B. Weeke

General Remarks on Principles, Equipment, Reagents and Procedures

B. Equipment for the immunoelectrophoreses . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Electrophoresis apparatus . . . . . . 2. Power supply . . . . . . . . . . . . . . . . 3. Voltmeter . . . . . . . . . . . . . . . . . . . 4. Wicks . . . . . . . . . . . . . . . . . . . . . . 5. Gel puncher . . . . . . . . . . . . . . . . . 6. Template . . . . . . . . . . . . . . . . . . . 7. Slit former . . . . . . . . . . . . . . . . . . 8. Glass plates . . . . . . . . . . . . . . . . . 9. Plexiholder for glass plates . . . . 10. Plastic vessels . . . . . . . . . . . . . . . . 11. Horizontal table . . . . . . . . . . . . . . 12. Water bath . . . . . . . . . . . . . . . . . 13. Electric heater . . . . . . . . . . . . . . . 14. Constriction pipettes . . . . . . . . . . 15. Measuring pipettes . . . . . . . . . . . 16. Razor blades and knives . . . . . . 17. Glassware . . . . . . . . . . . . . . . . . . . 15 16 17 17

Standardization of allergen extracts with appropriate methods. The combined use of skin prick testing and radio-allergosorbent tests.

This paper presents recommendations for standardization of allergen extract by the combined use of skin prick tests and radioallergosorbent tests. These tests provide appropriate means for quantitation of the total allergenic activity of an extract. Advantages and limitations are discussed with reference to the literature.

A New Lancet for Skin Prick Testing

A new lancet with a point length of 1.0 mm suitable for skin prick testing is described. The weal reactions appearing after the use of histamine chloride 1 mg/ml and an extract of Phleum pralense in a 1 HEP concentration (1 histamine equivalent prick) prove suitable as regards size and reproducibility compared with the reactions produced by a more conventional prick test method. The new lancet simplifies the prick test procedure and does not require as much experience as do needles and longer lancets.

Diagnosis and immunotherapy of mould allergy. V: Clinical efficacy and side effects of immunotherapy with Cladosporium herbarum

A placebo‐controlled, double‐blind study of immunotherapy with the mould species Cladosporium was performed in 22 adult asthmatics. The diagnosis of Cladosporium allergy was based on a combination of bronchial provocation test and daily symptom score in the Cladosporium season. An aqueous preparation of a potent, biologically standardized and purified extract was used in a clustered dose‐increase regimen. The clinical efficacy was evaluated by a combination of symptoms (asthma score + peak flow) and consumption of antiasthmatic medication. The mean changes in symptoms and medication consumption over a 10–week registration period (peak Cladosporium season) in 1982 after 5–7 months of immunotherapy were compared with the corresponding 1981 pretreatment 10‐week period A significant (P= 0.03) difference in terms of “improved”, “unchanged” and “deteriorated” patients in favour of Cladosporium treatment was found. Approximately 80% in the Cladosporium group showed improved/unchanged symptoms contrary to 30% of the placebo treated. Side effects were observed frequently but only in the Cladosporium‐treated. About 70% experienced a large local reaction and 100% had episodes of asthma during dose‐increase phase. Only a few severe systemic reactions occurred. Based on the clinical efficacy of the treatment we consider immunotherapy with Cladosporium feasible for highly specialized clinics.

Oral immunotherapy in birch pollen hay fever

Previous controlled trials with oral administration of allergen have not demonstrated any treatment effect in patients with allergic rhinoconjunctivitis or asthma. In the present double-blind, placebo-controlled trial, we have tested the effect of oral immunotherapy in adult patients with birch pollinosis. Thirty-nine patients completed this 18-month study comprising two birch pollen seasons. The patients received enterosoluble capsules daily, and the actively treated patients reached a cumulated dose of 280 times 10(6) biologic units of birch pollen extract, which is about 200 times higher than the dose used in conventional subcutaneous immunotherapy. We found a significant decrease in eye symptom scores and conjunctival sensitivity to birch pollen, as determined by conjunctival provocation test, as well as a numerical but nonsignificant decrease in nasal symptom scores, nasal sensitivity as determined by nasal provocation test, and antiallergic medication. The treatment was safe, and only a few slight side effects were observed. We thus conclude that our study demonstrates a clinical effect of oral immunotherapy in birch pollinosis.

