• Korean Journal of Veterinary Research
• /
• v.36 no.3
• /
• pp.681-692
• /
• 1996

The present study was conducted to isolate Babesia gibsoni by culture method of the microaerophilous stationary phase(MASP) and analyse the antigenic properties of the parasite by SDS-PAGE and immunoblot. The results obtained were summarized as follows. The protozoan parasite Babesia gibsoni multiplied in canine erythrocytes in RPMI 1640 medium(pH7.0) containing 20 40% normal canine serum under the MASP condition of 5% CO2 and 95% air at $37^{\circ}C$ incubator. The levels of parasitaemia in the erythrocytes were shown more higher by exchanging the medium at 24 hours interval. Under the above condition of MASP, the percentage of parasitized erythrocytes(PPE) after incubation for 8 days increased about 14 times more than that in the initiation of the 1% infected canine erythrocyte culture. The parasites were purely isolated from the MASP culture of red blood cells collected from dogs infected with Babesia gibsoni naturally or artificially. Among the total of 36 canine(Pit-bullterier) blood samples the parasites were isolated from 17 cases(47.2%) in the MASP culture while the parasites were detected from 20 cases(56%) and 12 cases(33.3%), respectively, by indirect fluorescent antibody(IFA) test and direct light microscopy(DLM). On the other hand, Babesia gibsoni was isolated by MASP culture from 15 cases(75%) and 11 cases(92%) of positive cases of IFA and DLM, respectively. In the analysis of the erythrocytic merozoite(AEOM) antigen derived from infected dog approximately 11 antigenic bands in molecular weight of 130, 120, 97.4, 92, 80, 52, 50, 42, 36, 30 and 29 KDa were observed on SDS-PAGE. Antigenic bands in the endoerythrocytic merozoite(CEOM) antigen derived from infected erythrocyte (sediment) in MASP culture were much similar to those of AEOM bands. In the exoerythrocytic merozoite(CEEM) antigen derived from supernatant of the infected erythrocyte culture approximately 20 antigenic bands were observed and the molecular weight of the major bands among these were 140, 120, 114, 105, 96, 93, 92, 80, 60, 52, 50, 38, 36, 30, 24, 18.5 and 16 KDa. In the protein patterns of AEOM and CEOM antigen by immunoblot 15 bands were observed and these patterns were much similar between each other. The molecular weight of the major bands in the both antigens were 130, 120, 80, 60, 52, 50, 42, 30, 29, 18.5 and 16 KDa. Approximately 21 bands were observed in CEEM antigen and the molecular weight of the major bands were 140, 120, 96, 92, 85, 80, 76, 60, 52, 50, 37, 30, 24, 16 and 15 KDa. The specific antigenic bands in the artificially infected dogs were firstly observed at 3 weeks afrer inoculation of infected blood and these antigenic bands were maintained up to 18 months after inoculation. In the immunoblot of the sera of the splenectomized dogs the specific antigenic bands with the molecular weight of 93 KDa and 52 KDa, respectively, were observed weakly comparing to those of non-splenectomized dog. In immunoblot of the sera collected from the naturally infected dogs the antigenic bands were observed as same as those of artificially infected dogs while antigenic band of 29 KDa in some individual dog showed strongly. In comparison of immunoblot of the sera collected from dogs non-treated and treated with diminazene aceturate(7mg/kg, IM) after artificial infection no differences of antigenic bands were observed. In analysis of antigenic bands by digoxigenin glycan/protein double labeling, antigenic bands in the molecular weight of 106, 60 58, 36, 30 and 29 KDa were determined as glycoproteins.

