Math-free guides for glycerin and allergens at variable subcutaneous injection volumes: How's my dosing? Update.

BACKGROUND: Current summaries of effective maintenance dose ranges for subcutaneous immunotherapy (SCIT) are based on administration of 0.5-mL volumes. Extract formulations delivering equivalent dose ranges for practices using different injection volumes have not been reported, and calculation of the final glycerin concentrations in these solutions remains an inconvenient and repetitive process. OBJECTIVE: To create math-free guides for allergen doses and glycerin concentrations that identify the extract concentrate volumes required to deliver doses within the ranges cited in the 2011 immunotherapy practice parameters for clinicians using 5.0-mL maintenance vials and injection volumes ranging from 0.2 to 1.0 mL. METHODS: Algebraic calculations were performed to determine the specific combinations of extract concentrate strengths, volumes of these products in patient vaccines, and injection volumes needed for administration of target allergen doses spanning the current SCIT practice parameter recommendations. RESULTS: For each product or group (nonstandardized extracts), tables were constructed to define the allergen doses provided by various combinations of extract concentrate volumes and injection volumes. The values within the effective dose ranges for each product were highlighted to facilitate comparisons of specific conditions relevant to allergy specialists. Glycerin tables were also created to permit convenient assessments of the final concentrations of this stabilizer in patient prescriptions. CONCLUSION: SCIT dosing and glycerin tables are useful tools to assist allergists with practice decisions that involve variable patient formulas and injection volumes and can help identify suitable conditions for treatment of patients presenting with diverse allergen sensitivities and specificity profiles.

Allergen stabilities and compatibilities in immunotherapy mixtures that contain cat, dog, dust mite, and cockroach extracts.

BACKGROUND: Indoor allergen mixtures that contain cat, dog, dust mite, and cockroach extracts are commonly used in allergy clinics for subcutaneous immunotherapy, but product-specific stabilities and mixing compatibilities in these complex patient formulas have not been determined. OBJECTIVES: To assess the recoveries of cat, dog epithelia, dog dander, dust mite Dermatophagoides farinae, and cockroach mix allergen activities in 5 component mixtures and 1:10 (vol/vol) dilutions stored for up to 12 months. METHODS: Concentrated stock mixtures, 10-fold dilutions of these mixtures in human serum albumin-saline diluent, and analogous single-extract controls were analyzed for major allergen concentrations (cat Fel d 1, dog dander Can f 1) and multiallergen IgE-binding potencies (dog epithelia, D farinae, cockroach mix) after storage for 3, 6, 9, and 12 months at 2°C to 8°C. RESULTS: The selected immunoassays were specific for individual target extracts in the 5-component mixtures and exhibited analytical sensitivities sufficient for evaluation of both the concentrated and diluted indoor allergen formulas. All control samples except diluted cockroach extract had near-complete stabilities during refrigerated storage. Mixtures that contained cat, dog epithelia, dog dander, and D farinae extracts exhibited favorable mixing compatibilities in 1:1 (vol/vol) concentrates (47.5% glycerin) and 1:10 (vol/vol) dilutions (4.75% glycerin), relative to corresponding control sample reactivities. Cockroach allergens in both 1:1 (vol/vol) and 1:10 (vol/vol) concentrations were stabilized significantly by mixing with the other 4 indoor allergen extracts. CONCLUSION: Extracts in mixtures that contained 5 common sources of indoor allergens possess favorable stabilities and mixing compatibilities and support the practice of combining these products in the same patient treatment formulations for subcutaneous immunotherapy.

Mixing compatibilities of Aspergillus and American cockroach allergens with other high-protease fungal and insect extracts.

BACKGROUND: Recent studies have shown that Alternaria and German cockroach allergens can be degraded by endogenous proteases from other insect and fungal extracts when combined for immunotherapy, but data supporting the compatibilities of other high-protease products in comparable mixtures have not been reported. OBJECTIVE: To assess the stabilities and compatibilities of Aspergillus fumigatus and American cockroach allergens after mixing with protease-rich extracts from other insects or fungi at concentrations similar to those recommended for subcutaneous immunotherapy. METHODS: Mixtures containing A fumigatus, American cockroach, and other fungal or insect extracts were evaluated by quantitative (enzyme-linked immunosorbent assays) and qualitative (immunoblotting) methods. Test mixtures and control samples at 10% to 50% glycerin concentrations were analyzed after storage for up to 12 months at 2°C to 8°C. RESULTS: Moderate to high recoveries of Aspergillus extract activities were retained in control samples and extract mixtures under all conditions examined. American cockroach extract controls were partly degraded at 10% to 25% glycerin, and cockroach allergen compatibilities were decreased significantly in mixtures with several fungal extracts at 25% glycerin. Mixing with other insects did not compromise the stability of American cockroach allergens at 25% to 50% glycerin. CONCLUSION: Aspergillus extracts exhibited favorable stabilities after mixing with other high-protease products. American cockroach extract potencies were unstable in less than 50% glycerin, even in the absence of other protease-containing allergens, and were destabilized in mixtures with several fungal extracts. Addition of fungal and insect extracts to separate treatment vials or preparation of fungal-insect mixtures at elevated glycerin concentrations might be necessary to produce compatible patient formulations for allergen immunotherapy injections.

