Species identification of Asini Corii Collas (donkey glue) by PCR amplification of cytochrome b gene.

Asini Corii Collas (ACC; donkey glue) is a crude drug used to promote hematopoiesis and arrest bleeding. Because adulteration of the drug with substances from other animals such as horses, cattle, and pigs has been found, we examined PCR methods based on the sequence of the cytochrome b gene for source species identification. Two strategies for extracting DNA from ACC were compared, and the ion-exchange resin procedure was revealed to be more suitable than the silica-based one. Using DNA extracted from ACC by the ion-exchange resin procedure, PCR methods for species-specific detection of donkey, horse, cattle, and pig substances were established. When these species-specific PCR methods were applied to ACC, amplicons were obtained only by the donkey-specific PCR. Cattle-specific PCR detected as little as 0.1% admixture of cattle glue in the ACC. These results suggest that the species-specific PCR methods established in this study would be useful for simple and easy detection of adulteration of ACC.

Standardization of animal epithelia.

The standardization of animal epithelia is warranted for an accurate diagnosis and safe and efficacious treatment of allergic respiratory diseases induced by the inhalation of mammalian aeroallergens. We have compared several sources of raw materials of cat, hamster, goat and rabbit hair and epithelia to establish differences between the protein and allergenic composition of these extracts. The main differences in these raw materials was that "epithelia" were supplied as a mixture of hair and epithelia previously treated with acetone, and that the hair was supplied and used untreated. A possible influence of the age of rabbits on the composition of rabbit hair extracts was also evaluated. Overall, important differences were detected in the composition of epithelia versus hair extracts. Epithelia extracts contained more irrelevant proteins and, in most cases, less amounts of major allergens. Important differences were also detected in the composition of the extracts prepared with hair of young versus adult animals. In general, the extracts derived from young animals contained less major allergen and more albumin than those derived from older animals. A greater effort should be made to identify the ideal sources of animal skin derived allergen extracts to provide the allergologists with better extracts for the diagnosis and treatment of allergic respiratory diseases. There is also a need for a consensus on the terminology applied to animal skin derived extracts. The use of the term dander extracts seems to be more appropriate. These extracts contain more relevant allergens and fewer albumins.

A comparison of two skin test methodologies and allergens from two different manufacturers.

BACKGROUND: Skin prick tests (SPTs) are a frequently used method for evaluation of atopy. A variety of standard allergen preparations are available, together with a number of different methods of application. OBJECTIVE: The objective of this study was to compare SPT reactivity 1) using Soluprick SQ allergens (ALK Allergologisk Laboratorium A/S, Hørsholm, Denmark) and Bayer allergens [Bayer Corporation, West Haven, CT], and 2) using two common methods of application, a standard prick lancet and a Quintest, both produced by Bayer. METHODS: SPTs were undertaken on 22 adult volunteers (mean age 40 years, 17 female, 5 male). Wheal size was recorded as mean diameters (mm) and area (mm2). RESULTS: Bayer allergens produced larger mean diameters and areas to dust mites than ALK allergens, with the differences significant when allergens were applied with the lancet. There was a tendency for the ALK cat allergen to produce larger reactions than the Bayer product. The method of application also influenced the wheal size, with the lancet producing significantly larger mean diameters than the Quintest for the histamine and allergens from both manufacturers, except the ALK cat allergen. There were similar differences between methods of application for reactions measured as an area. CONCLUSIONS: SPTs that use different allergens or different methods of application will not provide comparable assessments of atopy.

The stability of house dust mite allergens in glycerinated extracts.

