Human Immunoglobulin E Research Articles

Immunosensor for human IgE detection using electrochemical redox cycling with ferrocene‐mixed self‐assembled monolayers modified Au electrode

AbstractThe concentration of human immunoglobulin E (IgE) in blood is an indicator for diagnosing an allergy. For detecting human IgE by an electrochemical immunosensor, redox mediator molecules were modified on a gold electrode using thiolated organic molecules, which can form self‐assembled monolayers on the electrode surface. Ferrocene was used as a redox mediator, and the current signal was amplified via redox cycling using 4‐aminophenol. Measurements were obtained after the immobilization of the target‐captured antibody, target human IgE, biotin‐conjugated secondary antibody, and avidin alkaline phosphatase in a sandwich‐type immunosensor configuration. The prepared immunosensor exhibited a limit of detection of 1 IU/mL and dynamic range of 3–30 IU/mL.

Human IgE monoclonal antibody recognition of mite allergen Der p 2 defines structural basis of an epitope for IgE cross-linking and anaphylaxis in vivo.

Immunoglobulin E (IgE) antibody is a critical effector molecule for adaptive allergen-induced immune responses, which affect up to 40% of the population worldwide. Allergens are usually innocuous molecules but induce IgE antibody production in allergic subjects. Allergen cross-linking of IgE bound to its high affinity receptor (FcεRI) on mast cells and basophils triggers release of histamine and other mediators that cause allergic symptoms. Little is known about the direct allergen–IgE antibody interaction due to the polyclonal nature of serum IgE and the low frequency of IgE-producing B cells in blood. Here, we report the X-ray crystal structure of a house dust mite allergen, Der p 2, in complex with Fab of a human IgE monoclonal antibody (mAb) isolated by hybridoma technology using human B cells from an allergic subject. This IgE mAb, 2F10, has the correct pairing of heavy and light chains as it occurs in vivo. Key amino acids forming the IgE epitope on Der p 2 were identified. Mutation of these residues ablated their functional ability to cross-link IgE in a mouse model of passive systemic anaphylaxis. These analyses revealed an important conformational epitope associated with the IgE antibody repertoire to a major mite allergen.

Tracing Human IgE B Cell Antigen Receptor-Bearing Cells With a Monoclonal Anti-Human IgE Antibody That Specifically Recognizes Non-Receptor-Bound IgE.

Up to 30% of the population suffers from immunoglobulin E (IgE)-mediated allergies. Despite current stepwise gating approaches, the unambiguous identification of human IgE-producing cells by flow cytometry and immunohistology remains challenging. This is mainly due to the scarcity of these cells and the fact that IgE is not only expressed in a membrane-bound form on the surface of IgE-producing cells in form of the B cell antigen receptor (BCR), but is more frequently found on various cell types bound to the low and high affinity receptors, CD23 and FcϵRI, respectively. Here we sought to develop a sequential gating strategy for unambiguous detection of cells bearing the IgE BCR on their surface. To that aim we first tested the monoclonal anti-IgE antibody omalizumab for its ability to discriminate between IgE BCR and receptor-bound IgE using cells producing IgE or bearing IgE bound to CD23 as well as basophils exhibiting FcϵRI receptor-bound IgE. Using flow cytometry, we demonstrated that omalizumab recognized IgE producing cells with a high sensitivity of up to 1 IgE+ cell in 1000 human peripheral blood mononuclear cells (PBMCs). These results were confirmed by confocal microscopy both in cell suspensions as well as in nasal polyp tissue sections. Finally, we established a consecutive gating strategy allowing the clear identification of class-switched, allergen-specific IgE+ memory B cells and plasmablasts/plasma cells in human PBMCs. Birch pollen specific IgE+ memory B cells represented on average 0.734% of total CD19+ B cells in allergic patients after allergen exposure. Thus, we developed a new protocol for exclusive staining of non-receptor bound allergen-specific IgE+ B cell subsets in human samples.

Humanized Mediator Release Assay as a Read-Out for Allergen Potency.

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (FcεRI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of β-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.

