Rheumatoid factor interference in immunological methods.
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Humans↗
Biomedical subjects
R G Hamilton
Publications and source records attributed to R G Hamilton.
Two Ro (SS-A) autoantibody responses in systemic lupus erythematosus. Correlation of HLA-DR/DQ specificities with quantitative expression of Ro (SS-A) autoantibody.
Autoantibodies to Ro (SS-A), La (SS-B), and Sm/nuclear RNP were quantitated by enzyme-linked immunosorbent assay in 106 white patients with systemic lupus erythematosus. Two Ro autoantibody subgroups were identified that differed quantitatively, genetically, and clinically. The subgroup having anti-Ro only demonstrated significantly lower mean anti-Ro levels than did the subgroup with concomitant anti-La and showed a strong association with the linked HLA alleles DR2 and DQw1. The anti-Ro with anti-La subgroup was associated with the linked HLA alleles B8, DR3, DRw52, and DQw2 (DR3 was primary), and this subgroup consisted of patients with older ages at disease onset, sicca complex, and less renal involvement. Overall, the relative risk (RR) for having anti-Ro was highest in HLA-DR2/DR3 heterozygotes compared with non-DR2/DR3 heterozygotes (RR 15) and all other DR combinations (RR 7), suggesting a compound effect of 2 immune responses. Heterozygotes for HLA-DQw1/DQw2 demonstrated significantly higher mean levels of anti-Ro, which may be indicative of trans gene interaction at HLA-DQ. These data suggest the hypothesis that HLA genes exert their major effects on Ro/La autoantibody subsets of systemic lupus erythematosus.
Autoantibodies↗
Subclass distribution of human IgG autoantibodies in pemphigus.
The distribution of IgG subclasses in the intercellular substance (ICS) reactive autoantibodies in serum of 10 patients with pemphigus was analyzed by semiquantitative indirect immunofluorescence (IF) using the HP series of monoclonal antibodies specific for the four human IgG subclasses. IgG4 ICS specific autoantibody was present in all 10 sera at a titer of 10 to 320, while IgG1 antibodies were found in 9 of 10 sera at a seemingly lower level. IgG3 autoantibodies were detected in the serum of one patient, only after isolation of IgG using ion-exchange chromatography. Autoantibodies of IgG subclass 2 were not detectable in any of the 10 sera tested. One of the ten patients displayed circulating anti-ICS antibodies of only the IgG4 isotype.
Antibodies, Monoclonal↗
Monoclonal antibody-based immunoenzymetric assays for quantification of human IgG and its four subclasses.
A panel of 5 immunoenzymetric assays (IEMAs) has been developed for quantification of the total and four subclasses of immunoglobulin G (IgG) in human serum using IUIS/WHO documented monoclonal antibodies (MoAb). Human IgG specific MoAb was adsorbed to microtiter plates and used to capture IgG from serum. Peroxidase conjugated forms of polyclonal mouse anti-human IgG Fc or a mixture of 4 MoAb (anti-kappa, anti-lambda, anti-IgG Fc PAN and anti-IgG Fd PAN) were used as detection antibodies. Use of monoclonal antibody in chromatographically purified form was required for acceptable assay sensitivity (S) and working ranges (WR). All 5 IEMAs displayed good precision (intra-assay %CV less than 5%, inter-assay %CV less than 12%) and parallelism (inter-dilutional CV less than 20%). Both HP6069 or HP6070 (anti-IgG1 Fc) worked well alone or together as capture antibodies in the IgG1 IEMA: WR = 20-1250 ng/ml, S = 15 ng/ml. HP6002 (anti-gG2 Fc) alone or in combination with HP6014 (anti-IgG2 Fd) produced an IgG2 IEMA with a WR of 5-200 ng/ml and S of 5 ng/ml. HP6047 (anti-IgG3 hinge) alone generated a sensitive IgG3 IEMA with a narrow working range: WR = 2-50 ng/ml, S = 1.6 ng/ml. Both HP6025 and HP6023 (anti-IgG4 Fc) worked equally well alone and together to produce a useful IgG4 IEMA: WR = 8-250 ng/ml, S = 7.8 ng/ml. HP6017 (anti-IgG PAN Fc) was combined in an equal molar ratio with HP6046 (anti-IgG PAN Fd) to produce a total IgG PAN IEMA with a WR of 5-530 ng/ml and a sensitivity of 5 ng/ml. All 5 IEMAs fulfilled requirements for robust clinical immunoassays that permit the quantitation of human IgG and its 4 subclasses.
