Subcutaneous immunotherapy (SCIT) is a long-established treatment option for allergic rhinoconjunctivitis. Proper dosing of the allergens is critical for the efficacy and safety of SCIT. Of the hundreds of liquid allergen extracts in the United States, effective and well-tolerated SCIT dosing has only been established for a small number. Thus, SCIT dosing remains largely empiric and continues to be, by necessity, an art. To highlight the complexity of SCIT dosing, this review summarizes the historical and current landscape of U.S. allergen extracts, differences among U.S. and European allergen extracts, allergen selection for SCIT, considerations for compounding of allergen extract mixtures, and recommended dosing. As of 2021, 18 standardized allergen extracts are available in the United States; all other extracts remain unstandardized without characterization of allergen content or potency. U.S. allergen extracts differ from European extracts in formulation and potency characterization. There is no standardized methodology for SCIT allergen selection, and interpretation of allergen sensitization is not straightforward. Compounding of SCIT mixtures requires consideration of potential dilution effects, allergen cross-reactivity, proteolytic activity, and additives. Probable effective dose ranges for SCIT are recommended in U.S. allergy immunotherapy practice parameters, although there are few studies using U.S. extracts supporting these doses as therapeutic. In contrast, optimized doses of sublingual immunotherapy tablets have been confirmed in North American phase 3 trials. The SCIT dosing for each patient remains an art that requires clinical experience and consideration of polysensitization, tolerability, compounding of allergen extract mixtures, and the range of recommended doses within the context of extract potency variability.

Allergy/immunology specialists in the United States prescribing allergy immunotherapy (AIT) have placed a heavy value on practical experience and anecdotal evidence rather than research-based evidence. With the extensive research on AIT conducted in the last few decades, the time has come to better implement evidence-based medicine (EBM) for AIT. The goal of this review was to critically assess EBM for debated concepts in US AIT practice for respiratory allergies in the context and quality of today's regulatory standards. Debated topics reviewed were the efficacy and safety of AIT in various subgroups (eg, polyallergic patients, older patients, patients with asthma, and pregnant women), diagnosis topics (eg, skin prick test vs allergen-specific serum IgE, factors affecting skin prick tests, use of nasal or conjunctival allergen challenges, and telemedicine for diagnosis), and dosing topics (eg, optimal dosing for subcutaneous immunotherapy and sublingual immunotherapy tablets, US liquid allergen extract history, duration of treatment, and biomarkers of efficacy). In addition, EBM for patient-centered AIT issues (eg, adherence, use of practice guidelines, and pharmacoeconomics) and the approach to implementation of AIT EBM in future clinical practice were addressed. The EBM for each concept was briefly summarized, and when possible, a practical, concise recommendation was given.

Allergen immunotherapy (AIT) is thought to be clinically effective and safe in treating allergic rhinitis, asthma, and stinging insect allergy in Europe and North America. However, there are intercontinental differences in AIT therapeutic products in terms of their application and regulation. In North America unmodified standardized and nonstandardized aqueous aeroallergen extracts are approved and used almost exclusively for subcutaneous immunotherapy, whereas more product options are available in Europe, including adsorbed allergens, chemically modified allergens, or both. Both liquid extracts and tablets are approved for sublingual immunotherapy in Europe. Nevertheless, within the European Union, there are major differences in AIT products approved and used in individual countries. There are major differences in the clinical approach to subcutaneous immunotherapy in polysensitized patients; in the United States mixed extracts containing multiple aeroallergens are used, whereas European allergists preferably administer separate injections of single allergen sources or homologous groups deemed to be clinically relevant. Moreover, the regulatory approach differs between the European Union and United States. In contrast to the United States, where common allergen standards exist based on biologic activity, no common standards exist in Europe. In terms of development of new investigational products, the United States has followed the European example for phase II and III studies; no formal US Food and Drug Administration guidance has been issued.

