• BMB Reports
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• 제32권5호
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• pp.461-467
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• 1999

Recombinant Mycobacterium smegmatis expressing ovalbumin was used to immunize C57BL/6(H-$2^b$) mice, and the humoral immunity against recombinant ovalbumin was analyzed. Antibodies were purified by denatured ovalbumin-conjugated affinity chromatography. The epitopes of the antibodies were screened with a random peptide library displayed on the tip of fUSE5 filamentous phage pIII minor coat proteins. Two peptides, IRLADR and SPGAEV, were selected predominantly by the recognition of purified antibodies using biopanning methods. The composition of the peptide sequence with the primary structure of OVA revealed that the peptide sequence analogizes to INEAGR, part of the $^{323}ISQAVHAAHAEINEAGR^{339}$ sequence previously reported as the antigenic determinant for murine Band also Th cell epitopes (I-$A^d$ binding). Also, the structures of these mimotopes obtained from restrained molecular dynamic computations resulted in the formation of a $\beta$-turn proven to be a secondary structure of the parent peptide within the ovalbumin molecule, enabling us to confirm the structural similarity. This study demonstrates that immunization with recombinant M. smegmatis can generate neutralizing antibodies identical with those induced by the administration of natural antigenic proteins and supports the potential use of mycobacteria as vaccine delivery vehicles.

• The Plant Pathology Journal
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• 제20권4호
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• pp.252-257
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• 2004

We developed a polyclonal antibody based-ELISA system to monitor inocula accurately and rapidly before onset of anthracnose on soybean sprouts. Titer of mouse antisera against conidia of Colletotrichum gloeosporioides, determined by indirect ELISA, was high enough to be detectable up to ${\times}$25,600 dilutions. Both PAb1 and PAb2 had the highest level of reactivity to Colletotrichum gloeosporioides. Absorbance readings exceeded 0.15. Sensitivity of PAb to C. gloeosporioides was precise enough to detect spore concentration as low as 500 conidia/well by indirect ELISA. Both antibodies are very sensitive and highly specific to the target pathogen Colletotrichum gloeosporioides, apparently discriminating other unrelated pathogen, or epiphytes. This kit fulfills the requirements far detecting inocula before infection and onset of anthracnose. Our ELISA system should also be feasible to detect C. acutatum (Mungbean sprouts rot) and G. cingulata (C. gleosporioides), (apple, pepper). It was remarkable that absorbance value was not reduced even after 4 consecutive washings (Fig.4), suggesting that antigenic determinants are on the surface of conidia. Antigenic determinant was characterized by heating and enzyme treatment: Both PAb1 and PAb2 bind to protein epitope that does not contain residue of amino acid, arginine, and Iysine, even though more work needs to be done.

• 한국응용곤충학회지
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• 제27권4호
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• pp.211-218
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• 1988

담배거세미나방(Spodoptera litura: SI), 누에(Bombyx mori: Bm) 및 흰불나방(Hyphantria cunea : Hc)으로부터 분리된 핵다각체병 바이러스(nuclear polyhedrosis virus : NPV)의 다각체의 단백질의 특성과 그에 대한 alkaline protease의 영향을 SDS-PAGE 및 혈정학적 방법으로 분석했다. Alkaline protease를 부활화시킨 후 다가체 단백질을 SDS-PAGE한 결과, BmNPV의 경우 30kD, SINPV와 HcNPV는 31kD의 단일 major band 및 이것들의 종합체(polymer)인 57kD와 66kD의 minor band들이 관찰되었다. Alkaline protease의 부활화를 성략한 SI NPV 다각체를 알칼리 용액으로 시간 차이를 두고 처리한 후 SDS-PAGE한 결고, 알칼리 처리시간이 경과함에 따라서 alkaline protease활성에 의해 다각체 단백질이 일정한 pattern으로 저분자화됨이 뚜렷하였다. SINPV 와 BmNPV 다각체 단백질의 항체를 제조하여 SINPV, BmNPV및 HcNPV 다각체 단백질간의 혈정학적 상동성을 이중형역광산법과 Western blot으로 비교한 결과는 3종 모두에서 공통 antigenic determinants의 존재가 인정되었으며 major 다각체 단백질의 종합체 형성과 alkaline protease에 의한 분해를 확인했다.

