scholarly journals Establishment of international autoantibody reference standards for the detection of autoantibodies directed against PML bodies, GW bodies, and NuMA protein

2021 ◽  
Vol 59 (1) ◽  
pp. 197-207
Author(s):  
Bing Zheng ◽  
Rodrigo A. Mora ◽  
Marvin J. Fritzler ◽  
Minoru Satoh ◽  
Donald B. Bloch ◽  
...  
Keyword(s):  
Reference Materials ◽  
Antinuclear Antibodies ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Nuclear Staining ◽  
Mitotic Apparatus ◽  
Spindle Poles ◽  
Pml Bodies ◽  
Interphase Cells ◽  
Suitable Reference

AbstractObjectivesReference materials are important in the standardization of autoantibody testing and only a few are freely available for many known autoantibodies. Our goal was to develop three reference materials for antibodies to PML bodies/multiple nuclear dots (MND), antibodies to GW bodies (GWB), and antibodies to the nuclear mitotic apparatus (NuMA).MethodsReference materials for identifying autoantibodies to MND (MND-REF), GWB (GWB-REF), and NuMA (NuMA-REF) were obtained from three donors and validated independently by seven laboratories. The sera were characterized using indirect immunofluorescence assay (IFA) on HEp-2 cell substrates including two-color immunofluorescence using antigen-specific markers, western blot (WB), immunoprecipitation (IP), line immunoassay (LIA), addressable laser bead immunoassay (ALBIA), enzyme-linked immunosorbent assay (ELISA), and immunoprecipitation–mass spectrometry (IP-MS).ResultsMND-REF stained 6–20 discrete nuclear dots that colocalized with PML bodies. Antibodies to Sp100 and PML were detected by LIA and antibodies to Sp100 were also detected by ELISA. GWB-REF stained discrete cytoplasmic dots in interphase cells, which were confirmed to be GWB using two-color immunofluorescence. Anti-Ge-1 antibodies were identified in GWB-REF by ALBIA, IP, and IP-MS. All reference materials produced patterns at dilutions of 1:160 or greater. NuMA-REF produced fine speckled nuclear staining in interphase cells and staining of spindle fibers and spindle poles. The presence of antibodies to NuMA was verified by IP, WB, ALBIA, and IP-MS.ConclusionsMND-REF, GWB-REF, and NuMA-REF are suitable reference materials for the corresponding antinuclear antibodies staining patterns and will be accessible to qualified laboratories.

scholarly journals Interaction of NuMA protein with the kinesin Eg5: its possible role in bipolar spindle assembly and chromosome alignment

2013 ◽  
Vol 451 (2) ◽  
pp. 195-204 ◽  
Author(s):  
Yuko Iwakiri ◽  
Sachiko Kamakura ◽  
Junya Hayase ◽  
Hideki Sumimoto
Keyword(s):  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Mitotic Apparatus ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Interphase Cells ◽  
Correct Alignment ◽  
Spindle Microtubules

Bipolar spindle assembly in mitotic cells is a prerequisite to ensure correct alignment of chromosomes for their segregation to each daughter cell; spindle microtubules are tethered at plus ends to chromosomes and focused at minus ends to either of the two spindle poles. NuMA (nuclear mitotic apparatus protein) is present solely in the nucleus in interphase cells, but relocalizes during mitosis to the spindle poles to play a crucial role in spindle assembly via focusing spindle microtubules to each pole. In the present study we show that the kinesin-5 family motor Eg5 is a protein that directly interacts with NuMA, using a proteomics approach and various binding assays both in vivo and in vitro. During mitosis Eg5 appears to interact with NuMA in the vicinity of the spindle poles, whereas the interaction does not occur in interphase cells, where Eg5 is distributed throughout the cytoplasm but NuMA exclusively localizes to the nucleus. Slight, but significant, depletion of Eg5 in HeLa cells by RNA interference results in formation of less-focused spindle poles with misaligned chromosomes in metaphase; these phenotypes are similar to those induced by depletion of NuMA. Since NuMA is less accumulated at the spindle poles in Eg5-depleted cells, Eg5 probably contributes to spindle assembly via regulating NuMA localization. Furthermore, depletion of cytoplasmic dynein induces mislocalization of NuMA and phenotypes similar to those observed in NuMA-depleted cells, without affecting Eg5 localization to the spindles. Thus dynein appears to control NuMA function in conjunction with Eg5.

