Molecular and Cellular Biochemistry 275: 223–231, 2005. c ❣ Springer 2005 Tryptophan residue is essential for immunoreactivity of a diagnostically relevant peptide epitope of A. fumigatus Neel Kamal, 1,4 Shantanu Chowdhury, 1 Taruna Madan, 1 Deepak Sharma, 2,5 M. Attreyi, 2 Wahajul Haq, 3 Seturam Bandacharya Katti, 3 Anil Kumar 4 and P. Usha Sarma 1,6 1 Institute of Genomics and Integrative Biology, Mall Road, Delhi, India; 2 Department of Chemistry, University of Delhi, Delhi, India; 3 Biopolymers Division, Central Drug Research Institute, Lucknow, India; 4 School of Biotechnology, Devi Ahilya Vishwavidyalaya, Khandwa Road, Indore, India; 5 Present address: Biology Department, Boston College, Chestnut Hill, Massachusetts, U.S.A.; 6 Present address: Department of Plant Pathology, Indian Agricultural Research Institute, Delhi, India Received 22 December 2004; accepted 9 February 2005 Abstract The role of tryptophan (Trp 17 ) in immunoreactivity of P1, the diagnostically relevant peptide from a major allergen/antigen of Aspergillus fumigatus, was evaluated by chemically modifying tryptophanyl residue of P1. In BIAcore kinetic studies, unmodified P1 showed a 100-fold higher binding with ABPA (Allergic Bronchopulmonary Aspergillosis) patients’ IgG [K D (equilibrium dissociation constant) = 2.74 e −8 ± 0.13 M] than the controls’ IgG ( K D = 2.97 e −6 ±0.14 M), whereas chemically- modified P1 showed similar binding [K D patients’ IgG = 3.25 e −7 ± 0.16 M, K D controls’ IgG = 3.86 e −7 ± 0.19 M] indicating loss of specific immunoreactivity of P1 on tryptophan modification. Modified P1 showed loss of specific binding to IgE and IgG antibodies of ABPA patients in ELISA (Enzyme-Linked Immunosorbent Assay). The study infers that tryptophan residue (Trp 17 ) is essential for immunoreactivity of P1. (Mol Cell Biochem 275: 223–231, 2005) Key words: allergic bronchopulmonary aspergillosis, Aspergillus fumigatus, BIAcore, immunoreactivity, peptide, tryptophan Abbreviation: Afu: Aspergillus fumigatus; ABPA: Allergic bronchopulmonary aspergillosis; Asp f 1: An allergen/antigen/ cytotoxin of A. fumigatus; ELISA: Enzyme-linked immunosorbent assay; TFE: Tri fluoro ethanol; NBS: N-bromosuccinimide; K D : Equilibrium dissociation constant; k d : Dissociation rate constant; k a : Association rate constant. Introduction Aspergillus fumigatus (Afu), a ubiquitous fungus, induces a number of clinically distinct allergic and invasive forms of aspergillosis [1]. ABPA (Allergic bronchopulmonary as- pergillosis) is the most severe form of Afu-induced asthma [2]. Patients of ABPA show significantly high levels of specific Address for offprints: T. Madan, Molecular Biochemistry and Diagnostics Division, Institute of Genomics and Integrative Biology, Mall Road, Delhi-7, India (E-mail: taruna@igib.res.in) IgG and IgE antibodies against various allergens/antigens of Afu and hence, measurement of specific antibodies in ABPA has been recognized as one of the important diagnostic crite- ria [3]. However, standardized, pure, and relevant allergens for reliable immunodiagnosis and understanding pathogen- esis are not available till today. Hence, efforts were made to produce recombinant allergens and synthetic epitopic