Proteins Derived from Cnidium officinale Makino React with Serum IgE of Allergic Patients and Stimulate ERK/NF-kB Activation in Human Mast Cell Line HMC-1 Cells

Herbal products are the most representative alternative medicine, but most herbal drugs are taken orally and these can cause allergic reactions in the gastrointestinal tract such as food allergies. To confirm whether herbal proteins can cause allergic reaction by binding to human serum immunoglobulin E (IgE), we selected sera from 800 randomized human sera using Allergy-Q tests and investigated the allergenic reactivity of Cnidium officinale (CO) Makinoe extract with positive sera in HMC-1 cells. The proteins derived from CO extract were separated by SDS-PAGE, and identified these proteins using LC–MS/MS. The human sera IgE binding proteins were estimated at the four different predicted proteins such as FAR1-related sequence 5-like protein, a carrier protein, a hypothetical protein, and a predicted protein. The number of positive sera reacted with CO extract was 18 in 800 donor blood (2.25%). In HMC-1 cells, a co-treatment of the positive sera containing CO-specific IgE with CO extract induced synergy effects in ERK/NF-kB activation compared with a sole treatment of human sera, but negative sera did not show a significant change. This results imply that herbal allergens can cause allergic reaction through the crosslinking with human serum IgE, and it may trigger hypersensitivity of body immune system.

Evaluation of human allergen-specific immunoglobulin G, total immunoglobulin E, hematological parameters, and lung function in mill workers exposed to grain dust in South-East Nigeria

Background The incidence of allergy among mill workers has been a public health concern over the years. The human allergen-specific immunoglobulin G (IgG) and the total immunoglobulin E (IgE) are markers for allergic disorders and are important in health monitoring of individuals exposed to allergens. Nonadherence to health and safety precautions by grain workers has led to an increase in respiratory symptoms and other pathologies. Aim The aim is to assess human allergen-specific IgG and total IgE concentrations, hematological parameters, and lung functions in factory workers exposed to grain dust for effective health monitoring. Patients and methods A total of 69 patients were enrolled; 29 of them were controls whereas 40 were mill workers. Blood samples collected from the patients were analyzed using Mindray autoanalyzer for full blood count, whereas ELISA method was used for the estimation of human allergen-specific IgG and total IgE. The spirometric lung function test was evaluated using Micro 1 Spirometer. Results Lung function was significantly decreased compared with the control (P<0.05) in values of forced vital capacity (%Pred) and forced expiratory volume in 1 s (%Pred). But no significant difference was observed for the forced expiratory volume in 1 s/forced vital capacity ratio (%Pred). Additionally, the hematological parameters showed an increase in red blood cells, hemoglobin, packed cell volume, mean corpuscular volume, mean corpuscular hemoglobin concentration, and mean corpuscular hemoglobin of mill workers compared with the control group (P<0.05). The total IgE in mill workers and the control group showed no significant difference; however, the human allergen-specific IgG was increased in mill workers than in the control group. Conclusions Human allergen-specific IgG level may be a better indicator of allergy in mill workers.

Digitized microimmunoassays with nuclear antigens for multiplex quantification of human specific IgE to β-lactam antibiotics

Microanalytical systems have huge potential for prognosis and diagnosis of diseases where the quantification of biomarkers is the critical factor. Immunoglobulin E (IgE)-associated allergy is the most common immune disorder and reliable multiplex quantification of specific IgE antibodies is of key importance for in vitro allergy diagnosis. Here, we report an unprecedented highly sensitive multiplex colorimetric microanalytical system for reliable quantification of ß-lactam-specific IgE. The biosensor microsystem includes a regular digital versatile surface where nuclear antigens are immobilized in microarray format to determine simultaneously specific IgEs to amoxicillin, penicillin G, piperacillin and aztreonam, using a hacked disc drive as the optoelectrical sensor. The analytical microsystem was successfully tested in a cohort of 101 human serum samples, showing a limit of detection of 0.06 IU/mL (144 pg IgE/mL), a clinical sensitivity of 42.3 % for amoxicillin, and an excellent specificity (100 %). The analytical performances were good as compared and validated with both in vivo and in vitro reference clinical immunofluorescence assay, settling a good correlation between the techniques. The compact microanalytical system can be easily implemented in primary care laboratory settings without the need for any additional instrumentation, facilitating massive drug allergy screening and delabeling campaigns to better define drug sensitization profiles.

Electrochemical Immunosensor for Human IgE Using Ferrocene Self-Assembled Monolayers Modified ITO Electrode.

