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Particle counting immunoassay (PACIA) is shown to be a suitable nonradioisotopic method for the titration of anti-phospholipase A2 antibodies in the sera from honeybee venom-desensitized patients. The sensitivity of the method is equal to the RAST-IgG with a correlation coefficient between the two methods of r = 0.94. Within-day and between-day precisions were 2.5 and 9.2%, respectively. The main advantages of PACIA are: (i) the use of easily prepared non-radioactive reagents having a shelf-life of at least 1 year at -20 degrees C: (ii) partial automation; (iii) calibration with antisera of animal origin, and (iv) the possibility of allergen standardization of honeybee venom extracts.

The aim of biological standardization (BS) is to equilibrate the activity (potency) of allergen extracts from different source materials. This was done by performing skin prick tests (SPT) on patients who were sensitive to one of the following 10 allergens: Birch, alder, hazel, timothy, rye grass, velvet grass, cultivated rye, mugwort, D. farinae and Cladosporium herbarum. Patient sensitivity varied within a range of three to four powers of ten for each allergen investigated. The weal size in each patient corresponding to that elicited by histamine 1 mg/ml was calculated using the model log (mean weal diameter) = a + b log (concentration). The correlation coefficients of the regression lines of the allergen dose response relationship were found to be greater than 0.85 in most cases. The median slope for all extracts was 0.24. The slope for Cladosporium was significantly steeper than that for pollens. The amount of material in microgram dry weight (d.w./ml) equal to 1000 biological units/ml (BU/ml) varied within a factor of three between species for all tested purified allergen preparations but Cladosporium. For Cladosporium, about 30 times more material was needed than for D. farinae. When using crude rather than purified material, it was necessary to use five to ten times more to elicit a reaction corresponding to 1000 BU/ml, but the difference was significant only for Cladosporium. The narrow range of allergen concentrations used by us as well as other investigators does not assure positive skin prick test results in all patients with clinical symptoms due to the allergen in question. Skin prick testing should therefore be done over a wide range of concentrations to improve the methods for BS.

Most extracts used in hyposensitization are complex and ill-defined mixtures of a large number of antigenic components. A highly refined (purified) and well-characterized allergen preparation from Timothy pollen (Phleum pratense) is now available. This paper describes the results of hyposensitization for 3 years comparing the purified preparation Timothy N, the crude extract Timothy O and a four-grass mix in sixty patients with allergic rhinitis due to grass pollen. The sixty patients were randomized into three groups and compared with a control group not hyposensitized. All three groups showed a significant decrease in clinical symptoms compared with the control group. The Timothy N group had a significantly higher nasal tolerance shown by nasal challenge test after 3 years' treatment than the group treated with the crude extract (P = 0.05). In addition, the Timothy-N-treated patients needed significantly less antihistaminic medication than the patients having received the crude extract or the four-grass mix (P = 0.02 and P = 0.01, respectively).

The biological activity in the allergic patient is our first and most relevant concern. The physician using an extract must know the quality and degree of allergenic activity in the material. Preferably, it should also be possible for the physician to control such activity by simple techniques. No single test can fully satisfy these demands. The standardization which comes nearest to this is assessment of the biological activity and specificity by means of skin testing using histamine as a reference. Skin testing is, however, limited as regards precision and reproducibility. A more precise standardization may be achieved by means of certain in vitro methods. Relevant in vitro methods are suitable complements to the bio-assay. However, the in vitro methods chosen must be standardized themselves as compared to a stable reference, and their correlation with the biological reactions in man must be established before they can be introduced as possible means for extract standardization. Even then, bio-assays are necessary for control of content of irritants which may elicit non-immunological (non-specific) reactions in the hyperreactive tissues of allergic individuals. Methods for standardization are discussed. Examples of quality gradings of allergen extracts are given with emphasis on minimal requirements to be fulfilled by extracts in order to be accepted for clinical use.

The changes that occur in the antigens and allergens of a timothy grass pollen extract during storage were measured by radioallergosorbent test (RAST) inhibition, isoelectric focusing, (IEF), crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) and a dye-binding protein assay. Storage intervals varied from 7 to 92 days at temperatures of 4 degrees, 22 degrees, and 35 degrees C and the freeze-dried extract was rehydrated and stored in either phosphate-buffered saline with 50% glycerol (PBSG) or Coca's solution (CS). For extracts stored in CS at 35 degrees C, allergenic activity as measured by RAST inhibition dropped by almost one half at 7 days and certain of the IEF, CIE, ad CRIE bands were no longer visible, showing that these properties correlated with allergenic activity. After 65 dys, no IEF bands were visible but three CIE bands could be visualized. At 22 degrees the changes were slower and at 4 degrees C no changes were observed for the 92-day storage time. Extracts stored in PBSG showed some of the same changes but were much more stable at all temperatures. During storage, the protein content as measured by dye binding was also reduced, showing tha proteins were fragmenting. Because some of the allergenic proteins disappeared more rapidly than others, there was not only a loss in total allergenic activity but also a change in specificity. This raises serious questions concerning the use of such extracts, especially for diagnosis.

The choice of vehicle for patch test materials is important for the bioavailability and stability of the allergens. Examples are given from the standardization of the TRUE Test and include caine mix, Kathon CG, and fragrance mix.