Sensitive glass microfibre-based histamine analysis for allergy testing in washed blood cells. Results compared with conventional leukocyte histamine release assay

The new microfibre method for allergy testing is based on basophil histamine release after challenge with suspected allergens in samples of 50 μl washed blood cells. Released histamine is bound to microfibres and measured after removal of interfering substances by washing. The microfibre method was compared with the conventional leukocyte histamine release assay in 18 allergic patients tested with 10 different allergens. It was found that the same individuals responded with histamine release to the same allergens in both assays, and the number of responders was almost identical. Also the dose‐response curves and the cell sensitivity were almost identical, which further substantiated identity between the results obtained by the new microfibre method and the conventional assay. A comparison between the microfibre method and in vivo provocation tests showed good agreement when comparing the number of positive and negative responses in these test. The new method overcomes the problems in allergy testing, where only small amounts of blood are available and many tests have to be carried out.

A new method for detecting histamine release

Glass microfibres have been found to bind histamine with high affinity and selectivity. A new test for measuring basophil hisamine release has been developed using the glass microfibres as a solid phase. Glass microfibres are crushed and fixed to the bottom of microtitre plates with a water-soluble glue. Histamine release is performed in the glass microfibre-prepared microtitre plates by challenging 100 μl washed blood with 20 μl antigen per well for 90 min at 37°C. Released histamine is bound with high affinity to the glass microfibres, since 90% of histamine in the solution is adsorbed to the fibres. After incubation the microtitre plate is washed with H2O to remove cells and interfering substances. Fibre-bound histamine is detected by the fluorometrico-phthaldialdehyde method. The sensitivity of the assay is 0.63 ng histamine, 2 HCl and the histamine standard curve is linear up to at least 5 ng histamine, 2 HCl. Optimal conditions for the new assay are described. After challenge with anti-Ige a comparison with the conventional histamine release from Ficoll-Hypaque-isolated leukocytes showed almost identical results.

Identification and clinical significance of allergenic molecules of cat origin: part of the DAS 76 study

Freeze‐dried extracts from cat dander and the corresponding rabbit antibodies were used for establishing the CIE reference pattern for cat dander extracts. Anti‐Cat Ag 1 and anti‐cat albumin were used for identification of the corresponding antigens. CRIE on sera from selected groups of American and Danish cat‐allergic patients demonstrated antigen‐specific IgE binding to 10 of 15 cat dander antigens (Cat Ag 1 being the major allergen). Only minor differences were found between the two groups. Four of these allergens were serum proteins. Variable amounts of many of the 10 allergens were measured by QIE in saliva, serum, urine and three cat pelt extracts. However, extremely wide ranges for content of the serum allergens and the non‐serum allergens were found. This was exemplified by an albumin/cat Ag 1 ratio between 1 and 400, smallest in cat dander. Immunoabsorption using anti‐cat dander, anti‐cat albumin and anti‐Cat Ag 1 indicated that the anti‐cat dander, anti‐cat albumin, and the anti‐Cat Ag 1 absorbed approximately 90%, 25%, and 56%, respectively, of the dander RAST activity, and 87%, 11%, and 45%, respectively, of the saliva RAST activity, confirming the major importance of Ag 1. It is concluded that cat allergenic extracts should contain only modest amounts of serum albumin and other serum‐derived antigens and that any relevant standardization must include quantification of at least Cat Ag 1 and cat albumin.

21. Allergens Identified in Crossed Radioimmunoelectrophoresis

Summary. A timothy pollen extract was studied in crossed immunoelectrophoresis by means of precipitating antibodies from rabbits. Six precipitates appeared. Addition to the timothy precipitates of serum from patients with allergy to grass pollen caused a binding of IgE to some or all the precipitates. The IgE‐containing precipitates were identified by autoradiography after addition of 125I‐antihuman IgE.

Immunotherapy in hay fever with two major allergens 19, 25 and partially purified extract of timothy grass pollen. A controlled double blind study. In vitro variables, season i.

Forty grass pollen hay fever patients were randomly divided into two equal groups and treated from January 1978 with different timothy grass pollen extracts: whole pollen allergens (WPA) (Alutard® SQ), a partially purified extract and purified pollen allergens (PPA) consisting of the major allergens 19, 25. The effect was evaluated by symptom scores in grass pollen season 1, 1978, and titrated thresholds of skin prick test (SPT) and nasal provocation test (NFT) with extracts WPA, PPA, and FGM (five‐grass mixture).