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Notes: Extracts of Aspergillus fumigatus are required for the measurement of specific antibodies that are important indices in the diagnosis of allergic bronchopulmonary aspergillosis (ABPA). This study investigated the effect of different culture conditions on the production and release of antigenic and allergenic proteins of A. fumigatus. Increasing the incubation temperature from 25°C to 37°C altered the production of proteins by the mycelium which resulted in the release of a greater number of proteins that reacted with precipitating antibodies. Static sporulating cultures produced a much wider antigenic spectrum than shake cultures although the number of precipitating proteins (5 and 3 respectively) and major IgE binding proteins (5 and 3 respectively) was not greatly altered. The widest range of proteins bound by precipitating antibody or IgE from ABPA serum were released into the culture filtrate during 28 day static incubation at 37°C. The resultant extract proved useful for screening patients for specific IgE and will provide a starting material for the identification of individual antigens or allergens.

Notes: Precipitins against avian antigens in sera from patients with extrinsic allergic alveolitis, asymptomatic pigeon and chicken breeders and from control individuals were tested with different antigen extracts in six laboratories by a variety of different methods. Eighty percent of the results coincided in identifying the positive sera from patients and 90% in identifying the controls. It seems possible therefore to exchange results among experienced laboratories with fair confidence.

Notes: Precipitins against budgerigar serum proteins were not found in subjects without budgerigars or in healthy budgerigar fanciers. In budgerigar fanciers with respiratory disease, precipitins were found in sixteen out of nineteen suffering from allergic alveolitis, in four out of fourteen with asthma and two others in whom other respiratory diseases had been diagnosed. Specific IgE to budgerigar serum proteins was present in the atopic subjects with asthma due to budgerigar allergen but not in the nonatopic subjects with allergic alveolitis. Haemagglutination tests corresponded with the precipitin tests. Radio-immunoelectrophoretic tests showed that the precipitins belonged in the IgG, IgM and IgA classes. The presence of precipitins in budgerigar fanciers was closely associated with the presence of respiratory disease.

Notes: In vitro studies of T-lymphocyte responses using five different batches of Aspergillus fumigatus antigens, were undertaken in twelve patients with A. fumigatus related lung disease and in three normal controls.Using a leucocyte migration method, five of the twelve patients showed significant inhibition of leucocyte migration with a migration index of 0·80 or less to A. fumigatus, but in only three was this demonstrated with more than one batch of antigen (one patient with aspergilloma and two with bronchopulmonary aspergillosis).The same antigens were used in lymphocyte transformation tests. Only two patients, one with aspergilloma and one with bronchopulmonary aspergillosis, showed clearly significant transformation although there were several borderline results.Only three patients had evidence of delayed skin responses to A. fumigatus antigens in vivo, one with aspergilloma, one with bronchopulmonary aspergillosis and one with atypical bronchopulmonary aspergillosis. Two of these three patients also had one or more positive in vitro test results.Thus T-lymphocyte sensitization to A. fumigatus as demonstrated by these in vitro methods, although present in occasional patients, was not clearly related to any one particular clinical syndrome in this small group of patients with aspergillus related pulmonary disease. There was, however, one of the three aspergilloma patients with positive lymphocyte transformation to all five batches of antigens and having higher transformation indices than in any other patient. This suggests that lymphocyte studies should be extended in this group.In contrast to the frequent negative results using A. fumigatus antigens, evidence of T-lymphocyte sensitization to either Candida albicans or Mycobacterium tuberculosis or both, was shown by positive delayed hypersensitivity skin responses and in vitro inhibition of leucocyte migration in the majority of the patients, despite which lymphocyte transformation was often negative. The possibility of impaired capacity to transform is supported by the finding of an impaired response to phytohaemagglutinin (PHA) in four of the twelve cases.

