• Microbiology and Biotechnology Letters
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• v.16 no.5
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• pp.335-342
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• 1988

An anion exchange chromatography was employed for the purification of mouse monoclonal antibodies from ascitic fluid and in vitro cultivation media. After cultivation of hybridomas, Alps 25-3, HCGK, A4W, and KW, producing IgG1, the culture supernatants were harvested by centrifugation, precipitated with 50-60％ ammonium sulfate, and dialyzed against 0.025 M Tris-HCI buffer (pH 8.2). Then the dialyzed samples were loaded into a DEAE-Trisacryl M anion exchange column. Monoclonal antibodies bound to the DEAE-Trisacryl M were eluted with 0.025 M Tris-HCI buffer (pH 8.2) containing 30-40 mM NaCl. In ammonium sulfate precipitation, the recovery of the monoclonal antibody was shown to be 90％ and 84％ from in vitro culture media containing 10％ and 2％ fetal bovine serum, respectively. On the other hand, the pretreatment by ultrafiltration enhanced the yield up to 91％ whereas the purity was lower than that by ammonium sulfate treatment. Subsequently, in the DEAE-Trisacryl M chromatographic separation, the purities and recoveries of all the monoclonal antibodies from both the in vitro culture supernatants and ascitic fluids were 70-80％ and 65％ respectively. The monoclonal antibody, Alps 25-3 could be further purified with a purity of 95％ through an immunoadsorbent chromatography.

• Journal of the East Asian Society of Dietary Life
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• v.9 no.2
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• pp.200-206
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• 1999

This study was carried out to get a industrial information about a possibility of IgY antibody production, antimicrobial activity and Properties of IgY antibody in egg yolk. After the initial immunization the anti-Salmonella typhimurium IgY antibody level gradually were decreased from firth week to tenth week. On the other hand, the antibody level in the serum were increased from the first week, reaching its peak in the sixth week. Molecular weights of IgY were estimated approximately 72-75KD in a heavy chain and 30-40KD in a light chain by electrophoresis.

• 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.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.466-471
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• 1992

To determine the site at which -1 ribosomal frameshifting occurs within the gag-pro overlap of HTL V-I. DNA fragment corresponding to a portion of the gene overlap was cloned into a SP6 vector. The resultant plasmid harbors the hybrid gene consisting of a synthetic gene encoding 5 amino acids derived from chick prelysozyme including the initiator methionine plus 141 nucleotides of gag-pro overlapping region followed by Staphylococcus aurcus protein A gene fragment. In vitro transcription by SP6 RNA polymerase with this DNA template made an abundant amount of single species mRNA. Cell-free translation programmed with the RNA transcribed in vitro yielded a polypeptide of 21 kDal in size. which could be purified into homogeneity by IgG-Sepharose affinity chromatography. In vitro system described in this study must be useful for rapid purification and sequencing of the Gag-Pro transframe protein. allowing to determine the exact frameshift site on mRNA and to identify the tRNA involved in frameshifting event for the expression of pro gene.

• 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.

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

• 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.

• Food Science of Animal Resources
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• v.37 no.5
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• pp.743-751
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• 2017

During slaughtering, animal blood is typically discarded, resulting in water pollution. However, this discarded blood has valuable components, such as immunoglobulin (Ig). Although several studies have been conducted to develop methods for effective recycling of slaughterhouse blood, they have not been commercially utilized in Korea. Here, we extracted an Ig-rich fraction from porcine blood that was then subjected to various in vitro tests, including pathogen growth inhibition, antigenic cross-reactivity, and anti-toxin activity. The porcine immunoglobulin concentrate (PIC) was effectively purified by eliminating other components, such as albumin, and consisted of approximately $63.2{\pm}2.9%$ IgG and $7.2{\pm}0.4%$ IgM on a protein basis. The results showed that it significantly suppressed the growth of pathogenic bacteria, and bound to all tested pathogens, including both gram-positive and gram-negative species, although the degree of activity differed according to strain. The PIC bound to two types of lipopolysaccharide (LPS) obtained from Escherichia coli O111:B4 and Salmonella enterica serotype typhimurium in a concentration-dependent manner. In addition, the PIC restored the proliferation activity of the lymphoblast K-562 cells when co-incubated with pathogenic LPS. These results confirm that the PIC prepared in this study is a potentially valuable functional food material or diet supplement as an alternative to antibiotics that can protect animals from pathogenic bacteria.

• Clinical and Experimental Vaccine Research
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• v.12 no.4
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• pp.304-312
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• 2023

Purpose: Due to the many problems with commercially available vaccines, the production of effective vaccines against brucellosis is a necessity. The aim of this study was to evaluate the immune responses caused by the chimeric protein consisting of trigger factor, Bp26, and Omp31 (TBO) along with aluminum hydroxide (AH/TBO) and selenium (Se/TBO) nanoparticles (NPs) as adjuvants in mouse model. Materials and Methods: Recombinant antigen expression was induced in Escherichia coli BL21 (DE3) bacteria using IPTG (isopropyl-d-1-thiogalactopyranoside). Purification and characterization of recombinant protein was conducted through NiFe₃O₄ NPs, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blot. NP characteristics, including morphology and particle size, were measured in vitro. The recombinant TBO was loaded on to AH and Se NPs and were administered subcutaneously. After mice immunization, measurement of antibody titter and protection assay was performed. Results: The average sizes of AH and Se NPs were about 60 nm and 150 nm, respectively. The enzyme-linked immunosorbent assay results showed that the serum of mice immunized by subcutaneous injection with both nanovaccines produced significant immunoglobulin G (IgG) responses against the chimeric antigen. The results of TBO-specific IgG isotype (IgG2a/IgG1) analysis showed that both AH and Se NPs induced a type to T-helper immune response. In addition, the results of the challenge with the pathogenic strain of Brucella melitensis 16M showed that vaccinated mice with AH/TBO NPs indicated a higher reduction of bacterial culture than immunized mice with Se/TBO NPs and TBO alone. Conclusion: The results showed that AH NPs carrying chimeric antigen can be a promising vaccine candidate against brucellosis by producing protective immunity.

• Microbiology and Biotechnology Letters
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• v.17 no.1
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• pp.19-23
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• 1989

A sequential system composed of hydroxylapatite chromatography and gel permeation chromatography was developed to purify the IgM type monoclonal antibody against the colon cancer cell SC-1 from the ascitic fluid of mice injected with the murine hybridoma CH07E02. In the hydroxylapatite chromatographic step the band dilution could be reduced by controlling the gradient and flow rate of the eluent, the sodium phospate buffer, the optimum values for these variables being 5.82$\times$10$^{-3}$M/cm and 0.2$m\ell/\textrm{cm}^2$/min, respectively. A degree of purity better than 99.99% as judged from silverstaining of the SDS-PAGE bands, was obtained by adding the gel permeation chromatographic step in tandem.