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"In vitro assays in asthmatic patients have been exploring the "allergic" component of asthma. Solid-phase IgE tests can supplement or replace skin tests. Histamine release and determination are performed with automated, or RIA/ELISA; however, these assays are still research oriented. More recently, tryptase has been investigated, and the serum levels of this enzyme correlate well with mast-cell activation. But asthma is a multifactorial/facetted syndrome, and these assays provide minimal and specific informations which may just focus on limited etiological aspects of an elusive malady".

Modern approaches to the standardization of allergens require the use of not only biological methods, but also a wide range of physicochemical and immunological ones. To obtain Soviet standard samples, the optimum spectrum of methods for their characterization, evaluation and comparison with international standard samples should be definitely selected. After the preliminary study of a number of allergens by different immunochemical methods a set of such methods, including different kinds of electrophoresis, isoelectrofocusing, immunoblotting, etc., are proposed, these methods being considered obligatory for obtaining Soviet standard samples of allergens.

We compared in 50 asthmatic children, the cutaneous response by skin prick test to Dermatophagoides farinae with antigens from two different laboratories (Freeman and Hollister). In order to evaluate the importance of the selection, conservation and standardization of the allergic extracts. 27/50 children had a similar response, in those cases (6) where Freeman was doubtful and Hollister positive the allergic etiology was confirmed by RAST and Challenge tests. In 5 cases, Freeman was negative and Hollister doubtful we only confirm allergic etiology in 1. When both were positive all of them were allergic. The antigen Dermatophagoides farinae from Freeman laboratory needs another diagnostic method, when the cutaneous response is doubtful or negative and the clinical feature is highly suggestive. We don't have to forget the importance of the controls positive and negative.

Specific immunotherapy with standardized extracts can induce systemic reactions (SRs), possibly increased by a rush immunotherapy (RIT) protocol. A prospective study in 1152 mite-allergic patients (3 to 63 years of age) examined the incidence of SRs during an RIT or a step protocol. All patients received the same standardized extract of Dermatophagoides pteronyssinus with the same maintenance dose. In the first group, 290 patients had an RIT protocol without any preventive measure. In a second group, the prevention of SRs during RIT was attempted by pretreating 160 patients with methylprednisolone (0.5 mg/kg/day), ketotifen (2 mg/day), and theophylline (10 mg/kg/day). In a third group (479 patients), the same pretreatment was associated with preventive measures and with FEV1 results and the occurrence of large local reactions. A fourth group consisted of 223 patients who received a step protocol with the same pretreatment and preventive measures. The incidence of SRs per patient was 36.2% with RIT alone, 16.2% when the pretreatment was added, and 7.2% when pretreatment and preventive measures were used. Patients receiving the step protocol had 5.4% SRs. Adrenaline had to be used from 10.0%, 4.6%, 0.2%, and 0.2%, respectively. No reaction started 45 minutes or later after the last injection. Children younger than 5 years of age had a significantly greater number of SR.

Specific immunotherapy is ineffective with unstandardized mold extracts. A double-blind, placebo-controlled study was performed in 24 patients (5 to 56 years of age) only allergic to Alternaria. The extract was standardized by isoelectric focusing, crossed immunoelectrophoresis, crossed radioimmunoelectrophoresis, RAST inhibition, and skin tests and contained allergen Alternaria major allergen a I and antigen B. Thirteen patients received the active treatment, and 11 received the placebo. Immunotherapy was started by a 2-day rush protocol; maintenance injections were administered for 1 year. The patient's self-evaluation of the treatment was significantly (p less than 0.001) lower in the placebo-treated group. Global symptom-medication scores, including asthma and rhinoconjunctivitis, were significantly (p less than 0.005) lower in the actively treated group. Nasal challenges with Alternaria extract were performed before immunotherapy and after 1 year of treatment. There was no difference in the placebo-treated group and a significantly (p less than 0.01) increased mean provocative dose in the actively treated group. Skin tests were significantly reduced in the actively treated group. Specific IgG increased significantly in the actively treated group and were stable in the placebo-treated group. There was a significant correlation between nasal challenges and nasal symptom-medication scores (p less than 0.03) or the patient's self-evaluation of efficacy (p less than 0.05). This study demonstrated that patients only sensitized to Alternaria benefit from specific immunotherapy with a standardized Alternaria extract.

