• Title/Summary/Keyword: Antibody specificity

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Production and Isolation of IgY Antibody Raised Against a Lectin Obtained from Maackia fauriei (Maackia fauriei 유래 렉틴에 대한 IgY 항체의 생성 및 분리)

  • Chung Young Yun;Jung Eui Cha;Lee Hyun Jung;Kim HaHyung
    • YAKHAK HOEJI
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    • v.49 no.1
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    • pp.6-10
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    • 2005
  • Immunoglobulin Y (IgY) obtained from chicken as the immunization host brings several advantages to antibody production, such as improved yield, lower cost, longer stability and higher specificity than mammalian immunoglobulin. In the present study, we attempted to produce IgY against a sialic acid-binding lectin, Maackia fauriei agglutinin (MFA), from egg yolk of white Leghorn hens. For the isolation of IgY from egg yolk, we applied a water dilution method. The weekly yield of IgY was determined by enzyme-linked immunosorbent assay, with a final yield of anti-MFA IgY from total IgY of approximately $1\%$. The yielded IgY were used to prepare IgY-affinity column conjugated with CNBr-activated Sepharose 4B, which resulted in the lectin being successfully purified in a single step from Maackia fauriei. This purified lectin exhibited the same hemagglutination activity as lectin purified using conventional purification methods.

Expression of ORF6 gene of porcine reproductive and respiratory syndrome (PRRS) virus (돼지생식기호흡기증후군 바이러스의 ORF6 유전자 발현)

  • Bae, Su-Jung;Kim, Jin-Won;Yoon, Young-Sim;Kang, Shien-Young
    • Korean Journal of Veterinary Service
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    • v.32 no.1
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    • pp.19-25
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    • 2009
  • Porcine reproductive and respiratory syndrome (PRRS) virus is the etiological agent of diseases characterized by reproductive losses in sows and respiratory disorders in piglets. The PRRS virus is a small enveloped virus containing a positive-sense, single-stranded RNA genome. In the present study, ORF6 gene of Korean PRRS virus isolate, CNV, was cloned and expressed in baculovirus expression system. The ORF6 gene and expressed protein in the recombinant virus were confirmed by PCR/indirect fluorescence antibody (IFA) test and Western blotting, respectively. The recombinant protein with a molecular weight of approximately 24KDa was confirmed by Western blotting using His6 and PRRS virus-specific antiserum. Expressed ORF6 protein was applied for IFA to detect antibody against PRRS virus using field porcine sera. However, the sensitivity and specificity of developed IFA using expressed ORF6 protein were considerably low compared to those of commercial ELISA kit. This results suggest that IFA using expressed ORF6 protein could not be used as a diagnostic test for PRRS virus infection without further improvements.

TNF-${\alpha}$ Up-regulated the Expression of HuR, a Prognostic Marker for Ovarian Cancer and Hu Syndrome, in BJAB Cells

  • Lee, Kyung-Yeol
    • IMMUNE NETWORK
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    • v.4 no.3
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    • pp.184-189
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    • 2004
  • Background: Hu syndrome, a neurological disorder, is characterized by the remote effect of small cell lung cancer on the neural degeneration. The suspicious effectors for this disease are anti-Hu autoantibodies or Hu-related CD8+ T lymphocytes. Interestingly, the same effectors have been suggested to act against tumor growth and this phenomenon may represent natural tumor immunity. For these diagnostic and therapeutic reasons, the demand for antibodies against Hu protein is rapidly growing. Methods: Polyclonal and monoclonal antibodies were generated using recombinant HuR protein. Western blot analyses were performed to check the specificity of generated antibodies using various recombinant proteins and cell lysates. Extracellular stimuli for HuR expression had been searched and HuR-associated proteins were isolated from polysome lysates and then separated in a 2-dimensional gel. Results: Polyclonal and monoclonal antibodies against HuR protein were generated and these antibodies showed HuR specificity. Antibodies were also useful to detect and immunoprecipitate endogenous HuR protein in Jurkat and BJAB. This report also revealed that TNF-${\alpha}$ treatment in BJAB up-regulated HuR expression. Lastly, protein profile in HuR-associated mRNAprotein complexes was mapped by 2-dimensional gel electrophoresis. Conclusion: This study reported that new antibodies against HuR protein were successfully generated. Currently, project to develop a diagnostic kit is in process. Also, this report showed that TNF-${\alpha}$ up-regulated HuR expression in BJAB and protein profile associated with HuR protein was mapped.

