BACKGROUND: A fully automated enzyme-immunoassay (EIA) analyzer, Enzymun System, ES-300 (Boehringer Mannheim, Germany) uses streptavidin technology and performs single test or panels of up to 12 tests per run. We evaluated the results of ES-300 for anti-HCV by comparing the results with microplate-EIA, radioimmunoassay (RIA), and confirmatory test. METHODS: Total 79 sera (51 positive, 24 negative, 4 indeterminate results confirmed by Lucky HCD Confirm) were analysed. ES-300 with Enzymun-Test(R) Anti-HCV (Boehringer Mannheim, Germany) and microplate-EIA (Green Cross Center Innotest HCV 3.0(R)) were used. Fifty one sera were examined additionally by 2nd-generation RIA method, NANBDINE 125C(General Biologicals Corp., R.O.C.). And all results were compared to the results of Lucky HCD Confirm. RESULTS: The overall concordance rate of ES-300 and Innotest(R) was 72/79 (91.1%). The results of Lucky HCD Confirm on seven discrepant samples were five negative and two indeterminate. The results of ES-300 and NANBDINE 125C showed concordance rate of 90.2%. The sensitivity and specificity of ES-300 with regard to Lucky HCD Confirm were 94.5%, and 87.5%, respectively, and that of Innotest(R) were 98.2% and 66.7%, respectively. Clear distinction of positive and negative results by signal/cut off ratio was available in both EIAs. The positive predictive values of ES-300 and Innotest(R) were 94.5%, and 87.1%, respectively. CONCLUSIONS: ES-300 showed relatively good results in sensitivity and positive predictive value with regard to confirmatory test. In EIA-positive persons, however, follow-up study would be necessary for reliable evaluation of HCV infection.
Humans ; Radioimmunoassay ; Sensitivity and Specificity ; Streptavidin

Objective To compare the sensitivity and accuracy of amplified luminescent proximity homogeneous assay linked immunosorbent assay (AlphaLISA) and magnetic particles-based chemiluminescence immunoassay (MP-CLIA) for detection of staphylococcal enterotoxin C (SEC) in the simulated milk samples. Methods The AlphaLISA was constructed using goat anti-SEC polyclonal antibody-coupled receptor microspheres, biotin-labeled SEC monoclonal antibody and streptavidin-coupled donor microspheres. The MP-CLIA was constructed using goat anti-SEC polyclonal antibody conjugated alkaline phosphatase, biotin-labeled anti-SEC monoclonal antibody and streptavidin conjugated magnetic beads. Results The sensitivity of AlphaLISA to detect SEC content in simulated milk samples was 4.04 ng/L, and the coefficient of variation (CV) was 1.98%~9.82%. The sensitivity of MP-CLIA was 108.19 ng/L and CV was 4.63%~20.40%. Conclusion Compared with MP-CLIA, AlphaLISA is more sensitive and accurate to detecting SEC.
Animals ; Streptavidin ; Biotin ; Luminescence ; Milk ; Antibodies, Monoclonal ; Goats ; Immunoassay/methods*

BACKGROUND: Immuno-PCR has been known as a highly sensitive and specific method, yet no standardized protocol is available. We analyzed each step of immuno-PCR to develop a reliable standardized method. METHODS: We made a protocol modified from several methods reported previously, and performed immuno-PCR, but false positive reactions were noted. To reduce the false positivity, we investigated the buffer reagents and biotin-labelled oligo-nucleotide probe. Using a finally determined protocol, we compared the detection-limits of the immuno-PCR and ELISA methods. RESULTS: Streptavidin was identified as a main reagent causing a non-specific binding, thus it was replaced by neutravidin. The employment of CAS block as a dilution buffer for the biotin-labelled oligo-nucleotide probe and Casein block as a buffer for the detection antibodies resulted in a dramatic reduction in the false positive reactions. The standardized immuno-PCR detected angiogenin antigen at a concentration as low as 5 fg/mL, while an ELISA method detected 5 pg/mL. CONCLUSIONS: The immuno-PCR procedure newly described in this study was ultra-sensitive with no false positivity. This method can be utilized as an epochal tool for detection of a small amount of antigen which would not be discovered by ELISA method.
Antibodies ; Caseins ; Employment ; Enzyme-Linked Immunosorbent Assay ; False Positive Reactions ; Indicators and Reagents ; Limit of Detection ; Streptavidin