The immunoglobulin E (IgE) level in serum is an important factor in the examination of allergy. Ferrocene (Fc)-modified self-assembled monolayers (SAMs) were placed on an indium tin oxide (ITO) electrode as a sensing layer for the detection of human IgE. The Fc moiety in the SAMs facilitated the electron transfer through the organic SAMs layer and electrocatalytic signal amplification. The electrochemical measurement was accomplished after the sandwich type immobilization of the receptor antibody, target human IgE, and enzyme conjugated secondary antibody. The enzyme product, p-aminophenol, was quantitatively analyzed by redox cycling via Fc. In addition, the electrochemical impedance spectroscopy (EIS) was investigated for the detection of IgE. The limit of detection (LOD), limit of quantification (LOQ), and dynamic range of the electrochemical sensor were 3 IU/mL, 10 IU/mL, and from 10 IU/mL to 100 IU/mL, respectively.

Development and Utility of an ELISA Method for Sensitive and Specific Detection of IgE Antidrug Antibodies.

Biologics can potentially induce unwanted immune responses, leading to formation of antidrug antibodies (ADA) of various affinity, isotypes, and subclasses. Among them, antigen and drug-specific immunoglobulin E (IgE) antibodies have been reported to have potential correlation with hypersensitivity and anaphylaxis in particular. Recent regulatory guidance on immunogenicity testing has recommended the measurement of antigen-specific IgE antibodies for biologics with a reported high risk of anaphylaxis using assays with sensitivities in the high pg/mL to low ng/mL range. Nevertheless, IgE ADA remains challenging to detect due to their being the least abundant isotype in blood serum samples and the potential for interference in the bioanalytical methods due to high levels of endogenous immunoglobulin G (IgG) and immunoglobulin M (IgM) ADA, not to mention the nonspecific total serum IgE antibodies. Another challenge in developing IgE ADA assays is the need to create a surrogate drug-specific IgE antibody positive control to monitor the performance of the assay for the intended use. In this case study, utilizing a human IgE antidrug antibody positive control and a human IgE receptor as capture, an enzyme-linked immunosorbent assay (ELISA) method was developed for the measurement of IgE ADA, meeting the regulatory expectations, with excellent assay sensitivity, selectivity, specificity, and tolerance towards potential interference in serum samples. This assay format could be readily adapted and implemented to assess drug-specific IgE antibodies in the event of drug-related anaphylaxis in clinical and in nonclinical development programs.

In Vitro and In Vivo Validation of EP2-Receptor Agonism to Selectively Achieve Inhibition of Mast Cell Activity.

PurposeAgonism of the prostaglandin E2 receptor, E-prostanoid receptor 2 (EP2), may represent an alternative protective mechanism in mast cell (MC)-mediated diseases. Previous studies have suggested that activation of the MC EP2 receptor prevents pathological changes in the murine models of allergic asthma. This work aimed to analytically validate the EP2 receptor on MCs as a therapeutic target.MethodsMurine MC lines and primary cultures, and MCs bearing the human immunoglobulin E (IgE) receptor were subjected to IgE-mediated activation subsequent to incubation with selective EP2 agonists. Two molecularly unrelated agonists, butaprost and CP-533536, were tested either in vitro or in 2 in vivo models of allergy.ResultsThe diverse range of MC populations was consistently inhibited through selective EP2 agonism in spite of exhibiting a heterogeneous phenotype. Such inhibition occurred in both mouse and human IgE (hIgE)-mediated activation. The use of molecularly unrelated selective EP2 agonists allowed for the confirmation of the specificity of this protective mechanism. This effect was further demonstrated in 2 in vivo murine models of allergy where MCs are a key to pathological changes: cutaneous anaphylaxis in a transgenic mouse model expressing the hIgE receptor and aeroallergen-induced murine model of asthma.ConclusionsSelective EP2 agonism is a powerful pharmacological strategy to prevent MCs from being activated through IgE-mediated mechanisms and from causing deleterious effects. The MC EP2 receptor may be an effective pharmacological target in allergic and other MC-mediated conditions.

