Validation of an ELISA method for quantification of the major Timothy grass pollen allergen Phl p 5a (BSP090).

Progress towards standardisation of allergen products has been made in recent years. Nevertheless, no standardised test method to quantify the allergen content of grass pollen allergen products is available at present. One aim of the BSP090 project was to validate a quantitative assay for a major Timothy grass (Phleum pratense) pollen allergen, Phl p 5. Qualification of a candidate ELISA system was performed with regard to range, robustness and cross-reactivity in preliminary studies. The assay specifically detected Phl p 5 with a quantification range from 3.9 ng/mL to 62.5 ng/mL. Suitability to quantify recombinant and natural Phl p 5 was further assessed in a collaborative study including 14 laboratories in Europe and the USA. Precision and accuracy of the assay was satisfactory with 93% of calculated Phl p 5 concentrations and 100% of total recoveries being within the ± 30% acceptance range. Similar results were obtained for spike recoveries, with exclusion of the lowest concentration spike, showing spike recoveries exceeding the acceptance range for six laboratories. Inter-assay (repeatability) and inter-laboratory (reproducibility) variability were satisfactory, in the format used in the present study. Robustness towards different statistical methods for data analysis was demonstrated. In conclusion, the assay can easily be established in routine testing and results of the preliminary testing and collaborative study support the proposal of the assessed Phl p 5-specific ELISA as a European Pharmacopoeia general method.

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.

Minor allergen patterns in birch pollen allergen products-A question of pollen?

BACKGROUND: Contrary to the scientific differentiation between major and minor allergens, the regulatory framework controlling allergen products in the EU distinguishes relevant and non-relevant allergens. Given the lack of knowledge on their clinical relevance, minor allergens are usually not controlled by allergen product specifications. Especially, in birch pollen (BP) allergen products, minor allergens are commonly disregarded. OBJECTIVES: To quantify three minor allergens in BP allergen products from different manufacturers and to assess the influence of the utilized BP on minor allergen patterns. METHODS: Apart from common quality parameters such as Bet v 1 content, Bet v 4, Bet v 6 and Bet v 7 were quantified in 70 BP allergen product batches from six manufacturers, using ELISA systems developed in-house. Batch-to-batch variability was checked for agreement with a variability margin of 50%-200% from mean of the given batches for individual allergen content. Subsequently, minor allergen patterns were generated via multidimensional scaling and related to information on the pollen lots used in production of the respective product batches. RESULTS: Like the already established Bet v 4 ELISA, the ELISA systems for quantification of Bet v 6 and Bet v 7 were successfully validated. Differences in minor allergen content between products and batch-to-batch consistency were observed. Correlations between minor and major allergen content were low to moderate. About 20% of batches exceeded the variability margin for at least one minor allergen. Interestingly, these fluctuations could not in all cases be linked to the use of certain BP lots. CONCLUSIONS AND CLINICAL RELEVANCE: The impact of the observed minor allergen variability on safety and efficacy of BP allergen products can currently not be estimated. As the described differences could only in few cases be related to the used pollen lots, it is evident that additional factors influence minor allergens in BP allergen products.

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.

The influence of magnesium supplementation on magnesium and calcium concentrations in hair of children with magnesium shortage.

46 children, aged 2-6 years, with decreased magnesium concentrations in hair, were studied. Magnesium supplementation consisted of Asmag preparation for 3 months and multivitamin Multi-tabs preparation (containing magnesium, but without calcium) for the following 4 months. Control studies were performed again after 7 months of treatment, i.e. 12 months after the initial measurements (the same season of the year--early spring). The results proved increases of both magnesium (from 7.74 microg/g dry mass to 11.03 microg/g dry mass) and calcium (from 159.82 mg/g dry mass to 191.60 mg/g dry mass) concentrations in hair. Increased magnesium concentrations were observed in 40 studied children (86.95 per cent). Post supplementation magnesium deficiency was found in 22 children (47.83 per cent), and four children (8.70 per cent) showed further worsening of hypomagnesemia. Increased calcium concentrations were found in 42 children (91.30 per cent), while decreased Ca levels were found in 4 children (8.70 percent). The achieved results indicate a positive influence of that form of compensation of magnesium deficiency, and suggest the need of individual selection of doses and period of Mg supplementation. The initial level of hypomagnesemia, presence of factors that might inhibit intestinal absorption, accompanying diseases that might cause decrease in magnesium concentration and other factors that might influence the total body magnesium concentration should be taken into account while designing the supplementation therapy.

[An assessment of infant morbidity and antibiotic therapy according to the type and course of delivery between 1983 and 1993]. / Ocena zachorowalnosci i stosowanej antybiotykoterapii w latach 1983-1993 u dzieci w pierwszym roku zycia w zaleznosci od rodzaju i przebiegu porodu.

A comparative analysis of single and recurrent infant morbidity according to type and course of delivery and to kind of drugs used was performed in this study. A total of 3676 infants born between 1983-1993 was examined. This group was divided into four groups according to date of delivery 1983-1984, 1985-1987, 1988-1990 and 1991-1993). A steady were in the number of spontaneous and cesearian deliveries was noticed. The number of forceps and vacuum extractor deliveries was significantly decreased. A higher morbidity was observed among infants from breech and vacuum deliveries and among those from families with numerous children. Upper respiratory tract infections and allergic dermatitis occurred most frequently. The greathy declining trend of usage for the use of antibiotics in upper respiratory tract and urinary tract infections was observed. The use of two or more antibiotics per therapy was the most common in brachial and lung infections.

[The influence of the feeding type on the concentration of trace elements in the hair of infants]. / Wplyw sposobu karmienia na zawartosc wybranych biopierwiastków we wlosach u niemowlat.

The aim of this study was to assess hair magnesium, iron, copper, and zinc levels in breast-fed and formula-fed infants. Our findings showed that concentration of the assayed macro- and trace elements, except copper, were higher in breast-fed children and declined with age, faster in formula-fed children. The frequent occurrence of magnesium deficiency indicates that pregnant women should receive magnesium supplementation.