Artificial human sera: a breakthrough?
Authors: R. Crameri
Journal: Allergy (2016)

It is not common that a Brief Communication is chosen for an Editorial; however, the work presented by Offermann et al. 1 deserves to be commented. Obviously, the in vitro diagnosis of allergic sensitization is based on the determination of soluble allergen-specific IgE in serum 2. Although the correlation between sIgE and allergic sensitization might be disturbed by the presence of clinically irrelevant cross-reactive carbohydrate determinants 3, in most cases allergen-specific IgE in serum can be correlated with the symptomatic 4. Undoubtedly, biotechnology has strongly contributed to improve the in vitro diagnosis of allergy 5, but despite many efforts undertaken, not all problems have been solved. It is well known that the quality of allergen extracts is subject to huge variations depending, among others, on the starting raw material, extraction, standardization procedures and storage conditions used. This makes a comparison of extracts offered by different manufacturers difficult. Here, the development of component-resolved IgE tests and the use of monoclonal antibodies represent a promising approach to improve this unsatisfactory situation 6. However, at present, the recombinant allergens available are unlikely to cover the entire complexity of natural extracts, and monoclonal antibodies cannot cover the huge number of IgE-binding epitopes elicited during the development of allergic sensitization in predisposed individuals. During the past two decades, a major focus has been devoted to the development of international standards for recombinant allergens to be used for the standardization of allergen extracts summarized in the CREATE study 7. This pivotal project clearly showed the feasibility of producing fully standardized recombinant allergens under good manufacturing practice (GMP) conditions satisfying the requirements not only for diagnostic but also for therapeutic use. Of course, the production of standardized recombinant allergens as European Pharmacopoeia allergen reference standards characterized extensively at the physicochemical and immunological levels is tedious and time-consuming 8. However, concentrated efforts during the last few years have generated European Pharmacopoeia reference standards for the recombinant Phl p 5.0109 9 and Bet v 1.0101 allergens 10 which are available from the European Directorate for Quality of Medicine and Healthcare. The production of equivalent standards for the long list of major allergens needed to cover the most important allergenic sources including pets, foods, pollens and moulds, to mention only the most prominent ones, will require huge efforts in basic research and standardized industrial production of these allergens under GMP condition during the next years. This goal can only be reached with the support of the manufacturers, but is technically feasible. However, for the development and validation of standardized in vitro diagnostic tests, the availability of perfectly standardized recombinant allergens is essential but not sufficient. As pointed out in the CREATE report, the development of a serum bank from a broad spectrum of allergic patients with a wide range of IgE antibody levels is essential as consistent reference standard for the determination of sIgE levels in allergen preparations and is now available for research purposes 7. Obviously, such a serum pool is of limited size, unique because almost impossible to be reproduced, would require litre quantities of IgE positive sera for each specificity and, therefore, on a long term impracticable 11. Offermann et al. 1 propose the establishment of artificial human sera (ARTHUS) as a further step in the development of reproducible reagents for the standardization of allergen extracts. In a first step, they fused the extracellular domains of the human high-affinity IgG receptor FcγRI to the constant human IgE HCε1 to HCε4 domains to produce a recombinant adaptor molecule able to interact with IgG antibodies of different species. In a second step, polyclonal allergen-specific IgG antibodies were produced by immunization of rabbits either with pure recombinant or native allergens, as well as with whole allergen extracts. Finally, preincubation of purified polyclonal rabbit IgG with the adaptor molecule generated artificial allergen-specific reagents (ARTHUS), claimed to be useful as substitute for human serum pools. The concept might not be easy to understand for people not daily involved in molecular biology; therefore, I will try to explain it in more detail. The production of the adaptor molecule secreted into the supernatant of Hek-293 cells and the purification of the protein is straightforward and, if needed even under GMP conditions, completely reproducible. Immunization of rabbits with any protein or allergen extracts is a standard procedure to generate polyclonal IgG responses against the proteins of interest. Of course, the immune response will vary from animal to animal; however, methods for the purification of the IgG fraction from the sera as well as for their quantification using a wide variety of commercially available mono- and polyclonal antibodies are well-established procedures. Therefore, every new batch of rabbit anti-IgG allergen can be quantified and adjusted to the desired concentration resulting in a standardized reagent. Mixing defined quantities of the CD64-IgE adaptor molecule with purified rabbit-derived polyclonal IgG antibodies raised against the allergen of interest generates a reagent mimicking the polyclonal IgE response of allergic individuals to allergen exposure. The so-called artificial sera can be used in both, semi-automated direct and reverse assays to quantify single and/or multiple allergens present in extracts (Fig. 1). As expected, artificial sera raised against highly pure natural or recombinant allergens works better than those raised against allergen extracts. In fact, the immune response of rabbits against extracts will vary also in dependence of the quantity of the different allergens present which, as demonstrated for commercial preparations of different companies, is subject to huge variations also for major allergens as shown for example for the fungal allergens Alt a 1 and Asp f 1 12. Although the performance of artificial sera as a tool for the quality control of allergens and allergen extracts needs to be confirmed in independent studies, as usual for any new technology, I consider this work a further major step allowing quantification of the allergen content in extracts used in diagnosis and therapy of allergic diseases. If needed, the extracts can be spiked with pure native or recombinant allergens present in suboptimal concentrations to obtain reagents with a constant and optimized concentration of the most important single allergenic components 13. However, we should be aware of the fact of the lack of clinical symptoms in more than 20% of the individuals with allergen-specific serum IgE and/or positive skin test reactivity 13 cannot be overcome even with perfectly standardized allergen preparations. All in vivo and in vitro tests for ‘allergy’ are subject to inherent variation and potential interferences and, even if perfectly standardized, show only sensitization to allergens which is not necessarily linked to clinical symptoms 14. The laboratory of the author is supported by the Swiss National Science Foundation grants no. 3200_146247 and 32NM30_136032/1 (EuroNanoMed), and by the Swiss Commission for Technology Innovation (CTI) grant No. 16009.1 PFLS-LS. The authors declare that they have no conflicts of interest.