BACKGROUND: Primary targit injured from the immunologic mechanism of alopecia areata is not definitely confirmed although alopecia areata is regarded as a disease occuring from certain im munologic process. Recently, par ticular interest has been focused on the follicular keratinocytes which show morphologic and anigenic alterations in active lesions. Another important point is the subclinical state hypothesi. It is based upon the observation that the patterns of T lymphocytes infiltration and ultrastructural changes of dermal papilla cells are nearly identical in active lesions and in non-lesional areas of alopecia areata. OBJECTIVE: The purpose of this study is to determine and to compare the aberrant expression of HLA-DR, CD1 and ICAM-1 on the follicular keratinocytes from the active alopecia areata lesion, stationary lesion and non-lesional scalp. METHODS: We performed a n immunohistochemical study, using the streptavidin system to compare the patterns of HLA-DR, CD1 and ICAM-1 immunoreactivity in the follicular keratinocytes in five active alopecia lesions two stationary lesions and non-lesional scalp specimens among four active and two stationary patients, and two normal control scalp specimens. RESULTS: In the active aloecia lesions, the HLA-DR and ICAM-1 immunoreactivity was observed in some patients while CD1 immunoreactivity was observed in all the patients. In the sta tionary alopecia lesions, none of the HLA-DR or CD1 or ICAM-1 immunoreactivity was observed as was the case in the norrial control scalp specimens. In the non-lesional normal scalp specimens, the HLA-DR, CD1 and ICAM-1 immunoreactivity showed nearly the same patterns as those in the acute alopecia leions. CONCLUSION: The aberrant antigenic alterations, especially CD1 expression of follicular keratinocytes are likely to be asociated with the active progress of alopecia areata lesions. These antigenic alterations were also observed in non-lesional scalp as well as alopecia areata lesions in the very similar patterns.
Alopecia Areata* ; Alopecia* ; HLA-DR Antigens ; Humans ; Intercellular Adhesion Molecule-1 ; Keratinocytes* ; Scalp* ; Streptavidin ; T-Lymphocytes

BACKGROUND: Generally, there are few problems in the diagnosis of Paget's disease(PD) using the H&E stain. However, the differentiation of PD from the clonal type of Bowens disease and superficial spreading melanoma in situ that shows pagetoid spreading of tumor cells, may present diagnostic difficulties. In addition, the specia1 stains used for demonstrating the presenee of Pagets cells, such as PAS and mucicarmim, are non-specific and not always sensitive. So, inenunohistochemical stains with monoclonal antibodies against various antigens may be helpful for differentiating PD from ather morphologically similar skin lesions. OBJECTIVE: The purpose of this study was to investigate the diagnostic accuracy of immunohisto- chemical staining for diagnostic use in PD. METHODS: Immunohistochemical stains used in the biotin streptavidin amplificxl technique with monoclonal antibodies to several low rnolecular weight cytokeratin(CK)s, EMA and CEA, were performed on formalin-fixed, paraffin-embedded tissue. Twelve cases of PD(10 cases of extranmmmary PD and 2 cases of mammary PD), five cases of superficial spreading melanoma in situ and five cases of Bowens disease were investigated. RESULTS: The results were as follows. 1. Positive reactions with variable intensity using CK7, CKS, CK19 were seen in all cases(100%) of PD and the. staining intensity tor CK7 or CK19 was stronger than that of CKS. 2. Of the 12 cases of PD, both CK18 and CAM5.2 staining showed positivity in 11 cases(92%). 3. EMA and CEA staining showed positivity in 10(83%) and 9(75%) of 12 cases, respectively. 4. Some Pagets cells were negative for CK8, CK18 and EMA, although other positive cells were observed in the same sections. 5. All antigens were consistently negative in all cases of Bowens disease and superficial spreading melanoma in situ. CONCLUSION: The results show that moaoclonal antibodies to low molecular weight CKs are more sensitive than EMA or CEA in the demonstration of Pagets cells. Moreover, among the low molecular weight CK series, CK7 and CK19 are most useful for their high sensitivity and intensity.
Antibodies ; Antibodies, Monoclonal ; Biotin ; Bowen's Disease ; Coloring Agents ; Diagnosis ; Melanoma ; Molecular Weight ; Skin ; Streptavidin