scholarly journals Etiology, clinical characteristics and coinfection status of bronchiolitis in Suzhou

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiahong Tan ◽  
Jinfeng Wu ◽  
Wujun Jiang ◽  
Li Huang ◽  
Wei Ji ◽  
...  
Keyword(s):  
Virus Infection ◽  
Clinical Characteristics ◽  
Immunofluorescence Assay ◽  
Clinical Syndrome ◽  
Enzyme Linked Immunosorbent Assay ◽  
Direct Immunofluorescence ◽  
Increased Risk ◽  
Syncytial Virus ◽  
Direct Immunofluorescence Assay ◽  
Mixed Virus Infection

Abstract Background Bronchiolitis is a clinical syndrome commonly encountered in practice, particularly among infants and young children. To investigate the prevalence of pathogens in hospitalized children with bronchiolitis and study the clinical characteristics of bronchiolitis with or without coinfections. Methods We investigated the respiratory specimens and clinical data of 1012 children with bronchiolitis who were treated at the Children’s Hospital of Soochow University between November 2011 and December 2018. The nasopharyngeal aspirates were examined to detect viruses by direct immunofluorescence assay or polymerase chain reaction (PCR). Mycoplasma pneumoniae (MP) was tested by PCR and enzyme-linked immunosorbent assay. Results Of the 1134 children less than 2 years with bronchiolitis, 122 were excluded by exclusion criteria. Causative pathogen was detected in 83.2% (842 of 1012). The majority of these (614 [72.9%] of 842) were single virus infection. The most common pathogens detected were respiratory syncytial virus (RSV) (44.4%), MP (15.6%), and human rhinovirus (HRV) (14.4%). Coinfection was identified in 13.5% (137 of 1012) of the patients. Coinfection included mixed virus infection and virus infection with MP infection. Children with single virus infection had a higher rate of oxygen therapy compared with single MP infection. Conclusions The most common pathogen detected in children with bronchiolitis is RSV, followed by MP and HRV. Coinfection leads to a longer period of illness, increased severity of the symptoms and increased risk of hypoxemia.

scholarly journals The antinuclear antibody HEp-2 indirect immunofluorescence assay: a survey of laboratory performance, pattern recognition and interpretation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anne E. Tebo ◽  
Robert L. Schmidt ◽  
Kamran Kadkhoda ◽  
Lisa K. Peterson ◽  
Edward K. L. Chan ◽  
...  
Keyword(s):  
Indirect Immunofluorescence ◽  
Clinical Laboratory ◽  
Immunofluorescence Assay ◽  
Indirect Immunofluorescence Assay ◽  
Medical Laboratory ◽  
Nuclear Staining ◽  
International Consensus ◽  
In Vitro Diagnostic ◽  
Traditional Pattern

