: 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 immunoblotted. Out of 15 stained protein bands in SDS-PAGE, 7 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.
parasitology-helminth-trematoda ; Paragonimus westermani ; immunology ; antigen ; electrophoresis

In order to observe the protein compositions of soluble extracts of P. westermani, and their changes during early developmental stages, the crude saline extracts of 4, 6, 8, 10 and 12 week-old worms which were harvested from experimentally infected dogs were analysed by disc-PAGE. The results were as follows: A total of 15 bands were identified from electrophoregrams of respective developmental stages. Of them, 5 bands were recognized throughout the developmental stages. The number and protein amount of identified bands changed according to the worm development from 4 weeks to 12 weeks. However, the banding patterns of 4 and 6 week-old worms and 8 and 10 week-old worms were similar each other. Of 15 identified bands, band 1 was recognized only in 12 week-old worms whereas bands 3, 4, 8, 9, 10, 11 and 15 gradually lowered their amount according to development to disappear in 12 week-old. In addition, band 5 became a major band in 12 week-old while band 6 turned to a minor band at tha same age. The possible relations of changing patterns of protein bands with worm development were discussed.
parasitology-helminth-trematoda ; Paragonimus westermani ; immunology ; protein ; electrophoresis

Serological studies on toxoplasmosis were conducted with rabbits sera that were immunized with RH strain or infected with Beverley strain of Toxoplasma gondii. Complement fixation tests, agar-gel double diffusion tests and agar-gel immunoelectrophoresis were performed. Toxoplasma crude antigen was prepared from the organisms in mice peritoneal fluids, which were infected with RH strain of Toxoplasma gondii. The organisms were suspended in saline volume originally exudated and counted in hemocytometer for purity of the organisms over 99 per cent. These suspended organisms were prepared by sonication, and the solution was centrifuged for 30 min. at 10,000 rpm in 4C. These supernatant fluids were used as crude antigen. On the other hand, purified antigens were fractionated on Sephadex G-200 gel filtration. A Sephadex G-200 column, 80 by 4 cm, equilibrated with Tris-HCl-(0.1 M)-NaCl (1.0 M) buffer, pH 8.0 was used. The eluate fractions were collected in 3 ml per hour and the absorbance at 280 nm was measured with a Beckman Du-2 spectrophotometer. Each tube is pooled into 6 fractions by protein density graph. For immunization of rabbits, crude antigen of RH strain was emulsified with an equal amount of incomplete Freund's adjuvant and l ml of mixture was injected subcutaneously into them once a week for 5 successive weeks. Antisera were obtained at an interval of a week, beginning the first week after the last immunization, while several rabbits were infected with Beverley strain of Toxoplasma gondii by inoculating about 200 cysts and antisera were obtained from them serially at a week interval. The results were as follows: The sera from the rabbits immunized with the RH strain or infected with Beverley strain of Toxoplasma gondii againist the crude antigen showed the first positive reactions in 2 or 3 weeks after the administration or immunization in complement fixation tests. Maximum titers appeared in 4 or 5 weeks after immunization with RH strain and in 7 or 9 weeks after infection with Beverley strain respectively. Complement fixation tests showed the positive reactions in the rabbits sera immunized with RH strain against the purified antigens II, III, IV, V and VI: moreover, antigen IV fraction showed the highest titer. On the other hand in the rabbits sera infected with Beverley strain against the purifed antigens II, III and IV fractions showed the positive reaction; especially, antigen fraction IV showed the highest titer. In immuno-diffusion tests, the sera from the rabbits immunized with RH strain and infected with Beverley strain, against the crude antigen appeared the precipitin bands 2 weeks after the immunization or infection. And the former showed the 2 or 5 precipitin bands after 5-8 weeks and the latter showed the l or 2 precipitin bands after 6 weeks. The sera from the rabbits immunized with RH strain against the purified antigens II, III, IV,V and VI showed the precipitin bands, and the sera from the rabbits infected with Beverley strain against the purified antigens II, III and IV showed the precipitin bands in the immuno-diffusion tests. Especially antigen IV was the strongest reaction against the sera from RH strain and Beverley strain. In agar-gel immunoelectrophoresis, the immunized sera against the crude antigen showed 8 arcs. But the infected sera against the crude antigen showed 4 or 5 arcs. The immunized sera against the fractionated antigens II, III, IV, V, VI showed arcs, but against the fractionated antigen IV showed 6 arcs and in the antigens II, III, V, VI showed l or 2 arcs only. On the other hand, the infected sera against the fractionated antigens IV showed 4 arcs, II and III showed the l arcs, which was the most weak of all.
parasitology-protozoa-Toxoplasma gondii ; toxoplasmosis ; rabbit ; immunology ; electrophoresis

