• Parasites, Hosts and Diseases
• /
• v.35 no.4
• /
• pp.251-258
• /
• 1997

Tachyzoite antigens of Toxoplosnc gondii (RH) were partially purified by immunoaffinity chromatography. The cultivated ToxopLusmc in uiuo (mouse) and in nitro (Hep-2 cell) and peritoneal fluid of T. Bondii infected mice were collected for antigen analy- sis. Tachyzoite antigens collected from infected mouse showed positive bands of 76 kDa, 70 kDa,64 kDa, 53 kDa, 46 kDa, 44 kDa, 41 kDa, 35 kDa, 25 kDa, 18 kDa, and 13 kDa on immunoblot with anti-Toxoplcsmn rabbit sera, and those from infected Hep-2 cells revealed reactive bands of 70 kDa,64 kDa,53 kDa,35 kDa,28 kDa, and 13-10 kDa. After applying to an IgG-Sepharose column, two elusion peaks, E-1 and I-2 fractions, were obtained from both soluble antigen of T. gondii and the peritoneal fluid of infected mice, respectively. Immunoblots of soluble antigen with immunized rabbit sera revealed positive bands of 97 kDa, 63 kDa, 53 kDa and 35 kDa from I-1 fraction and 53 kDa and 35 kDa from I-2. In the case of the eluted peaks from mice peritoneal fluid, E-1 showed protein bands of 84 kDa,76 kDa,53 kDa and 29 kDa bands and 53 kDa and 45 kDa from I-2 on immunoblots. Serum IgG antibody titer of mice immunized with T gonnii tachyzoites was increased on 1 week after booster immunization when analysed by ELISA using crude antigen, while it was elevated on 3 weeks after booster immunization by ELISA using puri- fied antigen.

• Korean Chemical Engineering Research
• /
• v.44 no.1
• /
• pp.65-72
• /
• 2006

Recent technical advances in the biorecognition engineering and the microparticle fabrication may enable us to develop the single step purification using magnetic particle, because of its simplicity, efficacy, ease of automation, and process economics. In this study, we used commercial magnetic particles from Seradyn, Inc. (Indianapolis, USA). It was ca. 2.8 micron in diameter, consisted of polystyrene core and magnetite coating, and its surface had carboxyl groups. The model, capture protein was IgG and anti-IgG was used as the ligand molecule. We studied the different surfaces ('nude', ester-activated, and anti-IgG coated) for their biorecognition of IgG. At a high pH condition, we could reduce non-specific binding. Also anti-IgG immobilized magnetic particle could capture IgG more selectively. We attempted 'oriented immobilization' of anti-IgG, in which the polysaccharides moiety near the C-terminus was selectively oxidized and linked to the hydrazine-coated MP, to improve the efficacy of biorecognitive binding. Using this method, the IgG capturing ability was improved by ca. 2 fold. From the binary mixture of the IgG-insulin, IgG could be more selectively captured. In summary, the oriented immobilization of oxidized anti-IgG proved to be as effective as the streptavidin-biotin system and yet simpler and cost-effective. This immobilization method can find its applications in protein biochips and biotargeting.

• Korean Journal of Veterinary Research
• /
• v.25 no.1
• /
• pp.7-17
• /
• 1985

Many investigators have been pursuing various attempts so far to produce hepatitis B surface antigen(HBsAg) vaccines using the techniques such as isolation from plasma of chronic HBsAg carrier, recombinant DNA technique or preparation of synthetic peptides specific for immunogenic determinants. Hepatitis B virus can not grow on any cell lines by the tissue culture technique at the present time. The plasma of chronic HBsAg carrier is expensive and its source is limited. The HBsAg from the recombinant DNA technique gave still very low yield. Another approach, therefore, has been initiated to develop a synthetic hepatitis B virus vaccine. The possible use of several distinct synthetic vaccines in prophylaxis can be facilitated by availability of full synthetic immunogens. Peptides synthesized for potential application as antiviral vaccines have been mostly tested in the form of conjugates with carrier proteins, although the free synthetic peptide can be immunogenic. To understand basic knowledges on the antigenicity and immunogenicity of a synthetic peptide specific for major immunogenic determinant of HBsAg, a nonapeptide, $H_2N^{139}Cys-Thr-Lys-Pro-Thr-Asp-Gly-^{146}Asn-Aba$ COOH, which corresponds to HBsAg amino acid residues 139 to 147, was synthesized by the Merrifield's solid-phase method with a slight modification. The antigenicity and immunogenicity of this specific synthetic peptide were examined comparing with purified plasma-derived natural HBsAg. The results obtained are as follows; 1. The peptide synthesized showed the identical amino acid composition to the theoretical value. The degree of purification and molecular weight were acertained by methods of high performance liquid chromatography and mass spectrometry. 2. Using m-maleimidobenzoyl-N-hydroxysuccinimide ester as a conjugating agent, the synthetic peptide was conjugated to rabbit albumin and ${\gamma}$-globulin, tetanus and diphtheria toxoids, and keyhole limpet hemocyanin. Their conjugation yields were 8.3, 9.5, 15.8, 13.5, and 11.2%, respectively. 3. The natural HBsAg was purified from plasma of chronic HBsAg carrier. By the electron microscopic observation of the purified natural HBsAg preparation, no Dane particles were observed and the preparation showed negative DNA polymerase activity. 4. Antigenicity of the synthetic peptide and the plasma-derived natural HBsAg was determined by competition radioimmunoassay using $^{125}I$-natural HBsAg. Their 50% inhibitions appeared as $90{\mu}g/ml$ and $0.12{\mu}g/ml$ for the synthetic peptide and the natural HBsAg, respectively. This indicates that the former was about 750-fold less antigenic than the latter. 5. Immunogenicity of the synthetic peptide was determined by administering the peptide-carrier conjugates into rabbits with and without Freund's complete adjuvant. Regardless the carrier proteins and adjuvant, positive immune responses to the synthetic peptide were observed. The higher antibody titers, however, were shown in the groups administered with Freund's complete adjuvant. 6. Immunizing dose 50% in mice of the various peptide-carrier conjugates was 5.47, 6.00, 65.16, 31.25 and $13.03{\mu}g/dose$ for rabbit albumin and ${\gamma}$-globulin, tetanus and diphtheria toxoids, and keyhole limpet hemocyanin, respectively, while the natural HBsAg showed $0.65{\mu}g/dose$. 7. It was postulated that homologous proteins prefer to heterologous ones as the carriers.