Dermatophagoides dust mites are known to vary across North America depending on location with factors like elevation and humidity influencing their presence. These studies have included microspopic identification of mites, DNA analysis and allergen protein measurements. In order to develope a current mapping of allergen protein prevalence, we have studied dust from homes in 16 regions around the US for major dust mite allergens. In order to explore variability within a region, multiple homes from the same region were studied. Dust from vacuum cleaner bags from 24 homes was used to make 1:10w/v extracts using a 1% albumin, 0.9%saline phenol extraction fluid. The extracts were filtered using a 0.2micron filter then analysed for species specific Der p/f 1 using direct binding monoclonal ELISAs (ALK, Madrid). Nineteen of the homes had detectable mite allergens ranging from below 1 microgram/gram of dust to more than 20micrograms/gram. 12 of these homes showed D. farinae only, 1 in Southern California showed D. pteronyssinus only, and 6 homes contained both mites. The D. pteronyssinus containing homes were not confined to coastal areas. The homes in Spokane, Reno, Denver, and Sonoma showed no mites. Out of 9 homes in Texas that contained mite allergens,one had no detectable dust mites. This study confirmed widespread presence of dust mite allergens in homes across the US but are low in drier rocky mountain states. Many homes contain both species in New England and Southern regions.

BACKGROUND:Limited information is available for dogs on threshold concentrations (TCs), and the protein composition of common allergenic extracts produced by different manufacturers.HYPOTHESIS/OBJECTIVES:To characterize the protein heterogeneity of tree, grass, weed and mite allergens from different lots of allergenic extracts, and to determine intradermal TCs for healthy dogs using extracts from two manufacturers.ANIMALS:Twenty five privately owned, clinically healthy dogs and ten purpose-bred beagle dogs.METHODS AND MATERIALS:Protein concentration and heterogeneity of 11 allergens from two manufacturers were evaluated using a Bradford-style assay and SDS-PAGE. Intradermal testing was performed with 11 allergens from each company at four dilutions. Immediate reactions were subjectively scored (0 to 4+), and objectively measured (mm) and their percentage concordance evaluated. Model-based TCs were determined by fitting positive reactions (≥2+) at 15 min to generalized estimating equations.RESULTS:Allergen extract protein quantity and composition varied within and between manufacturers despite sharing the same PNU/mL values. Model-based TCs of one weed, five trees, two grasses and a house dust mite were determined for extracts from Manufacturer 1 (M1), and for extracts of three weeds, three trees and two grasses from Manufacturer 2 (M2). Receiver operating characteristic curve analyses determined a percentage concordance of the objective and subjective measurements of 77.3% for M1 and 75% for M2 allergens.CONCLUSIONS AND CLINICAL IMPORTANCE:Veterinary allergen extracts labelled as the same species and PNU/mL are not standardized; they show heterogeneity in composition and potency within and between manufacturers. Variability in extract content may require adjustment of intradermal testing concentrations.

Management of allergic rhinitis includes avoiding the exposure of allergens combined with drug therapy. For the patients that do not have substantial relief in allergic rhinitis symptoms or incur side effects, subcutaneous and sublingual immunotherapy can be a second line of treatment. This study looked at an alternative topical route of immunotherapy administration of allergen through the skin which may be beneficial in treating allergic rhinitis. IgE, IgG and lymph node T cell responses in mice were studied using ovalbumin antigen mixed with topical cream formulations with potential adjuvant properties applied to the skin. BALB/c mice were immunized with ovalbumin mixed with alum to induce an IgE response. After 14 days ovalbumin mixes with topical creams and controls were applied to the back skin of the mice and IgE, IgG2a and T Cell biomarkers from draining lymph nodes were measured up to 71 days and results compared with intraperitoneal injection controls. Mice developed IgE to ovalbumin after 14 days and application of cream formulations showed a dose dependent reduction in IgE compared to controls at 42 and 71 days. Topically applied ovalbumin showed an increase in sIgG2 comparable to intraperitoneal injection in some formulations and biomarkers such as CD69 indicated that the antigen reached draining lymph nodes. Initial results from this mouse study demonstrated that topical administration of allergens mixed in creams can have an immune response and may provide an alternative route for immunotherapy.

