One step sandwich enzyme immunoassay for human type IV collagen using monoclonal antibodies

Monoclonal antibodies were used in one step sandwich enzyme immunoassay (one step sandwich EIA) for human serum immunoreactive type IV collagen. The one step sandwich EIA using either polystyrene ball or microplate was characterized by carrying out two immunoreactions simultaneously, type IV collagen reacting with both a monoclonal antibody as a solid phase and a horseradish peroxidase-labeled monoclonal antibody (Fab') against human type IV collagen as a conjugate. Sensitivity of one step sandwich EIA system by using either polystyrene ball or microplate was 0.22 ng per tube or 0.04 ng per well for type IV collagen, and linearity was obtained between 0.22-40 ng/tube or 0.04-20 ng per well, respectively. Both methods gave reproducible quantitative analysis of immunoreactive type IV collagen levels in the sera of patients with hepatocellular carcinoma and patients with liver cirrhosis, which were apparently higher than the levels in the sera of healthy subjects. Protein immunoblotting shows that the immunoreactive type IV collagen trapped in our present one step sandwich EIA system was not the 7-S and NC1 domains of type IV collagen.