1.Study on the application of number fluorescence density in detecting autoantibodies titer.
Xiaodong PENG ; Ruiwei ZHANG ; Lanlan WANG ; Pingwu ZHANG
Journal of Biomedical Engineering 2002;19(2):221-224
This study was firstly conducted to detect antinuclear antibody(ANA) titer by using number influorescence density analysis assay instead of serum diluted assay. The best camera explore time was selected. Then 4,140 ANA positive sera were detected to determine the relationship between number influorescence density (detected by number camera system Spot 32 and computer analysis software ipwin32) and serum diluted titer. The consistent rates in different ANA patterns used by the two methods were compared. 4 seconds was found to be the best explore time and the relationship between number influorescence density and serum diluted titer was 29-50 vs 1:100, 51-85 vs 1:320, 86-175 vs 1:1000, 176-215 vs 1:3200, 216-237 vs 1:10,000. According to this standard we detected 3140 ANA positive sera by use of the two methods and observed a total consistency rate of 89.4%. The consistency rates of three ANA patterns including speckled, homogenous, mixture of speckled and homogenous were as high as 98.9%, 99.5%, 99.8% respectively. The lower consistency rate patterns included nucleolar (5.3%), centromere (1.8%), ribosome(12.6%) and other special patterns(0%). For practical purpose, number influorescence density analysis assay can be used in detecting the three main ANA patterns (speckled, homogeneous, mixture of speckled and homogenous) titer instead of serum diluted assay. The number influorescence density analysis assay is more objective, economical and simple than the serum diluted assay.
Antibodies, Antinuclear
;
analysis
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Fluorescence
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Fluoroimmunoassay
;
methods
;
Software
2.Comparison of indirect immunofluorescence assay and ELISA for detecting antinuclear antibodies and anti-double-stranded DNA antibodies.
Xue QIN ; Xia TAO ; Zhi-Jian CHEN ; Jie-Qiu JIANG ; Ming-Hui XU ; Ruo-Lin LI ; Tai-Jie LI ; Fa-Quan LIN ; Shan LI
Journal of Southern Medical University 2009;29(3):472-475
OBJECTIVETo compare indirect immunofluorescence assay (IIFA) and enzyme-linked immunosorbent assay (ELISA) for detecting antinuclear antibodies (ANA) and anti-double-stranded DNA antibodies (anti-dsDNA).
METHODSA total of 125 serum samples were obtained from patients with established or suspected autoimmune disease, and 82 samples were used for ANA detection and 57 for anti-dsDNA detection using both IIFA and ELISA. Fourteen samples were examined for both ANA and anti-dsDNA. In cases where discrepancy occurred in the results by the two methods, extractable nuclear antigens were detected using immunoblotting.
RESULTSThe positivity rate of ANA detected by IIFA and ELISA was significantly different (87.8% and 73.17%, respectively, P<0.01), but the positivity rate of anti-dsDNA was similar between IIFA and ELISA (77.19% and 71.93%, respectively, P>0.05). The percent agreement between the two testing methods with different cutoff values of ANA and anti-dsDNA showed significant differences (P<0.01), and for some uncommon patterns, the percent agreement of the two methods was lowered in ANA detection but remained unchanged for anti-dsDNA with different ANA patterns. High percent agreements of the two methods were obtained with the cutoff ANA titer of 1:100 and the cutoff anti-dsDNA value of weak positivity, but they demonstrated a significant difference in testing low-titer ANA and anti-dsDNA.
CONCLUSIONIIFA is more sensitive than ELISA in detecting the total ANA and anti-dsDNA. ELISA prescreening combined with IIFA can obtain the information of the nuclear pattern and allow the observation of the titer alterations. The combination of two or more testing methods can greatly enhance the accuracy of the results.
Antibodies, Antinuclear ; analysis ; DNA ; immunology ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Humans
3.Follow-up and outcome as well as the related biological factors on the cases with indeterminate HIV antibody level.
Yan LI ; Cai-yun LIANG ; Kai GAO ; Zhi-gang HAN ; Bi-lian LUO ; Hui-fang XU
Chinese Journal of Preventive Medicine 2011;45(10):916-919
OBJECTIVETo explore the follow-up visit, outcome and auxiliary diagnosis method on the cases with indeterminate antibody level measured by Western blotting as well as the related biological factors.