Abstract Background To evaluate the interpretation and reporting of antinuclear antibodies (ANA) by indirect immunofluorescence assay (IFA) using HEp-2 substrates based on common practice and guidance by the International Consensus on ANA patterns (ICAP). Method Participants included two groups [16 clinical laboratories (CL) and 8 in vitro diagnostic manufacturers (IVD)] recruited via an email sent to the Association of Medical Laboratory Immunologists (AMLI) membership. Twelve (n = 12) pre-qualified specimens were distributed to participants for testing, interpretation and reporting HEp-2 IFA. Results obtained were analyzed for accuracy with the intended and consensus response for three main categorical patterns (nuclear, cytoplasmic and mitotic), common patterns and ICAP report nomenclatures. The distributions of antibody titers of specimens were also compared. Results Laboratories differed in the categorical patterns reported; 8 reporting all patterns, 3 reporting only nuclear patterns and 5 reporting nuclear patterns with various combinations of other patterns. For all participants, accuracy with the intended response for the categorical nuclear pattern was excellent at 99% [95% confidence interval (CI): 97–100%] compared to 78% [95% CI 67–88%] for the cytoplasmic, and 93% [95% CI 86%–100%] for mitotic patterns. The accuracy was 13% greater for the common nomenclature [87%, 95% CI 82–90%] compared to the ICAP nomenclature [74%, 95% CI 68–79%] for all participants. Participants reporting all three main categories demonstrated better performances compared to those reporting 2 or less categorical patterns. The average accuracies varied between participant groups, however, with the lowest and most variable performances for cytoplasmic pattern specimens. The reported titers for all specimens varied, with the least variability for nuclear patterns and most titer variability associated with cytoplasmic patterns. Conclusions Our study demonstrated significant accuracy for all participants in identifying the categorical nuclear staining as well as traditional pattern assignments for nuclear patterns. However, there was less consistency in reporting cytoplasmic and mitotic patterns, with implications for assigning competencies and training for clinical laboratory personnel.

scholarly journals Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists

2020 ◽  
Vol 58 (9) ◽  
pp. 1489-1497 ◽  
Author(s):  
Lisa K. Peterson ◽  
Anne E. Tebo ◽  
Mark H. Wener ◽  
Susan S. Copple ◽  
Marvin J. Fritzler
Keyword(s):  
Indirect Immunofluorescence ◽  
Antinuclear Antibodies ◽  
Current Practice ◽  
Clinical Laboratory ◽  
Immunofluorescence Assay ◽  
Indirect Immunofluorescence Assay ◽  
Medical Laboratory ◽  
International Consensus ◽  
Clinical Laboratories ◽  
Reading Interpretation

AbstractBackgroundThe indirect immunofluorescence assay (IFA) using HEp-2 cell substrates is the preferred method by some for detecting antinuclear antibodies (ANA) as it demonstrates a number of characteristic staining patterns that reflect the cellular components bound as well as semi-quantitative results. Lack of harmonized nomenclature for HEp-2 IFA patterns, subjectivity in interpretation and variability in the number of patterns reported by different laboratories pose significant harmonization challenges. The main objectives of this study were to assess current practice in laboratory assessment of HEp-2 IFA, identify gaps and define strategies to improve reading, interpretation and reporting.MethodsWe developed and administered a 24-item survey based on four domains: educational and professional background of participants, current practice of HEp-2 IFA testing and training, gap assessment and the perceived value of International Consensus on Antinuclear Antibody Patterns (ICAP) and other factors in HEp-2 IFA assessment. The Association of Medical Laboratory Immunologists (AMLI) and American Society for Clinical Pathology administered the survey from April 1 to June 30, 2018, to members involved in ANA testing. This report summarizes the survey results and discussion from a dry workshop held during the 2019 AMLI annual meeting.ResultsOne hundred and seventy-nine (n = 179) responses were obtained where a significant number were clinical laboratory scientists (46%), laboratory directors (24%), supervisors (13%) or others (17%). A majority of respondents agreed on the need to standardize nomenclature and reporting of HEp-2 IFA results. About 55% were aware of the ICAP initiative; however, among those aware, a significant majority thought its guidance on HEp-2 IFA nomenclature and reporting is of value to clinical laboratories. To improve ICAP awareness and further enhance HEp-2 IFA assessment, increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials.ConclusionsBased on these suggestions, future efforts to optimize HEp-2 IFA reading, interpretation and reporting would benefit from more hands-on training of laboratory personnel as well as continuous collaboration between professional organizations, in vitro diagnostic manufacturers and clinical laboratories.