Identification of mono- or poly-specific monoclonal antibody to Porphyromonas gingivalis heat-shock protein 60

  • Choi, Jeom-Il;Lee, Sang-Yull;Kim, Koan-Hoi;Choi, Bong-Kyu;Kim, Myung-Jin
    • Journal of Periodontal and Implant Science
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    • v.41 no.2
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    • pp.54-59
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    • 2011
  • Purpose: The aim of this study was to define the immunoreactive specificity of Porphyromonas gingivalis (P. gingivalis) heat shock protein (HSP) 60 in periodontitis and atherosclerosis. Methods: In an attempt to define the cross-reactive bacterial heat-shock protein with human self-antigen at molecular level, we have introduced a novel strategy for cloning hybridoma producing anti-P. gingivalis HSP 60 which is polyreactive to bacterial HSPs or to the human homolog. Results: Five cross-reactive clones were obtained which recognized the #19 peptide (TLVVNRLRGSLKICAVKAPG) among 37 synthetic peptides (20-mer, 5 amino acids overlapping) spanning the whole molecule of P. gingivalis HSP 60. We have also established three anti-P. gingivalis HSP 60 monoclonal antibodies demonstrating mono-specificity. These clones recognized the #29 peptide (TVPGGGTTYIRAIAALEGLK). Conclusions: Peptide #19 and #29 of P. gingivalis HSP 60 might be important immunoreactive epitopes in the immuno-pathogenic mechanism of bacterial antigen-triggered autoimmune diseases.

A Study on the Validity of 'Hepa-S' Hepatitis B Antibody Detecting Reagent after Vaccination of 'Hepa-Vax' (B형 간염백신 'Hepa-Vax' 접종후 항체검사시약 'Hepa-S' kit의 정확도에 관한 연구)

  • Moon, In-Sook
    • Journal of Preventive Medicine and Public Health
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    • v.18 no.1
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    • pp.51-57
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    • 1985
  • To attempt to measure the effect of domestic product P.H.A. kit 'Hepa-S' after completion of 'Hepa-Vax' vaccination schedule, P.H.A. test and R.I.A. test on the 330 healthy adults were carried out. The results obtained were as follow ; 1. The positive anti HBs rate after completion of 'Hepa-Vax' vaccination were; in P.H.A. test with domestic product P.H.A. kit 81.2%, in P.H.A. test with foreign product P.H.A. kit 82.7%, and in R.I.A. test 95.8% 2. Using the result of R.I.A. test as the standard, sensitivity of P.H.A. test with domestic product P.H.A. kit was 84.8% and specificity was 100.0% 3. Using the result of R.I.A. test as standard, sensitivity of P.H.A. test with foreign P.H.A. kit was 86.4% and specificity was 100.0%. 4. The concordance rate of P.H.A. test with domestic product and foreign product kit was 98.5%. On the result of this study, there was no significant difference in the validity between the domestic product P.H.A. kit 'Hepa-S' and the foreign P.H.A. kit $'Hebsgencell^{TM}'$. So that it is recommendable to use domestic product P.H.A. kit instead of foreign product P.H.A. kit.

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Comparison of Gold Biosensor Combined with Light Microscope Imaging System with ELISA for Detecting Salmonella in Chicken after Exposure to Simulated Chilling Condition

