Annette W. Ford

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.

The collaborative study of the international standard of dog, Canis domesticus, hair/dander extract

A collaborative study was carried out to assess the suitability of a chosen preparation to serve as the international standard (IS) for dog (Canis domesticus) hair/dander extract. Sets of five coded preparations of dog extract, including the proposed IS, were delivered to each participating laboratory in glass-sealed ampules. Fifteen laboratories in nine different countries examined the preparations by RAST inhibition, crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis, histamine release, and by other methods. In spite of the use of different versions of methods and different reagents, quite consistent results were obtained, and the proposed IS was found to have a satisfactory activity in all assays and to be useful as a calibrator when allergenic potency of similar dog allergenic extracts was estimated. Four laboratories estimated the specific content of allergens Ag 3, Ag 13, and albumin and found the proposed IS to be a suitable standard for these assays. On the basis of the results from this study, the World Health Organization established the preparation as the international standard for dog hair/dander extract with an assigned unitage of 100,000 IU per ampule. The units refer to both the total allergenic activity of the ampule and to that of individual allergens.

The effect of carbohydrate additives in the freeze-drying of alkaline phosphatase.

Abstract— Alkaline phosphatase was used as a model in studies to assess the effects of lyophilization on biological activity and molecular integrity in the presence or absence of added carbohydrate. The stability of the activity of alkaline phosphatase, lyophilized in Tris buffer alone or in the presence of the carbohydrates mannitol, lactose or trehalose was examined. Enzyme activity in formulations with Tris buffer alone or with mannitol was considerably reduced by freeze‐drying and further storage at elevated temperatures; freeze‐drying with mannitol failed to maintain activity at a temperature of 37°C over 21 days, whilst the loss of activity was more gradual when freeze‐dried in buffer alone and stored at higher temperatures. Lactose and trehalose maintained the alkaline phosphatase activity after freeze‐drying and, furthermore, preparations containing trehalose retained activity even when the material was subjected to temperatures of up to 45°C for up to 84 days. At 56°C the alkaline phosphatase activity did not show a significant drop until 14 days with the lactose formulation or until 21 days with trehalose. After 84 days at 56°C, 30% of the activity still remained in the formulation containing trehalose. In addition to the changes in the enzyme activity, FPLC chromatographic traces and SDS‐PAGE gels demonstrated compositional differences between each formulation after storage.

Production and testing of an international reference standard of short ragweed pollen extract

A lyophilized candidate International Reference Standard of short ragweed pollen extract was prepared by use of defined source material. In preliminary experiments, this extract was demonstrated by RAST inhibition and crossed radioimmunoelectrophoresis assays to contain several well-characterized ragweed allergens and to contain multiple antigenic bands by crossed immunoelectrophoresis analysis. In a subsequent multinational collaborative study involving 12 laboratories in five countries, the candidate extract was compared with existing national reference or commercial ragweed extracts by a variety of immunochemical, biochemical, and physicochemical procedures. The candidate extract could be used to assign relative orders of potency to the comparison-test extracts. In separate studies, the candidate extract was demonstrated to be stable when it was stored at either -20 degrees C or +5 degrees C for at least 2 yr. The candidate extract has been accepted as an International Reference Standard with an assigned arbitrary potency of 100,000 units per ampule .

The allergens of dog. I. Identification using crossed radio‐immunoelectrophoresis

The antigens present in an extract of dog hair and dander were examined by crossed immunoelectrophoresis (CIE) and the IgE‐binding allergens by crossed radio‐immunoelectrophoresis (CRIE), respectively, using sera from 60 British and Finnish animal‐allergic subjects. The extract was comprised of a minimum of 28 antigens, 11 of which were common to dog serum. IgE antibody in the sera of the dog‐sensitive patients bound to 21 of the 28 antigens at varying frequencies and intensities. Binding of any intensity occurred most frequently to two serum proteins: antigen 23 (IgG) binding IgE in 88% of cases, and antigen 3 (dog serum albumin, DSA) in 77% of cases. Dander antigen 8 bound in 63% and antigen 1 in 42% of the sera. Strong IgE binding, however, was most commonly associated with dander antigen 8 followed by antigens 1 and 23 (IgG) then 3 (DSA). The ranking of the antigens as allergens was similar for the two populations except that DSA was more important for the British than for the Finnish subjects.