METHODSThe cases with indeterminate result were followed up according to the National Guideline for Detection of HIV/AIDS (2009) and samples were collected for HIV antibody detection, p24 antigen and nucleic acid were detected as a supplementary diagnosis at the same time. The samples were also be detected for HBV, HCV, TP, HTLV-I/II, ANA, and AFP, and the results were compared to that of screened positive and confirmed negative cases.
RESULTSA total of 73 were followed up successfully and taken a second HIV test, 25 cases were tested positive and 48 were tested negative for HIV during the follow-up period. For the 25 HIV positive cases, the HIV seroconversion rate was 100.00% at any time point when the interval between the first and returning detection was longer than 1 week. The major Western blotting bands for the cases with indeterminate result were p24 and gp160 and it was different between HIV positive and negative cases in Western blotting band profiles. The consistency and sensitivity of nucleic acid detection were higher than 90.00%, and were higher than that of p24 antigen (69.09% (38/55) and 27.27% (6/22)) (χ(2)(consistency) = 6.875, χ(2)(sensitivity) = 18.893, P < 0.05). The positive rates of ANA and AFP of indeterminate cases excluded from HIV infection were 20.83% (10/28) and 6.25% (3/48) and higher than that of screened positive and confirmed negative cases (0.00%), the difference had statistic significance (χ(2)(ANA) = 19.430, χ(2)(AFP) = 5.520, P < 0.05).
CONCLUSIONIt is critical to get timely diagnosis for the indeterminate cases according to the new national guideline for detection of HIV/AIDS. Nucleic acid detection has higher application value as auxiliary diagnosis for HIV infection than p24 antigen. The increased levels of ANA and AFP may be the factors resulting in the nonspecific indeterminate results.
Antibodies, Antinuclear ; blood ; Female ; Follow-Up Studies ; HIV Antibodies ; blood ; HIV Infections ; diagnosis ; immunology ; Humans ; Male ; alpha-Fetoproteins ; analysis
4.Characterization of DNA antigens from immune complexes deposited in the skin of patients with systemic lupus erythematosus.
Fan-qin ZENG ; Ruo-fei YIN ; Guo-zhen TAN ; Qing GUO ; De-qing XU
Chinese Medical Journal 2004;117(7):1066-1071
BACKGROUNDSkin lesions are common manifestations in systemic lupus erythematosus (SLE). It is still unknown what the definite pathogenesis of skin involvement was and whether DNA participated in it. Our study was designed to explore the pathogenetic role and nature of nuclear antigen (DNA) deposited in the skin lesions of patients with SLE.
METHODSThirty skin samples from patients with SLE and 2 normal skin samples were studied. Extracellular DNA was evaluated by indirect immunofluorescence methods. The deposited immune complexes were extracted by cryoprecipitation, and DNA was then isolated with phenol and chloroform. DNA fragment sizes were detected by agarose gel electrophoresis. Finally, 8 different probes were used to analyze the origin of these DNA molecules using Dot hybridization.
RESULTSExtracellular DNA staining was found only in skin lesions, mainly those located in the basement membrane zone, vascular wall, and hair follicle wall. Normal skin and non-lesion SLE skin showed no fluorescence at locations outside the nuclei. There were no differences in the rate and intensity of extracellular DNA staining when comparing active phase to remission phase patients. No relationship was found between extracellular DNA and circulating anti-dsDNA antibodies. Deposited DNA fragments clustered into four bands of somewhat discrete sizes: 20 000 bp, 1300 bp, 800-900 bp, 100-200 bp. Small sized fragments (100-200 bp) were positively correlated with disease activity (P < 0.05, r = 0.407). Dot hybridization showed significant homology of the various extracellular DNA fragments examined with human genomic DNA, but not with DNA from the microorganisms and viruses we examined. There were also homologies between DNA samples from different individuals.
CONCLUSIONSDNA and its immune complexes may contribute to the pathogenesis of skin lesions in SLE. These DNA molecules range in size from 100 bp to 20 kb and may be endogenous in origin.