Proteomics and polemic techniques are among the most valuable approaches in the research of life science in this new century, marking the beginning of a post-genome era. Two-dimensional electrophoresis and mass spectrometry, applied as key techniques in proteomic research, have given rise to new research strategies and improved the efficiency of researchers in exploring the unknown field. Its introduction into the exploration of spermatozoal proteins has given us so many pleasant surprises. This review presents some essential information about proteomics and two-dimensional electrophoresis and mass spectrometry, with a brief introduction of the recent progress in the researches on human sperm proteome, capacitation-related sperm proteins, sperm-egg interaction-related proteins, and sperm-immunity which has made great senses in the clinical problems.
Electrophoresis, Gel, Two-Dimensional ; Humans ; Male ; Proteome ; Spermatozoa ; immunology

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5'-nucleotidase subclass I and purine-specific 5'-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5'-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.
Animals ; Babesia/*immunology ; Babesiosis/blood/immunology/parasitology/*veterinary ; Cell Differentiation/immunology ; Dog Diseases/*blood/immunology/*parasitology ; Dogs ; Electrophoresis, Polyacrylamide Gel ; Erythrocytes/*immunology ; Reticulocytes/*immunology

OBJECTIVETo obtain egg yolk antibody in hen eggs laid by hens immunized with the protein of Porphyromonas gingivalis (Pg). To generate, purify IgY against Pg (anti-Pg-IgY) and identify its specificity.

METHODSPgATCC33277 was cultured under standard anaerobic conditions and harvested after proliferation. Then Pg was extracted by sonication until the cell pellets were shattered completely. After centrifugatiton, the supernatant was collected. Five-month-old Roman hens were immunized for egg antibody production. The antibody was inoculated intramuscularly and subcutaneously in the breast from multiple spot with 1.0 ml of a vaccine consisting of oil-adjuvant protein which was mixed with 1 ml protein of Pg and 1 ml Freund's adjuvant complete every 10 days, for 4 times. The eggs were collected after the first immunization and stored at 4°C. The anti-Pg-IgY was extracted and purified. The protein concentration was tested by bicinchoninic acid (BCA), the specificity of IgY analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE), the titre of IgY and its physicochemical character were evaluated by indirect enzyme-linked immunosorbent assay.

RESULTSThe concentration of obtained anti-Pg-IgY was 2.05 g/L. SDS-PAGE analysis of the anti-Pg-IgY showed that the molecular weight of IgY was consistent with the theoretical value. Protein of anti-Pg-IgY appeared approximately 5 days after the first immunization, and reached the peak at 50 - 55 days. Antibody titres reached 1:100 000. Each egg produced more than 10 mg IgY, and its purification was up to 95% as well.

CONCLUSIONSLayer hens immuned by Pg may provide specific IgY of high titre and high concentration. The antibody has high purity and is heat, acid and alkali-resistant.

Animals ; Antibodies ; chemistry ; immunology ; Chickens ; immunology ; Egg Yolk ; chemistry ; immunology ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Immunization ; Immunoglobulins ; chemistry ; immunology ; isolation & purification ; Porphyromonas gingivalis ; immunology

OBJECTIVETo identify cancer-derived immunoglobulin G (IgG) whole molecule-interacting proteins to provide important clues for studying IgG biological functions.

METHOSHeLa cell lysate was immunoprecipitated with rabbit antihuman IgG whole molecule antibody and normal rabbit IgG. The immunocomplex underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and was detected with silver staining. Three prominently enhanced bands were subjected to protein identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the MS data were analyzed with Swiss-Prot database. Cancer-derived IgG whole molecule-interacting proteins were screened and functionally annotated.