It is important to understand the scope of cross-reactivity among mold extracts when prescribing allergen immunotherapy (AIT) for mold allergic patients. Due to the large proportion of poly-sensitized patients in the clinical setting, we hypothesize that a high degree of cross-reactivity occurs between species within closely related taxonomic groups (e.g. Class, Order, and Family). Furthermore, we hypothesize that the potential for cross-reactivity exists between separate mold classes, due to protein homology. Therefore, we investigated commercial AIT extracts to better define the cross-reactivity relationships among clinically-relevant molds. The protein content and diversity of mold extracts were established by Bradford and gel electrophoresis assays. We evaluated cross-reactivity among 15 commercial mold extracts (ALK, Inc) using mono- and poly-sensitized sera derived from both rabbits and humans. Mold proteins were separated by SDS-PAGE, immobilized on nitrocellulose membranes, and immunoblotted using the above-mentioned sera. Human sera samples were purchased from Plasma Labs and screened for mold sensitivity by Thermo Fisher ImmunoCAP. Rabbit sera specific to single molds were generated by ALK (Denmark). In agreement with our hypothesis, molds probed with sera reactive to single or dual mold species produced strong banding patterns in multiple extract lanes. Cross-reactivity was consistently observed between related species within the same Class; sera reactive to Alternaria, Aspergillus, Cladosporium or Epicoccum reacted to all extracts present from molds in the class Dothideomycetes. Our results suggest an overlap in allergenic content of closely-related mold species. This finding may support the simplification of AIT formulations for treatment of mold sensitized patients.

ObjectiveTo review the methods and materials used for collection, purification, commercial production, and clinical application of Hymenoptera venoms.Data SourcesMost of the sources for this review are the experience and expertise of the authors. Published reports and review articles on Hymenoptera venom collection and production were identified through database searches (PubMed).Study SelectionsStudies describing the methods for Hymenoptera venom collection and production were selected for review.ResultsMeticulous methods for identification and collection of the insects are required. Collection and purification of the venoms from the insects are based on validated methods and result in a commercial extract that is standardized for the major allergenic proteins required for accurate diagnosis and safe and effective treatment of patients allergic to insect sting. The steps required for mixing, purifying, testing, and standardizing the products are described.ConclusionHymenoptera venom extracts were developed using many new methods for the collection, purification, and commercial production of the unique materials required for this product. Clinical applications for diagnosis and treatment are affected by the integrity and stability of the allergens after processing and purification.

Total protein extractability and IgE binding are useful methods for determining potency of allergenic extracts. These techniques were applied to investigate various production methods for walnut food extracts. English walnut food extracts were made to evaluate source material, defatting, extraction fluid, and processing. Protein content was tested by Bradford and SDS-PAGE. IgE binding was determined by Immunoblotting and microtiter plate ELISA titration using sera from walnut sensitive patients. Walnut food extracts can show a more than 100 fold variability in total protein depending on the extraction process. Aqueous extraction shows the lowest total protein content and 50% glycerin with blending greatly improves the extractability of protein. Total protein correlated to the ability to bind IgE with levels below 0.3mg/mL showing negative IgE binding with moderately sensitive serum (<17kU sIgE). Walnut extract IgE binding potency is dependent on the presence of glycerin during processing.

Purpose of reviewThe article reviews studies pertaining to US manufactured allergen extract stability that contribute to guidance for expiration dating for bulk concentrates, diluted patient testing and treatment vials, and adjustments following elevated temperature excursions.Recent findingsStudies on allergen stability were completed to satisfy the Food and Drug Administration requirements supporting labeled expiration dating for standardized short ragweed, dust mites, cat, grass, and venom extracts and are not published. These studies demonstrated the stability enhancing parameters of 50% glycerin and support the Food and Drug Administration mandated expiration of nonstandardized extracts allowing glycerin extracts twice the dating of nonglycerin aqueous extracts. Patient vials are commonly given 6-12 months dating. There is adequate evidence that human serum albumin stabilizes low protein diluted vials. High protease allergens such as molds and insects compromised potency of pollens when mixed. Subsequent work continued to define the effects of diluents on extract dilutions, temperature excursions that occasionally occur with shipping or refrigerator malfunctions, and allergen compatibility.SummaryPotency has been determined for allergen dilutions with diluents typically used for allergen immunotherapy. These studies along with changes of potency under various storage conditions and mixing designed to improve our guidance on expiration dating of allergen extracts will be discussed.