The allergens of dog II. Identification and partial purification of a major dander allergen

A dog hair and dander (DHD) extract was prepared from hair obtained from mixed breeds. By SDS‐polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting, using sera from 32 dog‐allergic subjects, a number of IgE radio‐staining bands could be seen. In 78% of sera a protein of molecular weight (MW) of 21 000 daltons, designated Ag X, was found to bind IgE and in 34% it did so strongly. This allergen was isolated from DHD by size‐exclusion and ion exchange chromatography. The final product was a single allergen of MW of 21 000 and an isoelectric point of approximately 5.2. An additional protein‐staining band could still be seen of MW of 24 000 daltons. Using a serum which contained IgE antibodies only to Ag X, this allergen was found only in DHD extract and dog saliva and was absent from dog serum and urine. It was the same dog allergen that we [1] reported as Ag 8 using crossed radio‐immunoelectrophoresis (CRIE) and that Blands et al. [2] and Løwenstein [3] described as Ag 13. We propose that this major dog allergen be given the title Can f I according to the new allergen nomenclature.

A Pig Collagen Peptide Fraction. A Unique Material for Maintaining Biological Activity During Lyophilization and During Storage in the Liquid State

There is frequent use of human and animal proteins as stabilizers during lyophilization of a variety of biological substances with a view to long term stable storage. This report describes the comparative excellent stabilizing effect of a porcine collagen peptide fraction (CPF) during the lyophilization and subsequent storage of three commonly used biological substances, alkaline phosphatase, tissue plasminogen activator and thrombin.

Studies on allergens of mammalian origin

Cat and dog saliva and extracts obtained from cat and dog hair have been examined by high-performance liquid chromatography (HPLC) to identify the likely allergenic molecules. Separation of the materials by size exclusion HPLC showed that several components in the range of apparent molecular weight from 35,000 to 200,000 had inhibitory activity in the radioallergosorbent test (RAST) when tested against pooled serum from donors hypersensitive to dogs and cats. One of these components active in our test system was identified as albumin. Despite differences in gross composition of the extracts no significant difference in the position of the active fractions was observed between cat hair and saliva, between dog hair and saliva or between hair extracts from different breeds of dog. Fractionation by anion-exchange HPLC, although promising, was complicated by problems of sensitivity.

Investigations of the allergens of cocksfoot grass (Dactylis glomerata) pollen.

The pollen of cocksfoot grass (Dactylis glomerata) is an important cause of allergic reactions in man. Preliminary studies, which established that constituents of an extract of this pollen could be separated, and then recovered efficiently, by size-exclusion chromatography on TSK G3000 SW, have been extended. A comparative examination has been made by this procedure of cocksfoot pollen extracts from different sources and of several batches of extract from one source. The recovery and distribution of biological activity has been assessed by the radioallergosorbent test, and the results have been used for the selection of fractions for further investigation by chromatography and electrophoresis. Two constituents active in the radioallergosorbent test have been purified from the extracts.

An investigation of alternatives to hog gastric mucin as virulence-enhancing agents in the cholera vaccine potency assay.

Discontinuance of the brand of mucin generally used heretofore as a virulence-enhancing agent in the potency assay of cholera vaccines stimulated a search for an effective alternative. Three new mucins and a number of other substances were tested for virulence-enhancing effects with cholera vibrios and of these ferric ammonium citrate was found to be the most active. When cholera vibrios were serially passaged in mice in the presence of ferric ammonium citrate the LD50 was reduced to levels similar to those observed with challenges enhanced with mucin. The mechanism of action of ferric ammonium citrate is discussed.