Correlation of pneumococcal antibody concentration and avidity with patient clinical and immunologic characteristics.

PURPOSE: The quality of an antibody response is determined by both the concentration and the strength of antigen-binding, or avidity, of the antibodies produced. Currently, only antibody concentration is routinely evaluated in the clinical assessment of humoral immunity. Here we studied correlations of avidities and concentrations of antibodies to pneumococcal polysaccharides with immunologic and clinical characteristics of patients with recurrent infections. METHODS: We measured concentration and avidity of antibodies to 12 pneumococcal serotypes in 78 children aged 0.6-18 years with recurrent bacterial respiratory infections, and in 80 individuals who were being tested for peanut allergy, ages 0.4-15 years, serving as a comparison group. Avidity was assessed by measuring antibody binding in the presence of thiocyanate. RESULTS: Antibody concentrations and avidities correlated positively for very few types contained in the conjugated pneumococcal vaccine (PCV7) in both patients and controls with some dependence on age; there were even fewer correlations for non-PCV7 types. Antibody concentrations and avidities negatively correlated with age for most of the PCV7 types. There was no consistent correlation of total IgG or IgG subclasses with either concentrations or avidities. Overall, antibody concentrations were higher and avidities were lower in patients compared to controls. Patients requiring chronic antibiotic use tended to have higher antibody concentrations and lower avidities for most serotypes than patients who did not. We identified several patients having many infections with apparent good antibody concentrations with low avidity for many types. CONCLUSION: Antibody concentration and avidity correlate with patient clinical characteristics and distinguish patients from controls. Measurement of antibody avidity may provide another dimension for the clinical assessment of pneumococcal polysaccharide antibody response.

Efficacy of intravenous gammaglobulin for immunoglobulin G subclass and/or antibody deficiency in adults.

BACKGROUND: The clinical significance and efficacy of treating patients who have immunoglobulin (Ig) G subclass deficiency and/or antibody deficiency with Ig-replacement therapy has been debated. There are no clear guidelines to recommend intravenous gammaglobulin (IgIV) in these patients as there are few published studies documenting its efficacy. METHODS: We studied in an open-label protocol 10 adult patients with recurrent respiratory infections and IgG subclass and/or antibody deficiency. All patients received monthly IgIV for 12 months and then were observed for 3 months without IgIV infusions. We studied quality of life, incidence of infections, need for antibiotics, frequency of hospitalizations due to infections, IgG subclass and antibody (tetanus and pneumococcal) levels. Innate immunity was evaluated by studying the status of Toll-like receptors and polymorphisms, mannan-binding lectin levels and genotypes. Correction of the immune defects during IgIV therapy was evaluated. RESULTS: Monthly IgIV significantly improved quality of life, decreased the number of infections and the need for antibiotics, and improved IgG subclass and antibody serum levels. No consistent finding of innate immunity could be detected. CONCLUSIONS: IgIV could be beneficial in patients with IgG subclass or antibody deficiency.

Application of photonic crystal enhanced fluorescence to detection of low serum concentrations of human IgE antibodies specific for a purified cat allergen (Fel D1).

We demonstrate the detection of low concentrations of allergen-specific Immunoglobulin E (IgE) in human sera using a Photonic Crystal Enhanced Fluorescence (PCEF) microarray platform. The Photonic Crystal (PC) surface, designed to provide optical resonances for the excitation wavelength and emission wavelength of Cy5, was used to amplify the fluorescence signal intensity measured from a multiplexed allergen microarray. Surface-based sandwich immunoassays were used to detect and quantify specific IgE antibodies against a highly purified cat allergen (Fel d1). A comparison of the lowest detectable concentration of IgE measured by the PC microarray system and a commercially available clinical analyzer demonstrated that the PCEF microarray system provides higher sensitivity. The PCEF system was able to detect low concentrations of specific IgE (~0.02 kU/L), which is 5-17-fold more sensitive than the commercially available FDA-approved analyzers. In preliminary experiments using multi-allergen arrays, we demonstrate selective simultaneous detection of IgE antibodies to multiple allergens.

Cross-reactivity between gutta-percha and natural rubber latex: assumptions vs. reality.

BACKGROUND: Immunological cross-reactivity between gutta-percha and natural rubber latex, or NRL, has not been demonstrated clearly despite recent concerns and several suspected cases reported in the literature. METHODS: The authors analyzed aqueous extracts of commercial gutta-percha points and raw gutta-percha samples for cross-reactivity to NRL by radioallergosorbent test, or RAST, inhibition; immunoblot inhibition; direct enzyme-linked immunosorbent assay, or ELISA; and ELISA inhibition using sera from NRL-allergic people as the source of anti-NRL immunoglobulin E, or IgE, antibodies. To confirm in vitro results, the authors conducted skin prick testing, or SPT, on a patient with type I NRL allergy using aqueous extracts from raw gutta-percha, ammoniated gutta-percha and gutta-percha points. RESULTS: Aqueous extracts from commercial gutta-percha points did not cross-react to NRL in RAST inhibition or immunoblot inhibition, ELISA or ELISA inhibition assays. However, three of 13 sera from subjects with type I NRL allergy exhibited IgE binding to raw gutta-percha extracts in direct ELISA. Moreover, in ELISA inhibition, the binding of IgE to raw gutta-percha extracts was inhibited in a dose-dependent manner by raw NRL and vice versa. SPT results from a subject with type I NRL allergy were positive for NRL and raw gutta-percha extracts but negative for gutta-percha point extracts. CONCLUSIONS: The authors found no detectable cross-reactivity between NRL and commercial gutta-percha points. However, their ELISA and SPT results demonstrated that some allergenic cross-reactivity exists between raw gutta-percha and raw NRL. CLINICAL IMPLICATIONS: Gutta-percha alone is not likely to induce symptoms in patients with type I NRL allergy. However, other materials used in obturating root canals may be irritating and potentially allergenic in patients with pre-existing allergies.