Background: Consistency in composition and potency, particularly regarding major allergens, is crucial for the quality of extracts for allergen immunotherapy.Objective: To characterize the major allergen composition of house dust mite (HDM) extracts commercially available in the United States and the SQ HDM sublingual immunotherapy (SLIT) tablet, and to relate the composition to patient sensitization patterns.Methods: Der 1/Der 2 ratios were determined in 10,000- and 30,000-AU/mL HDM extracts from 5 US companies and the SQ HDM SLIT-tablet. Allergen content was analyzed by enzyme-linked immunosorbent assay and compared with an in-house reference. Sensitivity toward Der p 1, Der p 2, and Der p 10 was determined in serum from randomly selected subgroups of 220 individuals from North American and European SQ HDM SLIT-tablet trials.Results: Mean Der 1/Der 2 ratios in US HDM extracts ranged from 0.4 to 20.5. For the SQ HDM SLIT-tablet (20 batches), variability did not exceed 12% regarding content of Der f 1 (SD, 11.9%; 95% confidence interval [CI], 0.94-1.06), Der p 1 (SD, 6.1%; 95% CI, 0.97-1.03), and combined Der 2 allergen (SD, 6.4%; 95% CI, 0.97-1.03), indicating a consistent Der 1/Der 2 ratio. High allergen sensitivity frequencies toward Der p 1 and Der p 2 were observed regardless of geographic region. Efficacy of the SQ HDM SLIT-tablet has been demonstrated in 5 clinical trials.Conclusion: The SQ HDM SLIT-tablet has efficacy potential for a broad range of patients because it includes a consistent 1: 1 ratio of the 2 major HDM allergens to which individuals were most frequently sensitized across geographic regions. Efficacy has been demonstrated. (C) 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Consistency in composition and potency, particularly regarding major allergens, is crucial for allergen immunotherapy efficacy and safety. We compared allergen sensitivity profiles for house dust mite (HDM) in North American and European patients with the allergen content of commercially available US HDM extracts and an SQ-HDM sublingual immunotherapy tablet (SLIT-tablet; MK-8237; Merck/ALK). Sensitivity towards Der p 1, Der p 2, and Der p 10 allergens was determined in randomly selected subgroups of 220 subjects from European and North American SQ-HDM SLIT-tablet trials. Der 1/Der 2 ratios were determined in 10,000 and 30,000 AU/mL HDM extracts from 3 US companies. SQ-HDM SLIT-tablets are produced by mixing body and fecal fractions of Dermatophagoides farinae and Dermatophagoides pteronyssinus to achieve a 1:1:1:1 ratio between Der f 1/2 and Der p 1/2 allergens. Allergen content was analyzed by ELISA and compared with an in-house reference. Subject sensitization profiles indicated high reactivity frequencies towards Der p 1 and Der p 2 regardless of geographic region. The average Der 1/Der 2 ratio in US extracts ranged from 1:1 to 5:1. For SQ-HDM SLIT-tablet (20 batches), normalized mean (SD) content for Der f 1, Der p 1, and combined Der 2 allergens was 1.000 (0.119), 1.000 (0.061), and 1.000 (0.064) units, respectively. Other HDM allergens were also present. Der 1/Der 2 are major HDM allergens in North American and European populations. The described process enables content control of each major allergen and results in SQ-HDM SLIT-tablets containing both mites, and consistent and equivalent Der 1/Der 2 content.

Panallergens are molecules that are highly homologous across plant species and represent potential cross-reacting allergens. Panallergens play a vital role in cellular processes, which explains their conserved occurrence throughout the plant kingdom.1 Profilin, a cellular actin-binding protein, and polcalcin, a calcium-binding protein, have been identified among other panallergens. We performed a case series analysis of 126 patients (2-66 years; mean age, 14 years; 60.3% males) who attended the University of Nevada, Reno Allergy Clinic (see Table I for summary of patient characteristics).

