• IMMUNE NETWORK
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• v.7 no.1
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• pp.26-30
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• 2007

Background: Mycobacterial antigens released as PIM, LM, LAM, lipoproteins and other cellular factors may contribute to macrophage and dendritic cell activation through pattern recognition receptors such as TLRs. In this study, we assessed cytokine production and ERK activation with stimulation of several major mycobacterial antigens. Methods: Purified mycobacterial antigens (10, 22, 30, 38kDa) and recombinant antigens (6, 16, 19, 38kDa, Ag85A antigen) were studied. The production of cytokines (TNF-${\alpha}$, IL-12, IL-6) was measured by ELISA. The ERK activation was detected by western blotting. The expression of TLR2 or TLR4 was measured by flow cytometry. Results: Among purified antigens only 30kDa antigen induced production of IL-6 or TNF-${\alpha}$ in THP-1 macrophage cells. When THP-1 macrophage cells were treated with 30kDa antigen, phosphorylation of ERK was detected. ERK activation also occurred in TLR2 transfectant HEK293 cells with 30kDa antigen stimulation. Conclusion: 30kDa antigen is one of the major mycobacterial antigens inducing cytokine production and MAP kinases phosphorylation in macrophages.

• Tuberculosis and Respiratory Diseases
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• v.45 no.2
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• pp.271-279
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• 1998

Background: Diagnosis by direct microscopy and/or by culture of the Mycobacterium tuberculosis from body fluids or biopsy specimens is "Gold standard". However, the sensitivity of direct microscopy after Ziehl-Neelsen staining is relatively low and culture of mycobacteria is time consuming. Detection of mycobacterial DNA in clinical samples by the polymerase chain reaction is highly sensitive but laborious and expensive. Therefore, rapid, sensitive and readily applicable new tests need to be developed. So we had evaluated the clinical significance of serologic detection of antibody to 38 kDa antigen, which is known as the most specific to the M. tuberculosis complex, and culture filtrate antigen by ELISA in sputum AFB smear negative patients. Method: In this study, culture tests for acid fast bacilli with sputa or bronchial washing fluids of 183 consecutive patients who were negative of sputum AFB smear were performed. Simultaneously serum antibodies to 38 kDa antigen and unheated culture filtrate of M. tuberculosis were detected by an ELISA method. Results: The optical densities of ELISA test with 38 kDa and culture filtrate antigen were significantly higher in active pulmonary tuberculosis cases than in non tuberculous pulmonary diseases (p<0.05), but in patients with active pulmonary tuberculosis, those of the sputum culture positive patients for M. tuberculosis were not significantly different from those of the sputum culture negative cases(p>0.05). In the smear-negative active pulmonary tuberculosis patients, the sensitivity of the ELISA using 38 kDa antigen and culture filtrate was 20.0% and 31.4%. respectively. The specificity was 95.3% and 93.9%. respectively. Conclusion : In active pulmonary tuberculosis but smear negative, the serologic detection of antibody to 38 kDa antigen and culture filtrate by ELISA cannot substitute traditional diagnostic tests and does not have clinically significant role to differenciate the patient with active pulmonary tuberculosis from other with non-tuberculous pulmonary diseases.

• The Journal of the Korean Society for Microbiology
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• v.34 no.6
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• pp.555-559
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• 1999

The 38 kDa protein of Mycobacterium tuberculosis, which was known previously as antigen 5, has been extensively used in the serodiagnosis of tuberculosis. In an attempt to develop and evaluate a serodiagnostic test using the antigen, we expressed the 38 kDa protein in BCG and its seroreactivity was compared to that expressed in Escherichia coli. The coding region of the 38 kDa protein was amplified by PCR, and the gene was cloned into a Mycobacterium-E. coli shuttle expression vector pYMC-his and pQE30 expression vector and expressed in BCG and E. coli, respectively. Both recombinant 38 kDa proteins showed strong seroreactivity against pooled serum from tuberculosis patients. There was no significant difference in seroreactivity between the two recombinant antigens in sera from the far advanced tuberculosis patients. However, of 25 tuberculosis patients graded as "minimal" by chest X-ray, 5 (20.0%) were seropositive by r38 kDa expressed in E. coli, while 8 (32.0%) by that expressed in BCG. Likewise, higher seroreactivity by r38 kDa expressed in BCG was found in sera from the moderately advanced tuberculosis. This study thus indicates that the recombinant 38 kDa expressed in BCG is more effective than that expressed in E. coli in detecting antibodies to the native 38 kDa protein of M. tuberculosis in sera from minimally affected tuberculosis patients.

