Validation of ELISA methods for quantification of the major birch allergen Bet v 1 (BSP090).

To date, the potency of allergen products in Europe is expressed in manufacturer-specific units relative to a product-specific in-house reference. Consequently, cross-product comparability of allergen products from different manufacturers with respect to strength and efficacy is impossible. The Biological Standardisation Programme (BSP) project BSP090 addresses this issue via the establishment of reference standards in conjunction with ELISA methods for the quantification of major allergens in allergen products. Since the initiation of BSP090, the recombinant major allergen Bet v 1 has been adopted by the European Pharmacopoeia Commission as a Chemical Reference Substance (CRS). In parallel, two sandwich ELISA systems for quantification of Bet v 1 were found suitable in preliminary phases of BSP090 to be validated in a large collaborative study. In this study, the candidate ELISA systems were compared with respect to accuracy, precision and variability. Thirteen participating laboratories tested model samples containing the CRS as well as spiked and unspiked birch pollen extracts. Both in pre-testing and in the collaborative study, the 2 candidate ELISA systems confirmed their suitability to quantify recombinant and native Bet v 1. As no clear-cut decision for one of the ELISA systems could be made based on the results of the collaborative study, a post-study testing was performed. Bet v 1 content of 30 birch pollen allergen products was determined in parallel in both ELISA systems. Consequently, 1 candidate ELISA system was selected to be proposed as the future European Pharmacopoeia standard method for Bet v 1 quantification.

Standardization of allergen products: 3. Validation of candidate European Pharmacopoeia standard methods for quantification of major birch allergen Bet v 1.

BACKGROUND: The BSP090 project aims at establishing European Pharmacopoeia Reference Substances in combination with the corresponding ELISA methods for the quantification of major allergens in allergen products. Two sandwich ELISAs proved suitable for quantification of Bet v 1, the major birch pollen allergen, in preceding phases of BSP090. METHODS: Two Bet v 1-specific ELISA systems were compared with respect to accuracy and precision in a ring trial including 13 laboratories. Model samples containing recombinant rBet v 1.0101 as well as native birch pollen extracts were measured independently at least three times in each facility. The assessment was completed with a comparative quantification of Bet v 1 in 30 marketed birch allergen products in one laboratory, simulating the future use as reference method. RESULTS: In the collaborative study, both candidate ELISAs confirmed their suitability to quantify recombinant and native Bet v 1. ELISA-A showed higher precision and lower interlaboratory variability, yet ELISA-B exhibited slightly higher accuracy. Subsequent parallel measurement of Bet v 1 in a panel of 'real-life' birch allergen products indicated better repeatability of ELISA-B. Both systems detected substantial differences in Bet v 1 content between allergen products, but the effect was more pronounced using ELISA-B due to persistently higher values compared to ELISA-A. CONCLUSIONS: In the collaborative study, no deciding differences were observed between the two candidate ELISAs. Further comparison under conditions simulating the intended use combined with the criterion of long-term availability enabled the selection of one Bet v 1-specific ELISA for proposal as European Pharmacopoeia standard method.

Thyroid cancer among Ukrainians and Belarusians who were children or adolescents at the time of the Chernobyl accident.

Our objective is to assess the regional and temporal dependences of the baseline cases contributing to thyroid cancer incidence among those exposed in childhood or during adolescence in Belarus and Ukraine after the Chernobyl accident. Data are analysed for Kyiv and Sevastopol City and the 25 oblasts (regions) in Ukraine, and for Minsk and Gomel City and the 6 oblasts in Belarus. Average thyroid doses due to the Chernobyl accident were assessed for every birth year in the period from 1968 to 1985. Case data pertain to people who underwent surgical removal of thyroid cancers during the period 1986 to 2001 and who were allocated to their place of residence at the time of the accident. The 35 oblasts/cities were subdivided into an upper, middle and lower group of baseline thyroid cancer incidence. Poisson regressions were performed to estimate age, time and gender dependences of the baseline incidence rates in the three groups. The majority of oblasts/cities with high average doses and the majority of Belarusian oblasts/cities belong to the upper group of baseline thyroid cancer incidence. The baseline in the upper group is estimated to be larger than in the middle group by a factor of 2.3, and by a factor of 4.0 when compared to the lower group. The baseline incidence increases with age and with time since exposure. Estimated baseline incidence rates were found to increase from 1988 to 1999 by factors of three and two for the upper and the two lower groups respectively. The estimated thyroid cancer incidence rates in Belarus and Ukraine, and their dependences on gender and age, are consistent with observed rates found in the larger cancer registries of other countries. In conclusion, the baseline cases are found to contribute about 70% to the thyroid cancer incidence in Ukraine, and about 40% to the incidence in Belarus.

Thyroid cancer risk in areas of Ukraine and Belarus affected by the Chernobyl accident.

