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.

Implications for the assay and biological properties of interleukin-3. Results of a WHO international collaborative study.

Five preparations of interleukin-3 (IL-3) have been evaluated by 28 laboratories in 12 countries for their suitability to serve as an international standard for this material in a joint international collaborative study for IL-3 and interleukin-4 (IL-4). The preparations were assayed in a wide range of in vitro bioassays and immunoassays. It is clear from the biological assays contributed to this study that different recombinant preparations of IL-3 can have very different biological specific activities, including those from the same source (i.e., E. coli). Biological assays of IL-3 were significantly more consistent in their estimates of levels of IL-3 than the immunoassays, suggesting an unusual pattern of epitope recognition amongst the antibodies included in the immunoassays. This study also illustrates the point that the level of cytokine measured by immunoassay does not necessarily reflect the biological potency of the cytokine. On the basis of results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-3 (91/510) was established as the international standard for interleukin-3 with an assigned unitage of 1700 IU/ampoule.

Implications for the assay and biological activity of interleukin-4. Results of a WHO international collaborative study.

Five ampouled preparations of interleukin-4 (IL-4) have been evaluated by 36 laboratories in 14 countries for their suitability to serve as an international standard for this material in a joint international collaborative study for interleukin-3 (IL-3) and IL-4. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. It is clear from the study that different recombinant preparations of IL-4 can have very different biological specific activities, including those from the same source (i.e., E. coli). In addition, immunoassay estimates of IL-4 levels did not correlate with those of bioassays, illustrating the fact that immunoassays do not necessarily measure biologically active cytokine. It is of interest that the estimates provided by the different bioassays were less variable than those produced by the immunoassays, suggesting that bioassays can be as accurate, if not more so, than immunoassays. The large reduction in the variability of estimates with the inclusion of a single reference preparation clearly illustrates the need for a single standard to assay IL-4. On the basis of the results reported here, with the agreement of the participants of the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) the preparation of IL-4 (88/656) was established as the international standard for interleukin-4 with an assigned unitage of 1000 IU/ampoule.

The international standard for granulocyte colony stimulating factor (G-CSF). Evaluation in an international collaborative study. Participants of the Collaborative Study.

Three ampouled preparations of granulocyte colony stimulating factor (G-CSF) have been evaluated by 29 laboratories in 11 countries for their suitability to serve as international standards for these materials. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here with the agreement of the participants in the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) one of the preparations (88/502) was established as the international standard for G-CSF.

The international standard for macrophage colony stimulating factor (M-CSF). Evaluation in an international collaborative study.

Three ampouled preparations of macrophage colony stimulating factor (M-CSF) were evaluated by 23 laboratories in nine countries for their suitability to serve as international standards for this material. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here, with the agreement of the participants in the study and with the authorization of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO), the preparation in ampoules coded 89/512 was established as the international standard for M-CSF.

The international standard for granulocyte-macrophage colony stimulating factor (GM-CSF). Evaluation in an international collaborative study. Participants of the Collaborative Study.

Two ampouled preparations of granulocyte-macrophage colony stimulating factor (GM-CSF) have been evaluated by twenty nine laboratories in eleven countries for their suitability to serve as international standards for these materials. The preparations were assayed in a wide range of in vitro bioassays and immunoassays. On the basis of results reported here, with the agreement of the participants in the study and with the authorisation of the Expert Committee on Biological Standardization (ECBS) of the World Health Organization (WHO) one of the preparations (88/646) was established as the international standard for GM-CSF.

WHO cytokine standardization: facilitating the development of cytokines in research, diagnosis and as therapeutic agents.