Allergen stabilities and compatibilities in mixtures of high-protease fungal and insect extracts.

BACKGROUND: Current practice guidelines state that protease-rich fungal and insect extracts can be combined when preparing immunotherapy vaccines, but data supporting the stability of allergens in these mixtures have not been reported. OBJECTIVE: To determine the stabilities and compatibilities of Alternaria alternata and German cockroach allergens in mixtures with other high-protease fungal and insect (cockroach, imported fire ant) extracts at final extract concentrations consistent with injection dose targets for maintenance immunotherapy. METHODS: Mixtures containing Alternaria, German cockroach, and other fungal and insect extracts frequently included in immunotherapy vaccines were analyzed by a combination of quantitative analyses (enzyme-linked immunosorbent assays for multiallergen immunoglobulin E [IgE]-binding potency, major Alternaria allergen Alt a 1, and major German cockroach allergens Bla g 1 and Bla g 2) and qualitative methods (immunoblotting). Mixtures and analogous single-extract controls containing 10 to 50% glycerin were evaluated after storage for up to 12 months at 2°C to 8°C. RESULTS: Mixtures of extracts within the same phylogenetic groups (fungal-fungal, insect-insect) retained favorable Alternaria and German cockroach allergen levels and activities under most conditions examined. For several cross-taxonomic (fungal-insect) extract combinations at 10 to 25% glycerin concentrations, different immunochemical test methods measuring single (major) or multiple allergens yielded threefold to 10-fold variations in allergen recoveries. CONCLUSION: Allergen compatibilities can be compromised in some fungal-insect extract mixtures, contrary to current immunotherapy practice parameter recommendations. Separation of these products into different treatment vials may be required to produce stable mixtures for subcutaneous immunotherapy. Data from assay methodologies with distinct binding specificities provide a critical assessment of allergen activities in high-protease extract mixtures.

How's my dosing? A one-step, math-free guide for comparing your clinic's maintenance immunotherapy doses to current practice parameter recommendations.

BACKGROUND: Immunotherapy dose recommendations for allergens specified in practice parameter updates have varied from version to version, and no convenient methods are available to incorporate dose changes or targets into maintenance vial formulations for patients. OBJECTIVES: To compare the allergen immunotherapy dose recommendations published between 2003-2011, and to provide math-free dosing tables that translate dose targets for single or multiple extracts into maintenance vial compositions. METHODS: Dose recommendations obtained from immunotherapy practice parameters published in 2003, 2007 and 2011, plus a worksheet created in 2004, were tabulated and compared. Conversion tables were created based on the fixed mathematical relationships between extract concentrate strengths and maintenance immunotherapy dose targets. Considerations of stock mixes, glycerin levels, and allergen compatibilities were applied using two examples of common extract formulations. RESULTS: Changes to immunotherapy dose ranges for standardized extracts included adjustment of upper limits (short ragweed), lower limits (pasture grasses), or both (cat, dust mites, Bermuda grass). Dose ranges for non-standardized products have also been modified over time. Conversion tables specified the extract concentrate volumes or percentages required to deliver minimum, midrange and maximum recommended doses with 0.5 mL injection volumes. CONCLUSIONS: These dosing guides, used in conjunction with cross-reactivity, compatibility and glycerin tolerance information, provide clinicians with a convenient and systematic method for determining the numbers and strengths of extracts that can be combined into treatment vials at various dose levels, and can facilitate optimization of maintenance immunotherapy mixtures for patients exhibiting a wide range of allergen sensitivities and specificities.

Allergen compatibilities in extract mixtures.

Stability studies with a few well-characterized allergen extracts have yielded useful information about the shelf-life of these products stored under various conditions. The development of validated stability-indicating tests and their clinical verification remains a fundamental challenge for extending this information to cover more products. This challenge becomes even greater for evaluations of more complex, multiextract mixtures that are used in clinical practice. Thus, the current approach for developing guidelines for extract expiration dating practices must rely on extrapolations of data obtained from a few well-controlled studies.

Stability and mixing compatibility of dog epithelia and dog dander allergens.

