Standardization of food allergen extracts for skin prick test.

The aim of the study was to standardize and evaluate technically optimized food allergen extracts for use in skin prick test (SPT). The standardization procedure comprised 36 allergic histories in 32 food allergic patients with 21 healthy, non-atopic individuals serving as controls. The patients had a history of allergic symptoms upon ingestion of either cow's milk (n=3), hen's egg (n=9), wheat (n=4), hazelnut (n=14) or cod (n=6). They also had specific IgE in serum to the food in question and a positive SPT with a fresh preparation of the food. The diagnosis had been confirmed by a double-blind, placebo-controlled food challenge, except for the hazelnut-allergic patients. The controls were subjected to an open food challenge with all the foods to ensure tolerance. The standardization was performed by means of titrated SPT in accordance with the guidelines on biological standardization from the Nordic Council on Medicine. Regression analysis of the skin wheal areas was performed for each patient and the median protein concentration of allergen preparation (median Ch10) eliciting a wheal area of the same size as histamine 10 mg/ml was calculated. The median Ch10 was 0.56 mg/ml for milk, 0.88 mg/ml for egg, 5.4 mg/ml for wheat, 2.1 mg/ml for hazelnut and 0.017 mg/ml for the cod extract. The sensitivity of the median Ch10 estimated from the SPT data was 1 for milk, 0.98 for egg, 1 for wheat, 1 for hazelnut and 0.87 for the cod extract. The allergenic activity of the hazelnut extract was further investigated by leukocyte histamine release (HR) and immunoblotting experiments using sera from 27 hazelnut allergic patients. The clinical sensitivity of the optimized hazelnut extract evaluated by HR was 0.78 compared to 0.30 for a commercially available hazelnut extract (Soluprick). Immunoblotting results showed a stronger IgE binding capacity and additional IgE-binding bands of the optimized hazelnut extract compared with the Soluprick extract.

Prevalence of sensitization to the storage mites Acarus siro, Tyrophagus putrescentiae, and Lepidoglyphus destructor in allergic patients with different degrees of sensitization to the house-dust mite Dermatophagoides pteronyssinus.

The prevalence of sensitization to the storage mites Acarus siro (AS), Tyrophagus putrescentiae (TP), and Lepidoglyphus destructor (LD) was studied in 250 sera of patients with different degrees of sensitization to the house-dust mite Dermatophagoides pteronyssinus (DP) by measuring IgE binding to extracts of the storage mites. Additionally, allergenic cross-reactivity between DP and the storage mite species was studied by RAST inhibition with five individual sera (and a pool of these sera) with moderate IgE levels to all three storage mites and to DP. Increased serum IgE to storage mites was found in 46% of the 200 patients sensitized to DP. Increased prevalence rates of IgE titers to storage mites were associated with higher IgE levels to DP. In 50 sera without sensitization to DP, only five sera showed increased IgE to one of the storage mites. Extracts of TP almost completely inhibited the IgE binding to AS, and vice versa. DP inhibited IgE binding to all storage mites up to 60%, whereas IgE binding to DP was only minimally inhibited by extracts of storage mites. In conclusion, cosensitization to storage mites is a frequent finding in patients sensitized to DP. Although this is largely the result of cross-reactivity between different mite species, it may nevertheless be of clinical significance in patients exposed to storage mites.

Determination of the stability of diluted allergen extracts using a concentration step prior to EAST inhibition.

BACKGROUND: Generally the stability of diluted allergen extracts, as used for skin testing, provocation testing and immunotherapy can not be measured using a normal enzyme allergosorbent test (EAST) inhibition method. OBJECTIVE: The aim of this study was to determine the stability of diluted allergen extracts using an ultrafiltration step prior to the standard EAST inhibition procedure, in which the allergen extract was concentrated 100-fold. METHODS: This concentration procedure was validated for Dermatophagoides pteronyssinus, timothy pollen, birch pollen and cat dander extracts and used in a stability study in which three batches were stored for 1 year at 6 degrees C and 25 degrees C. RESULTS: There was no difference in relative potency before and after concentration of birch and timothy pollen extracts. D. pteronyssinus and cat dander extracts showed a significant decrease of 25% and 35% of the relative potency after concentration. The mean coefficient of variation of 12 determinations of the stability study was 11.8%. CONCLUSION: For all allergens the 30 BU/mL or approximately 0.00025 mg/mL solution was stable for 12 months at both temperatures, except for D. pteronyssinus which declined rapidly at 25 degrees C.

