Fractionation of cocksfoot (Dactylis glomerata) pollen by preparative isoelectric focussing.

The technique of isoelectric focussing in polyacrylamide gel has been developed to a preparative mode. Using the improved technique cocksfoot (Dactylis glomerata) pollen has been split into many components, and the allergenic reactions of the preparations have been studied by passive transfer in animal skin and by RAST.

Assay of materials used in immunofluorescence techniques.

Samples of commercially available fluorescein isothiocyanate and conjugated antispecies globulins were evaluated. Only one of the samples was of very high quality. There was great variation in the quality of the conjugated globulin solutions and even the best of them showed defects.

Frequency of occurrence of IgG (S-TS).

A group of eighty-four sera from a selected population of patients suspected of hypersensitivity to food and environmental allergens was examined by both RAST and passive transfer in monkeys for IgE and IgT (S-TS) antibodies respectively. A high proportion (26%) showed IgG (S-TS) to milk, and this was more than four times the frequency (6%) for IgG (S-TS) to any other of the antigens tested IgE and IgG (S-TS) antibodies behaved as independent biological systems.

Profiles of allergen extract components by isoelectric focussing and radioimmunoassay.

When allergen extracts are subjected to isoelectric focussing in polyacrylamide gel, followed by radioimmunoassay different preparations of nominally the same allergen show markedly different concentrations of components. Not all the peaks are likely to be due to components of different antigenic specificity, but undoubtedly the sera from different patients contain different spectra of antibodies to the same preparation.

A new method of measuring histamine within single cells using a microspectrophotofluorimeter.

Methods of measuring histamine from large numbers of cells involve inaccuracies due to the difficulty of isolating a pure cell population without damage to the cells. A new method of measuring histamine within single cells by a microspectrophotofluorimetric method has been developed. Calibration of the microspectrophotofluorimeter used o-phtalaldehyde-conjugated-histamine absorbed to Sephadex particles. The concentration of histamine within single rat mast cells was measured and compared with the theoretical values obtained from measuring histamine released from large cell samples.

Familial human short-term sensitizing (IgG S-TS) antibody.

Skin tests on a middle-aged English housewife (the initial case) with a history of seasonal grass pollen rhinitis and asthma showed strong immediate and late reactions to grass pollens. Early in the grass pollen season, 1976, RAST showed normal levels of IgE antibodies in the serum. Passive transfer in Rhesus monkey skin was also negative for heat-labile IgE but gave a very vigorous reaction for short-term sensitizing heat-stable IgG antibody (IgG S-TS Ab). Skin tests and studies on sera from other members of the family showed that another five also formed IgG S-TS Ab, and indicated that the ability to form this antibody was familial. One member of the family had only an immediate reaction on skin testing, and much IgE to grass pollen. Towards the end of the grass pollen season the IgE titre in the initial case had been tripled and the IgG S-TS Ab had disappeared. By September the specific IgE titre had risen even further.

Human dander in house dust allergy.

The possible role of human dander in house dust allergy was investigated. Naturally shed human mite-free skin squames were collected from bedding and used to prepare a human dander extract. When the extract was coupled to cyanogen bromide-activated paper discs, and used in the RAST assay, IgE titres to the skin extract were observed in the sera from several patients with house dust allergy. The sera with IgE to the skin extract also had high IgE titres to either house dust, D. pteronyssinus or cat fur. RAST inhibition studies revealed cross-reaction between the human skin extract and both a D. pteronyssinus extract and a cat fur extract.

Simulated human skin scales.

Human skin scales which have been shed naturally bear a flora of microorganisms which is unknown until tested. To replace these scales in a study of the micro-environment of both the human body and of models a method has been devised of making synthetic scales which behave both physically and aerodynamically in a similar way to the natural material. The synthetic materials carry no natural flora and it is possible to include in them test markers of several kinds to assist in identification after dispersion.

Allergenic activity of fractions of cocksfoot (Dactylis glomerata) pollen. Definition of active components by skin testing, and inhibition RAST.

With the combined use of skin tests, RAST and isoelectric focusing, a potency profile of a cocksfoot ((Dactylis glomerata) pollen extract was prepared. It was found that two fractions with pI 4.5 and 6.0 were the most allergenic, had the highest protein concentration and were also the most easily extracted. The importance of converting mean weal sizes into potency units when comparing the allergenicity of pollen fractions is emphasized.