Notes: The homes of 68 atopic asthmatic children were studied to estimate the concentrations of perennial and seasonal aeroallergens (Der pl, Fel d1, grass pollen, tree pollen, Cladosporium, Aspergillaceae and Alternaria) to which children were likely to have been exposed during their first few months of life, by sampling in the child's month of birth. There was a strong association between the presence or absence of the house dust mite allergen Der p1 in the air with the skin test and IgE antibody test results (p 〈 0.001), with a similar association for cat allergen Pel d 1 (p 〈 0.01), when using a low volume sampler (equivalent to the minute tidal volume of a small baby). No significant correlation was found between levels of allergen in carpet dust and air in the same room. There was a strong indication that the presence of a cat at birth was linked with a higher risk of development of allergy to cat, but high levels of Fel d 1 were sometimes found in homes even when there was no cat present, indicating that allergen may be introduced from other sources. The levels of tree pollen were significantly higher in the homes of tree pollen-allergic children than in the homes of patients without this sensitivity (p 〈 0.01); and the degree of sensitivity, determined by RAST, correlated significantly to the level of tree pollen in the home (p 〈 0.001). However, no relationship was found between specific sensitivity and the levels of Cladosporium, Aspergillaceae, Alternaria or grass pollen measured in the homes. The effect of high allergen exposure was most prominent in children under 7 yr and not beyond that age.

Notes: Crossed immunoelectrophoresis and crossed radio-immunoelectrophoresis techniques have been employed for analysis of extracts of various rabbit source materials to identify rabbit allergens in addition to the already described Antigen R1 (AgR1). Urine and dander extracts were found to contain only low levels of AgR1 and its presence in urine was as a contaminant due to mode of collection – it was not present in urine collected directly from the bladder. Other allergens were only recognised by highly rabbit-sensitive individuals, one in particular (Ag2) being present in several source materials. Serum albumin proved to be of minor allergenic importance and except for dander its presence was only in minimal amounts. As both AgR1 and Ag2 are significant components in extracts of fur and dust these extracts are therefore most recommended for use in investigations of individuals sensitive to rabbits.

Notes: The appearance of IgG and IgE binding components in the medium of shake cultures of Aspergillus fumigatus has been studied. Cultures were grown in synthetic asparagine medium at 35°C and flasks harvested in duplicate 1, 2, 3, 4, 7 and 14 days after inoculation. The pH of the medium dropped from its initial value of 5.5 to pH 3, and then after 4 days gradually increased up to pH 7.5 in the 14-day medium. The weight of mycelium, after an initial peak followed by a slight decline, increased as the pH of the medium increased. Components able to bind IgG and IgE from pooled ABPA sera were detected by crossed immunoelectrophoresis/self-crossed radioimmunoelectrophoresis within 24 h of growth, but maximal release of both antigens and allergens coincided with the increase in pH of the medium and was seen in the 14-day culture filtrate. Two recognised “major” antigens, Ag 7 and Ag 13, detected using the relevant monospecific antisera, were present in the culture medium after 14 days of growth and similarly for Ag 3, the major allergen, although another allergen, Ag 1, was identified in the 1-day extract. None of the culture filtrates was found to contain the “C-substance” polysaccharide.

Notes: Investigations have been carried out into the presence of antibody light chains in rabbit allergenic extracts and the interference in RAST and crossed-radioimmunoelectrophoresis (XRIE) caused by antibodies directed against them- A “non-specific” uptake of radioactivity in XRIE has been demonstrated to be caused by direct cross-linking of the 12 5I rabbit anti-human IgE by the sheep antibodies in the Immunoprecipitate of rabbit light chains. Preincubation with normal rabbit serum blocked this direct uptake of the labelled antibody and enabled specific IgE uptake on the light chains to be demonstrated for rabbit allergic sera. Verification of the allergenicity of the light chains was obtained from a specific light chain RAST. Elution from a Sephacryl S-200 gel filtration column indicated a MW of approx. 50 Kd and confirmation of the component as light chain dimers, not Fab fragments, was obtained by allotyping for loci present on heavy chains and light chains in the Fab region. Light chains were detected in urine from rabbits of all ages and in extract of dust collected in a rabbit housing area. No background staining was observed in XRIE using rabbit antisera, either with rabbit allergic sera with specific IgE or with a human serum containing specific IgG antibodies to rabbit IgG. This latter serum also showed no evidence of uptake on all immunoprecipitates in systems using rabbit antisera, and did not give false positive RAST results when the labelled rabbit anti-human IgE contained unlabelled rabbit IgG. Those sera with specific IgE to light chains showed no uptake in XRIE using rabbit antisera, indicating that the IgE was possibly specific for epitopes revealed by the dissociation of the whole IgG molecule.