Imported fire ant whole-body extracts (IFAWBE) from three major U.S. commercial suppliers and a reference IFAWBE and imported fire ant venom (IFAV) preparation were compared by crossed immunoelectrophoresis and RAST-inhibition testing. Results of crossed immunoelectrophoretic studies showed major differences in antigen content between commercial preparations. IFAWBE and IFAV RAST-inhibition testing showed that no commercial extract had equivalent inhibitory activity to the reference IFAWBE. Reference IFAWBE reached 50% inhibition of IFAWBE and IFAV RAST at 52 and 492 micrograms/ml, respectively. IFAV gave 50% inhibition of IFAWBE and IFAV RAST at 5.9 and 58 micrograms/ml, respectively. Of the commercial extracts, only those from one supplier were able to reach 50% inhibition of the reference IFAWBE RAST, and no commercial extract from any supplier was able to reach 50% inhibition of IFAV RAST. Analysis of inhibition slopes and intercepts confirmed that there were both qualitative and quantitative differences among the commercial extracts and the reference IFAWBE and IFAV. Our findings suggest possible reasons why some individuals fail to respond to testing or immunotherapy with IFAWBE and show the urgent need for standardization of allergenic extracts for diagnosis and treatment of imported fire ant sensitivity.

Guidelines are proposed for determining the potency of allergenic extracts in relation to a reference extract using parallel line bio-assay. The practical performance, limitations, and advantages of skin prick test are discussed.

Sensitization to pollen from wind-pollinated trees causes significant allergy problems during springtime. The magnitude of prevalence is comparable with and, in some areas, even higher than grass pollen allergy. The symptom periods are often prolonged by cross-reactivity among species and sometimes families as well as by simultaneous sensitivity to several trees, pollinating at different times. There is a need for standardized extracts to improve diagnostic precision and treatment efficacy. A substantial amount of immunochemical data on the allergens from Betulaceae species, particularly Betula (birches), are available. Relevant methods to quantitatively and qualitatively measure the allergens of birch pollen extracts are clearly reproducible in laboratories around the world. By international collaboration, such work has generated an IS that has been accepted by the IUIS Subcommittee on Allergen Standardization. The IS is a yardstick for authorities, manufacturers, and researchers that is carefully specified in terms of 1 major and a few intermediate allergens, and also of total biologic activity by RAST inhibition. A meaningful declaration of biologic activity in vivo is awaited. As yet, only a few clinical trials have been undertaken with standardized tree pollen extracts. These are, however, highly interesting, particularly those that now have started with an oral route of administration.

Standardized dog and cat allergen preparations with defined allergen composition were used for immunotherapy (IT) in dog-sensitive and/or cat-sensitive adult patients. Seventeen patients completed 1 year of IT. Eleven patients were treated with dog allergen, five with cat allergen, and one with both dog and cat allergen. The treatment was well tolerated, with increased subjective tolerance. After 3 months of IT there was a significant decrease in conjunctival (p less than .05) and skin sensitivity (p less than .05). Specific IgE decreased significantly within 1 year as assayed by RAST and IgE-crossed radioimmunoelectrophoresis. Specific IgG increased in all patients. The results indicate that IT with potent and standardized dog and cat allergens influence the sensitivity to these allergens.

