Antinuclear antibody testing in a regional immunopathology laboratory

A systematic review has been undertaken of antinuclear antibody testing over a 6-year period in a regional immunotherapy laboratory servicing a population of 400 000. Twenty-eight per cent of the 20 205 antinuclear antibody tests performed on a hyperexpressing Ro transfected cellular substrate were positive (titre >/= 1 : 80) with the most common immunofluorescent patterns being homogeneous (39%), speckled (20%), mixed (17%), nucleolar (8%), Ro (7%) and centromere (4%). Ro antibody as detected by immunofluorescence was strongly concordant with anti-Ro detected by counter immunoelectrophoresis precipitation; of 261 anti-Ro counter immunoelectrophoresis precipitation positive patients surveyed, only 15 were missed and 20 masked (with homogenous pattern) by immunofluorescence. Ro antibodies were found in patients with a variety of immune disorders, particularly connective tissue disorders, whilst a clinical survey of the anticentromere sera revealed that 67% were derived from patients with limited scleroderma. Extractable nuclear antibodies and their characterization was performed on 10 939 occasions with 12.9% being positive with anti-Ro constituting 30.2%, anti-Ro/La 25.7%, unidentified precipitin line 17.8%, anti-ribo nuclear protein 12.5%, respectively, with anti-Scl70, anti-Jo-1 and anti-Sm and various combinations making up the remainder. Unidentified precipitin lines were particular prominent in patients with connective tissue disorders. DNA quantification was performed on 12 068 occasions with 11% giving elevated values, the majority from patients with systemic lupus erythematosus. Of these positive sera 44% also demonstrated one or more extractable nuclear antibodies and 25% anticardiolipin antibodies. Regular participation in a Quality Assurance Program revealed accurate and consistent performance of antinuclear antibody testing. In conclusion antinuclear antibody detection and characterization for systemic immune disorders can provide the clinician with useful diagnostic and prognostic information; it is important that the laboratory results are relevant, timely, accurate and precise. Systematic reviews as demonstrated in this report, can provide such evidence.     

Circulating Antinuclear Antibody and Rheumatoid Factor in Coal Pneumoconiosis

Circulating antinuclear antibody and rheumatoid factor have been measured in 109 coal miners with pneumoconiosis whose chest radiograph showed a range of abnormalities varying from simple pneumoconiosis of mild degree to advanced progressive massive fibrosis.At a screening dilution of 1/10 the overall incidence of antinuclear antibody was 17%. In almost half of the positive cases the titre was 1/40 or greater.The prevalence of antinuclear antibody was lowest in those with simple pneumoconiosis (9%) and highest in those with category C progressive massive fibrosis (27%). A similar but less striking trend was seen with rheumatoid factor, ranging from 6% in simple pneumoconiosis to 18% in category C progressive massive fibrosis. The trend of increasing frequency of autoantibodies with advancing radiographic category was most marked when the frequencies of antinuclear antibody and rheumatoid factor were combined. These autoantibodies were found in 13% of the miners with simple pneumoconiosis and 45% of those with category C progressive massive fibrosis (P for the trend=0·01).     

Hydralazine,antinuclear antibodies,and the lupus syndrome.

The incidence of patients with positive antinuclear antibody test results rose during three years of treatment with hydralazine. At the end of that period over half of the patients (both rapid and slow acetylators) had titres exceeding 1/20, but the rate of rise was faster in the slow acetylators than in the rapid. There was a significant relation between the cumulative dose of hydralazine and the proportion of patients found to have antinuclear factors. Fewer black patients had positive test results than white. Patients whose antinuclear antibody test results changed fron negative to positive during the study showed this change five to 26 months after beginning treatment. Some patients showed a substantial fall in antinuclear antibody titre even though hydralazine was continued. From these findings patients in whom test results for antinuclear antibody became positive during treatment with hydralazine need not have the drug stopped unless they have clinical features of the lupus syndrome.     

