Murine monoclonal antibody which can distinguish cystatins SA1 and SA2.
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.
Ito
Taichi
T
Oral Health Science Center, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan.
Komiya-Ito
Akiyo
A
Okuda
Katsuji
K
Minaguchi
Kiyoshi
K
Saitoh
Eiichi
E
Yamada
Satoru
S
Kato
Tetsuo
T
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D003429:Cross Reactions; D015891:Cystatins / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005260:Female; D014644:Genetic Variation; D006825:Hybridomas / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D012463:Saliva / Q000737:chemistry; D055333:Salivary Cystatins; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; C525172:CST2 protein, human; D015891:Cystatins; D000939:Epitopes; D011994:Recombinant Proteins; D055333:Salivary Cystatins