No evidence for IgE receptor FcεRI expression on bronchial epithelial cells of asthmatic patients

Immunoglobulin E (IgE) plays an important role in the pathogenesis of asthma and anti-IgE therapy was approved for treating patients with severe persistent allergic asthma. The exact target sites of anti-IgE therapy are not well characterized; however, it has been proposed that the therapeutic effects of anti-IgE come from its intervention in the airway remodeling process. To gain insights on how anti-IgE therapy improves asthma symptoms, we aimed to validate the expression of FceRI on airway epithelial cells and demonstrate its role in airway remodeling. The expression of FceRI was measured (1) in situ in bronchial biopsy tissues of asthmatic and control subjects using immunohistochemistry and (2) in vitro in primary bronchial epithelial cells obtained from asthmatic subjects, at baseline and after treatment with human IgE, using qPCR and flow cytometry. FceRI expression in situ was detected only in a very small number of cells in the epithelium of bronchial biopsies of asthmatic and control subjects. In vitro measurement revealed no expression of the receptor both at baseline and after stimulation with IgE. The release of transforming growth factor—beta (TGF)-β and thymic stromal lymphopoietin (TSLP) were examined by ELISA in bronchial epithelial cells after crosslinking of IgE. No significant differences in TSLP and TGF-β protein levels were detected between stimulated and unstimulated cells. Hence, our data conclusively indicate that bronchial epithelial cells have negligible expression of functional high affinity receptor for IgE. Taken together, anti-IgE therapy is very likely to exert its therapeutic effects via other structural cell types.

Immunogenicity of a peptide-based anti-IgE conjugate vaccine in non-human primates.

The anti‐human immunoglobulin E (IgE) monoclonal antibody, omalizumab (Xolair®, Genentech, South San Fransisco, CA), is effective in the treatment of poorly controlled moderate to severe allergic asthma and chronic idiopathic urticaria. It acts by specifically binding to the constant domain (Cϵ3) of free human IgE in the blood and interstitial fluid. Although efficacious, use of omalizumab is limited due to restrictions on patient weight and pre‐existing IgE levels, and frequent dosing (q2–4 weeks). A vaccine inducing anti‐IgE antibodies has the potential for similar clinical benefits with less frequent dosing and relatively lower cost of goods. We developed a vaccine containing two IgE peptide‐conjugates targeting the Cϵ3 domain of human IgE. As part of preclinical evaluation of the vaccine to optimize formulation and dose prior to initiating clinical studies, we evaluated the vaccine in non‐human primates, and demonstrate the induction of anti‐peptide antibodies that can bind to conformationally intact human IgE and are capable, at least in some animals, of substantial lowering circulating IgE levels.

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.

SplitCore Technology Allows Efficient Production of Virus-Like Particles Presenting a Receptor-Contacting Epitope of Human IgE.

Immunoglobulin E (IgE) plays a central role in type I hypersensitivity including allergy and asthma. Novel treatment strategy envisages development of a therapeutic vaccine designed to elicit autologous blocking antibodies against the IgE. We sought to develop an IgE-epitope antigen that induces antibodies against a receptor-contacting epitope on human IgE molecule. We designed the VLP immunogens which utilize hepatitis B virus core protein (HBcAg) as a carrier, and present arrays of the receptor-contacting epitopes of the human IgE on their surfaces. FG loop from the IgE domain Cε3 was engineered into the HBcAg. Two constructs explore a well-established approach of insertion into a main immunodominant region of the HBcAg. Third construct is different in that the carrier is produced in a form of an assembly of two polypeptide chains which upon expression remain associated in a stable VLP-forming subunit (SplitCore technology). No VLPs were isolated from E.coli expressing the IgE-epitope antigens with contiguous sequences. On the contrary, the SplitCore antigen carrying the FG loop efficiently formed the VLPs. Immunization of mice with the VLPs presenting receptor-contacting epitope of the IgE elicited antibodies recognizing the human IgE in ELISA.

Development of an antibody that neutralizes soluble IgE and eliminates IgE expressing B cells.

Immunoglobulin E (IgE) plays a key role in allergic asthma and is a clinically validated target for monoclonal antibodies. Therapeutic anti-IgE antibodies block the interaction between IgE and the Fc epsilon (Fcε) receptor, which eliminates or minimizes the allergic phenotype but does not typically curtail the ongoing production of IgE by B cells. We generated high-affinity anti-IgE antibodies (MEDI4212) that have the potential to both neutralize soluble IgE and eliminate IgE-expressing B-cells through antibody-dependent cell-mediated cytotoxicity. MEDI4212 variants were generated that contain mutations in the Fc region of the antibody or alterations in fucosylation in order to enhance the antibody's affinity for FcγRIIIa. All MEDI4212 variants bound to human IgE with affinities comparable to the wild-type (WT) antibody. Each variant was shown to inhibit the interaction between IgE and FcεRI, which translated into potent inhibition of FcγRI-mediated function responses. Importantly, all variants bound similarly to IgE at the surface of membrane IgE expressing cells. However, MEDI4212 variants demonstrated enhanced affinity for FcγRIIIa including the polymorphic variants at position 158. The improvement in FcγRIIIa binding led to increased effector function in cell based assays using both engineered cell lines and class switched human IgE B cells. Through its superior suppression of IgE, we anticipate that effector function enhanced MEDI4212 may be able to neutralize high levels of soluble IgE and provide increased long-term benefit by eliminating the IgE expressing B cells before they differentiate and become IgE secreting plasma cells.