• Parasites, Hosts and Diseases
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• v.32 no.1
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• pp.35-42
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• 1994

This study was designed to evaluate the humoral immune reactions in clonorchiasis before and after praziquantel treatment. Rabbits were infected with 150 or 450 metacercariae, treated on 4 and 8111 months after infection, and observed for 13 months of posttreatment. Infection controls were maintained for 22 months. Antigen was the metabolic product of worms incubated in physiologic saline. The immune reactions of anti-clonorchis IgG were observed using SDS-PAGE/immunoblot. During the Infection and Posttreatment, the antigenic Proteins of 66, 63, 54, 52, 50,47,42, 40, 38, 34,33,30, 27, 25, 23, 20, 19, 18, 17, 16, 15, 14, 13, 12.5 and 11.5 kDa were detected. Of them, 33,27, 13, and 12.5-kDa antigens were highly antigenic and observed predominently in infection controls. After the treatment, 13 and 12.5-kDa antigens faded in 6 months after the second treatment, but 33 and 27-kDa antigens were detected until 13 months of posttreatment. The results clearly demonstrate that 13 and 12.5-kDa antigens represent attenuated host immune reactions by praziquantel treatment. As the 12.5-kDa antigen had a large amount of protein in SDS-PAGE, it was designated as'K2-Ag'of C. sinenis.

• Parasites, Hosts and Diseases
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• v.31 no.3
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• pp.269-276
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• 1993

To ascertain that tegument of Pnragonimus westermoni has specific antigenic proteins, the tegumental fraction was isolated from 10-month-old worms by 0.1% digitonin solution, and subjected to SDS-PAGE and immunoblot. Component proteins of tegumental syncytium comprised of 94, 74 (76-66), 62, 54, 44, 42, 38, 28, 26, 25, 24, 17, 15.5 and 13.5 kDa proteins. Of them, the 94, 44 and 42kDa proteins were more specific to tegument, especially the 94 kDa protein was the most prevailing one. In immunoblot, antigens of the 94, 90, 78, 76, 74, 68, 65, 63, 60, 59 and 54 kDa proteins were commonly detected by 7 sera of 10 human paragonimlasis, but none of them reacted with 5 sera of clonorchiasis. In conclusion, the 94 kDa protein was the major tegumental protein, as well as the specific antigen. The 76 and 66 kDa proteins were the minor components of tegument, which were also specific antigens of R westermcni.

• Parasites, Hosts and Diseases
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• v.32 no.4
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• pp.249-258
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• 1994

Recently, the importance of toxoplasmosis is raised as a complication in immunosuppressed or AIDS patients. Our study focused on the identification of a variety of Toxoplasma antigens by immunoblotting. Rabbits and BALB/c mice were immunized with Toxoplosmo Iysate (RH strain) , frozen tachyzoites (RH strain) or cysts (Beverly and Fukaya strain) . Blood were collected from ear vein, heart or orbital plexus for detecting the serum antibody levels. For excretory-secretory (E.S) antigens, T gondii (RH) tachyzoite were cultured in CHL (Chinese hamster lung) cells with MEM containing of 5% FCS. After 72hrs, culture supernatant was collected. BALB/c mice were inoculated with RH tachyzoite intraperitoneally and peritoneal fluids were extracted three days later. E.S antigens were detected in culture supernatant and infected mouse peritoneal fluid by EITB. Serum IgG levels in rabbit were 1 :512 of 10 days after primary immunization, 1 : 2,048 of 10 days after secondary immunization, 1: 1,024 of 20 days after secondary immunization by IFAT, respectively. Serum IgG levels of immunized mice were 1:128 after 7 weeks. Tachyzoite antigens of the RH strain were detected 25 protein bands ranging 10 kDa-220 kDa of molecular weights with Coomassie blue stain. Toxoplcsma major antigens corresponding to n of 24 kDa, 27 kDa,30 kDa, 35 kDa, 38 kDa were recognized by IgG and IgM antibodies. Excretory-secretory antigens present in culture supernatant with M. W. of 20, 30 kDa and in infected mouse peritoneal fluid with M.W. of 33 (P30), 45 kDa. When RH tachyzoite antigen was probed with different mice sera immunized with 2 strains of T gondii, the IgG antibody bud of Fukaya and Beverly strain (8 week-serum) is identical to those of RH strain. It is considered that the 30 kDa polypeptide detected in excretory- secretory materials and Iysate was important major antigen of T gondii (RH).