  • Mi-Kyung Park
    • Journal of Microbiology and Biotechnology
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    • v.33 no.2
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    • pp.228-234
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    • 2023
  • In this study, the performance of a gold biosensor combined with light microscope imaging system (GB-LMIS) was comparatively evaluated against enzyme-linked immunosorbent assay (ELISA) for detecting Salmonella under simulated chilling condition. The optimum concentration of antiSalmonella polyclonal antibodies (pAbs) was determined to be 12.5 and 100 ㎍/ml for ELISA and GBLMIS, respectively. GB-LMIS exhibited a sufficient and competitive specificity toward three tested Salmonella among only. To mimic a real-world situation, chicken was inoculated with Salmonella cocktail and stored under chilling condition for 48 h. The overall growth of Salmonella under chilling condition was significantly lower than that under non-exposure to the chilling condition (p < 0.05). No significant differences in bacterial growth were observed between brain heart infusion and brilliant green broth during the enrichment period (p > 0.05). Finally, both GB-LMIS and ELISA were employed to detect Salmonella at every 2-h interval. GB-LMIS detected Salmonella with a competitive specificity by the direct observation of bacteria on the sensor using a charge-coupled device camera within a detection time of ~2.5 h. GB-LMIS is a feasible, novel, and rapid method for detecting Salmonella in poultry facilities.

Evaluation for detection of Cryptosporidium oocysts in diarrheal feces of calves (야외 송아지 설사변에서 작은와포자충 검출에 대한 평가)

  • Wi, Seong-Hwan;Ju, Hu-Don;Gang, Yeong-Bae
    • Parasites, Hosts and Diseases
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    • v.34 no.2
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    • pp.121-126
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    • 1996
  • For the detection of Cwptospori,mum oocysts, fecal samples were collected from 201 calves which showed diarrhea. Among the 201 samples, 29 samples (14.4%) were positive for Cwptosporinium spry. by the DMSO-modified acid-fast stain (MAFS) , 23 samples (11.4%) were positive by commercial kit (Meridian Diagnostics, Cincinnati, Ohiol and 23 by the indirect immunofluorescence antibody (IFA )assay employing the monoclonal antibody (mAb C6). When tested by both IFA and MAFS, 20 fecal samples were positive for Cwptosporinium oocysts whereas 169 fecal samples were negative. If the MAFS is considered a standard method for oocyst detection, the IFA showed 69% of sensitivity and 98% of specificity. When tested by both IFA and commercial kit, 22 fecal samples were positive for Cwptospori,mum oocysts while 177 samples were negative. One sample tested by IFA was found to be false negative, when compared with the results by commercial kit. The sensitivity of IFA was calculated as high as 96%; the specificity as 99% and the predictive value was also 99%. In the present study, IFA employing the nAb C6 revealed that 23 samples (11.4%) were positive among the 201 calves showing diarrhea. Of 23 IFA positive samples, 4 samples (5%) showed cryptosporidial oocysts more than 105 OPG Therefore. it is concluded that the calves showing cryptosporidial oocysts more than 105 OPG in the feces were highly associated with clinical cryptosporidiosis.

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Serological Diagnosis of Human Sparganosis by means of micro-ELISA (효소면역측정법을 이용한 스파르가눔증의 혈청학적 진단)

  • Hyuck Kim;Suk-Il Kim;Seung-Yull Cho
    • Parasites, Hosts and Diseases
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    • v.22 no.2
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    • pp.223-228
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    • 1984
  • Seven cases of surgically proven sparganosis were serologically tested by means of microELISA for their specific IgG antibody levels. For that purpose, crude saline extract of spargana from snake, Natrix tigrina lateralis was prepared and used as antigen. The sparganosis sera were also tested with Paragonimus and Cysticercus antigens to observe the cross reactivity. A total of 71 sera from normal control, ectopic and pulmonary paragonimiasis, clonorchiasis, cysticerCOSIS and Taenia saginata cases were also included. Except for one case of old calcified infection, all of 6 human sparganosis showed higher serum levels of specific IgG antibody when the differential point of positive reaction was set at the absorbance value of 0.25 (the sensitivity being 85.7%). In control and other helminthic infections, all except 3 cases of T. saginata infection showed negative reaction to sparganum antigen (the specificity being 90.7%). None of sparganosis cases showed cress reactivity to Paragonimus and Cysticercus antigens. Undiluted cerebrospinal l1uid also showed high levels of antibody when central nervous system was invaded. The serologic diagnosis by means of micro ELISA could be a useful tool in epidemiological study of human sparganosis in susceptible population, as well as in individual diagnosis.