Comparison of the in vivo autologous skin test with in vitro diagnostic tests for diagnosis of chronic autoimmune urticaria.

Previous studies indicate that 30-50% of chronic urticaria patients have an autoimmune etiology. Clinical diagnosis of autoimmune urticaria is supported with the autologous serum skin test. The purpose of this study was to compare two laboratory tests for measurement of IgG autoantibodies to IgE or IgE receptors and compare the results with the autologous serum and plasma skin tests. We performed skin tests and two functional in vitro tests, basophil histamine release, and CD63 up-regulation to detect autoantibodies relevant to autoimmune urticaria. Both sera and citrated plasma were evaluated in the autologous skin test and histamine release assay. Thyroid autoantibodies were also measured. Basophils were incubated with patient plasma, sera, buffer, or anti-IgE. The cells were analyzed for CD63 expression and the supernatants were recovered for histamine analysis. There was high correlation between CD63 up-regulation and histamine release assays, but histamine release was more sensitive. There was a high concordance between sera and citrated plasma for the skin test. Sera from chronic urticaria patients produced higher mean histamine release (23%) compared with citrated plasma (12%). Thirty-one percent of patients positive in the histamine release assay were also positive for thyroid autoantibodies. This compares with 12% who were negative in the histamine release assay. These data show that in vitro basophil histamine release can be used to measure antibodies to FceRI, FceRII/CD23, or IgE and identify patients with autoimmune urticaria.

Measurement of cytokine production using whole blood.

Whole blood (WB) ex vivo stimulation assays are useful for measuring cytokine responses due to the easy access of samples from healthy donors and patients and the minimal processing of the sample required. Because the assay mimics the natural environment, WB culture may be the best milieu in which to study cell activation and cytokine production in vitro. Whole blood stimulation has been used to investigate the cellular responsiveness to a variety of stimuli, including bacterial endotoxin (LPS), antigens, allergens, and antibiotics. Various clinical uses of whole blood stimulation assays have been suggested, including the assessment of autoimmune diseases, the monitoring of drug and vaccine efficacy, and immunotoxicity. Thus, whole blood cell culture may be useful in studying the biological effects of potential allergenic and/or antigenic substances or drugs on immune cell activation and cytokine secretion.

Proficiency testing of allergen measurements in residential dust.

BACKGROUND: Analyses of household dust for allergens are more common with increased efforts to reduce and control asthma. Currently, no laboratory accreditation or quality assurance program specific to household dust allergen analyses exists. Moreover, there is an absence of peer-reviewed data on within-laboratory and between-laboratory variability that is achievable for these analyses. OBJECTIVES: The purpose of this study was to characterize the levels of intralaboratory and interlaboratory variability in analyses of allergen concentrations in residential dust and to investigate the utility of quality control samples for monitoring laboratory performance. METHODS: Aliquots from homogeneous batches of dust and dust extracts were provided to 8 commercial, academic, and municipal laboratories to be analyzed for as many as 6 allergens (Der p 1, Der f 1, Fel d 1, Can f 1, Bla g 1, Mus m 1) by using ELISA techniques. RESULTS: Coefficients of variation on the estimated geometric means from the analytical results ranged between 61% and 93%. In most cases, between-laboratory variability was the dominant component of total variability. In spite of this between-laboratory variability, reasonable agreement was observed between the means of allergen levels in the reference laboratory characterizations and the estimated geometric means from the model fitting of results across participating laboratories. CONCLUSION: The results from this study indicate that, in most cases, participating laboratories could measure the concentrations of allergens by using ELISA procedures with a level of accuracy and precision that may be acceptable in many situations.

Utilization of serum tryptase and immunoglobulin e assay in the postmortem diagnosis of anaphylaxis.

The postmortem diagnosis of anaphylaxis is difficult. Serum concentrations of tryptase (a mast cell product released during anaphylaxis) have been used after death as an indicator of possible antemortem anaphylaxis. However, studies have indicated that tryptase may be elevated with increasing postmortem interval (PMI), or in nonanaphylactic deaths with significant atherosclerosis or chest trauma. Serum total IgE has been used by some to confirm anaphylaxis when tryptase is elevated. Serum levels of tryptase from 57 decedents with varying PMI, all dying of presumed nonanaphylactic causes, were determined. In cases with elevated levels (>11.4 ng/mL), an assay of total serum IgE was also performed. Both tryptase and IgE demonstrated significant elevations with increasing PMI. Decedents were categorized according to presence of cardiovascular disease, chest trauma, or both; many demonstrated elevation of 1 or both markers, without statistically significant differences between categories. Postulated mechanisms for nonanaphylactic elevations of these markers are reviewed. The possible utility of allergen-specific IgE or allergen panels is discussed.