Antibodies, Antinuclear ; blood ; Antigen-Antibody Complex ; analysis ; DNA ; analysis ; immunology ; Humans ; Lupus Erythematosus, Systemic ; immunology ; Skin ; immunology ; Staining and Labeling
5.A Prospective Study on the Prevalence and Clinical Significance of Autoantibodies in Patients with Suspected Nonalcholic Fatty Liver Disease.
Dae Hyeon CHO ; Moon Seok CHOI ; Dong Hee KIM ; Do Young KIM ; Sang Goon SHIM ; Joon Hyeok LEE ; Kwang Cheol KOH ; Seung Woon PAIK ; Byung Chul YOO ; Jong Chul RHEE
The Korean Journal of Hepatology 2005;11(3):261-267
BACKGROUND/AIMS: Exclusion of liver disease from other causes such as autoimmune hepatitis is necessary for diagnosis of nonalcoholic fatty liver disease (NAFLD). However, there has been no study on the prevalence and significance of autoantibodies in the patients with clinically suspected NAFLD in Korea, where hepatitis B is endemic and autoimmune hepatitis is relatively uncommon. METHODS: We prospectively tested for anti-nuclear antibody (ANA), anti-smooth muscle antibody (ASMA), and anti-mitochondrial antibody (AMA) in 135 serially enrolled patients with suspected NAFLD. We compared the clinical characteristics and biochemical indices of the ANA-positive or ASMA-positive group with those of the autoantibody-negative group. RESULTS: Sixteen patients (11.8%) had serum autoantibodies; there was ANA in 8 patients (5.9%), ASMA in 7 (5.1%), and AMA in 2 (1.5%). Both ANA and AMA were positive in one patient. The ANA-positive or ASMA-positive group showed an older age (49.5+/-13.0 vs. 42.0+/-10.9 years, respectively, P=0.018) and higher levels of serum globulin (3.1+/-0.4 vs. 2.9+/-0.4 g/dL, respectively, P=0.037), compared with the autoantibody-negative group. Two cases with positive ANA or ASMA fulfilled the diagnostic criteria for probable autoimmune hepatitis and two cases with positive AMA were suspected as primary biliary cirrhosis. CONCLUSIONS: These findings suggest that autoantibodies could be found in some patients with suspected NAFLD in Korea, AMA-positivity or ASMA-positivity could be associated with old age and high serum globulin, and some of the autoantibody-positive cases could be diagnosed as autoimmune hepatitis or primary biliary cirrhosis. Further studies are necessary to clarify the clinical significance of autoantibody positivity in those patients.
Adult
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Aged
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Antibodies, Antinuclear/analysis
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Autoantibodies/*blood
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English Abstract
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Fatty Liver/*immunology
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Female
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Humans
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Male
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Middle Aged
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Muscle, Smooth/immunology
6.Clinical and pathological features of primary biliary cirrhotic patients with negative anti-mitochondria antibody.
Qi-xia WANG ; Lei SHEN ; Xiao-yu CHEN ; De-kai QIU ; Xiong MA
Chinese Journal of Hepatology 2011;19(5):340-344
OBJECTIVETo explore the clinical and pathological features of primary biliary cirrhosis (PBC) patients with negative anti-mitochondria antibody (AMA).
METHODSTwo hundreds and eight PBC patients were enrolled. The clinical and histological data of the negative AMA cases were compared with the AMA/AMA-M2 positive cases.
RESULTS30 out of the 208 cases (14.4%) were AMA negative patients in our study. The general status, biochemical tests and histological findings between the two groups had no significant difference (P > 0.05). The Gamma-globulin, IgG, IgM and IgA levels of AMA/AMA-M2 positive PBC patients were higher than that of the AMA negative cases (P < 0.05). The abnormal rate of cholesterol in AMA negative PBC patients was 65.4% as compared to 50.4% in AMA/AMA-M2 positive cases, no significant difference existed between (P > 0.05). Anti-nuclear antibody (ANA) was observed in 29 (96.7%) AMA negative PBC patients, including 14 (48.3%) with granular pattern, 8 (27.6%) with nuclear membrane pattern, 6 (20.7%) with kinetochore pattern and 1 (3.4%) with homogeneous pattern. AMA negative PBC patients had elevated serum ALP, GGT, IgM and cholesterol levels, and decreased serum AST, IgG and IgA levels as compared with that of autoimmune hepatitis patients (P < 0.05, respectively).