BACKGROUND: Mite allergen vaccines are important diagnostic and immunotherapeutic reagents. Previous studies on mite allergen stability under different storage conditions have yielded contradictory results. OBJECTIVE: We sought to compare, over a 12-month period, the stability of mite allergens reconstituted in 50% glycerol and stored at different temperatures and to examine the role of protease inhibitors in enhancing allergen stability. METHODS: Lyophilized allergen extracts were reconstituted in 50% glycerol, with and without protease inhibitors, and stored at -70 degrees C, -20 degrees C, 4 degrees C, or 37 degrees C for 12 months. At 6 and 12 months, the extracts were compared with freshly dissolved extracts by competition ELISA with pooled allergic sera, 2-site ELISA with mite-specific mAbs, and immunoblot analyses. RESULTS: The overall potencies of the stored extracts measured by competition ELISA were stable at -20 degrees C and 4 degrees C. As determined by means of the immunoblot and 2-site ELISA, Der f 1 levels decreased at 4 degrees C. Levels of Der f 2, Der p 1, and Der p 2 decreased in at least one of the allergen-specific assays. Storage at 37 degrees C led to overall loss of potency and allergen content, whereas storage at -70 degrees C was associated with a moderate loss of potency that increased with multiple freeze-thaw cycles. Protease inhibitors had no effect on allergen stability. CONCLUSION: Although overall potency of the extracts, as measured by competition ELISA, was preserved at -20 degrees C and 4 degrees C, allergen-specific assays indicated loss of allergens. These findings suggest that the competition ELISA is insensitive to decreases in the concentrations of individual allergens.

Regulating human-tissue products in France.

The absence of a pan-European regulatory framework for medical devices incorporating human tissues is causing difficulties for the growing number of manufacturers of these products. France has implemented strict legislation in this area and is spearheading an effort for Member States to adopt similar measures. This article looks at the status of French regulations, which form the basis of its recent proposal to the European Commission for a new Directive on human tissues.

Criteria for the choice of viruses in validation studies.

The criteria for the selection of viruses in validation studies are based on the nature of the virus contamination which may be encountered. A range of viruses with a spectrum of properties should be used, and may include those known or suspected to be present as well as those included to test the rigour of the process.

Use of beef extract as a diluent in direct agglutination test for visceral leishmaniasis.

Direct Agglutination Tests (DAT) have been developed for sero-diagnosis of visceral leishmaniasis (VL). The objective of this study was to find a more suitable diluent for serum dilution in a Direct Agglutination Test. 1% of beef extract (BEX) was used as a diluent in the Direct Agglutination Test; 1% foetal bovine serum (FBS) and 0.2% gelatine (Gel) were included for comparison as alternative diluents. Serum from VL patients, individuals suffering from hydatidosis, syphilis and from healthy adult Kenyans was used as control. Beef extract was found to be easy to handle, less expensive and results comparable to those obtained from the other diluents.

A collaborative study on the first international standard of Dermatophagoides pteronyssinus (house dust mite) extract.

A collaborative study was carried out to assess the suitability of a preparation to serve as the International Standard for Dermatophagoides pteronyssinus (house dust mite) extract. The proposed international standard of D. pteronyssinus, two additional freeze-dried extracts, and a commercially available skin testing solution were tested in the study. Nineteen laboratories in 11 different countries participated. The assay methods used included RAST inhibition, crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis, isoelectric focusing, quantitative skin testing, and various other methods for assessing total allergenic activity. In addition, six laboratories measured the quantity of antigen P1, and three laboratories measured antigen DpX in each of the preparations. On the basis of the results from this study, the World Health Organization established the preparation as the International Standard for D. pteronyssinus extract with an assigned unitage of 100,000 IU per ampule. The units refer both to the total allergenic activity of the ampule and to that of the individual allergens, such as P1 and DpX.

Standardization of house dust mite extracts. Liberation of allergenic and antigenic components in relation to extraction conditions.

The spontaneous release of house dust mite components from cultures of Dermatophagoides pteronyssinus into slightly buffered water was studied against time, using both continuous and discontinuous extraction procedures. It was shown that proteins, carbohydrates, IgE binding components and precipitating antigenic components were rapidly released from the house dust mite cultures, reaching a maximal liberation within 1 h of extraction. Repeated extractions of house dust mite cultures (discontinuous extraction) showed an additional release of IgE components but the IgE binding potency declined after successive extractions, while showing increasing release of immunological inactive components. IgE binding to antigens immobilized to polystyrene surfaces (IgE-ELISA) appeared to be less sensitive compared with cyanogen-bromide activated discs (IgE-RAST). It was concluded that extraction procedures of house dust mite cultures with short incubation time of 1 h or less are to be preferred.