Asymmetric cell division in fucoid algae: a role for cortical adhesions in alignment of the mitotic apparatus

2001 ◽  
Vol 114 (23) ◽  
pp. 4319-4328
Author(s):  
Sherryl R. Bisgrove ◽  
Darryl L. Kropf
Keyword(s):  
Cell Division ◽  
Asymmetric Cell Division ◽  
Cell Cortex ◽  
Mitotic Apparatus ◽  
Pharmacological Agents ◽  
Division Plane ◽  
Adhesion Sites ◽  
Spindle Poles ◽  
Pelvetia Compressa ◽  
Two Phases

The first cell division in zygotes of the fucoid brown alga Pelvetia compressa is asymmetric and we are interested in the mechanism controlling the alignment of this division. Since the division plane bisects the mitotic apparatus, we investigated the timing and mechanism of spindle alignments. Centrosomes, which give rise to spindle poles, aligned with the growth axis in two phases – a premetaphase rotation of the nucleus and centrosomes followed by a postmetaphase alignment that coincided with the separation of the mitotic spindle poles during anaphase and telophase. The roles of the cytoskeleton and cell cortex in the two phases of alignment were analyzed by treatment with pharmacological agents. Treatments that disrupted cytoskeleton or perturbed cortical adhesions inhibited pre-metaphase alignment and we propose that this rotational alignment is effected by microtubules anchored at cortical adhesion sites. Postmetaphase alignment was not affected by any of the treatments tested, and may be dependent on asymmetric cell morphology.

scholarly journals An intrinsic B cell defect is required for the production of autoantibodies in the lpr model of murine systemic autoimmunity.

1991 ◽  
Vol 173 (6) ◽  
pp. 1441-1449 ◽  
Author(s):  
E S Sobel ◽  
T Katagiri ◽  
K Katagiri ◽  
S C Morris ◽  
P L Cohen ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cells ◽  
Lupus Erythematosus ◽  
Immunofluorescence Assay ◽  
Total Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Phenotype ◽  
Autoantibody Production ◽  
Systemic Autoimmunity

Mice homozygous for the gene lpr develop marked lymphadenopathy and a spectrum of autoantibodies closely resembling that of human systemic lupus erythematosus. The unusual T cell phenotype of the expanded lymphocyte population and the T-dependence of several antibodies in this strain have suggested that primary T cell abnormalities underlie the autoimmune syndrome. Using double chimeras, we now show that expression of the lpr gene in B cells is absolutely necessary for autoantibody production. Combinations of anti-Thy 1.2 + C' treated bone marrow from congenic strains of C57BL/6 mice, differing only at the immunoglobulin heavy chain (Igh) and lpr loci, were transferred into lethally irradiated B6/lpr mice. Double chimerism was documented by allotype-specific surface IgD and IgM immunofluorescence assay of peripheral blood and by allotype-specific enzyme-linked immunosorbent assay for total IgM in serum. Despite the presence of both +/+ and lpr B cells, IgM and IgG2a anti-chromatin as well as IgM anti-IgG were entirely the products of lpr B cells. Total serum IgG2a and IgG1 were also dominated by the lpr phenotype but not to the same extent. A similar experiment using B6/lpr-Igha recipients confirmed these findings. Additional experiments in which B6/lpr recipients were infused with ratios of donor bone marrow favoring B6.C20 +/+ over B6/lpr showed that even though +/+ B cells were overrepresented, autoantibodies were only of the lpr allotype. In addition, in the presence of lpr B cells, normal B cells showed little response to an exogenous, T cell-dependent antigen. The data thus indicate that lpr B cells manifest an intrinsic abnormality which is essential for autoantibody production in the lpr model.

scholarly journals Humoral Autoimmune Responses to Insulin-Like Growth Factor II mRNA-Binding Proteins IMP1 and p62/IMP2 in Ovarian Cancer

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Xinxin Liu ◽  
Hua Ye ◽  
Liuxia Li ◽  
Wenjie Li ◽  
Yi Zhang ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Growth Factor ◽  
Binding Proteins ◽  
Immunofluorescence Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Insulin Like Growth Factor ◽  
Mrna Binding Proteins ◽  
Normal Individuals ◽  
Factor Ii ◽  
Mrna Binding

