• Proceedings of the PSK Conference
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• 2003.04a
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• pp.316.2-317
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• 2003

Immunoglobulin Y (IgY) obtained from chicken as the immunization host brings several advantages to antibody production, such as improved yield, lower cost, longer stability, and higher specificity than mammalian immunoglobulin. In the present study, we attempted to purify Maackia fauriei lectin using antilectin IgY-affinity chromatography in order to produce a good yield and to reduce the purification time. (omitted)

• Korean Journal of Microbiology
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• v.42 no.4
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• pp.294-298
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• 2006

An immunoaffinity chromatography for the specific binding of Streptomyces somaliensis phospholipase D (PLD) that is considered as an industrially potential enzyme was developed. By using the protein structure prediction programs and the X-ray crystal structure of a Streptomyces PLD, 5 different epitopes with high antigenicity that are predicted to locate on the surface of the S. somaliensis PLD were selected and then synthesized for the preparation of antipeptide antibodies. Each purified rabbit IgG was coupled with NHS-activated Sepharose to prepare the immunoaffinity resins. After one-step purification of the culture concentrate on the antipeptide IgG-coupled Sepharose column, SDS-PAGE and the Western blot analysis of the purified samples showed that purification of PLD on the affinity columns was satisfactory, indicating that the peptide design using the structural information of Streptomyces PLDs was rational. However, the purified PLD in the solution aggregated rapidly, which resulted in poor specific activity and low purification yield.

• Korean journal of food and cookery science
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• v.18 no.1
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• pp.94-100
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• 2002

The dietary carbohydrates are mainly digested and adsorbed at small intestine. We developed a new food additive as an egg yolk antibody(1gY) against maltase, sucrase and sodium dependent g1ucose cotransporter(SGLT) for the regulation of blood glucose level and weight control. The maltase, sucrase and SGLT were purified from porcine small intestine which is very similar to that of human in physiological characteristics. The purification step contained an ultracentrifugation, ion exchange chromatography and hydrophobic chromatography. The hens were immunized by purified protein and the IgY activities against immunized antigens were determined. This antibody obtained from the immunized hen's egg yolks directly inhibited the activities of maltase and sucrase in vitro. And the IgY delayed and decreased the increment of blood g1ucose level after administration of maltose, sucrose and glucose in rat about 30 to 60%. The results of this study suggest that the IgY inhibiting the carbohydrate digestion could be used as functional food materials for weight control and regulation of blood glucose level in diabetes.

• Journal of Life Science
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• v.13 no.1
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• pp.67-72
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• 2003

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a major virulence factor and a potent immunogen. Recombinant H. pylori urease expressed in E. coli was purified by simple purification procedures utilizing (CNBr-activated Sepharose-anti-urease IgG immunoaffinity chromatography or epoxy- activated Sepharose-urea affinity chromatography. Urease was apparently bound so tightly to the anti-urease IgG resin that it could not be eluted at various elution conditions except at certain extreme pH 1, including 100 mM carbonate (pH 10.5) buffer solution, which was shown to elute slightly inactivated but relatively pure enzyme. Urease eluted from the epoxy-activated Sepharose-urea affinity column showed higher activity, but the smaller UreA subunit of the enzyme appeared as a Fainter band of diminished intensity when subjected to SDS-polyamide gel electrophoresis.

• Microbiology and Biotechnology Letters
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• v.24 no.1
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• pp.9-18
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• 1996