CONCLUSIONIn cholestatic patients with elevated IgM and cholesterol levels, ANA positive with non-homogeneous pattern, the diagnosis of PBC should be suspected, albeit AMA negative. The clinical, biochemical and histological features of the AMA negative PBC patients were similar to classic PBC patients, but quite different from autoimmune hepatitis.
Adult ; Antibodies, Antinuclear ; analysis ; Female ; Humans ; Liver Cirrhosis, Biliary ; immunology ; pathology ; Male ; Middle Aged ; Mitochondria ; immunology ; gamma-Globulins ; metabolism
7.A Case of Anti-LKM 1 Positive Autoimmune Hepatitis Accompanied by Systemic Lupus Erythematosus.
Dae Han CHOI ; Hae Kyung KIM ; Tae Il PARK ; Byung Min JOHN ; Sung Hwan KANG ; Yoon Serk LEE ; Tae Hyun KIM ; Uh Joo LEE ; Tae Seung LEE ; Gwi Ok YOON
The Korean Journal of Gastroenterology 2008;51(3):190-193
Overlap of autoimmune hepatitis and systemic lupus erythematosus (SLE) is a comparatively rare condition. Although both autoimmune hepatitis and SLE can share common autoimmune features such as polyarthralgia, hypergammaglobulinemia and positive ANA, it has been considered as two different entities. We report a case of anti-LKM1 positive autoimmune hepatitis who developed SLE two years later. The presence of interface hepatitis with lymphoplasma cell infiltrates and rosette formation points to the autoimmune hepatitis rather than SLE hepatitis. Autoimmune hepatitis is infrequently accompanied by SLE, therefore, it could be recommended to investigate for SLE in patients with autoimmune hepatitis.
Antibodies, Antinuclear/analysis
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Autoantibodies/*analysis
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Echocardiography
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Female
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Hepatitis, Autoimmune/complications/*diagnosis/immunology
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Humans
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Liver/pathology
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Lupus Erythematosus, Systemic/complications/*diagnosis/immunology
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Young Adult
8.Significance of autoantibodies in rheumatic diseases.
Chinese Journal of Pediatrics 2004;42(4):315-317
Antibodies, Antineutrophil Cytoplasmic
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analysis
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immunology
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Antibodies, Antinuclear
;
analysis
;
immunology
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Antigens, Nuclear
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immunology
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Arthritis, Juvenile
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immunology
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Autoantibodies
;
analysis
;
immunology
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Humans
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Monitoring, Physiologic
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methods
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Nucleosomes
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immunology
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Phospholipids
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immunology
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Rheumatic Diseases
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immunology
;
physiopathology
10.A radioimmunoassay method for detection of DNA based on chemical immobilization of anti-DNA antibody.
Seoung Kyo YOO ; Myung Ok YOON ; Ul Jae PARK ; Hyon Soo HAN ; Jeong Hee KIM ; Hyun Jin HWANG
Experimental & Molecular Medicine 1999;31(3):122-125
High selectivity provided by biomolecules such as antibodies and enzymes has been exploited during the last two decades for development of biosensors. Of particular importance are efficient immobilization methods for biomolecules in order to preserve their biological activities. In this study, we have evaluated immobilization strategies for an anti-DNA antibody on a self-assembled monolayer of omega-functionalized thiols. The antibody was immobilized via peptide bond formation between the primary amines in the antibody and the carboxyl groups on the self-assembled monolayer. The peptide bond coupling was achieved by activating COOH groups on the surface through N-Hydroxysuccimide (NHS)-ester formation, followed by acylation of NH2 group in the antibody. DNA binding activity of the immobilized antibody was examined by counting beta emission from 35S-labeled DNA.
Antibodies, Antinuclear*
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DNA/immunology
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DNA/analysis*
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DNA-Binding Proteins/chemistry
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Gold
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Membranes, Artificial
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Polymerase Chain Reaction
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Polyvinyls/chemistry
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Radioimmunoassay/methods*
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Thioctic Acid/chemistry