Journal of Clinical Immunology ( C  2007) DOI: 10.1007/s10875-007-9078-y Stability of Protease-Rich Periplaneta americana Allergen Extract During Storage: Formulating Preservatives to Enhance Shelf Life V. THANGAM SUDHA, 1,2 DEEPSIKHA SRIVASTAVA, 1,2 NAVEEN ARORA, 1 SHAILENDRA NATH GAUR, 3 and BHANU PRATAP SINGH 1,4 Received October 13, 2006; accepted January 26, 2007 Allergenic proteins in extracts degrade rapidly and lose po- tency on storage. Hence, formulation of optimum conditions is required to enhance shelf life of extracts for proper allergy diagnosis and immunotherapy. In the present study, allergenic potency of P. americana proteins was evaluated after storage with ε-aminocaproic acid (EACA), sucrose, glycerol, pepstatin A, and aprotinin, individually for 1, 3, 6, and 12 months at 4, 25, and 37 ◦ C. P. americana extract stored with EACA and sucrose individually retained potency comparable to proteins in standard extract (freeze-dried extract, stored at −70 ◦ C) upto 6 months at 4 ◦ C. The extracts without preservatives or with glyc- erol, pepstatin A, aprotinin, or stored at 37/25 ◦ C were severely degraded and lost potency by 3 months. A formulation contain- ing a combination of EACA and sucrose enhanced the shelf life of P. americana proteins upto 12 months at 4 ◦ C. Hence, EACA and sucrose together show better potential for stabilization of protease-rich extracts. KEY WORDS: Periplaneta americana; cockroach extract; stability; ε-aminocaproic acid; immunoblot; ELISA inhibition. INTRODUCTION Extracts used for allergy diagnosis and therapy are het- erogeneous mixture of proteins. Allergen extract(s) are required to contain all the defined major, minor allergens and be biologically potent (1). The extracts show maxi- mum potency when they are freshly made but deteriorate on storage. The loss in potency affects the results of allergy diagnosis and immunotherapy (2). Therefore, stability is 1 Institute of Genomics and Integrative Biology, Delhi, India. 2 Department of Biotechnology, University of Pune, Pune, India. 3 V.P. Chest Institute, Delhi University, Delhi, India. 4 To whom correspondence should be addressed to Allergy and Immunol- ogy Section, Room 509, Institute of Genomics and Integrative Biology, Delhi University Campus, Mall Road, Delhi, 110007, India; e-mail: singhbp1951@yahoo.com. an important parameter for an allergenic product under various conditions of storage such as temperature, light, solvent, dilution, etc. (1). Aqueous allergen extracts are transported long dis- tances under variable temperatures and kept at room tem- perature while testing. These extracts contain proteases that can cleave self-proteins and other extracts in vaccines. Proteases are present maximally in the extracts of fungi, insects, and house dust mites (3–5). P. americana extract is rich in proteases and reduces the potency of timothy grass, Bermuda grass, white oak, box alder, Russian thistle, and cat extracts when mixed for immunotherapy prescription (6). Similarly, there was considerable loss of potency in rye grass extract when mixed with Helminthosporium and cockroach extract (7). Various preservatives and stabiliz- ers such as ε-aminocaproic acid (EACA), sucrose, glyc- erol, human serum albumin (HSA), phenol, Tween 80, etc., were used to maintain the potency of allergenic ex- tracts (8–11). Cockroach allergens play an important role in the etiol- ogy of allergic bronchial asthma (12, 13). Commercially available cockroach extracts (aqueous/glycerinated) are highly variable in protein content and allergenic potency (14). Whole body cockroach extracts are widely used but studies defining shelf life based on real-time stability stud- ies are lacking. Each indigenous preparation should be checked for degradation because extracts from different sources behave differently under storage. In the present study, the effect of various preservatives was investigated on Periplaneta americana extract under different storage conditions. MATERIALS AND METHODS Preparation of Cockroach Extract Mixed culture of male and female cockroaches (Peri- planeta americana) were freeze-dried and ground to a fine 0271-9142/07 C  2007 Springer Science+Business Media, LLC