To develop a model for mold allergen extract standardization, we studied eight commercial Alternaria extracts from various suppliers by a variety of immunochemical and physicochemical techniques, including measurement of Alt-I, a purified allergenic fraction of Alternaria. Wide variations were noted in the allergenic and antigenic potencies of these extracts. Estimates of Alt-I content measured by Alt-I RAST inhibition and by radioimmunoassay correlated significantly (p less than 0.05), but Alt-I activity by either method could not be correlated with allergenic potency as measured by RAST inhibiton using solid-phase Alternaria. Each test extract produced unique and differing patterns of Coomassie blue-stained bands in isoelectrofocusing gels and in crossed immunoelectrophoresis gels using rabbit antibodies to Alternaria. The optimal method for mold allergen standardization involves a combination of RAST inhibiton, isoelectrofocusing, and crossed immunoelectrophoresis techniques, and, if possible, quantitation of individual allergens.

Optimal concentrations of honeybee, hornet and yellow jacket venoms were used in the radioallergosorbent test (RAST) before and after dialysis. RAST titers in the majority of sera from insect-allergic patients increased significantly with the dialyzed yellow jacket and hornet venom-coupled discs. Of 26 sera which had negative RASTs with crude venom, 20 became positive with dialyzed venom. No change in RAST results was found with crude and dialyzed honeybee venom. The low molecular weight component in the vespid venoms removed by dialysis had no specific activity in RAST inhibition experiments, suggesting nonspecific blocking of binding sites. These results indicate the importance of the appropriate antigen preparation coupled to discs in the RAST.

Oral immunotherapy (IT) was evaluated in a pilot study in two centres in children aged 8-15 years with allergic rhinoconjunctivitis. High doses (up to 20 X 10(6) BU monthly) of a defined freeze-dried birch pollen extract administered in enteric-coated gelatine capsules were given either daily for seven consecutive days every month or once weekly. Symptom scores, as assessed by sneezing, dripping and blockage of the nose, and redness, itching and swelling of the eyes, were significantly lower in treated patients compared to untreated, or placebo treated controls after 3 to 5 months of therapy. In all the 16 treated, but only in three of eight untreated patients, the scores were lower during the pollen season 1982 than during the pollen season preceding the treatment period, despite comparable pollen counts during the two seasons. One year after beginning treatment the reactivity in conjunctival provocation tests was decreased about 10-fold (P less than 0.001) in the patients receiving more than 2 X 10(5) BU monthly compared to about two-fold in patients receiving lower doses, or placebo. Increased levels of IgE antibodies directed against birch pollen were recorded in the serum and saliva of most patients after 3-4 months of active IT. In contrast, IgG antibody responses were poor in most of the patients. Side effects, particularly from the gastrointestinal tract, appeared in all treated children. In one of them a systemic reaction occurred during IT. The study indicates that properly performed oral IT with a potent birch allergen extract in enteric-coated capsules may be effective.

House dust mites of the species Dermatophagoides pteronyssinus (Dpt) represent one of the major allergens inducing asthma. However, a strict allergy to Dpt is not always observed in Dpt-allergic patients, since nonallergic and other allergic triggering factors often coexist. It was suggested that specific immunotherapy with house dust-mite extracts is more effective in children than in adults. A controlled study was undertaken in 215 Dpt-allergic patients with asthma ranging in age from 3 to 72 years (mean +/- SD, 28.2 +/- 10.9 years) to investigate parameters that might predict the efficacy of specific immunotherapy before it is started; 171 patients received a rush immunotherapy protocol with a standardized Dpt extract and, subsequently, maintenance injections with 3000 BU of this extract, and 44 patients served as a control group. Before immunotherapy, all patients had a complete evaluation of the severity of asthma by symptom-medication scores and a pulmonary function test, as well as a study of other triggering factors by checking on seasonal and perennial allergens, sinusitis, and other possible causes, such as aspirin intolerance; 196 patients were investigated between 9 and 12 months after the onset of the survey by means of pulmonary function tests and symptom-medication scores. Medications were adapted according to peak flow rates. It was observed that patients with chronic sinusitis, and/or aspirin intolerance, and/or other perennial allergies did not benefit from specific immunotherapy; therefore, the treatment of a major etiologic factor did not demonstrate improvement in the patients.(ABSTRACT TRUNCATED AT 250 WORDS)