• 미생물학회지
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• 제28권1호
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• pp.13-18
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• 1990

adr아형 B형 간염바이러스의 preS2유전자 부위를lacZ 유전자의 5말단에 연결하여 preS2-$\beta$-galactosidase 융합단백질을 생성하는 플라스미드, pTSZ를 건설하였다. 갈본된 preS2 유전자의 3' 및 5발단을 결손시켜 얻은 재조합 플라스미드를 발현시킨 후 결손된 preS2를 포함하는 융합단백질의 항원성을 단일클론항체 H8을 사용하여 비교하며 보았다. 양말단에서 일정부위까지의 결손은 항원성에 영향을 미치지 않았지만 그 이상의 결손에 의하여는 항윈성이 소실됨을 볼 수 있었다. 이상의 항원성 전한부위를 DNA 염기서열 분석에 의하여 결정할 수 있었다. 그 결과 항원결정인자의 양말단은 preS2 서열 중 아미노산 전기 130-132와 140→142 사이에 각각 존재함을 알 수 있었고, 아미노산 143번의 결손은 항원성의 부분적인 감소를 초래하는 것으로 보아 항원성 결정에 보충적 역할을 한다고 생각된다. 한편 adr과 adw2아형 간의 아미노산서열의 차이가 항원결정부위 중 130, 132 및 141번 위치에 존재하며 단일를론항체 H8이 adr아형에만 특이하게 결합하는 것으로 부터, 세 잔기 중 하나 혹은 그 이상이 아형특이성에 관여한다고 추정된다.

• BMB Reports
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• 제28권4호
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• pp.353-358
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• 1995

Epitope tagging is the process of fusing a set of amino acid residues that are recognized as an antigenic determinant to a protein of interest. Tagging a protein with an epitope facilitates various immunochemical analyses of the tagged protein with a specific monoclonal antibody. The monoclonal antibody H8 has subtype specificity for an epitope derived from the preS2 region of hepatitis B virus surface antigen. Previous studies on serial deletions of the preS2 region indicated that the preS2 epitope was located in amino acid residues 130~142. To test whether the amino acid sequence in this interval is sufficient to confer on proteins the antigenicity recognizable by the antibody H8, the set of amino acid residues in the interval was tagged to the amino terminal of ${\beta}$-galactosidase and to the carboxyl terminal of the truncated $p56^{lck}$ fragment. The tagged ${\beta}$-galactosidase, expressed in Escherichia coli, maintained the enzymatic activity and was immunoprecipitated efficiently with H8. The tagged $p56^{lck}$ fragment, synthesized in an in vitro translation system, was also immunoprecipitated specifically with H8. These results demonstrate that the amino acid sequence of the preS2 region can be used efficiently for the epitope tagging approach.

• Parasites, Hosts and Diseases
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• 제41권3호
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• pp.175-177
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• 2003

An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the C-terminal of the molecule was expressed as a $6{\;}{\times}{\;}His$ tagged protein (Ncp43p) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1％) were found to react with Ncp43p positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43p could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T.gondii infections in other mammals.

• Journal of Microbiology and Biotechnology
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• 제9권6호
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• pp.832-838
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• 1999

Two different types of lipases (lipase I and lipase II) secreted into culture medium by Rhizopus sp. L-I were purified using a hydrophobic chromatography and were partially characterized. Both enzymes were monomeric as revealed by SDS-PAGE and gel filtration. The molecular masses of the enzymes were identified as 45 kDa (lipase I) and 69 kDa (lipase II). The isoelectric points were estimated to be 3.6 and 5.2 for lipase I and lipase II, respectively. pH and temperature activity optima for lipase I were as 7.5 and $50^{\circ}C$, respectively, whereas the corresponding parameters for lipase II were 6.0 and $45^{\circ}C$. The amino terminal sequences of lipase I and lipase II, determined by Edman degradation, were found to be Leu-Val-Met-Ile-Gln-Arg and Leu-Val-Met-Lys-Gln-Arg, respectively. By western blotting analysis, the two lipases were found to have a common antigenic determinant. Immuno-electron cytochemistry conducted with polyclonal anti-lipase I antibody indicated the enzyme located in both the periplasm and the adjacent vesicles of fungal hyphae. Fortunately, the sites on the cell envelope where lipase was exported into the culture medium was also identified.

