Human serum substitution by artificial sera of scalable allergen reactivity based on polyclonal antibodies and chimeras of human FcγRI and IgE domains

Abstract

Human sera are the first choice as controls for diagnostic applications such as immunoassays, but are limited regarding availability, varying quality, and high costs. In this study, we aimed to circumvent these limitations by the use of a chimeric adaptor molecule comprising the extracellular domains of the human FcγRI (CD64) fused with human IgE Fc domains (CD64-IgE Fc). Allergen-specific antibodies were produced in rabbits using eight different allergens, extracts, and allergen mixtures including mites, pollen, drugs, and food. Preincubation of polyclonal IgG with CD64-IgE Fc established allergen-specific artificial sera that showed comparable results for more than 20 allergens and allergen extracts in three diagnostic systems for the determination of specific IgE. The agreement for these artificial sera is within ±1 radioallergosorbent test (RAST) class. Hence, rabbit IgG complexed with the IgG-specific CD64-IgE Fc adaptor molecule could provide a substitute for human reference sera with specificity for virtually any protein of interest.

Evaluation of the manufacturer-dependent differences in specific immunoglobulin E results for indoor allergens

“…The allergens are not the only stepping-stone toward improving the standardization of in vitro allergy diagnostics: The generation of molecularly well-defined reference samples could be a strong tool for external quality as well as the manufacturers' internal quality control. This could be achieved by using artificial constructs as recently published by Offermann et al 28 As proposed in our last paper, 14 we believe that a molecularly well-characterized serum would be the most natural and reliable sample, despite the higher production costs. Some voices believe precise characterization is impossible, but we think that the combination of a patient's clinical history and a quantitative evaluation of the IgE antibodies using different methods could be successful.…”

Bispecific Single-Domain Antibodies as Highly Standardized Synthetic Calibrators for Immunodiagnosis

“…The titration curve of the Az/Om calibrator was compared to that obtained with an artificial human serum (cIgE) (Dr. Fooke Laboratorien, Neuss, Germany), which was obtained by combining a fusion of the Fc domain of IgE and FcγRI (CD64) with aztreonam-specific polyclonal rabbit IgG. 7 One unit (1 IU/mL) of the Arthus aztreonam is equivalent to 1 IU/ mL of aztreonam-specific IgE.…”

“…The strategy commonly used for this purpose has been to combine the human Fc region of interest with the antigen-binding site of heterologous antibodies obtained by hyperimmunization with the test antigen. Initially, this approach was achieved by chemical conjugation of whole antibodies, but later these calibrators were produced as recombinant chimeric antibodies by joining the targeted human Fc region with Fab or scFv fragments specific for the antigen of interest. − The Fc region can also be combined with Fc receptors (FcγR) to build (FcγR) 2 Fc chimeras that can be loaded with hyperimmune polyclonal antibodies to the target antigen . While providing a solution to the problem of standardization, these constructs need to be expressed in eukaryotic systems and are laborious and expensive to produce.…”

“…4−6 The Fc region can also be combined with Fc receptors (FcγR) to build (FcγR) 2 Fc chimeras that can be loaded with hyperimmune polyclonal antibodies to the target antigen. 7 While providing a solution to the problem of standardization, these constructs need to be expressed in eukaryotic systems and are laborious and expensive to produce. In addition, the scFv components often present difficulties associated with their low stability and high propensity to aggregate, 8 which may require additional stabilization steps through optimization of the linkers, framework region, introduction of interchain disulfide bonds, etc.…”

Standardizing In Vitro β-Lactam Antibiotic Allergy Testing with Synthetic IgE

“…Conversely, establishing a serum bank from a diverse range of allergic patients with substantial levels of IgE antibodies is impractical due to limitations in size, reproducibility of serum pools, and the significant quantity of IgE-positive sera required for each specificity . Previous attempts to develop artificial human sera, such as chimeric adaptor molecules and specific bi-nanobodies, have involved animal immunization and lack the functional structure of IgE. − …”