Development of human IgE biosensor using Sezawa-mode SAW devices

This paper reports Sezawa-mode surface acoustic wave (SAW) devices with via-isolated cavity to construct the allergy biosensor. To fabricate Sezawa-mode SAW devices, the RF magnetron sputtering method for the growth of piezoelectric ZnO thin films are adopted and influences of the sputtering parameters are investigated. The optimal substrate temperature of 300 °C, RF power of 120 W and sputtering pressure of 2 Pa were used to deposit piezoelectric ZnO films with a smooth surface, uniform grain size and strongly c-axis-orientated crystallization. A back-etched SAW resonator is used in this study. The wet etching of (100)-oriented silicon wafers is used to form a back-side cavity which is critical to the formation of a hopper cavity for holding bio-analytes. The remaining membrane structure silicon thickness was 25 μm. In this report, the chrome (Cr, 12 nm)/gold (Au, 66 nm) layer was initially deposited onto the sensing area of SAW devices as the binding layer for biochemical sensor. The resonance frequency of the Sezawa-mode SAW device is 1.497 GHz. The maximum sensitivity of the Sezawa-mode is calculated to be 4.44 × 106 cm2/g for human immunoglobulin-E (IgE) detection. The stability for human IgE detection is calculated to be 80% and the variation of the stability ±3% was obtained after several tests.

Human IgE flips between two acutely bent structuresviaan ensemble of extended conformations

Immunoglobulin E (IgE) antibodies mediate allergic reactions, and their binding to FcaRI is responsible for longterm sensitisation of mast cells and basophils [1]. Disruption of this interaction is a validated strategy for therapeutic intervention in allergic diseases including asthma [2]. IgE is known to display an acutely and asymmetrically bent conformation in the Fc region through which it binds to FcaRI; this bend becomes even more acute upon receptor engagement, as shown both crystallographically and in solution [3-7]. We report the crystal structure of a complex formed between IgE-Fc and two bound Fab fragments of a inhibitory anti-IgE antibody, and show that IgE-Fc can also adopt a totally extended conformation. The IgE-Fc has adopted a completely symmetrical conformation that requires an “unbending” of approximately 120° from the previously characterised structure. Molecular dynamics simulation reveals a series of stable conformations for free IgE-Fc that suggest a pathway from the acutely bent crystal structure through stable, extended conformations close to that seen in the Fab complex. We show by ITC, stoppedflow kinetic and FRET analyses that IgE-Fc adopts extended conformations in solution, and that these are an intrinsic property of IgEFc, not induced by Fab binding. We propose that IgE-Fc passes through these extended conformations as it flips between two bent conformations in which the CΣ2 domains fold back on opposite faces of the CΣ3-CΣ4 domains. The ability of IgE to exist in both bent and extended conformations may be essential for allergen recognition by IgE-Fc when bound to FcΣRI on the surface of mast cells, and as the B cell receptor respectively. Understanding the full range of conformations accessible to the free IgE molecule is also key to developing IgE-targeted therapies for allergic disease.

Effect of filgrastim (recombinant human granulocyte colony stimulating factor) on IgE responses in human asthma: A case study