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Thyroglobulin Measurement in Fine Needle Aspirates for Diagnosing Cervical Lymphnode Metastasis from Differentiated Thyroid Malignancy (갑상선암의 경부 림프절 전이 진단을 위한 미세침세척액 티로글로불린 측정법)

  • Ko, Hee-Young;Kim, Seung-Su;Lee, Chun-Ho
    • The Korean Journal of Nuclear Medicine Technology
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    • v.14 no.2
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    • pp.181-185
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    • 2010
  • Purpose: Several studies report that detection of thyroglobulin (Tg) in fine-needle aspiration (FNA) biopsy washout fluid from lymph nodes identifies recurrences or metastases of differentiated papillary thyroid cancer (DPTC) in the neck with higher sensitivity and specificity than fine-needle aspiration cytology (FNAC). We evaluate the diagnostic efficacy and usefulness of Tg measurement in FNA washout fluid (FNA-Tg) and compare with FNAC. Materials and Methods: Forty-eight FNA samples of 37 patients who undergone ultrasonography to detect cervical lymph node metastasis of DPTC, were included for this study. Lymph node metastasis was confirmed by histopathologic examination or long-term imaging follow-up. Sensitivity, specificity and accuracy of FNA-Tg and FNAC were calculated. In 34 patients, we evaluated diagnostic accuracy of FNA-Tg according to the presence or absence of Tg antibody. Results: Sensitivity, specificity and accuracy of FNAC were 75.0%, 97.2% and 91.7%, respectively, and those of FNA-Tg were 100%, 88.9% and 91.7%, respectively. The presence of Tg antibody was not relevant to the diagnostic accuracy of FNA-Tg. Conclusion: FNA-Tg is a as accurate as FNAC with higher sensitivity. FNA-Tg and FNAC are complement techniques for diagnosing lymph node metastasis of DTPC.

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Expression of Hepatitis B Virus S Gene in Pichia pastoris and Application of the Product for Detection of Anti-HBs Antibody

  • Hu, Bo;Liang, Minjian;Hong, Guoqiang;Li, Zhaoxia;Zhu, Zhenyu;Li, Lin
    • BMB Reports
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    • v.38 no.6
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    • pp.683-689
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    • 2005
  • Antibody to hepatitis B surface antigen (HBsAb) is the important serological marker of the hepatitis B virus (HBV) infection. Conventionally, the hepatitis B surface antigen (HBsAg) obtained from the plasma of HBV carriers is used as the diagnostic antigen for detection of HBsAb. This blood-origin antigen has some disadvantages involved in high cost, over-elaborate preparation, risk of infection, et al. In an attempt to explore the suitable recombinant HBsAg for the diagnostic purpose, the HBV S gene was expressed in Pichia pastoris and the product was applied for detection of HBsAb. Hepatitis B virus S gene was inserted into the yeast vector and the expressed product was analyzed by sodium dodecyl sulphate polyacrolamide gel electrophoresis (SDS-PAGE), immunoblot, electronic microscope and enzyme linked immunosorbent assay (ELISA). The preparations of synthesized S protein were applied to detect HBsAb by sandwich ELISA. The S gene encoding the 226 amino acid of HBsAg carrying ahexa-histidine tag at C terminus was successfully expressed in Pichia pastoris. The His-Tagged S protein in this strain was expressed at a level of about 14.5% of total cell protein. Immunoblot showed the recombinant HBsAg recognized by monoclonal HBsAb and there was no cross reaction between all proteins from the host and normal sera. HBsAb detection indicated that the sensitivity reached 10 mIu (micro international unit)/ml and the specificity was 100% with HBsAb standard of National Center for Clinical Laboratories. A total of 293 random sera were assayed using recombinant S protein and a commercial HBsAb ELISA kit (produced by blood-origin HBsAg), 35 HBsAb positive sera and 258 HBsAb negative sera were examined. The same results were obtained with two different reagents and there was no significant difference in the value of S/CO between the two reagents. The recombinant HBV S protein with good immunoreactivity and specificity was successfully expressed in Pichia pastoris. The reagent for HBsAb detection prepared by Pichia pastoris-derived S protein showed high sensitivity and specificity for detection of HBsAb standard. And a good correlation was obtained between the reagent produced by recombinant S protein and commercial kit produced by blood-origin HBsAg in random samples.