Development, characterization, and technical applications of a fish lysozyme-specific monoclonal antibody (mAb M24-2)

Abstract

Lysozyme is one of several humoral and cellular factors associated with front line, innate immunity in all vertebrates. Historically, circulating lysozyme has been quantified in teleosts by measuring enzymatic activity against heat-killed Mycococcus lysodieticus using whole serum or plasma at a low pH. However, the amount of serum or plasma required for standard lysozyme activity exceeds that which can be easily acquired from small fish, thus making lysozyme a difficult endpoint to measure in limited sample volumes. Moreover, while circulating lysozyme is considered to be an indicator of proinflammatory phagocyte activity, the cellular source of this protein is not easily detected in fish. While several antibodies against lysozyme are commercially available for use in higher vertebrates, neither reacts with lysozyme in fish. In this study, a monoclonal antibody (mAb) for detecting and quantifying lysozyme was developed from mummichog, Fundulus heteroclitus, myeloid cells that also recognizes hen egg lysozyme (HEL), then tested for cross-reactivity in different species of teleosts. A single protein of ≈14-15 kDa mass was identified by the mAb in fish cell lysates and plasma samples, as well as denatured HEL. Total circulating lysozyme protein was compared to lysozyme activity using standard ELISA procedures and was found to correlate with enzymatic activity. Using mAb M24-2, intracellular lysozyme protein was detected in formalin-fixed and permeabilized lymphoid cells adhered to glass cover slips. Moreover, mAb M24-2 localizes lysozyme to myeloid cells. Finally, it was demonstrated that mAb M24-2 is suitable for immunohistochemistry in that lysozyme could be detected in plastic-embedded lymphoid tissues.