The allergenic potencies of samples of two commercial Alternaria tenuis extracts stored under various conditions were compared at 3 months intervals over a period of 1 year. Aliquots of the extracts at 5.12 mg/ml were maintained in: saline; 50% glycerol-saline mixture; 0.4% phenol-saline; 0.4% phenol-saline containing 0.03% human serum albumin, and samples were also kept in lyophilized form. The samples were stored at -20, 4, 24, 37 and 56 degrees C. Their potency was measured by radioallergosorbent inhibition assay and by mouse IgE passive cutaneous anaphylaxis (PCA) tests. The lyophilized extracts fully maintained their activity at all temperatures for 12 months. Aqueous extracts with or without preservatives also maintained their potency if stored at -20, 4 and 24 degrees C, but showed a significant loss of PCA activity at 56 degrees C, after 3 months. A deleterious effect of phenol was observed in one of the extracts stored at -20 degrees C for 3 months, by PCA tests. The use of preservatives to retain potency over a period of 12 months is therefore unnecessary and may be deleterious. The results indicate that, while storage of A. tenuis in a lyophilized form is best at all temperatures, aqueous extracts are also remarkedly stable when stored at -20, 4 and 24 degrees C, over a 12-month period.

Components of an extract of Dermatophagoides pteronyssinus were electrophoretically separated, transferred to nitrocellulose membranes, and then probed with a variety of antibodies and lectins. Using mite-allergic human sera as the first antibody source, IgE-binding components were identified. Binding patterns by monoclonal and heterologous sera were also obtained. The method is suitable for the qualitative, and ultimately the quantitative, characterization of allergen extracts. The electroblotting procedure employing specific probes, therefore, promises to be a valuable aid for allergen standardization.

Atopic allergens can readily be shown to inactivate hemolytic complement in human serum. This property has been explored as a means of in vitro allergen quantitation and standardization. The purification of house dust allergen, guided by skin tests and complement inactivation, demonstrates that the in vitro technique is clinically relevant. Since human sera differ in their sensitivity to complement inactivation by allergens, it is desirable to prepare a purified house dust allergen as a standard. By referring to this standard, other house dust extracts may then be standardized by using any given serum sample of human origin, provided that such extracts have undergone some degree of purification. Kinetic studies demonstrate that other allergens of the house dust group may be standardized in similar fashion; it was noted that the ratio of the complement inactivating powers of these respective allergens remains constant whatever the serum used. The method is not suited for the quantitation of pollen allergens.

The technique proposed by the National Institute for Biological Standards and Control, London (NIBSC, 1977) for the control of allergenic extracts for non-immunological activity in skin tests has proved to be both sensitive and useful. We have observed 'false positive' results to substances such as merthiolate or glycerine and also to lyophilised preparations with sugar, notably glucose, as the carrier substance. Other factors studied in 'false positive' reactions to a given extract were pH, osmotic pressure and the presence of traces of endotoxins or histamine. Individually none of these provided satisfactory explanations for the false positive reactions and the use of antagonists or inhibitors of the release of chemical mediators only confirmed the complexity of the non-immunological reaction and the participation of factors other than histamine.

Twelve whole mite extracts (WME) from seven sources were examined for their content of the allergen Dpt 12, protein content, and the inhibitory capacity in RAST by use of paper discs coupled with either WME or Dpt 12. Linear regression analysis demonstrated that the concentration of Dpt 12 determined either by single radial immunodiffusion or by RAST inhibition did not correlate with either the RAST-inhibition titer with use of discs coupled with WME or with protein content, suggesting that Dpt 12 content does not reflect the overall potency of WMEs. However, statistically significant correlations were obtained between Dpt 12 content and whole mite-RAST inhibition titers if extracts from the same manufacturer were examined or if data obtained from extracts containing large Dpt 12 to protein concentrations (greater than 25%) were excluded from the analysis. In contrast, statistically significant correlations between protein concentration and WME-RAST inhibition titers (p = 0.00037) and between Dpt 12 content and Dpt 12 RAST-inhibition titers (p = 0.00001) were found. Thermal stability studies demonstrated that Dpt 12 per se and WMEs were stable at 4 degrees C for up to 3 mo. Storage at 23 degrees C for 3 mo, however, resulted in partial loss of both Dpt 12 and whole-mite activity. Substantial losses occurred at 36 degrees C for both 1-month and 3-month incubation periods. Esterase, esterase lipase, and alkaline phosphatase were found in three WMEs studied. Dpt 12 was biochemically unrelated to these enzymes.

The production and standardization of the bacterial allergens are very difficult and connected with many unknown and disputable problems. The authors have carried out a study with three basic bacterial allergens produced by themselves: bacterial extracts, bacterial suspensions and a mixture of both from Streptococcus hemolyticus and Staphylococcus albus. The specific activity of all allergens in different concentrations is determined by skin intradermal tests simultaneously on 22 patients sensitized to Str. hemolyticus. 23 patients sensitized to Staph. albus, as well as on 10 healthy persons. The results of the study show that the bacterial extracts give predominantly immediate type skin allergic reaction while the bacterial suspensions lead to delayed type reactions. The mixed allergens give both type skin reactions, and reveal more specifically sensitization against the bacteria. The authors suggest that the optimal concentration of the mixed bacterial allergens for diagnosis by intradermal test is 10,000 PNU/ml and 100,000,000 bacterial/ml. The advantages and disadvantages of the three bacterial allergens are discussed.