One-step sandwich enzyme immunoassay using monoclonal antibodies for detection of human enamelysin (MMP-20).
A one-step sandwich enzyme immunoassay (EIA) system for human matrix metalloproteinase 20 (MMP-20, enamelysin) was established by use of a solid-phase monoclonal antibody and a separate peroxidase-labeled monoclonal antibody. The EIA system was shown to be sensitive and quantitative for the detection of MMP-20. As little as 1.0 ng/ml (50 pg/assay) of MMP-20 protein could be reliably detected. The EIA system was linear over a range of 2.5-160 ng/ml (125-8,000 pg/assay), and the EIA system was versatile in that it was capable of detecting with equal sensitivity proMMP-20, active MMP-20, and MMP-20 with COOH-terminal deletions. The EIA system was validated by the successful detection of MMP-20 in the culture medium of Chinese hamster ovary cells (CHO-K1) that were transfected with an MMP-20 expression vector. No MMP-20 was detected in normal human serum, normal saliva, or in selected tumors. However, when recombinant human MMP-20 was added to human saliva, the EIA system did detect quantifiable amounts of the MMP-20, indicating that the system will work within the framework of complex in biological fluids.
Wang
T
T
Biopharmaceutical Department, Fuji Chemical Industries, Ltd, Toyama, Japan.
Aoki
T
T
Iwata
K
K
Takata
T
T
Uchida
T
T
Knäuper
V
V
Llano
E
E
Okada
Y
Y
Bartlett
J D
JD
D000230:Adenocarcinoma / Q000201:enzymology; D000818:Animals; D000911:Antibodies, Monoclonal; D016466:CHO Cells; D002294:Carcinoma, Squamous Cell / Q000201:enzymology; D004396:Coloring Agents; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D007124:Immunoenzyme Techniques / Q000379:methods*; D008175:Lung Neoplasms / Q000201:enzymology; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases / Q000032:analysis* / Q000097:blood / Q000235:genetics; D009062:Mouth Neoplasms / Q000201:enzymology; D011994:Recombinant Proteins; D012463:Saliva / Q000201:enzymology; D012680:Sensitivity and Specificity; D013045:Species Specificity; D014162:Transfection
D000911:Antibodies, Monoclonal; D004396:Coloring Agents; D011994:Recombinant Proteins; C507111:MMP20 protein, human; D053519:Matrix Metalloproteinase 20; D020782:Matrix Metalloproteinases