The purpose of the present study was to analyze the thyroid cancer incidence risk after the Chernobyl accident and its degree of dependence on time and age. Data were analyzed for 1034 settlements in Ukraine and Belarus, in which more than 10 measurements of the (131)I content in human thyroids had been performed in May/June 1986. Thyroid doses due to the Chernobyl accident were assessed for the birth years 1968-1985 and related to thyroid cancers that were surgically removed during the period 1990-2001. The central estimate for the linear coefficient of the EAR dose response was 2.66 (95% CI: 2.19; 3.13) cases per 10(4) PY-Gy; for the quadratic coefficient, it was -0.145 (95% CI: -0.171; -0.119) cases per 10(4) PY-Gy(2). The EAR was found to be higher for females than for males by a factor of 1.4. It decreased with age at exposure and increased with age attained. The central estimate for the linear coefficient of the ERR dose response was 18.9 (95% CI: 11.1; 26.7) Gy(-1); for the quadratic coefficient, it was -1.03 (95% CI: -1.46; -0.60) Gy(-2). The ERR was found to be smaller for females than for males by a factor of 3.8 and decreased strongly with age at exposure. Both EAR and ERR were higher in the Belarusian settlements than in the Ukrainian settlements. In contrast to ERR, EAR increases with time after exposure. At the end of the observation period, excess risk estimates were found to be close to those observed in a major pooled analysis of seven studies of childhood thyroid cancer after external exposures.

A radioecological model for thyroid dose reconstruction of the Belarus population following the Chernobyl accident.

A radioecological model was developed to estimate thyroid exposures of the Belarus population following the Chernobyl accident. The input of the model includes an extensive data set of the (137)Cs activity per unit area deposited during the Chernobyl accident, the rainfall data for different regions of Belarus, the (131)I/(137)Cs ratio in the deposit and the start of the grazing period in Belarus in April/May 1986. The output of the model is the age-dependent thyroid exposure due to the intake of (131)I with fresh milk. Age-dependent average thyroid doses were assessed for selected regions of Belarus. The maximum thyroid doses were estimated for the inhabitants of Gomel oblast where the highest deposition was observed among the regions considered here. The lowest doses were estimated for Vitebsk oblast with the lowest level of depositions. The mean exposures for the oblasts of Grodno, Minsk, Mogilev and Brest were very similar. The results were compared with estimations of thyroid exposure that were based on (131)I measurements in human thyroids, and they are in good agreement. The model may be used for the assessment of thyroid doses in Belarus for areas where no (131)I measurements are available.

Time trends of thyroid cancer incidence in Belarus after the Chernobyl accident.

The rates of childhood thyroid cancer incidence observed in Belarus during the period 1986 to 1995 are described as a function of time after exposure, age at exposure, and sex. Conclusions are drawn for the excess absolute risk function. After a minimum latent period of about 3 years after exposure, this risk function has a linear increase with time for at least 6 years. After correction for the dependence of average doses on age, the radiation-induced absolute thyroid risk in Gomel is about a factor of 3 higher for children up to age 10 at exposure compared to older ones; this may be due in part to different case-collection quality. In addition, in the group up to 10 years at exposure, the thyroid of girls is more sensitive to radiation by a factor of about 1.5 than the thyroid of boys on an absolute scale. Risk estimates from external exposure are consistent with risk estimates from Gomel assuming that the increase in excess cases reaches a plateau soon.

Cancer risk estimation in Belarussian children due to thyroid irradiation as a consequence of the Chernobyl nuclear accident.

The thyroid doses received by the juvenile population of Belarus following the Chernobyl accident ranged up to about 10 Gy. The thyroid cancer risk estimate recommended in NCRP Report No. 80 was used to predict the number of thyroid cancer cases among children during 1990-1992 in selected Belarussian regions and cities. The results obtained using this risk estimate show an excess of thyroid cancer cases being registered vs. the predicted cases. Thyroid cancer incidence rate among boys under investigation is higher than among girls in the postaccident period. The excess of the observed over the expected incidence in the general juvenile population is caused by the high thyroid cancer incidence rate among boys. These results, which can be considered part of the first stage of a thorough thyroid cancer risk estimation after the Chernobyl accident, demonstrate the critical need to complete these studies in depth.

Radiation effects on the population of Belarus after the Chernobyl accident and the prediction of stochastic effects.

Evaluation of conditions of exposure during the post-accident period makes it possible to identify two periods in the radiation exposure of Belarus's population. As a result of our investigations we obtained data about doses for four different categories in the exposed population: people who lived in the contaminated territories without evacuation and relocation; evacuated people: cleanup workers ("liquidators"); and people who were exposed in childhood, especially for thyroid exposure. The total doses for these categories in different time periods were analysed. Evaluation of doses received by the Belarusian population due to the Chernobyl accident shows no evidence of doses, that could lead to the deterministic consequences of radiation exposure. For all exposed groups we made predictions about different types of stochastic consequences of exposure.