Streptococcus mutans has been implicated as primary causative agents of dental caries by insoluble glucan (IG) in human and experimental animals. An attempt was made to search for the ${\alpha}$-1,3 glucanase that degrades IG produced by S. mutans. ${\alpha}$-1,3 glucanase was detected in the culture supernatant of microorganisms, which are isolated from soils on agar medium containing IG as a sole carbon source. This Streptomyces sp. hydrolysed IG produced by immobilized S. mutans and was named as Y9373. This enzyme required ${\alpha}$-1,3 glucan (IG) as an inducer. The optimum conditions for enzyme production were studied. The enzyme was purified by 30~70% $(NH_4)_2SO_4$ precipitation, anion exchange chroma tography on DEAE-cellulose and gel filtration on Sepadex G-75. The purified enzyme has a specific activity of 7840.0 U/mg protein giving 32.1-fold purification and final yield of 0.53%. The molecular weight was estimated to be about 22.5 kDa by SDS-PAGE. The optimum pH and temperature for enzyme reaction were 6.5 and 37$^{\circ}C$, respectively and the enzyme was relatively stable at the temperature below 60$^{\circ}C$. The activity of purified enzyme was enhanced by adding $Co^{2+},\;Mn^{2+}\;and\;Mg^{2+}$ into the medium, whereas inhibited by adding $Hg^{2+},\;Zn^{2+}$ and SDS. The $K_m\;and\;V_{max}$ value of ${\alpha}$-1,3 glucanase for IG were estimated to be 2.50 mM and 0.0431 mM/min, respectively. The thin layer chromatographic analysis of hydrolysates from IG with ${\alpha}$-1,3 glucanase showed that glucose was the main product of reaction. This enzyme activity was about 14 times higher than marketing dextranase as preventive agent against artificial dental caries by S. mutans in TH medium including 5% sucrose after 30 minutes.

• Parasites, Hosts and Diseases
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• v.45 no.4
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• pp.287-293
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• 2007

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electro transfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.

• Journal of Marine Bioscience and Biotechnology
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• v.1 no.3
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• pp.218-223
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• 2006

The present study evaluated effect of egg yolk immunoglobulin (IgY) from the hen immunized with Edwardsiella tarda. The purification of anti-E. tarda IgY was performed by polyethylene glycol (PEG). Purified IgY had heavy chain of 64 kDa and light chain of 27 kDa size. The IgY was instable against olive flounders digestive factors and artificially modulated pH 2 and 3. Nevertheless, some activity of IgY appeared in intestine. IgY was orally administered with viable E. tarda to the olive flounders and the efficacy of protection against E. tarda infection was evaluated. Orally administered IgY at a dose of 20 mg/fish delayed infection period of E. tarda cannulated at $10^{6{\sim}8}CFU/fish$ to small size (30~40 g) and middle size (110~120 g) flounder. Moreover, orally administered IgY at dose of 20 mg/fish inhibited the penetration of E. tarda cannulated at $10^8CFU/fish$ into the liver, kidney, spleen and gill via intestine. The fish orally administered with IgY showed increased survival rate. These results suggest that egg yolk containing anti-E. tarda IgY is effective in preventing edwardsiellosis.

• Parasites, Hosts and Diseases
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• v.29 no.1
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• pp.1-8
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• 1991

Out of many component proteins in crude saline extract of Spirometra mansoni plerocercoid (sparganum) , 36 kDa and 29 kDa proteins were found to be the most antigenic and were already purified by immunoaffinity chromatography using monoclonal antibody as a ligand. In this study, a single step purification of these potent antigenic proteins of sparganum extract was investigated. When the crude saline extract was charged to gelatin-Sepharose 4B affinity column, 36 kDa and 29 kDa protein fractions were bound. SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE/immunoblot confirmed that the bound protein to gelatin was serologically pure. When evaluated by ELISA with patients sera, the purified protein of 36 and 29 kDa also showed improved antigenicity.

• Journal of Animal Science and Technology
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• v.45 no.3
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• pp.491-498
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• 2003

The purpose of this study is to observe purification and properties of osteopontin(OPN) from bovine milk. The purification of osteopontin from bovine milk was performed by using ion-exchange and hydrophobic chromatography. SDS-PAGE analysis revealed that the protein migrated at Mw. 60,000. NH2-terminal sequence analysis of the first seven amio acids revealed the protein to be identical to that previously reported for bovine OPN. 35-wk-old chickens, including 3 Single Comb White Leghorn (SCWL), were used to produce egg yolk antibody(IgY) against OPNas a antigen. However, the anti-OPN antibody activities determined by ELISA. Immunological assy of OPN in milk was performed using radial immunodiffusion test based on the standard curve of pure OPN. The radial precipitation lines of four different milk samples indicated that the concentrations of OPN in the milk samples were within the range of 31.7 to 39.7${\mu}g$/ml. On inhibition with OPN on precipitation of calcium phosphate, OPN was slightly higher than casein phosphopeptide(CPP) and poly-glutamic acid.

• Parasites, Hosts and Diseases
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• v.52 no.2
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• pp.137-142
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• 2014

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.