Animals↗
Increased frequencies of Sm and nRNP autoantibodies in American blacks compared to whites with systemic lupus erythematosus.
Frequencies of autoantibodies to Sm, nRNP, Ro (SSA) and La (SSB) were determined by countercurrent immunoelectrophoresis (CIE) and/or enzyme linked immunosorbent assays (ELISA) in 106 whites and 60 blacks with systemic lupus erythematosus. Anti-Sm occurred significantly more frequently in blacks (25%) than whites (10%) (p = 0.02), as did anti-nRNP (40% versus 23%; p = 0.03). By CIE, anti-Sm and/or nRNP occurred in 47% of blacks and 24% of whites (p = 0.004), and by ELISA the Sm/nRNP complex was detected in 52% of blacks versus 26% of whites (p = 0.003). Antibodies to Ro and La occurred with equal frequencies between the races.
Autoantibodies↗
Analysis of paired serum, urine and filter paper blood specimens for presence of filarial antigen by immunoradiometric assay.
Paired serum, urine, and finger-prick whole blood dried on filter paper were analyzed by immunoradiometric assay (IRMA) for filarial antigen using Brugia malayi-specific rabbit antibody. Nine sera and 6 urines from the 10 paired serum-urine samples obtained from individuals with microfilaremia contained IRMA detectable filarial antigen. In contrast, all serum and urine specimens from patients with chronic infections, endemic and non-endemic controls were negative. Whole blood eluted from filter paper spots contained IRMA detectable material; their degree of positivity agreed well with IRMA binding levels obtained with paired urines. Reduced recovery of antigen dried on filter paper was observed at antigen levels less than 10 ng/ml equivalents, presumably due to irreversible absorption onto the filter paper. Urine and finger-prick filter paper blood specimens can be used in the diagnosis of microfilaremic infections that have been associated with circulating antigen in the blood.
Animals↗
Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis.
Quality control of murine hybridoma secretory products was performed using two variations of the isoelectric focusing affinity immunoblot analysis. The first approach employed antigen-coated nitrocellulose placed on top of an acrylamide gel containing isoelectrically focused ascites to bind antigen specific monoclonal antibody (MoAb). Murine antibody bound to the insolubilized antigen was then detected with enzyme-conjugated anti-mouse IgG. In a second variation, focused ascites proteins were passively blotted onto nitro-cellulose and specific monoclonal antibody was detected with enzyme-conjugated antigen. Several batches of ascites containing anti-human IgG antibodies that were produced by 6 hybridomas over a 1-3 year period were assessed by IEF-affinity immunoblot analysis. Both immunoblot approaches permitted effective monitoring of immunoreactive antibody for pI microheterogeneity. IEF-affinity immunoblot patterns of unprocessed ascites displayed specific MoAb banding patterns with narrow pI ranges (less than or equal to 0.6 pH units), in contrast to the reported 5.5-8.0 pI range of polyclonal mouse IgG. Banding patterns obtained in the IEF affinity immunoblot typically displayed 3-5 major dense bands flanked by 2-4 minor fainter bands. Batches of ascites obtained years apart produced similar immunoblot patterns, indicating constant antibody production and confirming the stability of these hybridoma clones. Minor bands appeared in 2 earlier lots of ascites, suggesting possible modification of antibody during storage. IEF affinity immunoblot analysis is a useful tool for monitoring MoAb pI microheterogeneity as an indicator of antibody quality without the need for isolation of monoclonal antibody from culture medium or ascites.