Background: Otolaryngologists commonly use glycerin and normal saline with phenol (NSP) in diluting solutions to help preserve allergenicity in immunotherapy vials. Studies have shown that diluting with human serum albumin (HSA) may provide better allergen stability. The purpose of this study was to assess the ability of various diluents to preserve allergen content in testing/treatment boards (TTBs) and immunotherapy treatment vials (ITVs) at multiple time points.Methods: TTBs with 4 allergens were prepared with HSA, NSP, 10% glycerin, and 50% glycerin. The major allergen content of the TTBs was measured at creation (time 0), 3 days, 8 weeks, and 18 weeks. Multiallergen ITVs containing the 4 allergens were prepared from the NSP board (diluted to 10% glycerin) and the HSA board (diluted in HSA) at the time of making the freshly prepared TTBs and again 8 weeks later, simulating the creation of ITVs from a "new" and an "old" TTB. The major allergen content of these ITVs was determined at creation and at interval time points thereafter.Results: TTBs and ITVs showed a substantial loss of allergen at day 3. The loss of allergen was more pronounced in the more dilute ((#)4, (#)5, and (#)6) vials. HSA and 50% glycerin showed superior allergen preservation compared to NSP and 10% glycerin in TTBs. HSA showed superior allergen preservation compared to 10% glycerin-NSP in ITVs.Conclusion: The use of HSA as a diluent in TTBs and ITVs showed superior allergen preservation compared to NSP and 10% glycerin, particularly for more dilute vials. (C) 2015 ARS-AAOA, LLC.

Tropomyosin is a heat stable, major allergen of shellfish and other crustacea. Dust mite and cockroach tropomyosins are also allergenic. Cross-reactivity between tropomyosins may cause allergic symptoms related to allergen exposure. Our aim was to develop a sensitive ELISA that could be used to measure tropomyosin for both exposure assessment and allergen standardization. A two-site ELISA was developed using a monoclonal antibody raised against dust mite tropomyosin, with polyclonal anti-shrimp tropomyosin for detection. The assay was calibrated using purified natural shrimp tropomyosin, with protein content determined by amino acid analysis. The ELISA standard curve ranged from 0.1-50ng/ml, with a limit of quantitation of 0.2ng/ml. The assay readily detected tropomyosin in allergen extracts from shellfish (shrimp, crab, clam, lobster, crawfish, oyster, n=16) 0.1-297μg/ml) and dust mite species (n=4, 2-72μg/ml), as well as tick and yellow mealworm extracts. Extracts of fish, mammals and birds did not react in the assay (<0.002μg/ml). A sensitive ELISA with defined specificity for invertebrate tropomyosins has been developed. This assay will allow standardization of tropomyosin levels in diagnostic or therapeutic allergen extracts, as well as providing a method for monitoring tropomyosin exposure in the environment and as a potential source of shellfish contamination in the food industry.

Background Panallergens like profilin are proteins that have very similar sequences and structure. People can develop IgE specific to these highly cross reactive panallergens. The purpose of this study was to determine the prevalence of profilin sensitivity and the contribution to multiple allergen skin test responses. Sensitivity to profilin was established by IgE binding to purified profilins. Possible effects on skin test results were studied by including Queen Palm (QP) allergen in the skin prick testing panel as a possible source of profilin. QP is a species with a low probability of pollen exposure.

There is growing evidence that the mouse allergen is a major causative factor for allergic rhinitis, conjunctivitis, and asthma in children and adults of urban and rural populations. [1] Matsui E.C. Role of mouse allergens in allergic disease. Curr Allergy Asthma Rep. 2009; 9: 370-375 Crossref PubMed Scopus (16) Google Scholar Furthermore, exposure to mice by laboratory animal workers is associated with a high risk of developing occupational allergies. [2] Bush R.K. Stave G.M. Laboratory animal allergy: an update. ILAR J. 2003; 44: 28-51 Crossref PubMed Scopus (76) Google Scholar Our allergy practice at Vanderbilt University has recognized a number of patients with poorly controlled allergies to the mice with which they work. This clinical observation prompted an investigation for a better diagnostic and therapeutic option for our patients.

