omatic hypermutation (SHM) is a hallmark of antibody-secreting B cells used to produce mature and high affinity antibodies. SHM takes place in the variable region and within the 1.5 kb downstream region of the immunoglobulin (Ig) gene promoter but no mutations have been thus far reported in the constant region. This study reports the characterization of DNA immunization-generated hybridoma clones secreting functional and Blo t 11-specific monoclonal antibodies but with mutations on the constant region of the Ig gene. The nucleotide sequence of the constant region of the Ig gene of four mutant hybridoma clones secreting Blo t 11-specific monoclonal antibodies that are total Ig-positive was characterized. The antibodies had no detectable isotype/subclass by enzyme-linked immunosorbent assay (ELISA). A series of transition, transversion, deletion and insertion mutations were detected on exons 3 and 4 but not on exons 1 and 2 of the Ig gene. Activation-induced cytidine deaminase (AID) transcripts were observed in all mutant hybridomas examined. Sequence analysis showed that 7 and 9 of the observed mutations in clones P1C9 and P1A6, respectively, were located on RGYW and WRCY hotspots where AID have been reported to occur at high frequencies. The results presented in this study may represent a novel observation related to Ig gene SHM and maturation that may be associated with DNA immunization. S INTRODUCTION The generation of enormous antibody diversity by the mammalian immune system is a result of complex genetic diversification processes undergone by immunoglobulin (Ig) genes. In response to a given antigen, the Ig genes of B cells undergo somatic hypermutation (SHM), gene conversion and class-switch recombination (Weill and Reynaud 1996). These specialized genetic events are essential to produce mature and high affinity antibodies. Antibody-diversification mechanisms in B cells involve the action of activation-induced cytidine deaminase (AID) and mismatch repair (MMR) systems (Martin and Scharff 2002). SHM takes place in the variable region and within the 1.5 kb downstream region of the Ig gene promoter (Rada et al. 1997). Although the activity of AID to initiate point mutations has been reported to occur in the variable region and its vicinity, AID-induced mutations have been reported in other genes and even in non-B cells (Yoshikawa et al. 2002; Ramiro et al. 2002). AID expression in bronchial biopsies with asthma (Takhar P et al. 2007) and in nasal mucosa cells from allergic rhinitis patients (Coker et al. 2003) were likewise implicated in class switch recombination to IgE antibodies. Positive association between polymorphisms in the 5’-flanking and coding regions of the AID gene, the symptoms of atopic asthma and the regulation of total serum IgE were similarly established (Noguchi et al. 2001). We reported previously the production of monoclonal antibodies by DNA immunization (Ramos et al. 2004) against an important high molecular weight house dust mite (HDM) allergen Blo t 11 (Ramos et al. 2001). Six Blo t 11 monoclonal antibodies were generated and used for the immunoaffinity purification of native Blo t 11 and the development of sandwich enzyme-linked immunosorbent assay (ELISA) for Blo t 11 detection and quantitation. However, a significant number of the generated hybridoma clones secreted monoclonal antibodies were total Ig-positive but with no detectable isotype/subclass by ELISA (JD Ramos, unpublished observations). We report in this paper the characterization of DNA immunization-generated hybridoma clones secreting functional and antigen specific monoclonal antibodies but with mutations on the constant region of the Ig gene. 38 Philippine Science Letters Vol.2 | No.1 | 2009 *To whom the correspondence should be addressed. Email: jaramos@mnl.ust.edu.ph Received: July 18, 2009 Revised: September 19, 2009 Accepted: September 22, 2009 Editor-in-Charge: Eduardo A. Padlan ARTICLES Characterization of Blo t 11 Monoclonal Antibodies with Constant Region Mutations John Donnie A. Ramos 1* , Nge Cheong 2 , Kaw Yan Chua 2 1 Research Center for the Natural Sciences, University of Santo Tomas, España St., Manila 1008, Philippines 2 Department of Paediatrics, Yong Yoo Lin School of Medicine, Lower Kent Ridge, National University of Singapore, Singapore 119074