To evaluate prognostic significance of p53 gene mutation and p53 protein overexpression, 83 cases of primary gastric carcinoma (stage 1b, II, and III) were analyzed. Immunohistochemical assay using labeled streptavidine biotin method and mouse Anti-P53 was done on paraffin embedded tissues. The overall 5-year survival rate was 54.2%. Depth of invasion, lymph node metastasis, and stage were the prognostic factors (p<0.05). p53 was positive in 33 cases(39.8%). There were no significant association between p53 expression and other parameters such as age, sex, tumor location, size, gross finding, histologic type, depth of invasion, lymph node metastasis, and stage. The 5-year survival rates of p53 positive and negative groups were 62% and 42.4% respectively (p=0.0657). Survival distribution for p53 adjusted for location and depth of invasion showed significant difference. Five-year survival rates were 55%, 25%, 0% in p53 positive groups and 65.4%, 65%, 28% in negative groups at the lower, middle, and upper portion of the stomach, and 53.9%, 36.9%, 0% in p53 positive and 77.3%, 53%, 0% in negative groups in t2, t3 and t4. In comparison of 5-year survival rates of p53 positive and negative groups in the same stage, p53 positive groups showed worse prognosis than the negative, but they were not significant statistically. There were 57.1%, 92.9% in stage 1b (p=0.0678), 50%, 61.1% in stage II, 42.9%, 42.9% in stage IIIa, and 14.3%, 25% in stage IIIb respectively. So overexpression of p53 protein has little prognostic significance.
Animals ; Biotin ; Genes, p53 ; Lymph Nodes ; Mice ; Neoplasm Metastasis ; Paraffin ; Prognosis ; Stomach ; Streptavidin ; Survival Rate

PURPOSE: There has been a renewal of interest in Macrophage migration inhibitory factor (MIF), especially correlation in pathogenesis of sepsis by many infectious diseases and in regulation of host inflammatory and immune response. We developed immunoradiometric assay (IRMA) to determine serum human MIF concentration. MATERIALS AND METHODS: The IRMA system utilizes solid phase bound monoclonal anti-recombinant human MIF (rhMIF) antibody as a capture antibody, biotinylated polyclonal anti-rhMIF antibody as a detector antibody. We applied with rhMIF that concentration of standard solutions increased from 0 ng/ml to 100 ng/ml. We used 125I-streptavidin (SA) as radiotracer to determination of rhMIF concentration. Streptavidin was labeled with 125I by Chloramine-T method and 125I-SA was purified by ultracentrifugation. 125I-SA stability was evaluated by ITLC analysis at 4 degrees C and room temperatures until 60days. To validate IRMA system for MIF, we experimented intra-assay and inter-assay coefficients of variation, recovery test and dilution test. RESULTS: Radiolabeling yield of 125I-SA was 87% and purified 125I-SA retained above 99% radiochemical purity. 125I-SA showed above 93% stability in 4 degrees C until 60days that it is good for immunoradiometric assay as radiotracer. Plotted standard dose response curve showed that increased concentration of rhMIF linearly correlated (R2=0.99) with bound radioactivity of 125I-SA. The highest intra- and inter-assay coefficients of variation were 5.5% and 7.6%, respectively. The average of recovery of MIF in samples was 102%. In dilution test, linear response curves were obtained (R2=0.97). CONCLUSION: Radioimmunoassay using 125I-SA as radiotracer thought to be useful for the determination of serum MIF concentration, and further, its data will be used to evaluate the correlation between clinical significance and serum MIF concentration in patients with various inflammatory diseases.
Communicable Diseases ; Humans ; Immunoradiometric Assay ; Macrophages* ; Radioactivity ; Radioimmunoassay* ; Sepsis ; Streptavidin ; Ultracentrifugation