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.328-335
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• 1998

Outer membrane proteins(OMPs) of Brucella abortus 1119-3 strain were extracted by Triton X-100 treatment, and fractionated by DEAE-cellulose column chromatography and Sephacryl S-300 column chromatography. The antigenic proteins in these fractions were identified by Western blot analysis. In Western blot analysis, a single band(38kDa) was observed in the DEAE fractions from 36th fraction to 38th fraction against sera of cattle infected with B abortus. And other fractions have several bands. However, the Sephacryl S-300 fractions exhibited a total of 3 peaks of proteins with a broad range from about 30 to 116kDa. In order to characterize further, the extracted OMPs and the DEAE fractions were analyzed by two dimensional polyacrylamide gel electrophoresis(2-DE) and Western blot using serum from naturally infected cattle with Brucella spp. The 2-DE immunoblots of DEAE fraction showed immunoreactive spots more than twenty two. The major protein spots have ranging from about 32 to 47kDa. The pI values of the spots were detected from pH 4.7 to 5.4. Among the major protein spots, the 38kDa protein which is a specific antigen, located at the point of approximately a pI 4.8.

• Clinical and Experimental Pediatrics
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• v.54 no.10
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• pp.414-421
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• 2011

Purpose: Recently, an increase in the number of patients sensitized to rice allergen with or without clinical symptoms has been reported. This study was designed to determine the major allergens in rice and their clinical significance. Methods: Twenty-four children (15 boys and 9 girls; mean age, 16.3 months) with allergic disease, who were sensitized to rice antigen (by UniCAP) in the Pediatric Allergy Respiratory Center at Soonchunhyang University Hospital, were enrolled in this study. The allergenicity of various types of rice (raw, cooked, and heat-treated, simulated gastric fluid [SGF], and simulated intestinal fluid [SIF]) was investigated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoglobulin E (IgE) immunoblots. The patients' medical records, including laboratory data and allergy symptoms after ingestion of rice were reviewed. Results: Patients were sensitized to an average of 13.5 food antigens and their mean total IgE was 6,888.7 kU/L. In SDS-PAGE, more than 16 protein bands were observed in the raw rice, whereas only 14-16 kDa and 31-35 kDa protein bands were observed in cooked rice. The common SDS-PAGE protein bands observed in SGF-, SIF-, and heat-treated rice were 9, 14, and 31 kDa. In a heated-rice IgE immunoblot, protein bands of 9, 14, and 31-33 kDa were found in 27.8%, 38.9%, and 38.9% of all sera, respectively, and in 50%, 50%, and 75%, of ser a from the 4 symptomatic patients, respectively. Conclusion: The 9-, 14-, and 31-kDa protein bands appeared to be the major allergens responsible for rice allergy symptoms.

• Parasites, Hosts and Diseases
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• v.51 no.6
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• pp.755-757
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• 2013

Gnathostoma spinigerum can cause subarachnoid hemorrhage (SAH). The detection of specific antibodies in serum against G. spinigerum antigen is helpful for diagnosis of neurognathostomiasis. There is limited data on the frequency of G. spinigerum infection in non-traumatic SAH. A series of patients diagnosed as non-traumatic SAH at the Srinagarind Hospital, Khon Kaen University, Thailand between January 2011 and January 2013 were studied. CT or MR imaging of the brain was used for diagnosis of SAH. Patients were categorized as aneurysmal subarachnoid hemorrhage (A-SAH) or non-aneurysmal subarachnoid hemorrhage (NA-SAH) according to the results of cerebral angiograms. The presence of specific antibodies in serum against 21- or 24-kDa G. spinigerum antigen was determined using the immunoblot technique. The detection rate of antibodies was compared between the 2 groups. Of the 118 non-traumatic SAH patients for whom cerebral angiogram and immunoblot data were available, 80 (67.8%) patients had A-SAH, whereas 38 (32.2%) had NASAH. Overall, 23.7% were positive for specific antibodies against 21- and /or 24-kDa G. spinigerum antigen. No significant differences were found in the positive rate of specific antibodies against G. spinigerum in both groups (P-value=0.350).

• Parasites, Hosts and Diseases
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• v.38 no.4
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• pp.245-249
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• 2000

A total of 492 cattle sera was screened by IgG-ELISA against Neospora caninum (Nc-1 strain and a Korean isolate, KBA-2) and Toxoplasna gondii. Out of 492, 113 sera (23.0%) reacted positively to either Nc-1 or KBA-2 strains of N. caninum. Among the 113 positive sera, 92 sera (81.4%) reacted with antigens of both strains, but 6 sera (5.3%) with Nc-1 and 15 sera (13.3%) with KBA-2 strain only And with T. gondii antigen, 6 sera (1.2%) were positive but all reacted with N. caninum antigen also. Western blot revealed typical binding pattern according to ELISA values, such that high OD group reacted specifically to the major surface proteins including 43 kDa protein. Seroprevalence of 23.0% indicates that neosporosis seemed to be one of major causes of abortion in cattle. It is suggested here to establish more epidemiological researches nationwide systematically.