BACKGROUND: Little information or data are available concerning the stability and compatibility of dog epithelia and dog dander allergens. OBJECTIVE: To determine the immunochemical reactivities of commercial, nonstandardized dog epithelia and dog dander extracts after exposures to various temperatures or after mixing with high-protease fungal and cockroach extracts at concentrations recommended for maintenance immunotherapy (IT) injections. METHODS: Quantitative enzyme-linked immunosorbent assay and qualitative (immunoblot) analyses were performed to compare specific compositional changes with total or individual allergen activities. Assays for dog allergens Can f 1 and Can f 3 (albumin) used specific mouse or rabbit antibodies. Multiallergen enzyme-linked immunosorbent assay inhibition and immunoblot methods were conducted using a human serum pool with high levels of IgE to dog allergens. RESULTS: Dog allergen recoveries ranged from 22% to 134% after short exposures to moderate or extreme temperatures and from 28% to 118% after mixing with fungal or insect extracts and storage for up to 15 months at 2 degrees C to 8 degrees C. Recoveries in dog dander extracts varied up to 2.5-fold with different test methods. Immunoblots revealed partial degradation of dog albumin molecules to discrete fragments that retained antibody-binding activities. In most cases, recoveries improved at elevated glycerin concentrations. CONCLUSIONS: Dog allergens in epithelia and dander extracts exhibited favorable temperature stabilities. Compatibilities with fungal or insect extracts may be compromised or at risk in some combinations. These data support current IT practice parameter recommendations of separating high-protease extracts from other products if possible; they also demonstrate that dog extracts possess allergen stabilities suitable for many IT formulations.

Compatibility of imported fire ant whole body extract with cat, ragweed, Dermatophagoides pteronyssinus, and timothy grass allergens.

BACKGROUND: Recommendations regarding the administration of imported fire ant whole body extract (IFA WBE) combined with aeroallergens or environmental allergens in a single immunotherapy injection are lacking. OBJECTIVE: To evaluate the degradative effect of IFA WBE on cat, ragweed, Dermatophagoides pteronyssinus, and timothy grass allergens. METHODS: Imported fire ant whole body extract was combined with extracts of cat, ragweed, D pteronyssinus, and timothy grass. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on each sample after storage for 0, 1, 3, and 6 months at 4 degrees C. In addition, cat and ragweed combinations were evaluated by radial immunodiffusion (RID); D pteronyssinus by enzyme-linked immunosorbent assay (ELISA) inhibition; and timothy grass by ELISA inhibition and Western blot. RESULTS: Imported fire ant whole body extract combined with timothy grass demonstrated degradation of timothy grass allergens by SDS-PAGE, ELISA inhibition, and Western blot results. Cat and ragweed allergens were stable after mixing with IFA WBE, based on SDS-PAGE and RID analyses. Stability of D pteronyssinus allergens with IFA WBE was evident from SDS-PAGE and ELISA inhibition data. CONCLUSIONS: Imported fire ant whole body extract combined with timothy grass resulted in significant and rapid timothy protein degradation. Imported fire ant whole body extract mixed with cat, ragweed, or D pteronyssinus revealed aeroallergen stability, yielding the possibility of combining these extracts in a single immunotherapy injection. Compatibilities of IFA WBE with other common aeroallergens remain undetermined and thus are not recommended for single-injection immunotherapy formulations.

Stability of standardized grass, dust mite, cat, and short ragweed allergens after mixing with mold or cockroach extracts.

BACKGROUND: Limited data are available on the immunochemical compatibilities of standardized and nonstandardized allergen extracts in immunotherapy vaccines. Extract combinations recommended in immunotherapy practice parameters are based primarily on theoretical considerations rather than on actual product compatibilities. OBJECTIVES: To determine the stabilities of standardized grass, short ragweed, dust mite, and cat extracts after mixing with fungal and cockroach extracts at final product concentrations similar to those recommended for maintenance immunotherapy injections. METHODS: Mixtures were prepared using individual products from multiple sources at variable glycerin concentrations and were analyzed after storage for up to 1 year at 2 degrees C to 8 degrees C. Quantitative analyses included radial immunodiffusion assays for cat Fel d 1 and short ragweed Amb a 1 and human IgE enzyme-linked immunosorbent assay inhibitions for meadow fescue grass and dust mite allergens. Immunoblot analyses provided qualitative patterns of IgE binding. RESULTS: Meadow fescue grass allergens were unstable after mixing with fungal or cockroach extracts but were highly compatible with dust mite extracts from numerous commercial sources. Fescue and dust mite allergen recoveries varied considerably when mixed with different mold extracts. The presence of cockroach extracts reduced dust mite allergen potencies but retained moderate levels of cat and short ragweed allergen activities. In all cases examined, glycerin provided concentration-dependent improvements in allergen recoveries. CONCLUSIONS: Several allergen extract combinations generally regarded as unstable by current practice parameters seem to possess considerable biochemical compatibilities. Use of these mixtures in immunotherapy vaccines is supported for practitioners seeking to optimize formulations, doses, and treatment regimens for their patients.