RationaleFood diagnostic extracts used in the USA are non-standardized with no measurement of potency. Most food diagnostics have not been extensively characterized or allergen proteins identified. This study analyzed the protein composition of several foods extracted under varying conditions.MethodsAnimal and plant foods were defatted with acetone and extracted overnight in 50% glycerin or aqueous extraction fluid using multiple extraction techniques. Extractions were performed at 4°C or room temperature and blended or stirred. Following extraction, all samples were adjusted to contain 50% glycerin, centrifuged and sterile filtered. The extracts were then evaluated for protein profiles and concentrations using Coomassie or silver stained SDS-PAGE and Bradford methods.ResultsThe food extracts showed a wide range of protein content. For example, tree nut allergens, almond and Brazil nuts were over 10 mg/mL whereas pecan and walnut were 1-3 mg/mL. For most foods the protein profiles within the same species were similar for the differing extraction conditions and correlated with the final protein concentrations. Walnut extraction, however, produced final protein concentrations that varied significantly according to the extraction method. Glycerin extraction resulted in an average final concentration of 2-3mg/ml, versus 0.3mg/ml for aqueous extraction. Room temperature extraction resulted in higher protein yields (2.8mg/ml), when compared to extraction at 4°C (1.7mg/ml), and persistent stirring was the superior method of agitation.ConclusionsSDS-PAGE profiles and total protein content were obtained for many food extracts in several food groups in order to optimize extraction conditions and to eventually monitor for allergen content, potency and stability. RationaleFood diagnostic extracts used in the USA are non-standardized with no measurement of potency. Most food diagnostics have not been extensively characterized or allergen proteins identified. This study analyzed the protein composition of several foods extracted under varying conditions. Food diagnostic extracts used in the USA are non-standardized with no measurement of potency. Most food diagnostics have not been extensively characterized or allergen proteins identified. This study analyzed the protein composition of several foods extracted under varying conditions. MethodsAnimal and plant foods were defatted with acetone and extracted overnight in 50% glycerin or aqueous extraction fluid using multiple extraction techniques. Extractions were performed at 4°C or room temperature and blended or stirred. Following extraction, all samples were adjusted to contain 50% glycerin, centrifuged and sterile filtered. The extracts were then evaluated for protein profiles and concentrations using Coomassie or silver stained SDS-PAGE and Bradford methods. Animal and plant foods were defatted with acetone and extracted overnight in 50% glycerin or aqueous extraction fluid using multiple extraction techniques. Extractions were performed at 4°C or room temperature and blended or stirred. Following extraction, all samples were adjusted to contain 50% glycerin, centrifuged and sterile filtered. The extracts were then evaluated for protein profiles and concentrations using Coomassie or silver stained SDS-PAGE and Bradford methods. ResultsThe food extracts showed a wide range of protein content. For example, tree nut allergens, almond and Brazil nuts were over 10 mg/mL whereas pecan and walnut were 1-3 mg/mL. For most foods the protein profiles within the same species were similar for the differing extraction conditions and correlated with the final protein concentrations. Walnut extraction, however, produced final protein concentrations that varied significantly according to the extraction method. Glycerin extraction resulted in an average final concentration of 2-3mg/ml, versus 0.3mg/ml for aqueous extraction. Room temperature extraction resulted in higher protein yields (2.8mg/ml), when compared to extraction at 4°C (1.7mg/ml), and persistent stirring was the superior method of agitation. The food extracts showed a wide range of protein content. For example, tree nut allergens, almond and Brazil nuts were over 10 mg/mL whereas pecan and walnut were 1-3 mg/mL. For most foods the protein profiles within the same species were similar for the differing extraction conditions and correlated with the final protein concentrations. Walnut extraction, however, produced final protein concentrations that varied significantly according to the extraction method. Glycerin extraction resulted in an average final concentration of 2-3mg/ml, versus 0.3mg/ml for aqueous extraction. Room temperature extraction resulted in higher protein yields (2.8mg/ml), when compared to extraction at 4°C (1.7mg/ml), and persistent stirring was the superior method of agitation. ConclusionsSDS-PAGE profiles and total protein content were obtained for many food extracts in several food groups in order to optimize extraction conditions and to eventually monitor for allergen content, potency and stability. SDS-PAGE profiles and total protein content were obtained for many food extracts in several food groups in order to optimize extraction conditions and to eventually monitor for allergen content, potency and stability.