抗中性粒细胞胞浆抗体、抗核抗体联合检测对类风湿关节炎的诊断价值

目的：探讨抗中性粒细胞胞浆抗体（ANCA）与抗核抗体（ANA）联合检测对类风湿关节炎的临床意义。方法：采用IIF法对82例RA患者（RA组）、74例非RA自身免疫疾病患者（非RA组）和52例健康体检者（正常对照组）的血清ANCA和ANA谱进行了检测分析,并用ELISA法进行抗丝氨酸蛋白酶3（PR3）、抗髓过氧化物酶（MPO）、ANA谱的定量检测。结果：RA组82例患者中,64例ANCA阳性,阳性率为78．08％,其中核周型（PANCA）37例,阳性率为45．1％,胞浆型（CANCA）27例,阳性率为32．9％;非RA组74例患者中有7例ANCA阳性率分别为9．4％;正常对照组50例中没有一例ANCA阳性。利用Elisa法对患者血清进行检测,分别能够特异的检测到PR3、MPO、抗双链DNA抗体（抗ds—DNA抗体）、抗SS—A等抗体、抗ss—A抗体、抗PM—SCL抗体的存在。结论：联合ANCA、ANA检测有助于提高类风湿关节炎的诊断。     

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.     

False-Positive Anti-Toxoplasma Fluorescent-Antibody Tests in Patients with Antinuclear Antibodies

The indirect fluorescent-antibody (IFA) method for diagnosis of toxoplasmosis is widely used and is considered to be as specific as the Sabin-Feldman dye test. After observing a patient with systemic lupus erythematosus (SLE) who had a positive toxoplasma IFA test but a negative dye test, we studied sera with high titers of antinuclear antibodies from 16 SLE patients and from 2 with rheumatoid arthritis for Toxoplasma antibodies in the immunoglobulin G and M (IgG and IgM) IFA tests and the dye test. Results of these tests were compared with titers of antinuclear antibodies, precipitating antibodies to single-strand deoxyribonucleic acid (DNA), and binding antibodies by use of DNA labeled with (3)H-actinomycin D. Of 18 patients, 11 had IgG and 4 had IgM IFA Toxoplasma antibodies; only 2 had antibodies detectable in the dye test. The immunofluorescence patterns in the Toxoplasma IFA test were indistinguishable from those obtained in patients with toxoplasmosis without antinuclear antibodies. Absorption of SLE sera with DNA did not result in a decrease in Toxoplasma IFA titers. When SLE sera were absorbed with live T. gondii, a marked drop in IgG IFA titer was observed as well as a decrease in titers of antinuclear antibodies and (3)H-DNA binding. Treatment of Toxoplasma cells with deoxyribonuclease and ribonuclease did not decrease their fluorescence. These results suggest that T. gondii nuclear antigens can absorb antinuclear antibodies but do not have exposed substrates for deoxyribonuclease. Tests in which organisms containing "nuclear" antigens for IFA detection of antibodies to these organisms are used may result in "false-positives" with sera containing antinuclear antibodies.     

Proteomic analysis of purified Newcastle disease virus particles

Background

Recent advances in liquid chromatography-mass spectrometry (LC-MS) technology have led to more effective approaches for measuring changes in peptide/protein abundances in biological samples. Label-free LC-MS methods have been used for extraction of quantitative information and for detection of differentially abundant peptides/proteins. However, difference detection by analysis of data derived from label-free LC-MS methods requires various preprocessing steps including filtering, baseline correction, peak detection, alignment, and normalization. Although several specialized tools have been developed to analyze LC-MS data, determining the most appropriate computational pipeline remains challenging partly due to lack of established gold standards.

Results

The work in this paper is an initial study to develop a simple model with "presence" or "absence" condition using spike-in experiments and to be able to identify these "true differences" using available software tools. In addition to the preprocessing pipelines, choosing appropriate statistical tests and determining critical values are important. We observe that individual statistical tests could lead to different results due to different assumptions and employed metrics. It is therefore preferable to incorporate several statistical tests for either exploration or confirmation purpose.