Effect of dilution, temperature, and preservatives on the long-term stability of standardized inhalant allergen extracts.

BACKGROUND: Although documented stability of allergens used for diagnosis is important, research in this area has been limited. Most studies on extract stability have been of limited duration and discrepancies have been reported between stability test results of in vivo and in vitro methods. OBJECTIVE: In this study we determined the stability of allergenic extracts, comparing the intracutaneous test and enzymallergosorbent test inhibition method and determining the effect of temperature, dilution, and preservatives. METHODS: Three formulations of timothy pollen, birch pollen, house dust mite (D. pteronyssinus) and cat dander extracts, as used for bronchoprovocation, skin prick testing and intracutaneous testing, were stored for 24 months at 6 degrees C. The influence of temperature on various formulations was determined using the enzymallergosorbent test inhibition technique during storage for up to 36 months. RESULTS: Most formulations were found to be stable for 24 (intracutaneous test) or 36 (enzymallergosorbent test inhibition) months at 6 degrees C. At 25 degrees C, most formulations showed a decrease in relative potency, which remained above the limit of 0.3 times the in-house-reference for the bronchoprovocation formulation of timothy pollen, birch pollen, and house dust mite and for the skin prick test formulation of cat dander. CONCLUSIONS: Cat dander was remarkably stable at 6 and 25 degrees C in glycerine and birch pollen was very susceptible to phenol. This destructive effect of phenol could be prevented by adding human serum albumin. The discrepancy between in vivo and in vitro tests reported by others was confirmed for house dust mite and timothy pollen.

Optimization of skin testing. II. Evaluation of concentration and cutoff values, as compared with RAST and clinical history, in a multicenter study.

In this multicenter study we evaluated the results of a previous study (part I) in a relatively large Dutch patient population, using the two previously tested allergens (house-dust mite and grass pollen) and two other standardized allergens (tree pollen and cat dander). The obtained skin test results were expressed as a histamine ratio and compared with RAST and clinical history (CH). The sensitivity and specificity were calculated at different cutoff values of the skin tests. The optimum cutoff values of 0.7 intracutaneous tests (ICT) and 0.4 skin prick tests (SPT) resulted in a predictive value for the detection of allergic sensitization of 83% (RAST) and 77% (CH), and 91% (RAST) and 86% (CH), for the ICT and SPT, respectively. As the ICT and SPT were performed in different centers, the results of these methods cannot be compared. No systemic side-effects of the skin tests were recorded. These results generally correspond well with the predictions regarding safety and predictive value of part I of this study, in which a limited number of patients was studied. In conclusion, through the use of a limited number of standardized allergens in a small group of patients, it may be possible to predict a safe and efficacious concentration for routine skin testing and to extrapolate from these results to other standardized allergens.

Optimization of skin testing. I. Choosing allergen concentrations and cutoff values by factorial design.

Standardized extracts of Phleum pratensis (grass) and Dermatophagoides pteronyssinus (house-dust mite) were used as test allergens for multiple regression in order to determine optimum concentrations and cutoff values with regard to diagnostic capacity and safety. If a RAST value of class 1 or more was taken as an indication of sensitization, it was found that the optimum concentrations for the detection of sensitization are in the range 10-100 BU/ml and 1500-10,000 BU/ml for intracutaneous tests (ICT) and skin prick tests (SPT), respectively. The skin test results were expressed as histamine ratios. Using allergen concentrations of 30 and 3,000 BU/ml, we found cutoff values of 0.87 and 0.53 and predictive values of 87.1% and 79.1% for ICT and SPT, respectively. The maximum wheal size (mean wheal size + 2 SD) to be expected in 95% of the population was 26.6 mm (ICT) and 10.9 mm (SPT), sizes regarded as safe by most clinicians. In conclusion, by using this method with a limited number of patients, one can probably improve the diagnostic precision and safety of the skin test. In the second part of this study, these hypotheses were prospectively tested in a multicenter study.