Aluminum sulfate significantly reduces the skin test response to common allergens in sensitized patients.

BACKGROUND: Avoidance of allergens is still recommended as the first and best way to prevent allergic illnesses and their comorbid diseases. Despite a variety of attempts there has been very limited success in the area of environmental control of allergic disease. Our objective was to identify a non-invasive, non-pharmacological method to reduce indoor allergen loads in atopic persons' homes and public environments. We employed a novel in vivo approach to examine the possibility of using aluminum sulfate to control environmental allergens. METHODS: Fifty skin test reactive patients were simultaneously skin tested with conventional test materials and the actions of the protein/glycoprotein modifier, aluminum sulfate. Common allergens, dog, cat, dust mite, Alternaria, and cockroach were used in the study. RESULTS: Skin test reactivity was significantly reduced by the modifier aluminum sulfate. Our studies demonstrate that the effects of histamine were not affected by the presence of aluminum sulfate. In fact, skin test reactivity was reduced independent of whether aluminum sulfate was present in the allergen test material or removed prior to testing, indicating that the allergens had in some way been inactivated. CONCLUSION: Aluminum sulfate was found to reduce the in vivo allergic reaction cascade induced by skin testing with common allergens. The exact mechanism is not clear but appears to involve the alteration of IgE-binding epitopes on the allergen. Our results indicate that it may be possible to diminish the allergenicity of an environment by application of the active agent aluminum sulfate, thus producing environmental control without complete removal of the allergen.

Deleterious effects of electron beam radiation on allergen extracts.

BACKGROUND: The recent threat to the public posed by the dissemination of Bacillus anthracis through the US postal system has resulted in increased security measures, including electron beam irradiation for the sterilization of some mail. The deleterious effects of electron beam radiation on biological products are not fully understood. OBJECTIVE: The purpose of this investigation was to assess the effect of electron beam radiation, as currently used to sterilize packages and mail in the United States, on several standardized or characterized allergen extracts. METHODS: Selected irradiated extracts were analyzed for allergen content and potency by SDS-PAGE, immunoblot, and ELISA (including inhibition) and compared with untreated extracts. RESULTS: The compositions and immunochemical potencies of these products were altered significantly by irradiation treatment. Physical changes to native protein structures observed after electrophoretic separations coincided with near-complete loss of allergenic and antigenic epitopes present on major and minor allergens, according to ELISA and immunoblot comparisons with untreated extracts. CONCLUSIONS: These results indicate that extracts subjected to electron beam sterilization conditions are likely to contain modified component structures and properties that might compromise the clinical effectiveness of these products.

Major allergen measurements: sources of variability, validation, quality assurance, and utility for laboratories, manufacturers, and clinics.

The isolation and characterization of prominent allergenic proteins or glycoproteins is an important step in the development of allergenic extracts exhibiting improved definition, consistency, and clinical utility. Quantitative analyses specific for major allergenic components currently are being performed in numerous corporate and academic laboratories but have not been validated within or across laboratories in a systematic manner. In our laboratory, validation of double-bind (sandwich) ELISA assays for a diverse group of major allergens or extract components revealed a number of critical assay variables and reagent incubation conditions that directly influenced the precision, accuracy, specificity, and robustness of these tests. Data from ELISA methods for six allergens (Dermatophagoides farinae Der f 1, Alternaria Alt a 1, dog albumin, dog Can f 1, fire ant Sol i 3, and yellow jacket venom Ves 5) showed that up to twofold differences in results were observed when analysts or microplates were varied. Analyses of dog allergens using multiple reagents and concentrations indicated that twofold variations in results also can be produced by distinct combinations of materials or incubations from different assay steps. Data from Can f 1 and egg white analyses produced up to fivefold differences in antigen concentrations based on changes in the capture antibody source (mouse monoclonal versus rabbit polyclonal) or storage buffer. These results suggest that differences in major allergen concentrations reported by different testing laboratories may be related to assay differences as well as extract variations and raise questions as to the accuracy of major allergen concentrations and therapeutic dose recommendations reported at regional and national allergy meetings. Validated double-bind ELISA methods may be well suited for consistency monitoring and standardization of extracts provided that reference materials, reagent qualifications, and interlaboratory comparability are defined precisely.