Ovarian cancer is one of the leading causes of cancer-related deaths among women. There is an urgent need of better approaches for the identification of appropriate biomarkers in the early detection of ovarian cancer. The aim of this study was to elucidate the significance of autoantibodies against insulin-like growth factor II mRNA-binding proteins (IMPs) in patients with ovarian cancer. In this study, autoantibody responses to two members (IMP1 and p62/IMP2) of IMPs were evaluated by enzyme-linked immunosorbent assay (ELISA), western blotting, and indirect immunofluorescence assay in sera from patients with ovarian cancer and normal human individuals. The results have demonstrated that both IMP1 and p62/IMP2 can induce relatively higher frequency of autoantibody responses in patients with ovarian cancer (26.5% and 29.4%) compared to normal individuals(P<0.01). Our preliminary data suggest that IMP1 and p62/IMP2 can stimulate autoimmune responses in ovarian cancer, and anti-IMP1 and anti-p62/IMP2 autoantibodies could be used as potential biomarkers in immunodiagnosis of ovarian cancer.

scholarly journals Reference standards for the detection of anti-mitochondrial and anti-rods/rings autoantibodies

2018 ◽  
Vol 56 (10) ◽  
pp. 1789-1798 ◽  
Author(s):  
S. John Calise ◽  
Bing Zheng ◽  
Tomoko Hasegawa ◽  
Minoru Satoh ◽  
Natasa Isailovic ◽  
...  
Keyword(s):  
Western Blotting ◽  
Reference Materials ◽  
Staining Pattern ◽  
Pyruvate Dehydrogenase Complex ◽  
Reference Standards ◽  
Enzyme Linked Immunosorbent Assay ◽  
Interferon Α ◽  
Primary Biliary Cholangitis ◽  
Inosine Monophosphate Dehydrogenase ◽  
Clinical Laboratories

Abstract Background: Anti-mitochondrial antibodies (AMA) are found in >90% of primary biliary cholangitis patients. Anti-rods/rings antibodies (anti-RR) are most commonly associated with interferon-α and ribavirin treatment in hepatitis C patients. Clinical laboratories routinely screen for AMA and anti-RR using indirect immunofluorescence on HEp-2 cells (HEp-2-IFA). Therefore, we sought to establish reference materials for use in AMA and anti-RR testing. Methods: AMA-positive and anti-RR-positive human plasma samples (AMA-REF and RR-REF), identified as potential reference materials based on preliminary data, were further validated by multiple laboratories using HEp-2-IFA, immunoprecipitation (IP), western blotting, IP-western, line immunoassay (LIA), addressable laser bead immunoassay (ALBIA) and enzyme-linked immunosorbent assay (ELISA). Results: AMA-REF showed a strong positive cytoplasmic reticular/AMA staining pattern by HEp-2-IFA to ≥1:1280 dilution and positive signal on rodent kidney/stomach/liver tissue. AMA-REF reacted with E2/E3, E3BP, E1α and E1β subunits of the pyruvate dehydrogenase complex by IP and western blotting and was positive for AMA antigens by LIA, ALBIA and ELISA. RR-REF showed a strong positive rods and rings staining pattern by HEp-2-IFA to ≥1:1280 dilution. RR-REF reacted with inosine monophosphate dehydrogenase by IP, IP-western and ALBIA. RR-REF also produced a nuclear homogenous staining pattern by HEp-2-IFA, immunoprecipitated proteins associated with anti-U1RNP antibody and reacted weakly with histones, nucleosomes, Sm and nRNP/Sm by LIA. Conclusions: AMA-REF and RR-REF are useful reference materials for academic or commercial clinical laboratories to calibrate and establish internal reference standards for immunodiagnostic assays. AMA-REF and RR-REF are now available for free distribution to qualified laboratories through Plasma Services Group.

Sign in / Sign up

Export Citation Format

Share Document