• 미생물학회지
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• 제44권3호
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• pp.221-227
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• 2008

Bordetella pertussis는 유아의 호흡기감염질환인 백일해의 원인균으로 배신도입에 의해 발생률이 크게 낮아졌다. 그러나 최근에 일부 백신접종률이 높은 국가에서 증가된 백일해 발생건수가 보고되고 있으며, 그 추정 원인 중에 하나는 백신주와 유행주 사이의 유전형 흑은 혈청형 변이 이다. 따라서 분리주에 대한 변이 현황을 유전형 혹은 혈청형 분석을 통하여 확인하는 것이 필요하고 국내에서 백일해 발병증가에 대한 가능성을 추정해야 한다. 이에 본 연구에서는 $1999\sim2006$년 사이에 분리된 국내 백일해 분리균주의 변이 양상을 agglutinogen과 fimbriae에 대한 혈청형 조사를 통해 확인하였다. 그 결과 agg 1과 fim 2가 국내 분리주에서 가장 많이 발견되는 혈청형이었고, 두 항원 모두 시대에 따른 혈청형 변이 현상이 확인되었으며, 특히 agglutinogen의 경우는 백신균주(agg 1,2)와는 다른 유형(agg 1)이 증가되는 유형으로 나타났다. 그러나 fimbriae의 경우는 백신균주(fim 2)와 동일한 유형이 증가되어 차이를 나타내었다. 이러한 결과는 백일해 발병증가가 나타나는 국가들에서도 확인되는 현상으로 국내에서의 백일해 발병증가에 대한 보다 정확한 예측을 위해서는 항원결정기 유전자에 대한 변이정도 및 정상인에서의 항체가 분포양상 등에 대한 연구가 필요할 것으로 사료된다.

• 한국가축번식학회지
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• 제9권2호
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• pp.133-139
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• 1985

An immunoassay may be defined as an analytical procedure involving the competitive reaction between a limiting concentration of specific antibody and two populations of antigen, one of which is labelled or immobillized. The advent of immunoassay has revolutionised our knowledge of reproductive physiology and the practice of veterinary and clinical medicine. Radioimmunoassay (RIA) was the first of these methods to be developed, which meausred the analyte with good sensitivity, accuracy and precision (1,2). The essential components of RIA are:-(i) a limited concentration of antibodies, (ii) a reference preparation, and (iii) an antigen labelled with a radioisotope (usually tritium or iodine-125). Most procedures invelove isolating the antibody-bound fraction and measuring the amount of labelled antigen. Good facilities are available for scintilltion counting, data reduction nd statistical analysis. RIA is undergoing refinement through:-(i) the introduction of new techniques to separate the antibody-bound and free fractions which minimize the misclassification of labelled antigen into these compartments, and the amount of non-specfic binding. (3), (ii) the development of non-extration for the measurement of haptens (4), (iii) the determination of a, pp.rent free (i.e. non-protein bound) analytes (5), and (iv) the use of monoclonal antibodies(6). In 1968, Miles and Hales introduced in important new type of immunoassay which they termed immunora-diometric assay (IRMA) based on t도 use of isotopically labelled specific antibodies(7) in a move from limited to excess reagent systems. The concept of two-site IRMAs (with a capture antibody on a solid-phase, and a second labelled antibody to a different antigenic determinant of the analyte) has enabled the development of more sensitive and less-time consuming methods for the measurement of protein hormones ovar wide concentration of analyte (8). The increasing use of isotopic methos for diverse a, pp.ications has exposed several problems. For example, the radioactive half-life and radiolysis of the labelled reagent limits assay sensitivity and imposes a time limit on the usefulness of a kit. In addition, the potential health hazards associated with the use and disposal of radioactive cmpounds and the solvents and photofluors necessary for liquid scientillation counting are incompatable with the development of extra-laboratory tests. To date, the most practical alternative labels to radioisotopes, for the measurement of analytes in a concentration > 1 ng/ml, are erythrocytes, polystyrene particiles, gold sols, dyes and enzymes or cofactors with a visual or colorimetric end-point(9). Increased sensitivity to<1 pg/ml may be obtained with fluorescent and chemiluminescent labels, or enzymes with a fluorometric, chemiluminometric or bioluminometric end-point. The sensitivity of any immunoassay or immunometric assay depends on the affinity of the antibody-antigen reaction, the specific activity of the label, the precision with which the reagents are manipulated and the nonspecific background signal (10). The sensitivity of a limited reagent system for the measurement of haptens or proteins is mainly dependent upon the affinity of the antibodies and the smalleest amount of reagent that may be manipulated. Consequently, it is difficult in practice to improve on the sensitivity obtained with iodine-125 as the label. Conversely, with excess reagent systems for the measurement of proteins it is theoretically possible to increase assay sensitivity at least 1000 fold with alternative luminescent labels. To date, a 10-fold improvement has been achieved, and attempts are being made to reduce the influence of other variables on the specific signal from the immunoreaction.

• 대한수의학회지
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• 제25권1호
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• pp.7-17
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• 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.