This study was designed to evaluate the expression of growth factor in periodontal tissue during the experimental movement of rat incisors by LSAB(Labelled streptavidine Biotin) immunohistochemical staining for EGF(Epidermal growth factor). 23 Sprague-Dawley rats were divided into a control group(3rats) and experimental groups(20rats), where a force(75g) from helical springs across the maxillary incisors was applied. Experimental groups were sacrificed at 12 hours, 1, 4, 7 and 14 days, after force application, respectively. And Tissue slides of control and experimental groups were studied immunohistochemically and histologically. The results were as follows: 1. In 14days after force application, periodontal fibers were strectched on the tension side, and compressed in pressure side of all experimental groups, and the arrangement of periodontal fibers was not recovered yet. 2. The degree of EGF expression in control group was strongly positive in the oral epithelium, predentin, capillaries in pulp and periodontal spaces. But osteoblasts and osteoclasts were stained mildly positive. 3. EGF expression was mild and diffuse in 12 hours, 1, 4 and 7 days of experimental groups and was not significantly different between the tension and pressure sides. 4. The degree of EGF expression in the 14-day experimental group was higher than any other group. And the tension side showed a more positive EGF expression than the pressure side. The apical area revealed a more positive EGF expression than the cervical area.
Animals ; Capillaries ; Epidermal Growth Factor ; Epithelium ; Incisor* ; Osteoblasts ; Osteoclasts ; Rats* ; Rats, Sprague-Dawley ; Streptavidin ; Tooth Movement

OBJECTIVE: Angiogenesis, the proliferation of capillary endothelial cells, is a vital component in the development, progression, and metastasis of many human tumors. Vascular endothelial growth factor(VEGF) is an endothelial cell-specific mitogen and induces angiogenesis and vascular permeability. The features of glioblastoma, distinct from low grade astrocytomas, are the presence of necroses and vascular endothelial proliferation. In this study, we investigated VEGF expression in the different grades of astrocytomas and determined whether VEGF expression correlates with development of glioblastoma and progression of astrocytomas. PATIENTS AND METHODS: Forty seven patients with astrocytic tumors(24 males and 23 females), aged 3 to 65 years, were evaluated. Immunohistochemical staining was carried out using labelled streptavidin biotin method and primary antibody was a antirabbit polyclonal Ab against N-terminus region of VEGF165(Oncogene research product, MA, USA). Immunoreactivity(IR) was classified into no IR(absent or a trace of stain), moderate IR and intense IR by level of staining amount and intensity. RESULTS: Six pilocytic astrocytomas showed 3 no IR and 3 moderate IR, 10 astrocytomas showed 2 no IR, 6 moderate IR and 2 intense IR, 12 anaplastic astrocytomas showed I no IR, 7 moderate IR and 4 intense IR and 19 glioblastomas showed 1 no IR, 11 moderate IR and 7 intense IR. Immunoreactivity was significantly different between low and high grade of tumors but there was no significant difference between anaplastic astrocytomas and glioblastomas. Gemistocytic tumor cells represented the predominent VEGF-immunoreactive cell types, as compared with compactly-arranged small tumor cells. In glioblastomas VEGF IR was observed in both perinecrotic and vital tumor areas. CONCLUSION: VEGF seems to be a important angiogenic factor in anaplastic astrocytomas and glioblastomas and VEGF expression may contribute to neovascularization of human astrocytomas.
Angiogenesis Inducing Agents ; Astrocytoma ; Biotin ; Capillary Permeability ; Endothelial Cells ; Glioblastoma ; Humans ; Male ; Necrosis ; Neoplasm Metastasis ; Streptavidin ; Vascular Endothelial Growth Factor A*

PURPOSE: To determine whether intracellular localization of intravenously injected infection specific MoAb can be localized in the infected cells, immunohistochemical staining was obtained in animals infected with Mycobacterium tuberculosis. MATERIALS AND METHODS: One mg of intact mouse MoAb against mwobacteria (group I, n=10) and F(ab')2 (group II, n=6) were intravenously injected to the rabbits of each groups infected with M. tuberculosis H37RV. Immunohistochemical staining using an anti-mouse antibody was obtained at the 1,3, 5, 7 and 8th days in Group I and at the 1, 2, 3rd days in group II by streptavidin biotin method. For the control study, I mg of non specific polyclonal human IgG (group III, n=10) and 100 microgram of normal rabbit IgG F(ab')2 (group IV, n=6) were injected to the rabbits and guinea pigs respectively. RESULTS: Both groups (group I & II) showed a positive Ag-Ab reaction within the cytoplasm of monocytes. A weak but positive reaction was observed intracellularly in group III, however no positive reaction in group IV. CONCLUSION: Our results suggest intracellular Ag-Ab reaction plays an important role in the localization of infection by immunoscintigraphy using a specific MoAb fragments.
Animals ; Biotin ; Cytoplasm ; Guinea Pigs ; Humans ; Immunoglobulin G ; Mice ; Monocytes ; Mycobacterium tuberculosis ; Rabbits ; Streptavidin ; Tuberculosis ; Tuberculosis, Pulmonary*