Age-dependency of sensitization to aero-allergens in asthmatics.

Skin reactivity (intracutaneous test) to histamine and allergens was studied cross-sectionally in a Dutch asthmatic patient population from childhood to old age (4-75 years). It was found that the histamine skin reactivity rose significantly (p less than 0.05) during childhood, was significantly higher in the 10-15-year age group, and was constant between 20 and 75 years of age. The mean wheal index (histamine ratio) of all allergens was constant during childhood, and decreased after the age of 25 for grass pollen and house-dust mite and after the age of 15 for the other allergens. The prevalence of a positive skin test decreased with age, except for grass pollen. During childhood the indoor allergens, cat dander and house-dust mite, were the most important, while after the age of 15 sensitivity to an outdoor allergen, grass pollen, increased markedly. At all ages house-dust mite was the most important allergen. After the age of 25 the prevalence of every allergen declines. The prevalence of a positive skin test to Cladosporium was unexpectedly high in childhood (10-40%). It can be concluded that the prevalence of a positive skin test declines with age, except for grass pollen. The degree of sensitization in asthmatics peaked in the age groups between 20 and 40 and sensitivity to indoor allergens developed earlier than sensitivity to outdoor allergens.

Glucuronidation of labetalol at the two hydroxy positions by bovine liver microsomes. Isolation, purification, and structure elucidation of the glucuronides of labetalol.

Glucuronidation is known to be a major metabolic pathway for labetalol. As the drug contains a phenolic and an alcoholic hydroxy group, in principle two regio isomeric glucuronides can be formed. By incubating the substrate labetalol with bovine liver microsomes, in the presence of the co-substrate UDP-glucuronic acid, both hydroxy positions were glucuronidated. The different glucuronides were isolated from the incubation mixture using Bond-Elut extraction cartridges and separated by means of high performance liquid chromatography. The formation of glucuronides was confirmed by performing reference incubations using radiolabeled UDP-glucuronic acid. Structure elucidation of the various glucuronides was done by nuclear magnetic resonance, ultraviolet spectroscopy, and mass spectrometry.

Optimization of the in vitro glucuronidation of ibuprofen using factorial design.

Ibuprofen was chosen as a test compound to perform a multivariate design in order to determine the highest yield of the in vitro glucuronidation reaction in relation to the concentration of reacting and activating substances. Preliminary studies with a univariate design indicated that the concentration of Mg2+ had no significant effect on the glucuronidation and that Triton X-100 could be omitted as it appeared to inhibit the glucuronidation. In a 3(3) factorial design the influence of concentrations of ibuprofen, UDP glucuronic acid and enzyme, respectively, on the yield of ibuprofen glucuronide was established. It was concluded that the highest amount of ibuprofen glucuronide formed (within the limits of this design) was achieved with an ibuprofen concentration of 486 microM, a UDP-glucuronic acid concentration of 3 mM and an enzyme concentration of 3.57 mg/ml. Using this methodology it is possible to optimize the glucuronidation yield in a more rational way, which can be useful in the upscaling of enzymatic reactions.

Determination of enantiomeric purity of the new D-2 dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437) by reversed-phase high-performance liquid chromatography after pre-column derivatization with D(+)-glucuronic acid.

This paper describes an enzymic derivatization procedure that allows accurate determination of very small amounts of enantiomeric impurities in the D-2 dopamine agonist 2-(N-propyl-N-2-thienylethylamino)-5-hydroxytetralin (N-0437). After pre-column glucuronidation of the individual enantiomers, two diastereoisomers were formed which were separated by reversed-phase high-performance liquid chromatography. An enantiomeric purity of 99.84% was calculated for the (-)-enantiomer, against 99.89% for the (+)-enantiomer. The assay was validated by spiking 1% of the (-)-enantiomer in the (+)-enantiomer. A high accuracy (error 4.5%) and precision (coefficient of variation 2.9%, n = 5) of the method were established.