Investigators: K Maghni1, DSc, PhD, A Cartier1, MD, D Gautrin1, PhD, R Castaño1, MD, MSc,, J H Bonlokke2, MD, PhD, T Sigsgaard2, MD, PhD. 1Asthma in the Workplace Centre; 2 Aarhus University, Denmark. Summer student: Marie-Eve Rochon, first year medical school program, Université de Sherbrooke.
Introduction: The detection of specific IgEs is an important step in the diagnosis of sensitization to occupational agents. RIA and ELISA quantification of specific IgEs consist on the coating of the occupational agent (purified recombinant proteins, protein extracts or chemical conjugated to a carrier) on a solid-phase (e.g. into microplate wells for ELISA) followed by the incubation with dilutions of sera, and finally the detection and quantification of specific IgEs bound to the coated occupational agent. High molecular weight (HMW) sensitizing occupational agents (e.g. snow crab) induce both a specific IgE and IgG antibody response whereas low molecular weight (LMW) agents produce mainly an IgG antibody response, the production of specific IgE being still controversial for some agents (e.g. diisocyanates). The production of specific IgEs over specific IgGs (including IgG1, IgG2, IgG3 and IgG4) is in a proportion of 1:100 to 1:1,000 depending on the chemical nature of the sensitizing occupational agent examined and differences in antibody responses amongst workers. Thus, based on a simplified scenario where IgGs and IgEs have similar affinities for antigenic epitopes, the probability for serum specific IgEs to bind the coated occupational agent (and to be detected) is 1:100 to 1:1,000 over the binding of specific IgGs. Therefore, it is likely that the low or the absence of detection of sera specific IgEs in solid-phase-based assays is attributable to their “weak” competition with IgGs for binding coated occupational agents. Dr. Cartier and co-investigators have been funded by the AWPC in 2007 for a study aiming at better understanding snow crab asthma and allergy in Greenland snow crab processors. Preliminary results show that a high proportion of workers had skin test reactivity to snow crab allergens; sera were also collected from these participants for total and specific IgEs analysis.
Objectives. To develop and validate a novel procedure to increase sensitivity threshold detection of specific IgEs in solid-phase-based assays. This procedure will consist on the removal of total (including specific) IgGs prior to assay sera for the quantification of specific IgEs. Sera obtained from snow crab processors will be analysed using this novel procedure.
Methods: This novel procedure will include the following steps: 1) quantification of total IgGs and IgEs in sera, 2) specific depletion of total IgGs, 3) quantification of total IgGs and IgEs after IgGs removal and 4) quantification of snow crab specific IgEs in sera and IgG-depleted sera by ELISA. 1- Quantification of total IgGs and IgEs in sera. Total levels of IgEs and IgG isotypes (IgG1, IgG2, IgG3 and IgG4) will be quantified using the Bio-Plex® Pro assays human isotyping panel kit (Bio-Rad) with our Bio-Plex workstation based on the Luminex technology. 2- Specific depletion of total IgG. This will be performed using the ProMax serum IgG removal kit according to Polysciences Inc.’s instructions. This kit is based on the use of BioMag® superparamagnetic particle technology (ProMax IgG removal particles) and the application of a magnetic field for the removal of particles bound-IgG from human sera. 3- Quantification of total IgGs and IgEs after IgGs removal. The efficiency of the IgG depletion process (without affecting IgE levels) will be determined by measuring total IgEs and IgGs in IgG-depleted sera using the Bio-Plex workstation as described above. Because levels of IgGs are likely to be different amongst workers, and thus, may be over the ProMax serum IgG removal kit dynamic range, a second run of IgGs depletion will be performed in samples still containing IgGs after the first depletion. IgG-re-depleted sera will be then re-assayed for total IgGs and IgEs levels. 4- Quantification of snow crab specific IgEs in sera and IgG-depleted sera by ELISA. In the context of Dr. Cartier’s 2007 AWPC project, we have already prepared concentrated extracts from raw and cooked snow crabs, and cooking water. Dr. Cartier’s project aiming quantification and comparison of specific IgEs’ reactivity toward proteins of raw versus cooked snow crab and cooking water, three ELISA will be built for each extracts. Briefly, extracted snow crab proteins will be diluted in carbonate buffer and coated in high binding ELISA plates (Microlon) overnight at 4 °C. Following washing steps, dilutions of sera and IgG-depleted sera will be incubated in the ELISA microplate, and bound IgEs will be detected with horseradish peroxidase (HRP)-labelled goat anti-human IgE and quantified by adding HRP soluble substrate TMB. Optical densities will be then recorded for the relative quantification of IgEs.
Significance: Depleting IgGs in sera is likely to increase sensitivity threshold detection of specific IgEs in solid-phase-based immunoassays, using snow crab allergy as a model for HMW agents. This novel procedure could then be applied for the quantification of specific IgEs to other HMW and also to LMW sensitizing occupational agents.
Budget: 20,000 $ is requested for technician time (including benefits), the cost of the BioMag Microcentrifuge Tube Separator (Polysciences Inc.) requested to run the ProMax serum IgG removal kit, and costs for kits and consumables.