The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

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.

Allergy vaccines: a need for standardisation in mass units of major allergen.

Treatment of respiratory allergies can be performed with allergen-specific immunotherapy using allergen extracts. These products are biologicals with an extremely complex and variable composition. Only a few components are of major importance for the disease, the so-called major allergens. At present, standardisation of allergen extracts is dominated by techniques that aim at establishing their overall IgE-binding potencies using pooled sera of allergic patients. Each company in the market uses its own type of units to express potencies, thus hampering comparability. Another disadvantage is that the major allergen composition is not determined. Most companies have introduced assays for the measurement of major allergens in their quality control systems, but these data are not yet used for labelling purposes. The need to include major allergen content in standardisation protocols is now widely accepted. To support future labelling on the basis of major allergen content the European Union has funded the multidisciplinary multicentre project CREATE. This project aims at developing international certified references for the most important major respiratory allergens and at evaluating the performance of available ELISA for their measurement. The project will facilitate expression of potencies by active ingredient (major allergen) content and will allow direct comparison of competitor products.

A new approach to sterilization conditions. The IMO concept.

The sterilization requirements for medical/pharmaceutical applications are traditionally based on an extensive overkill. In the past few years, however, an evolution towards bioburden related sterilization processes has been started (F0 theory). Especially manufacturers of large volume parenterals have - forced by the thermolability of the product - contributed to this development. In this paper both philosophies are combined, resulting in a concept in which the bacteriological and physical bases of the sterilization process are mathematically related by using the F0 theory and by introducing an Imaginary Micro-Organism (IMO). The IMO concept provides the opportunity for anyone in the field of sterilization to raise the quality control level, which can be achieved by: - selecting optimum sterilization conditions without performing pre-sterilization counts: - step by step introducing the pre-sterilization count which results in even more favourable sterilization conditions.

Analysis of factors influencing the Bowie-Dick-type test.

Investigations were conducted to determine the air-detecting capacity of the Bowie-Dick-type test for prevacuum steam autoclaves. The results indicate that the limitations in air detection are related to the specific type of sterilizer and test procedure being used, rather than some theoretical consideration. The chemical indicators on the test sheet appear to respond to incomplete steam penetration, rather than to the presence of air. The so-called small-load effect should be attributed to other than the commonly accepted causes. It appears that the causes of the small load effect are (a) air trapped by a condensate layer, and (b) the reluctance of steam a and air to mix.

Transport of amino acids in membrane vesicles of Rhodopseudomonas spheroides energized by respiratory and cyclic electron flow.

Active transport of amino acids in whole cells and membrane vesicles from the facultative photo-synthetic bacterium Rhodopseduomonas spheroides is coupled to electron flow in the respiratory chain and in the cyclic electron transfer system. In vesicles made from cells grown aerobically in the dark transport of amino acids is most effectively energized by the oxidation of NADH and to a lesser extent by ascorbate or succinate in the presence of N,N,N',N'-tetramethyl-1, 4-phenyldiamine dihydrochloride or by ascorbate + phenazine methosulphate via the respiratory chain with oxygen as terminal electron acceptor. In membrane vesicles from cells grown anaerobically in the light the energy for active transport of amino acids is supplied upon illumination by photo-oxidation of bacteriochlorophyll and subsequent electron flow through the cyclic electron transfer system. The initial rate of transport increases with the light intensity upto saturation levels. In both vesicle preparations, inhibitors of electron transfer flow inhibit amino acid uptake. In order to obtain light-driven amino acid transport the isolation of membrane vesicles needs to be performed in a medium with a redox potential between 0 and 100 mV. Illumination of vesicles made from cells grown anaerobically in the light results in the generation of a membrane potential as is indicated by the uptake of the lipophylic cation triphenyl-methylphosphonium.