Murine monoclonal antibody which can distinguish cystatins SA1 and SA2

Abstract

To develop a diagnostic trial enabling the selective examination for a target cystatin in human body fluids, we attempted to prepare monoclonal antibodies against human cystatin SA1 (originally cystatin SA) and its variant form (cystatin SA2). BALB/c mice were immunized with recombinant (r-) cystatins SA1 and SA2. Two monoclonal antibodies designated Cys3F11 and Cys2E5 were selected. By ELISA analyses, the Cys2E5 was shown to react with r-cystatin SA2 but also somewhat with r-cystatin SA1 (22% cross-reactivity) and with plasma cystatin C (18% cross-reactivity), indicating a high specificity for cystatin SA2. The Cys3F11 reacted not only with r-cystatin SA1 but also with r-cystatin SA2 (89% cross-reactivity) and plasma cystatin C (47% cross-reactivity). This finding was further emphasized by immunoblotting of human submandibular-sublingual saliva samples. ELISA additivity test suggests that the two monoclonal antibodies bind to distinct epitopes. In conclusion, we have succeeded in producing two antibodies that discriminate the structural differences between salivary cystatins S and SN, which share more than 90% identity in amino acid sequence with cystatin SA.

Fig. 1

ELISA reactivities of mouse antiserum against r-cystatin SA1. The recombinant and native cystatins were used as coating antigens. The wells of 96-well microtiter plates were coated with 10 μg of either antigen. Antiserum was serially diluted with PBS.

Fig. 2

Detection of S-type cystatins by Western blot. Western blot of human submandibular–sublingual saliva was performed as described by Shintani et al. (1994). The first antibodies used are: P; anti-r-cystatin SA1 antiserum, A; Cys3F11, B; Cys2E5.