Conclusions

The LC-MS data from our spike-in experiment can be used for developing and optimizing LC-MS data preprocessing algorithms and to evaluate workflows implemented in existing software tools. Our current work is a stepping stone towards optimizing LC-MS data acquisition and testing the accuracy and validity of computational tools for difference detection in future studies that will be focused on spiking peptides of diverse physicochemical properties in different concentrations to better represent biomarker discovery of differentially abundant peptides/proteins.     

The development of a monoclonal antibody specific for a 2(')-C-allyl modification of uridine,and its use in the localization of ribozymes in vivo

Ribozymes are catalytically active RNA molecules that cleave other RNA molecules in a sequence-specific fashion, with significant turnover. The successful design and synthesis of ribozymes with modifications to increase their stability in biological fluids, while maintaining catalytic activity, has been instrumental in moving this technology from the laboratory into clinical trials. With the entry of ribozymes into the clinical setting, the need has arisen for reagents and/or assays to detect these drugs in tissues. We have developed a monoclonal antibody to the 2(')-deoxy-2(')-C-allyl uridine modification present in our synthetic hammerhead ribozymes. The monoclonal antibody, termed CA1USR, is a murine IgG1(k), whose epitope appears to involve both the 2(')-C-allyl modification, and the uridine base. Use of CA1USR for immunohistochemical detection of ribozymes in the tissues of mice which were administered two structurally different ribozymes has demonstrated its utility as a reagent for in vivo localization of ribozymes containing the 2(')-C-allyl uridine modification.     

Diagnostic possibilities of serology in diseases caused by members of the genus Candida berkhout

Up to now, the diagnosis of candidiases has been an intriguing problem chiefly because the detection of Candida in the sick organism is no conclusive evidence of its etiological involvement. The endeavour to use serological methods in the diagnosis has not been successful and their use has so far practically been limited to taxonomic studies. In an effort to find a serological approach to the diagnosis, we found the agarimmunodiffusion assay of Ouchterlony to be the most promising method and used it in our own modification. The first stage of the work was to select and prepare the most suitable type of antigen for this reaction (described in the present paper). Sixteen antigen types classifiable into four groups were prepared: simple water-extracted antigens, alcohol-extracted antigens, antigens obtained on disrupting cells by repeated freezing and thawing, and antigens prepared by boiling cultures. Cultures for antigen preparation were grown on Salvin's medium or on liquid Sabourad's medium and the antigens obtained were either used in their native form or dialysed. Serological tests with hyperimmune rabbit sera prepared by our own schedule were done repeatedly. By far the best were simple water-extracted antigens, nondialysed, from cultures grown on Sabouraud's medium. They reacted the most sensitively, gave high antibody detection rates and assessable precipitin reactions and showed high species specificity. Tests with positive human sera fully confirmed these findings; in fact, species specificity was even somewhat higher here.     

Chronic Liver Disease: Differences in Autoimmune Serological Reactions between Australians and Asians

A total of 164 patients from Australia, Ceylon, India, Singapore, and Thailand were studied for the prevalence of autoantibodies associated with “idiopathic” chronic liver disease—namely, antinuclear antibody, smooth muscle antibody, and mitochondrial antibody. The prevalence of these autoantibodies was high among patients from Australia (55%), but was low among patients from Ceylon (14%), India (11%), Singapore (0%), and Thailand (8%). There are variations in types of hepatitis and cirrhosis between races, and this applies particularly to the type associated with autoimmune markers. This may be related to genetic differences that have evolved between peoples of European and Asian descent.     

The relevance of various tests for the study of specificity in immunocytochemical staining: a review

Factors determining the specificity of immunocytochemical (ICC) tissue stainings as well as the various tests to study these factors are discussed. Since every specificity test only deals with particular aspects of the ICC procedure, a practical sequence of known test methods is proposed, which enables the determination of the specificity of the ICC tissue staining and, after possibly needed antiserum purification steps, may result in a monospecific staining. It is made clear that such a sequence has always to include a tissue-spectrum affinity test, in which the spectrum of tissue antigens is controlled for antibody binding. A variety of such tests, consisting of separation of tissue compounds, fixation, and ICC detection, are discussed as well as their pros and cons with respect to their predictability for the actual serum specificity in the tissue section.     

