Allergens should be stored frozen in a -20ºC freezer. Avoid repeated freeze-thaw cycles.
Affinity chromatography, ion exchange or Immobilized Metal Affinity Chromatography are used in combination with size-exclusion chromatography for purification of natural, recombinant and LoTox™ allergens.
The mission of IBI is to provide its customers with the highest quality biologics. Our manufacturing laboratory operates under ISO 9001:2015 International Standard for Quality Management Systems certification.
Natural and recombinant allergens are supplied in various buffers (please see individual CoAs included with the product shipment).
LoTox™ allergens contain very low endotoxin levels below 0.03 EU/µg protein and are ideal for human and murine cellular studies using T-cells, APC’s or dendritic cells.
The LoTox™ product is ideal because it has very low endotoxin levels below 0.03 EU/µg protein and avoids the issue of whether the T-cell responses may be driven by endotoxin.
Allergen extracts are heterogeneous and contain non-allergenic proteins and other macromolecules. Investigators often use extracts because they tend to give high T cell responses, but the interpretation of these responses is difficult. The specificity of the response cannot be verified and it is quite possible that the responses could be driven by non-allergenic proteins or other substances. Thus, we recommend using purified allergens to measure, for example, IgE and IgG antibody responses, histamine release experiments and animal models. We strongly recommend LoTox™ products for any cellular studies, where endotoxin may influence the cellular response.
Recombinant allergens compare structurally very well to their natural counterparts and show comparable binding in IgE antibody assays. Pichia pastoris expressed recombinant proteins tend to be hyperglycosylated which may interfere with IgE binding. Thus, recombinant allergens expressed in Pichia pastoris are often engineered with a minor amino acid exchange to silence the glycosylation motif. Examples include rFel d 1 (RP-FD1D), rBla g 2 (RP-BG2) and rDer p1 (RP-DP1D.)
Der p 1 is activated by 5 mM cysteine or 1mM Dithiothreitol to regenerate its thiol group, which becomes oxidized during purification (Gough et al. Journal of Experimental Medicine, 1999).
DTT-dependent cysteine protease activity can be measured in a fluorescent assay using the fluorescent peptide substrate Boc-QAR-MCA.
DTT-independent serine protease activity can be measured in a fluorescent assay using the fluorescent peptide substrate Boc-QGR-MCA.
The shrimp used for manufacturing of the shrimp tropomyosin are Carolina shrimp sourced from a local seafood market and are boiled before extraction.
The seasons for white (Litopenaeus setiferus) and brown shrimp (Litopenaeus aztecus) in the Carolina’s overlap and thus the shrimp we buy are likely a mixture of these two species. This is why we do not use the allergen nomenclature for this product.
Yes, the validation process for each new allergen product includes testing for IgE binding using allergic patient sera.
Purity of manufactured allergen products is assessed using an in-house mass spectrometry facility. In addition, allergens will be analyzed by SDS-PAGE.
Recombinant allergens are expressed mainly using Pichia pastoris and E.coli. A mammalian expression system has recently been established as well.
