Improving Safety and Potency Testing of Allergen Extracts

Principal Investigator: Jay E. Slater, MD
Office / Division / Lab: OVRR / DBPAP / LI

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Overview

Public Health Issue: Allergen extracts are used for the diagnosis and treatment of allergic disease, such as cockroach allergy. Diagnosing allergies using allergen skin tests is rapid, accurate and economical. Specific treatments, i.e., allergen immunotherapy, have been shown to be curative for several allergic diseases. Currently, a large majority of allergen extract products are non-standardized, i.e., they are labeled in units but no standards exist to establish fixed biological potency of the extracts. Thus, although these products are safe and effective for diagnosis and/or treatment, dosing of these products can be difficult. Cockroach allergy appears to be a major cause of inner city asthma. Like other non-standardized products currently available cockroach allergen extracts are of variable potency. This laboratory is developing novel tests to accurately assess the potency of cockroach extracts.

Regulatory Contribution: Existing allergen extract potency assays estimate extract potency by one of two methods. When the specific allergen is known, it is determined using specific assays. When the specific allergen is not yet known, overall potency is estimated using a polyclonal human IgE antiserum. The strength of the latter approach is that potency may be estimated even when the specific allergens are unknown. However, data from our laboratory have shown that the overall potency approach may be insensitive to clinically significant changes in individual allergens. Thus, large fluctuations within an extract of potentially important specific allergens may go undetected if overall potency assays are used. To address this issue, our laboratory is developing the multiplex allergen extract potency assay (MAEPA). The purpose of MAEPA is to assess the overall potency of complex allergen mixtures as the integral of multiple discrete allergen assays. The design of MAEPA will be the conjugation of anti-allergen scFvs to specific Luminex microbeads; incubation of the conjugated microbeads with - in sequence - with allergen extract, polyclonal rabbit anti-allergen serum, biotinylated anti-rabbit IgG, and avidin-fluorophore; and quantification of antigen binding to specific beads using the Luminex model 200 microbead reader. In order for this multiplex assay to be validated, several steps will need to be achieved: 1. The immediate target allergen for MAEPA is German cockroach allergen extract. German cockroach allergy has been shown to be important in the pathogenesis of inner city asthma, and currently available German cockroach allergen extracts are unstandardized, highly variable, and generally low in potency. Known allergens include Bla g 1, Bla g 2, Bla g 4, Bla g 5. We recently demonstrated that none of these four known allergens was immunodominant in an inner city asthma and allergy population. Thus, German cockroach allergen extract is a good candidate for standardization, and the MAEPA approach appears to be a reasonable alternative to the other approaches available.

Research Approach: Although we intend to focus on German cockroach allergen extracts, we will - if successful - turn subsequent attention to other multi-allergen extracts: dust mites, grass pollens, tree pollens, weed pollens, foods, and molds. Another important problem with allergen extracts is the presence of endotoxins, especially in D farinae mite extracts. In prior work, we showed that standardized D. farinae mite extracts contain endotoxin, and that endosymbiotic Bartonella DNA could be isolated from fresh frozen D farinae mite bodies. In 2007, we expanded these studies by isolating DNA from commercial mite extracts, and demonstrated similar bacterial DNA in these sterile extracts. We will explore these findings using an independent scientific approach, to produce an unbiased population profile of endosymbionts in multiple species of dust mites.

Mission Relevance & Outcomes: Outcomes of this improve the safety and efficacy of allergenic extracts.

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Publications

Clin Exp Allergy 2007 Jul;37(7):1033-9
Biological potency of German cockroach allergen extracts determined in an inner city population.
Slater JE, James R, Pongracic JA, Liu AH, Sarpong S, Sampson HA, Satinover SM, Woodfolk JA, Mitchell HE, Gergen PJ, Eggleston PA

J Mol Biol 2007 May 4;368(3):742-52
Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab.
Padavattan S, Schirmer T, Schmidt M, Akdis C, Valenta R, Mittermann I, Soldatova L, Slater J, Mueller U, Markovic-Housley Z

Allergy Asthma Proc 2007 Mar-Apr;28(2):210-5
Characterization of the N-glycans of recombinant bee venom hyaluronidase (Api m 2) expressed in insect cells.
Soldatova LN, Tsai C, Dobrovolskaia E, Markovic-Housley Z, Slater JE

J Endotoxin Res 2006;12(4):241-5
beta-Glucans in standardized allergen extracts.
Finkelman MA, Lempitski SJ, Slater JE

Clin Exp Allergy 2006 Apr;36(4):525-30
Competition enzyme-linked immunosorbant assay (ELISA) can be a sensitive method for the specific detection of small quantities of allergen in a complex mixture.
Dobrovolskaia E, Gam A, Slater JE

J Allergy Clin Immunol 2005 Dec;116(6):1296-1300
Bacterial 16S ribosomal DNA in house dust mite cultures.
Valerio CR, Murray P, Arlian LG, Slater JE

Dev Biol 2005;122:145-52
Characterization of allergen extracts.
Slater JE

Clin Exp Allergy 2005 Aug;35(8):1040-8
Exploiting the avian immunoglobulin system to simplify the generation of recombinant antibodies to allergenic proteins.
Finlay WJ, Devore NC, Dobrovolskaia EN, Gam A, Goodyear CS, Slater JE

Clin Allergy Immunol 2004;18:421-32
Standardized allergen extracts in the United States.
Slater JE

Clin Allergy Immunol 2004;18:369-86
Latex allergens.
Slater JE

Methods 2004 Mar;32(3):209-11
Recombinants allergens in the US
Slater JE