RESULTS AND CONCLUSIONWe identified 6 potential cancer-derived IgG whole molecule-interacting proteins with co-immunoprecipitation combined with LC-MS/MS, which provides valuable clues for studying the function of cancer-derived IgG.

Antibodies, Neoplasm ; immunology ; Chromatography, Liquid ; Electrophoresis, Polyacrylamide Gel ; HeLa Cells ; Humans ; Immunoglobulin G ; immunology ; Neoplasms ; immunology ; Proteins ; immunology ; Tandem Mass Spectrometry

OBJECTIVETo purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies.

METHODCaprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.

RESULTSAntibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 microg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 microg/mL and 1.03 microg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 microg/mL, and the linear working range and LOD were 10.91-11412.29 microg/mL and 3.42 microg/mL, respectively.

CONCLUSIONAntibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.

Antibodies, Monoclonal ; immunology ; Chromatography, Affinity ; Chromatography, Agarose ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Food Contamination ; Fruit ; Insecticides ; immunology ; Organothiophosphorus Compounds ; immunology ; Pesticide Residues ; analysis ; immunology ; Vegetables

Antigen Rv2628 of Mycobacterium tuberculosis is associated with latent tuberculosis infection. In this study, Rv2628 was prokaryotic expressed and purified, its immunological characteristics was evaluated with macrophage cell line RAW264.7 and BALB/c mice. The results show that Rv2628 was mainly expressed in form of inclusion body confirmed by SDS-PAGE, and could react with rabbit anti-H37Rv polyclonal antibody detected by Western blotting assay, indicating that the protein had an effective immunoreactivity. The interactions between Rv2628 and macrophage cell line RAW264.7 confirmed that it could effectively induce cells to produce pro-inflammatory cytokines, the relative expression level of IL-6 mRNA was higher than the control group in 1-12 h. BALB/c mice were subcutaneously immunized with Rv2628 protein, the production of IFN-gamma and IL-4 in the spleen cells was determined by Sandwich ELISA, in the Rv2628 immunized group, the level of IFN-gamma was significantly higher than that of IL-4 (P < 0.000 1). It indicated the protein induced Th1-tendency immune responses. At the same time, Rv2628(11-30) peptide used as coating antigen, the murine serum antibody titer detected by indirect-ELISA was 1:1 600, which demonstrated that Rv2628 could also induce humoral immune responses. In summary, Rv2628 could induce specific pro-inflammatory cytokines, affectively induce strongly Th1-tendency immune response and humoral response, it could be a potential target for developing subunit vaccine against TB. In addition, it laid foundation for probing the cross-talk between M. tb and host.
Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; immunology ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Interferon-gamma ; immunology ; Interleukin-4 ; immunology ; Interleukin-6 ; immunology ; Macrophages ; immunology ; Mice ; Mice, Inbred BALB C ; Mycobacterium tuberculosis ; Th1 Cells ; immunology ; Tuberculosis ; immunology

The pollen of Artemisia has been considered as the main late summer-autumn allergen source in this country. To identify its allergenic components, Artemisia princeps pollen extracts were separated by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membrane, where IgE binding components were detected by the reaction with sera of twenty Artemisia-allergic patients and 125I-anti-human IgE, sixteen components in the molecular range of 10,000 and 85,000 daltons were detected. Twelve bands bound to IgE from 50% of the sera tested, and two bands (37,000, 23,000 daltons) showed the highest (85%) frequency of IgE-binding in twenty sera tested. When the gel of SDS-PAGE with Artemisia pollen extracts was sliced into 11 allergenic groups (AG) and the protein of each AG was obtained by the gel elution method, the wormwool-RAST inhibition test showed that the AG 10 demonstrated to be the most potent, and the AG 7 was the next. Six AGs showed significant responses (more than 100% of wheal size to histamine, 1 mg/ml) on the skin prick test in more than 50% of the patients tested. It is suggested that electrophoretic transfer analysis with SDS-PAGE may be a valuable method for Artemisia allergen identification, and the possibility of partial purification of allergens by employing gel elution is discussed.
Blotting, Western/methods ; Electrophoresis, Agar Gel/methods ; Human ; Korea ; Lymphokines ; Plants/immunology ; Pollen/analysis/*immunology ; Skin Tests/methods