Activated paper as an immunoabsorbent.

Some workers when using cyanogen bromide activated paper as a solid immunoabsorbent have reported variable success in radio immuno assays. A quantitative investigation has been made of some of the factors which effect the activation of various papers with CNBr, and also of the subsequent coupling reaction with the sensitizing antigen. Different papers have been found to react in different ways, and some papers take up so little antigen that they are unsuitable for use for this purpose.

Activity units for allergen extracts.

The two present systems of units for the strength of allergen extracts, Noon and p.n.u. are both unsatisfactory. A new activity unit, based on skin prick in humans, is described, and its relationship to in vitro tests is explored. Potency limits for provocation materials used for skin prick tests are suggested.

'Barn allergy': asthma and rhinitis due to storage mites.

The differential diagnosis of asthma in farm workers is between a type I hypersensitivity to a pollen or dust allergen and a type III hypersensitivity to mould antigens. We describe asthma and rhinitis, and not farmer's lung, in a farming population due to the non-pyroglyphid grain storage mite. Before a diagnosis of farmers' lung due to mouldy hay is made in any patient whether or not precipitins to Micropolyspora faeni are present, skin tests for storage mite should be made. If these are positive a diagnosis of 'barn allergy' should be considered and a trial of sodium cromoglycate be given.

Allergenic activity of fractions of cocksfoot (Dacylis glomerata) pollen.

A cocksfoot (Dactylis glomerata) pollen extract has been fractionated by isoelectric focusing in polyacrylamide gel, the separated components extracted from the gel and assayed by three methods: prick tests in human skin, RAST and PCA tests in monkey skin. Prick testing in human skin showed that subjects responded differently to the separated components, indicating the presence of more than one antigenic determinant, and a profile of antibody activity against each fraction could be constructed. In general, good agreement was observed between RAST profiles and skin test profiles, although some patients who gave positive skin tests lacked circulating IgE and consequently did not give a RAST profile or a monkey PCA. These studies emphasize the current difficulty in attempting to isolate a single pure allergen from cocksfoot which can be used either diagnostically for the detection of pollen allergy, or for the standardization of diagnostic extracts.

Complement in the induction of IgE antibody formation.

The IgE antibody response of NIH mice to ovalbumin was suppressed by in vivo complement depletion with cobra factor. This observation extends to IgE the finding that complement participates in thymus-dependent antibody formation. Control mice producing IgI anti-ovalbumin antibody after boosting all developed marked peripheral eosinophilia, whereas mice in which the IgE antibody response had been suppressed by cobra factor did not. Administration of cobra factor itself had no effect on peripheral eosinophil levels.

Controlled activity mite vaccines for 'barn allergy'.

Three species of mites, Tyrophagus longior, Glyciphagus destructor, and Acarus farris have been isolated from the dust of barns of farms in Orkney. The mites were cultured in amount sufficient for the production of bulk extracts. These were then tested by skin prick in a population of 30 inhabitants of Orkney and potency was assigned in units of biologic activity. The concentrations of the bulk extracts from the different species were adjusted to the same biologic potency and then used to make delayed-release vaccines for injection therapy and aqueous solutions for provocation tests and therapy by the nasal route. The vaccines were then administered by injection to nine subjects at weekly intervals over a period of 2 months. Assessment by questionnaire suggested that there was a significant improvement in the symptoms of eight of the nine subjects. A course of nasal therapy with the aqueous solutions produced no improvement in four out of five subjects.

Antibodies mediating type I skin test reactions to polysaccharide and protein antigens of Candida albicans.

Immediate, type I, prick test reactions in man to purified fractions of Candida albicans group A were mediated by different classes of antibody. The allergens were the cell-wall polysaccharide, mannan, and 'purified' cytoplasmic protein fraction, free of mannan, prepared by passage through Sepharose-concanavalin-A. Passive transfer tests in the monkey gave immediate reactions, (1) to the purified, cytoplasmic, protein mediated by heat-labile long-term sensitizing antibody, and (2) to the polysaccharide, mannan, mediated by heat-stable short-term sensitizing antibody.