP)and an indirect competitve enzyme-linked immunosorbent assay(ELISA). The assay is based on competitve binding of guinea pig anti-SMZ with SMZ in smaple and SMZ-gelatin conjugate(SMZ.GEL). Percent binding (B.Bo$\times$100) was calculated from the absorbance in the absence (B0) and presence (B) of SMZ. By the sandard curve prepared by plotting log(SMZ) vs percent binding of each known reference solution, the detection limit was 1.0ppb or less. Cross reacton with sulfadimethoxine, sulfaguaniding, sulfamerazine, sulfamthoxpyridazine, sulfanilamide, sulfisomidine and sufisoxazole were not observed. But sulfamerazine crossreacted in the test. The EC-50 value (concentration causing 50% inhibition of color development compared with blank) of sulfamerazine was 2.0 ppm. Further quality control will make the ELISA system ideal for the detection of SMZ in meat or feeds.