RationaleThe role of peripheral blood progenitor cell mobilization on Immunoglobulin E (IgE) responses has not been studied. MethodsDistributions of blood lymphocytes (CD4+, CD8+, CD8+CD60+, CD19+, CD23+, CD16/56+, CD25, CD45RA+, CD45RO+, CD34+), and levels of serum immunoglobulins (IgM, IgG, IgA, IgE) were studied in an allergic asthmatic serum IgE+ (181IU/mL) adult (m/45 y/o) donor undergoing routine stem cell mobilization protocol (American Society of Hematology) before (day-30), during (day 4), and after (1wk post last dose) filgrastim (subcutaneous, 480 mcg, 2qd) treatment (flow cytometry, nephelometry, UniCAP Total IgE Fluoro enzyme immunoassay). ResultsOn day 4 of filgrastim treatment, numbers of CD8+CD60+T cells and CD23+ blood cells dramatically increased (98% and 240% respectively) compared with pre treatment. In contrast on day 4 of treatment, serum IgE levels decreased (>50%) compared with pre treatment. CD8+CD60+T cells and CD23+ blood cells and serum IgE levels approached pre-treatment levels at 1week post treatment. ConclusionsFilgrastim treatment transiently increases numbers of CD8+CD60+T and CD23+ expressing cells, which are known to regulate human IgE responses, while also transiently suppressing ongoing IgE responses. These results suggest that filgrastim affects IgE related responses, and may be useful in modulating allergic responses.

Doxycycline suppresses Chlamydia pneumoniae-mediated increases in ongoing immunoglobulin E and interleukin-4 responses by peripheral blood mononuclear cells of patients with allergic asthma

Chlamydia pneumoniae, an obligate intracellular bacterium, has been associated with asthma and the induction of immunoglobulin E (IgE) responses. Whereas tetracyclines have anti-chlamydial activity, their effect on human IgE responses to C. pneumoniae has not been studied. Peripheral blood mononuclear cells (PBMCs) from serum IgE+ allergic asthmatic subjects (n = 11) and healthy controls (n = 12) were infected with C. pneumoniae and cultured for 12 days with or without doxycycline (0.01-1.0 mg/L). IgE, interferon (IFN)-γ and interleukin (IL)-4 levels in supernatants were determined on days 1-12 post-infection, and C. pneumoniae DNA copy numbers in PBMC culture were measured on day 2 (quantitative PCR). C. pneumoniae-infected PBMCs from allergic asthmatic individuals had increased levels of IgE in supernatants compared with uninfected PBMCs (520% on day 10 post-infection, P = 0.008). IgE levels in PBMC cultures from controls were undetectable (<0.3 ng/mL). Increases in C. pneumoniae-induced IgE in asthmatics correlated with those of C. pneumoniae-induced IL-4 (r = 0.98; P < 0.001), but not with IFN-γ. The addition of doxycycline (1.0 mg/L) to the culture strongly suppressed the production of IgE (>70%, P = 0.04) and IL-4 (75%, P = 0.018), but not IFN-γ. The suppressive effect on IL-4 production remained significant even at concentrations of doxycycline that were subinhibitory (0.01 mg/L) for C. pneumoniae. In both asthmatic participants and controls, no significant effect of doxycycline on DNA copy numbers of C. pneumoniae was observed. Doxycycline suppressed the C. pneumoniae-induced production of IgE and IL-4, but not IFN-γ, in PBMCs from IgE+ allergic asthmatic subjects. These findings resulted from the immunomodulatory anti-allergic properties of tetracyclines.

Image-based ELISA on an activated polypropylene microtest plate—A spectrophotometer-free low cost assay technique

In this communication, we report ELISA technique on an activated polypropylene microtest plate (APPµTP) as an illustrative example of a low cost diagnostic assay. Activated test zone in APPµTP binds a capture biomolecule through covalent linkage thereby, eliminating non-specific binding often prevalent in absorption based techniques. Efficacy of APPµTP is demonstrated by detecting human immunoglobulin G (IgG), human immunoglobulin E (IgE) and Aspergillus fumigatus antibody in patient's sera. Detection is done by taking the image of the assay solution by a desktop scanner and analyzing the color of the image. Human IgE quantification by color saturation in the image-based assay shows excellent correlation with absorbance-based assay (Pearson correlation coefficient, r=0.992). Significance of the relationship is seen from its p value which is 4.087e-11. Performance of APPµTP is also checked with respect to microtiter plate and paper-based ELISA. APPµTP can quantify an analyte as precisely as in microtiter plate with insignificant non-specific binding, a necessary prerequisite for ELISA assay. In contrast, paper-ELISA shows high non-specific binding in control sera (false positive). Finally, we have carried out ELISA steps on APPµTP by ultrasound waves on a sonicator bath and the results show that even in 8min, it can convincingly differentiate a test sample from a control sample. In short, spectrophotometer-free image-based miniaturized ELISA on APPµTP is precise, reliable, rapid, and sensitive and could be a good substitute for conventional immunoassay procedures widely used in clinical and research laboratories.