Animals↗
Human IgG subclass measurements in the clinical laboratory.
Complement activation, cell surface-receptor binding, blocking activity, and possibly placental transfer are among the biologically important functional differences that have been detected between the four human IgG subclasses by use of polyclonal antisera. In 1985, a IUIS/WHO panel of immunologists, using eight immunological methods, documented the specificity of select monoclonal antibodies for the IgG subclasses. Clinical assays have been developed involving these monoclonal antibodies that allow quantification of the concentration of IgG subclass protein and distribution of the IgG subclass antibodies in human immune responses. This review addresses issues of concern to investigators who are evaluating and (or) developing quantitative human IgG subclass assays in the clinical laboratory. Unique physical (structural) and biological (functional) properties of human IgG subclasses are summarized, with a focus on aspects pertinent to their clinical importance and in vitro quantification. The HP-series monoclonal antibodies with documented specificity are examined within the context of their application to several immunological methods. I describe unique technical aspects of total and antigen-specific IgG-subclass immunoassays involving these monoclonal antibodies. Finally, this report outlines clinical applications and indications for IgG-subclass measurements in the study of human health and disease.
Adult↗
Dirofilaria immitis: diethylcarbamazine-induced anaphylactoid reactions in infected dogs.
The immunopathogenesis of the anaphylactoid Mazzotti reactions has been studied by comparing physiologic and immunologic aspects of diethylcarbamazine-induced shock in Dirofilaria immitis infected dogs with antigen induced anaphylaxis in infected and uninfected controls. Filarial antigen, specific host IgG antibody, and C1 and C3 complement levels were quantitatively measured over time in relation to the levels of histamine and prostaglandin D2 in the blood and changes in mean blood pressure. D. immitis antigen injected into uninfected dogs having no detectable IgG antibody to D. immitis or Toxocara canis produced a rapid drop in blood pressure that paralleled a drop in C1 and C3 levels and an increase in prostaglandin D2. Antigen injected into infected dogs with IgG antibody produced a similar drop in blood pressure and complement and increase in prostaglandin D2 which differed from the uninfected group only in the slower clearance of antigen from the blood. Diethylcarbamazine alone produced no measurable changes in blood pressure or complement in uninfected hosts. Diethylcarbamazine, however, administered into skin test positive infected dogs, produced a temporally slower but quantitatively similar loss in blood pressure, drop in complement, and increase in prostaglandin D2 and histamine to that induced by antigen injection. Complement activation and immune complex formation are initiated by antigen release, and subsequent vasoactive mediator release leads to shock with prostaglandin D2 being quantitatively higher in blood than is histamine.
Airway Resistance↗
Humoral immune responses in Peyronie's disease patients receiving clostridial collagenase therapy.
Human IgG and IgE antibody responses to clostridial collagenase (Nucleolysin) were monitored in 150 untreated healthy blood donors and 44 patients receiving intrapenile injections of collagenase for treatment of Peyronie's disease. Pre, 1 and/or 2 month post treatment sera were analyzed in radioimmunoassays for human IgG and IgE antibodies using solid phase purified Cl. histolyticum collagenase to extract antibodies from serum and 125I-Protein A or rabbit anti-human IgE to detect bound IgG and IgE, respectively. IgG and IgE antibody levels greater than 2.5 Units and 0.2 per cent Bmax, respectively, were considered positive if the binding was greater than 80 per cent inhibitable with soluble collagenase. IgG anti-collagenase was detected in 34 per cent of the 150 healthy controls and 58 per cent (24/41) of untreated Peyronie's disease patients. Intralesional injection of 3,000 to 12,650 Units of collagenase induced an increase of two- to 10-fold in the IgG levels at one to two months in 88 per cent of patients. Of the 186 individuals tested, only one (0.5 per cent) had detectable collagenase-specific IgE antibody in his serum as defined by positive threshold and antigen inhibition criteria.
