Hybridoma Passage In Vitro May Result in Reduced Ability of Antimannan Antibody To Protect against Disseminated Candidiasis
- 1 July 2006
- journal article
- research article
- Published by American Society for Microbiology in Infection and Immunity
- Vol. 74 (7), 4310-4321
- https://doi.org/10.1128/iai.00234-06
Abstract
We previously reported the enhanced resistance of monoclonal antibodies B6.1 (an immunoglobulin M [IgM]) and C3.1 (an IgG3) against experimental candidiasis. Both MAbs recognize the same fungal epitope. We have since found that a highly passaged B6.1 hybridoma (hp-B6.1) resulted in antibody that has little protective potential. The potential clinical applicability of the antibody and our interest in understanding antibody protection against candidiasis led us to investigate an explanation for this phenomenon. Antibody genetic structure of hp-B6.1, the original hybridoma clone (ori-B6.1) stored frozen since 1995, a subclone of hp-B6.1 that produces protective antibody, the IgG3-producing hybridoma, and a nonprotective IgG1-producing hybridoma were compared. Variable region gene sequences of heavy (VH) and light chains showed genetic instability of VHchains with only the hp-B6.1; the VHsequences from ori-B6.1 and the subclone were, however, identical. Activation-induced cytidine deaminase levels were greatest in the B6.1 hybridomas, which may explain the instability. The constant region CH3 domain remained unchanged, implying normalN-glycation and complement-fixing potential, and antibody binding affinities appeared unchanged. Complement fixation assays surprisingly showed that ori-B6.1 antibody fixes C3 more rapidly than does hp-B6.1 antibody. The VHregion primary structure may affect complement activation, which could explain our result. Indeed, antibody from the hp-B6.1 subclone fixed complement like antibody from ori-B6.1. These results show that the greatest protection occurs when antimannan antibodies possess the dual abilities of recognizing the appropriate carbohydrate epitope and rapidly fixing complement; loss of the latter property results in the loss of protective potential by the antibody.Keywords
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