• Biomedical Science Letters
• /
• v.2 no.1
• /
• pp.1-12
• /
• 1996

Antigenic components reacting with IgE and IgG antibodies were localized in muscular layer of adult and of larva, sparganum. But the antigenic components inducing IgG were localized at tegument and parenchyma in addition to muscular layer in adult and sparganum. Also in sparganum, the surface of calcareous corpuscles of parenchyma showed immunoreactivity to IgG antibody. However antigenic components inducing IgE antibody were not localized in tegument and parenchyma, but in adult worm, we observed the immunopositive reaction at the lining of vitelline follicles in mature proglottis and on surface of egg shell within uterus of graved proglottis. By the method of immunogold-labelling, we observed the location of antigenic particles in tegument of sparganum. The density of antigenic particles inducing IgG was higher than that of antigen particles inducing IgE in syncytial tegument, tegument cells. A total of 43 and 36 protein bands were resolved from crude extracts of adult and sparganum, respectively, by SDS-PAGE. 34 bands from crude extracts of adult and larva were migrated to same positions. By EITB, 21 bands of 44 bands in adult were recognized with IgG antibody, and also 21 bands of 36 bands in sparganum. 13 bands of them were common antigenic components both in the adult worm and sparganum. Because 19 bands of 44 bands in adult worm were reacted with IgE antibody, they were IgE antigenic component. In sparganum, 13 bands were IgE antigenic components. 9 bands of them were common antigenic component inducing IgE antibody in both a-dult and sparganum. 3 bands of antigenic component recognized by IgE and IgG antibody were nonspecific antigen in both adult and sparganum of Spirometra erinacei.

• The Korean Journal of Zoology
• /
• v.32 no.4
• /
• pp.429-440
• /
• 1989

Spirometru erinocei의 유충인 sparganum에서 추출한 조항원 단백질을 항원으로 하여 sparganosis, cysticercosis, hydatidosis환자의 IgG 항체와 정상인의 IgG 항체를 혈청반응시켜 ELISA와 EITB에 의해 교차반응을 일으키는 비 특이항원 성분과 종특이항원 성분을 추구하였다. 1. sparganum에서 0.01 M PBS (PH 7.4)로 추출한 조항원 단백질을 SDS-PAGE로 전개하여 290 Kd에서 23 Kd범위의 분자량을 가진 25개의 단백질이 분획되었다. 2. sparganum의 조항원을 항원으로하여 ELISA로 sparganosis, cysticercosis, hydatidosis 환자의 190 항체와의 혈청 반응치는 sparganosis환자에게서는 0.44 $\pm$ 0.07에서 1.90 $\pm$ 0.03으로 negative control normal sera의 0.D (0.15 $\pm$ 0.03)를 기준으로 하였을 때 모두 양성이며 민감도(sensitivity)가 100 %이었으며 cysticercosis, hydatidosis환자혈청에서 양성반응이 나타났으며 교차반응도 있었다. 3. EITB에서는 spargancsis환자의 IgG항체에 의해 16개의 항원성분이 인지되었으며 이 중 6개의 항원성분이 정상인의 혈청에서도 인지되어 교차반응을 일으키는 항원성분이었으며 cysticercosis환자혈청에서 인지된 4개의 항원성분 중 2개의 항원성분이 sparganosis 환자혈청에서 인지된 것과 같았으며 hydatidosis환자의 190항체에 의해 인지된 19개의 항원성분 중 12개의 항원성분이 sparganosis환자혈청에서 인지된 항원 성분과 같았다. 4. 290 Kd, 200 Kd, 28 Kd의 항원성분은 sparganosis환자의 196항체에서만 인지되었고 228 Kd, 152 Kd, 66 Kd항원성분은 hydatidosis환자의 19G항체에서만 인지되었으며 66 Kd항원성분은 sparganosis, cysticercosis, hydatidosis, 정상인의 혈청에서 모두 인지되었다. We studied the serological reaction between the specific and nonspecific antigenic components from metacestode (plerocercoid) of spiromeko erimacei and IgG antibodies in sparganosis, cysticercosis. hydatidosis patients and normal human sera by ELISA and EITB. We prepared the crude extracts of sparganlim from snake, Matrix tigrina laterolis and used as antigenic components. By SDS-PAGE, we detected a total 25 peptide bands (fractions) with 290 Kd to 23 Kd molecular weight, and 8 bands of these detected bands developed strongly by silver stain. In serological test, ELISA, we recognized the cross-reaction of antigenic components reacting with IgG antibodies in heterogenous sera, cysticercosis and hydatidosis patients sera. The crude antigenic components of sparganum showed the high sensitivity in sparganosis, hydatidosis patients sera, but showed lower sensitivity in cysticercosis patients sera than the sparasanosis, hydatidosis patients sera. Sixteen antigenic components of these 25 separated bands were recognized by antibodies In sparsanosis patients sera,8 antigenic components in normal human sera, 4 antigenic components in cysticercosis patients sera and 19 antigenic compoenents in hydatidosis patients sera. The crude antigenic compnenets with 290 Kd, 200 Kd, 125 Kd and 28 Kd molecular weight was only recognized in sparganosis patients sera, but 64 Kd antigenic component was nonspecific antigenic components which were also cross-reacted with sparganosis, hydatidosis, cysticercosis patients sera and normal human sera.