Adult↗
Clinical laboratory methods for the assessment and management of human allergic diseases.
The biologic role and clinical relevance of IgE (reaginic) antibodies in immediate-hypersensitivity (allergic) reactions are reviewed, and methods useful in the laboratory diagnosis and management of human allergic diseases are discussed. Quantitative immunoassays for total and allergen-specific IgE antibodies are examined within the context of their use in diagnosis. The radioallergosorbent test inhibition assay and specific IgG antibody immunoassays are described as useful methods in the planning and monitoring of immunotherapy.
Allergens↗
Disc/stick ELISA for diagnosis of bancroftian filariasis.
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Antibodies↗
Naturally occurring carbohydrate antibodies: interference in solid-phase immunoassays.
Antibody assays utilizing carbohydrate matrices (agarose, cellulose) for protein insolubilization are subject to non-specific and specific interfering factors. This report examines factors which diminish the quality of particle-based solid-phase radioimmunoassays (SPRIAs) for antigen-specific human IgG. Interfering factors are divided into (a) constant non-specific binding which is similar with all human sera and appears related to simple adherence of IgG to agarose and cellulose, and (b) absorbable binding which varies considerably among sera in agarose and cellulose-based assays, and results from the presence of IgG antibody specific for the carbohydrate matrix. Constant non-specific binding is predictably 1-3% Bmax (maximum binding) in all human and rabbit sera. In contrast, the absorbable binding levels vary widely: 0.75-29% Bmax in 58% (study 1, n = 50) and 40% (study 2, n = 200) of normal individuals for agarose, and 3-30% Bmax in 70% of the population for microcrystalline cellulose. IgG anti-agarose antibodies were found in 13 of 16 rabbit sera examined. Ultracentrifugation and immune-complex studies demonstrated that aggregated or immune-complexed IgG does not contribute to the absorbable IgG binding. Inhibition with acid hydrolyzed soluble agarose and provided evidence for a specific IgG anti-agarose antibody that causes variable background binding. Pre-absorption of sera with agarose prior to analysis in the agarose-based SPRIA removed greater than 90% of anti-agarose antibodies and eliminated false positive results. These studies suggest rabbit and perhaps other heterologous antibodies prepared by protein-agarose affinity column chromatography may contain significant levels of naturally occurring antibodies against agarose or cellulose. These naturally occurring carbohydrate antibodies may interfere in solid-phase carbohydrate-based immunologic methods and immunoassays.
Antibody Specificity↗
Limitations of the radioimmunoprecipitation polyethylene glycol assay (RIPEGA) for detection of filarial antigens in serum.
The performance of the radioimmunoprecipitation polyethylene glycol assay (RIPEGA) was examined for quantitation of filarial antigens (Brugia malayi and Dirofilaria immitis) in serum from infected human and animal hosts and non-infected controls. Multiple PEG concentrations were employed to determine the level of non-specific binding (NSB) in non-exposed human sera (NEHS) containing no filarial antigen. The NSB observed when 3 different 125I-labeled IgG antibodies were added to 26 NEHS varied 3-fold and was correlated significantly with total serum IgM (r = 0.80, P less than 0.005, n = 24) but not with serum IgA (r = 0.37) or IgG (r = 0.45). NSB levels were significantly reduced when a Fab'2 fragment of the 125I-labeled antibody was used, but the correlation of NSB with total serum IgM remained significant (r = 0.57, P less than 0.01). The presence of rheumatoid factor in NEHS sera also significantly increased NSB by an average of 3-fold. These effects eliminated the assay's ability to detect in sera from infected hosts filarial antigen the presence of which could be readily demonstrated by an immunoradiometric assay. The RIPEGA's precision (intra-assay coefficient of variation (CV) = 21% at 35% Bmax) and reproducibility (inter-assay CV = 29% at 35% Bmax) are less satisfactory than many alternative immunoassays. In many cases, positive sera failed to dilute out in parallel with each other or with an antigen-spiked standard reference curve. We conclude that poor performance characteristics currently limit the utility of the RIPEGA for quantitating filarial antigen in human and animal serum.