To standardize the Dermatophagoides pteronyssinus extracts used in clinical allergy practice, the relative potencies of several purified extracts were estimated by radioallergosorbent test (RAST) inhibition, histamine release, and intracutaneous titration testing, and the results were compared. The potencies of nine independently prepared D. pteronyssinus extracts were assayed with respect to an extract adopted as a reference. In vivo the cutaneous wheal surface after intradermal injections was measured on a population of D. pteronyssinus-sensitive subjects. In vitro RAST inhibition was performed with the reference extract as allergosorbent and with a pool of human IgE-rich sera from patients sensitized to D. pteronyssinus but untreated. For histamine release human basophils from patients allergic to D. pteronyssinus were used. In the three method dose-response curves were plotted for the reference extract and the other extracts. A comparison of the measurement of in vitro and in vivo potencies is reported. Simialr results were obtained with the three methods: all the extracts but one were as potent as the reference preparation. However, for routine purposes RAST inhibition appears to be the most convenient and reliable procedure.

An IgE FAST(TM) inhibition protocol has been developed as a rapid in vitro method for determining relative potencies of in vivo allergenic extracts. Results are obtained within one day and correlate well with those of the RAST inhibition protocol (r = .96), making the FAST assay a preferable procedure for inprocess potency determination during the manufacture of standardized allergenic extracts.

Skin prick tests (SPT) were carried out on 164 asthmatic children using dog dander allergen preparations (A, B1, B2 and D) from three manufacturers. Bronchial and/or conjunctival provocation tests with allergen A were performed on all patients, as well as the Phadebas RAST (allergen e5). There was a close agreement between the clinical history and the SPT with allergens A and D. The provocation tests and RAST gave results in good agreement with the SPT reactions to all allergens except B1. The overall agreement between the SPT reactions to the different allergens was good. However, significant differences were observed in the allergenic activity of the different preparations and between different batches from the same manufacturer (allergens B1 and B2).

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.

Wide variations are observed in the potency of allergen preparations available for use on humans. There are numerous methods of in vivo or in vitro testing: skin testing, passive anaphylaxis testing on animals, study of precipitins, histamine release tests on human leukocytes, radio-immunological assay test (RAST), blasticlymphocyte transformation test (TTB). The first investigations seemed to show that the allergenic potency of a large number of inducing substances was due to proteic activity; this gave rise to the theory that potency could be evaluated by protein nitrogen units (PNU). However, the protein content of an allergenic extract is not always active. In addition, natural specific proteins are altered during the extraction process or subsequently during preservation. Where the active antigen is known, comparison of different testing with various preparations may be made. The results show excellent correlation between the two biological results (cutaneous reaction and histamine release) and the content of E antigen. No relation with PNU has been observed. Certain authors propose the following: skin testing, PNU titration, dosage of certain elements (polysaccharides, lipids, phosphates, etc.), RAST. The great differences observed between the value of skin testing and the RAST may be due to the large number of active antigens in different 'dusts' and to the fact that the RAST method measures only IgE whereas allergic activity of these allergens is no doubt associated with other types of antibodies. The complexity of the problem shows that a great many studies are still necessary to provide a satisfactory solution.

Six candidate extracts of Lolium perenne (rye-grass) pollen have been studied in 6 laboratories using a variety of immunochemical and physicochemical techniques. Radioallergosorbent test inhibition, crossed immunoelectrophoresis, crossed radio-immunoelectrophoresis, sodium dodecyl sulphate-polyacrylamide gel electrophoresis combined with immunoblot, thin-layer isoelectric focusing and enzyme-linked immunosorbent assay inhibition were used to evaluate each of the coded extracts. The source materials were also studied for identity and possible contamination by light microscopy. On the basis of these data, the Rye-Grass Working Party recommended to the Steering Committee of the Allergen Standardization Subcommittee of the International Union of Immunological Societies that the extract coded C be chosen as the candidate international reference preparation.