Abstract Tissue-nonspecific alkaline phosphatase (TNSALP) is an enzyme that catalyzes the hydrolysis of phosphomonoesters and promotes mineralization of bone. TNSALP contains five N-glycosylation sites: N140, N230, N271, N303, and N430. Bone alkaline phosphatase (BALP) and liver alkaline phosphatase (LALP) are isoforms of TNSALP. Despite sharing identical amino acid sequences derived from the single gene ALPL, these isoforms exhibit different glycosylation patterns. As osteoblasts express BALP, serum BALP levels reflect osteoblastic activity and are used as markers of bone formation. However, distinguishing serum BALP from coexisting LALP is challenging, as the two isoforms differ only in carbohydrate composition. Moreover, while BALP-specific immunoassays using BALP-selective monoclonal antibodies (mAbs) are currently used in clinical laboratories, they exhibit significant cross-reactivity with LALP, highlighting the need for more selective assays for BALP. In the present study, we developed mAbs against BALP with minimal cross-reactivity to LALP. Using these mAbs, we generated an in-house BALP-specific immunoassay that exhibited high correlation with a clinically approved BALP test kit. Using a series of single N-glycosylation site mutants of TNSALP, we found that all six clones of BALP-selective mAbs recognized an epitope relevant to N303-linked glycans. These mAbs could facilitate the development of improved BALP assays with reduced cross-reactivity to LALP, enabling more accurate diagnostic evaluation, particularly in patients with liver disease.
Kimura et al. (Mon,) studied this question.