Animals↗
Mechanisms of acute allergic reactions.
Acute allergic reactions range from mild conditions of local tissue swelling and pruritus to severe multisystem syndromes including asthma, urticaria and/or angioedema, gastrointestinal distress, and vascular collapse. Such reactions share a common pathophysiology characterized by vasodilation and postcapillary permeability, resulting in increased extravasation of fluid within minutes after exposure to an eliciting substance. Smooth muscle contraction of the respiratory or gastrointestinal tracts may also be involved. Most of these changes can be explained by the release of chemical mediators from circulating basophilic leukocytes and tissue mast cells. Human basophils and mast cells can be activated to release chemical mediators by several known pathways: crosslinking by allergens of specific immunoglobulin E antibodies attached to basophils and mast cells; anaphylatoxin formation following immune complex activation of the classical complement pathway; anaphylatoxin formed from direct activation of the alternative complement pathway by negatively charged surfaces; non-complement-, non-antibody-mediated direct histamine release; and idiosyncratic mechanisms involving physical exercise, psychological stress, or aspirin intolerance. Any or all of these mechanisms could be operative in patients experiencing acute allergic reactions at the commencement of hemodialysis.
Anaphylaxis↗
Serological methods in the diagnosis and management of human allergic disease.
The diagnosis and management of human allergic disease is aided by serological measurements involving the quantitation of total human serum IgE, allergen-specific IgE and allergen-specific IgG. Initially, a brief overview of immediate hypersensitivity reactions will be presented with a focus on the role of "reaginic" allergen-specific IgE antibody. The principles and design of immunoassay methods employed in the measurement of total serum IgE and allergen-specific human IgE will then be examined with a discussion of the pros and cons of different radioallergosorbent test (RAST) reporting schemes. Next, Hymenoptera venom will be used as a model allergen system for exploring the clinical utility, principles and design of immunoassays used to quantitate allergen-specific IgG antibody in human serum. Examples of the utility of specific IgG measurements will be provided with recent clinical data. Finally, an overview will be presented on more research-based techniques used in the study of human allergic responses such as cross-immunoelectrophoresis and immunoassays for leukotrienes, prostaglandins, platelet Factor 4 and cyclic AMP.
Allergens↗
Immunoradiometric assay for detection of filarial antigens in human serum.
The immunoradiometric assay (IRMA) for detection of filarial antigens in the serum of patients infected with Brugia malayi (Bm) or the closely related filarial parasite Wuchereria bancrofti (Wb) was investigated, and its performance and clinical utility were examined. Reference sera prepared by the addition of crude Bm antigen (BmA) to negative control human sera provided a reproducible reference curve. The IRMA displayed acceptable precision and reproducibility. Agreement between dilutions (parallelism) was good in sera without specific antibody, but the presence of even modest levels of antibody resulted in nonparallelism in about one-half of the tested sera from endemic areas. Significant reduction in detectable BmA occurred when low levels of specific antibody (less than 1 microgram/ml) were added to BmA containing sera. Thus, antibody interference limited absolute quantitation of antigen in the IRMA. Results were therefore expressed in a semi-quantitative manner by using the mean + 3 SD of the binding of nonexposed human sera as the positive threshold. The frequency of reliable filarial antigen detection in individuals from the Wb endemic areas of India and the South Pacific was the following: microfilaremia, 15 out of 15; elephantiasis, 2 out of 18; tropical pulmonary eosinophilia, 2 out 8. These findings show clearly that a two-site IRMA can effectively detect circulating antigen (and thus be diagnostic of infection) in a great many patients with filariasis, but to enhance the sensitivity of the assay to the point where all patients can be diagnosed, a number of suggested modifications will be necessary.
Animals↗
Detection of filarial antigen in urine of humans with Wuchereria bancrofti infection by immunoradiometric assay.
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Antigens, Helminth↗