Differences Between a Monoclonal Anti-NC1 Antibody (12D) That Selectively Stains Injured Kidneys and Other Anti-NC1 Antibodies

Background Type IV collagen is a major structural component of the renal basement membrane and plays a critical role in glomerular filtration. Its C-terminal noncollagenous domain is referred to as the NC1 domain. In indirect immunohistochemical staining of frozen sections, a mouse-derived monoclonal anti-NC1 antibody (12D) stains basement membranes in injured kidneys but does not stain those in normal kidneys. In contrast, a rabbit-derived polyclonal anti-NC1 antiserum (polyclonal NC1 antibodies) stains basement membranes in both injured and normal kidneys. In the present study, to clarify the distinctive characteristics of 12D, we investigated differences between 12D and other anti-NC1 antibodies, including analyses involving nephritic urine. Methods Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting (WB) were performed. NC1 treated with the reducing agent 2-mercaptoethanol (2ME) (reduced NC1) and NC1 not treated with 2ME (non-reduced NC1) were subjected to electrophoresis, and their reactivity with 12D, nephritic urine, polyclonal NC1 antibodies (polyclonal NC1 antibodies), and an anti-yp08 polyclonal antiserum (yp08 antibodies) was analyzed by WB. The antigen for the anti-yp08 antibody was a synthetic peptide, yp08 (PFISR CAVCE APAVV IAVHS), corresponding to an amino acid sequence within the NC1 domain of the type IV collagen α5 chain. Results 12D reacted with non-reduced NC1 but did not react with reduced NC1, in which disulfide bonds had been cleaved by 2ME. The two polyclonal antibodies (polyclonal NC1 antibodies and yp08 antibodies) reacted with both non-reduced NC1 and reduced NC1. Among 23 nephritic urine samples, 13 reacted with non-reduced NC1, whereas 20 samples, including these 13, reacted with reduced NC1. Conclusion Unlike polyclonal NC1 antibodies or factors present in nephritic urine, 12D requires intact disulfide bonds for its interaction with NC1. These results demonstrate that the binding of 12D to NC1 is disulfide bond-dependent. Because disulfide bonds are formed through oxidative processes, these findings suggest that kidneys stained by 12D in indirect immunohistochemistry are in an oxidized state.