Two-step sandwich enzyme immunoassay using monoclonal antibodies for detection of soluble and membrane-associated human membrane type 1-matrix metalloproteinase.
A two-step sandwich enzyme immunoassay (EIA) system for the detection of human membrane Type 1-matrix metalloproteinase (MT1-MMP) was established by using two monoclonal antibodies against recombinant MT1-MMP. MT1-MMP in which samples were reacted with solid-phase antibody and then detected with peroxidase-labeled second antibody. At least 1.25 ng/mL was detected by the EIA system, and linearity was obtained between 1.25 and 160 ng/mL. This EIA system is specific for MT1-MMP and did not show cross-reactivity against several other MMP's examined. Shedding of soluble MT1-MMP into the medium by some cancer cell lines was also detected by this system. However, soluble MT1-MMP in serum from normal and cancer patients was under the detection limit. Membrane-associated MT1-MMP of cancer cell lines was also detected after solubilization of the membranes with extraction buffer containing detergent. Additionally, MT1-MMP in clinical samples was examined. Elevated levels of MT1-MMP were detected in homogenate of cancer tissue compared with the levels for normal tissue and the level of MT1-MMP in tumors correlated with the rate of metastasis to the regional lymph nodes. Thus, we demonstrated that this EIA system is the first to measure MTI-MMP in clinical specimens, thus suggesting its useful for diagnosis of cancer or prediction of malignancy.
Aoki
Takanori
T
Biopharmaceutical Department, Fuji Chemical Industries, Ltd, Takaoka, Toyama, Japan. t-aoki@fuji-chemi.co.jp
Yonezawa
Kayoko
K
Ohuchi
Eiko
E
Fujimoto
Noboru
N
Iwata
Kazushi
K
Shimada
Taketoshi
T
Shiomi
Takayuki
T
Okada
Yasunori
Y
Seiki
Motoharu
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006258:Head and Neck Neoplasms / Q000201:enzymology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D020781:Matrix Metalloproteinase 1 / Q000032:analysis*; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000032:analysis; D015203:Reproducibility of Results; D012995:Solubility
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D020781:Matrix Metalloproteinase 1