The development and widespread application of recombinant DNA technology has dramatically increased the number of cytokines available for clinical evaluation. New and novel cytokines are being discovered, cloned and entered into clinical trials at such a rate that it is often the case that the biological activities of these proteins are poorly understood during their development as therapeutic agents. In addition, manufacturers of any one cytokine can produce the protein from different cellular sources resulting in materials that exhibit markedly different specific activities. When estimating the amount of biological activity of different preparations with different specific activities by bioassay, mass units cannot be used and biological activity is therefore expressed as 'biological potency units'. The biological unit requires definition by a standard that is assay-independent (especially when measuring a particular type of biological activity). In many cases, a variety of assay methods will be available and the material chosen for a standard should ideally be suitable for use with as many of them as possible. Once the unit is defined, this can be used in any laboratory, thus providing a means of ensuring uniformity throughout the world in the designation of potency of different biological preparations. The World Health Organisation (WHO) standardization programme involves the production of biologically stable, well characterised potency and immunoassay standards that are available world-wide using a single international unitage. Over the years, WHO international standards have been used to dramatically reduce the variation in estimates of cytokine preparations within and between laboratories for immunoassays and bioassays. WHO international standards are primary reference preparations against which secondary, or working standards (including regional standards, national standards, pharmacopoeial standards and in-house working standards) can be calibrated.

Catalytic properties and stability of lipase purified from human pancreatic juice.

Catalytic properties of a preparation of human pancreatic lipase purified from pancreatic juice have been compared to those of the enzyme present in pooled plasma from patients suffering from acute pancreatitis. They were very similar as regards influence of effectors (sodium deoxycholate, colipase and Ca2+), optimal pH and apparent KM in optimized conditions. The stability of the preparation appeared to be satisfactory. It was found to be stable for at least 200 days in a liquid form at +4 degrees C and predictive degradation rates per year of the lyophilized form at +4 degrees C and -20 degrees C were 0.06% and 0.00%, respectively. The close similarity of properties of this preparation with those of a recombinant human pancreatic lipase produced in V79 Chinese hamster lung cells suggests that both approaches (purification from human pancreatic juice and gene transfer technology) could be used to produce a suitable reference material for this enzyme.

Biological standardization of cytokines and growth factors.

To quantify the biological activity of different cytokine preparations with different specific activities by bioassay, mass units cannot be used and biological activity has to be expressed as << biological potency units >>. The biological unit requires definition by a standard that is assay-independent (especially when measuring a particular type of biological activity). Once the unit is defined, it can be used in any laboratory, thus providing a means of ensuring uniformity throughout the world in the designation of potency of different biological preparations. The World Health Organisation (WHO) standardization programme involves the production of biologically stable, well-characterised potency and immunoassay standards that are available world-wide using a single international unitage. These international standards have been used to reduce dramatically the variation in estimates of cytokine preparations within and between laboratories for immunoassays and bioassays.

The development of a World Health Organisation international standard for islet cell antibodies: the aims and design of an international collaborative study.

Islet cell antibodies (ICA) are a specific marker for Type 1 (insulin-dependent) diabetes mellitus. ICA are found in the serum of over 80% of newly diagnosed patients and the levels of ICA are directly of prognostic value. Standardisation of ICA and the uniform reporting of ICA levels in international units is critical to preclinical/clinical research and the development of assays for ICA as diagnostics, in particular for the differential diagnosis of late onset Type 1 and Type 2 diabetes. Proficiency studies carried out by the Immunology of Diabetes Workshops on Standardization have clearly shown that a single reference material, serum sample 673, obtained by Dr J. Ludvigsson, has significantly reduced inter- and intra-assay variability in the reporting of ICA levels. Nevertheless, this material is a frozen serum of limited shelf-life and is difficult to distribute on a worldwide and routine basis. Therefore, the Immunology of Diabetes Workshop Standardization Committee and the Juvenile Diabetes Foundation International requested that the National Institute for Biological Standards and Control (NIBSC) organise an international collaborative study to compare the activities of lyophilised, stable ICA preparations. In addition, the purpose was to investigate if sample 673 could also serve as a standard for GAD65 and IA-2 antibodies. Twenty participants in eight countries have been recruited to the study.

Refinement and validation of an alternative bioassay for potency testing of therapeutic botulinum type A toxin.

The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurones. This property has led to the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms. At present, the only recognised assay with the specificity and sensitivity to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the end point. Refinement of this assay, with respect to the end point, was explored on the basis of the development of flaccid paralysis of muscles following subcutaneous injection of the toxin at the inguinocrural region. Potency estimates, relative to in house reference preparations, for different therapeutic preparations obtained using flaccid paralysis as a scored response gave excellent agreement with estimates obtained in independent assay using the currently required control method. This study demonstrates that an alternative, more humane bioassay for potency testing of clostridia neurotoxins gives valid estimates equivalent to those currently in use.