• Journal of fish pathology
• /
• v.28 no.3
• /
• pp.133-143
• /
• 2015

The antigenic proteins of Lymphocystis disease virus (LCDV) from tumors of olive flounder, Paralichthys olivaceus, are described following characterization by mass spectrometry. In SDS-PAGE, predominant protein bands were observed at 114, 88, 70, 54, 52, 47, 42 and 24 kDa. Western blot analysis showed that antisera reacted strongly at molecular weights of 114, 67 and 54 kDa, and reacted weakly at molecular weights of 74, 70, 36, 24 and 22 kDa. In the identification of LCDV antigenic proteins by matrix-assisted laser desorption ionization (MALDI) TOF mass spectrometry, 10 of 14 excised bands consisted mostly of proteins with amino acid sequences that matched LCDV-C (lymphocystis disease virus isolate China) ORFs. Strong antigens with molecular weights of 114, 67 and 54 kDa were identified as LDVICp236 (chromosome segregation ATPase), LDVICp033 (membrane bound metallopeptidase) and LDVICp157 (hypothetical protein), respectively. Minor antigens with molecular weights of 70, 36, 24 and 22 kDa proteins were identified as LDVICp160 (acetyl-coA hydrolase), LDVICp213 (hypothetical protein), LDVICp039 (hypothetical protein) and LDVICp213 (hypothetical protein). However, the major capsid protein (LDVICp043) did not react with the polyclonal antibody.

• Microbiology and Biotechnology Letters
• /
• v.23 no.1
• /
• pp.110-117
• /
• 1995

This study was carried out to immunologically characterize Bacillus thuringiensis (B.t) antigens. Protein patterns of ultrasonicated- antigens of B. thuringiensis subspecies using SDS- PAGE revealed marked similarities among all the strains analyzed except for the difference between quantative variations of bands and some protein antigens. The comparison of the protein patterns showed that the protein antigen of 45 kilodalton (kd) was common in 11 strains and that the difference between B. thuringiensis subsp. canadensis and galleriae was noticed in quantative variations of bands despite of ambiguous serogrouping, suggesting a useful method for identification. All strains examined showed similar antigenic patterns in SDS-PAGE, while immunodominant bands differed in antigenic reactivity in western blot using polyclonal antibodies. Polyclonal antibody to B. thuringiensis subsp. thuringiensis and israelensis in indirect immunofluorescence assay reacted with flagella and cell surface antigens. The present study indicates that SDS-PAGE and western blot analysis may be used as tools for differentiation and identification of B. thuringiensis subspecies.