Comparison of different approaches for evaluation of the detection and quantitation limits of a purity method: a case study using a capillary isoelectrofocusing method for a monoclonal antibody

Several different techniques suggested by the International Conference on Harmonization (ICH) Q2R1 guideline were used to assess the signal and concentration at the limit of detection (LOD) and limit of quantitation (LOQ) for a purity method. These approaches were exemplified with a capillary isoelectrofocusing (cIEF) method, which has been developed to quantify the distribution of the charge isoforms of a monoclonal antibody. The charge isoforms are the result of incomplete posttranslational processing of C-terminal lysine residues of the heavy chain by carboxypeptidase. Results showed no significant discrepancy between LOD/LOQ obtained by the different techniques. Validation experiments corroborated the calculated LOQ. The results indicate that any single technique can provide meaningful values for the LOD and LOQ. Finally, important points to consider when applying these techniques to purity methods are discussed.     

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

Immunoglobulin G (IgG) crystallizable fragment (Fc) glycosylation is crucial for antibody effector functions, such as antibody-dependent cell-mediated cytotoxicity, and for their pharmacokinetic and pharmacodynamics behavior. To monitor the Fc-glycosylation in bioprocess development, as well as product characterization and release analytics, reliable techniques for glycosylation analysis are needed. A wide range of analytical methods has found its way into these applications. In this study, a comprehensive comparison was performed of separation-based methods for Fc-glycosylation profiling of an IgG biopharmaceutical. A therapeutic antibody reference material was analyzed 6-fold on 2 different days, and the methods were compared for precision, accuracy, throughput and other features; special emphasis was placed on the detection of sialic acid-containing glycans. Seven, non-mass spectrometric methods were compared; the methods utilized liquid chromatography-based separation of fluorescent-labeled glycans, capillary electrophoresis-based separation of fluorescent-labeled glycans, or high-performance anion exchange chromatography with pulsed amperometric detection. Hydrophilic interaction liquid chromatography-ultra high performance liquid chromatography of 2-aminobenzamide (2-AB)-labeled glycans was used as a reference method. All of the methods showed excellent precision and accuracy; some differences were observed, particularly with regard to the detection and quantitation of minor glycan species, such as sialylated glycans.     

Multiple independent origins of a protease inhibitor resistance mutation in salvage therapy patients

With the continuing march of the AIDS epidemic and little hope for an effective vaccine in the near future, work to develop a topical strategy to prevent HIV infection is increasingly important. This stated, the track record of large scale "microbicide" trials has been disappointing with nonspecific inhibitors either failing to protect women from infection or even increasing HIV acquisition. Newer strategies that target directly the elements needed for viral entry into cells have shown promise in non-human primate models of HIV transmission and as these agents have not yet been broadly introduced in regions of highest HIV prevalence, they are particularly attractive for prophylaxis. We review here the agents that can block HIV cellular entry and that show promise as topical strategies or "virustats" to prevent mucosal transmission of HIV infection     

Prevention of systemic lupus erythematosus in children born to mothers treated for this disease

Genetic predisposition and environmental factors (physical, chemical, hormonal and drugs inducing collagen-like syndrome) play an important role in the pathogenesis of the systemic lupus erythematosus. Elimination of these factors from the environment of the genetically predisposed individuals may prevent part of them against the disease. Basing on the above assumption, a chart of prophylaxis has been constructed and distributed among the mothers with the systemic lupus erythematosus, recommending prophylactic measures in both mothers and children. Within 1977-1987, 50 children were examined from time to time. Basic laboratory tests, phenomenon LE, antinuclear antibodies and antibodies anti-DNA have been determined. Transient presence of antinuclear antibodies was seen in 23 children. A tendency to an increase in the antibody titre was observed in girls of this group whereas a decrease in the titre was noted in the boys with the time lapse. Systemic lupus erythematosus prophylaxis in both mothers and children is uncomplicated and favourable for children. Regular determination of antibodies enables early diagnosis of the disease.