Laboratory testing of whole cell pertussis vaccine: a WHO proficiency study using the Kendrick test.

Whole cell pertussis vaccine (WCV), commonly in combination with vaccines for diphtheria and tetanus, has an important role in reducing morbidity and mortality among children in most parts of the world. Testing to assure the efficacy of such vaccines is essential. We have, therefore, carried out, under the Global Training Network (GTN) of the Department of Vaccines and Biologicals at the World Health Organization (WHO), a proficiency study involving 13 laboratories in 12 countries that routinely test WCV. Two vaccine samples were tested in this study and represented samples which were expected clearly either to pass (sample B, a full strength vaccine) or to fail (sample A, 1/8 strength of vaccine B). Data from this study showed good performance by the majority of participants. Most assays were statistically valid and were carried out to the level of precision achieved for these assays in previous studies. This study also indicated that, relative to the assay precision, the in-house reference (IHR) preparations are in general accurately calibrated. Statistically valid assays of the sub-potent vaccine, A, showed it to fail in all except one laboratory. Statistically valid assays of the potent vaccine, B, showed it to pass in all laboratories. Nevertheless, the between laboratory variability of estimates for vaccine B, and for comparisons of the two vaccine samples suggested that there are some differences in results in different laboratories. The introduction of a common working standard may assist in reducing inter-laboratory variation. This study has shown clearly satisfactory performance by most laboratories. However, a serious problem was detected in one laboratory where the sub-potent vaccine A could have been passed and was not distinguished from the eight-fold more potent vaccine B. There were also indications of possible problems in several other laboratories, where IHR preparation may not be accurately calibrated or where vaccine samples A and B may not be completely distinguished. Although this study provides reassurance that most laboratories perform well, it demonstrates the essential role of ongoing proficiency studies in high-lighting problems.

Aluminium-adjuvanted vaccines transiently increase aluminium levels in murine brain tissue.

Aluminium is widely used as an adjuvant in human vaccines, and children can often receive up to 3.75 mg of parenteral aluminium during the first six months of life. We show that intraperitoneal injection of aluminium adsorbed vaccines into mice causes a transient rise in brain tissue aluminium levels peaking around the second and third day after injection. This rise is not seen in the saline control group of animals or with vaccine not containing aluminium. It is likely that aluminium is transported to the brain by the iron-binding protein transferrin and enters the brain via specific transferrin receptors.

Neutralizing antibodies to granulocyte-macrophage colony-stimulating factor, interleukin-1alpha and interferon-alpha but not other cytokines in human immunoglobulin preparations.

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-alpha2a (IFN-alpha2a) and interleukin-1alpha (IL-1alpha) in immunoglobulin products. We found no neutralization of granulocyte colony-stimulating factor, macrophage colony-stimulating factor, stem cell factor, IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-12, tumour necrosis factor-alpha, oncostatin M (OSM) and IFN-gamma. Most batches which neutralized IFN-alpha2a activity also neutralized other IFN-alpha subtypes, IFN-omega and IFN-beta. Most products (94%) neutralized the biological activity of GM-CSF. No correlation between batches and their ability to neutralize bioactivities of GM-CSF, IFN-alpha2a and IL-1alpha 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-1alpha, or IFN-alpha2a in immunoglobulin products.

An aerosol challenge model of Bordetella pertussis infection as a potential bioassay for acellular pertussis vaccines.

Whole cell and five different types of acellular pertussis vaccine were assayed using a mouse aerosol challenge model which permitted delivery of a controlled, consistent dose of Bordetella pertussis to the lower respiratory tract. Using this system, the viable counts in the lungs of vaccinated mice were immunisation dose-dependent and allowed the protective capacity of different vaccine preparations to be distinguished. This model may thus provide the basis for a protection assay for pertussis vaccines. Comparison of acellular vaccines with a whole cell pertussis vaccine showed that the latter gave better active protection in mice but with a different dose-response relationship. Thus the two types of vaccine are not directly comparable in the same assay and require different reference standards. A pentavalent type acellular vaccine is suggested as a possible candidate standard for the acellular vaccine potency test. The results suggest that this aerosol challenge model has potential for use as a potency test for acellular pertussis vaccines.