• Parasites, Hosts and Diseases
• /
• v.32 no.2
• /
• pp.111-116
• /
• 1994

Immunoblot analysis utilizing bovine sera from naturally or experimentally infected with Theileria sergenti were used to determine the immunodominant polypeptides of T sergenti (Korean isolated. The previously recognized major bands, 18 kDa,29 kDa, 34 kDa and 45 kDa, were excised after electrophoresis and transfer to PnF membrane. The individual bands were sequenced. The 34 kDa polypeptide which was the most antigenic and immunogenic peptide was observed in the Western blot. However, Chou-Fasman prediction sites (antigenic site) for antigen determinants of the 45 kDa, 34 kDa, 29 kDa and 18 kDa polypeptide were 6, 4, 2 and 0, respectively. However, the 45 kDa polypetide showed no reaction with anti- T sergenti hyperimmune serum.

• Parasites, Hosts and Diseases
• /
• v.26 no.3
• /
• pp.163-168
• /
• 1988

To observe the antigenic protein fractions in saline extract of Spirometra mansoni plerocercoid (sparganum), the crude extract was separated in reducing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE). The proteins, transferred by celctrophoresis to introcillulose paper, were reacted with sera from 15 surgically confirmed sparganosis and 24 cysticercosis patients for immunoblotting. Out of 30 identified protein bands in the extract, bands of 29 and 36 kilodaltons (kDa) were the strongest and the most frequently reacting with specific antibody (IgG) in sparganosis sera. Bands of highter molecular weight also reacted with the sera but their frequency of reactions was lower. Sera of cysticercosis reacted with different protein bands in saline extract of sparganum, but the cross reactions were observed in strong antigenic bands of 29 and 36 kDa.

• Parasites, Hosts and Diseases
• /
• v.26 no.4
• /
• pp.239-244
• /
• 1988

In order to observe the antigenic fractions in saline extract of adult Paragonimus westermani, proteins in the crude extract were separated by sodium dodecyl sulfate-polyacylamide gel electrophoresis(SDS.PAGE) in reducing conditions. The separated protein fractions were transferred to nitrocellulose paper on which 20 sera from human paragonimiasis were reacted and immunoblottrd. Out of 15 stained protein bands in SDS-PAGE, 7 reacted with infected sera while 8 did not. Additionally, 7 unstained protein bands in SDS-PAGE reacted with the sera. Of 14 reacted bands, 30 kilodalton(kDa) band was the most frequently reacted (95%) and was a strong antigen. Protein bands of 23 and 46 kDa were also strong antigens. Bands of over 150 kDa, 120 kDa, 92 kDa, 86 kDa, 74 kDa, 62 kDa, 51 kDa, 32 kDa, 28 kDa, 16.5 kDa and 15.5 kDa were also reactive but their frequencies of the reaction were variable. Key words: Paragonimus westermani, human paragonimiasis, antigenic proteins, SDS-PAGE/ immunoblot

• Journal of Plant Biology
• /
• v.36 no.1
• /
• pp.91-96
• /
• 1993

Phycoerythrins from the ceramiaceous red algae Campylaephora crassa (Okamura) Nakamura and related species, C. hypnaeoides J. Agardh and Ceramium kondoi Yendo, were investigated for absorption spectra, protein bands by gel electrophoresis and antigenic reactivity to anti-phycoerythrin using Ouchterlony double diffusion and immunoblot. Similarities in absorption spectra, showing peaks at ca. 566 nm>534 nm>495 nm, were found between C. crassa and Cm. kondoi, while C. hypnaeoides differed slightly. There were no differences in fluorescence emission spectra and protein bands between C. crassa and related species tested. Since Ouchterlony double diffusion, however, showed that phycoerythrins from C. crassa and Cm. kondoi were similar in antigenic reactions, and differed from that of C. hypnaeoides, the taxonomic position of C. crassa should be reinvestigated using other experimental approaches.

• Parasites, Hosts and Diseases
• /
• v.53 no.1
• /
• pp.85-93
• /
• 2015

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.