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Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Summary Human immunoglobulin preparations are used therapeutically for various disorders. Such therapy is generally safe but adverse effects occasionally occur in recipients. It has been suggested that antibodies to cytokines present in clinical immunoglobulin products may contribute to undesirable effects in recipients. Therefore, we investigated intravenous and intramuscular immunoglobulin products for the presence of cytokine‐specific neutralizing antibodies. Using validated bioassays, we detected neutralizing activity against human granulocyte–macrophage colony‐stimulating factor (GM‐CSF), interferon‐α2a (IFN‐α2a) and interleukin‐1α (IL‐1α) in immunoglobulin products. We found no neutralization of granulocyte colony‐stimulating factor, macrophage colony‐stimulating factor, stem cell factor, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐6, IL‐9, IL‐10, IL‐12, tumour necrosis factor‐α, oncostatin M (OSM) and IFN‐γ. Most batches which neutralized IFN‐α2a activity also neutralized other IFN‐α subtypes, IFN‐ω and IFN‐β. Most products (94%) neutralized the biological activity of GM‐CSF. No correlation between batches and their ability to neutralize bioactivities of GM‐CSF, IFN‐α2a and IL‐1α was found. This neutralizing activity could be traced to plasma pools used for manufacture of immunoglobulins. The neutralization was mediated by specific cytokine antibodies contained within immunoglobulin products as it was present in specific immunoglobulin G (IgG) fractions eluted from cytokine affinity chromatography columns. Specific binding of such IgG fractions to cytokines in immunoblots and in enzyme‐linked immunosorbent assays (ELISAs) was observed. This contrasts with the broad non‐specific recognition of cytokine proteins observed using unfractionated immunoglobulins in ELISAs. This is the first comprehensive study showing the presence of neutralizing antibodies against GM‐CSF, IL‐1α, or IFN‐α2a in immunoglobulin products.

Intraperitoneal (i.p.) injections are widely used in laboratory animal experiments. This technique has a failure rate that is typically reported to be of the order of 10-20%. It is not apparent that failures of i.p. injection and their consequences for the experimental results are as widely recognized as the use of the technique. We illustrate the consequences of i.p. injection failure for the analysis and interpretation of several bioassays. We suggest approaches to data analysis that should be considered, and emphasize the need to recognize and allow for the possibility of i.p. injection failure in the analysis and interpretation of laboratory animal experiments involving this technique.

These results show that markers of cytokine release from both WBCs and platelets are useful indicators of the performance and efficacy of the WBC-reduction process and of platelet quality.