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Background: Although alanine aminotransferase (ALT) is a widely used indicator of liver function, ALT enzymatic activity may not always reflect the degree of liver damage. Improved methods or approaches would be useful. Methods: Monoclonal antibodies (mAbs) to ALT were generated to develop a sandwich enzyme immunoassay system. We used an immunoassay to measure ALT mass concentration and a common biochemical analyzer to assay ALT enzymatic activity in serum samples from patients with liver diseases and healthy individuals. The results from the 2 methods were compared and analyzed by ROC curve analysis. Results: The ALT sandwich enzyme immunoassay system demonstrated reliable performance in linearity, recovery, and imprecision studies. The ALT activity assay exhibited a higher diagnostic accuracy in acute hepatitis (AH) patients, but the ALT immunoassay exhibited higher sensitivity and specificity in patients with chronic liver diseases. The areas under the ROC curve for ALT mass and enzymatic activity were 0.82 and 0.98, respectively, in AH, 0.99 and 0.52 in hepatocellular carcinoma (HCC), and 0.94 and 0.45 in liver cirrhosis (LC). Serum samples from HCC and LC patients had higher amounts of ALT–immunoglobulin complexes [median A450, 1.7 (interquartile range, 1.4–1.9)] than the other groups [1.3 (interquartile range, 0.9–1.6)]. Conclusions: Our analysis of sera from the HCC and LC patient groups revealed considerable amounts of immunologically active but catalytically inactive ALT. The amount of the ALT–immunoglobulin complex increased with the severity of the liver disease. The 2-site immunoassay method may be useful in the differential diagnosis of some causes of liver disease.

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Lysosomes are responsible for the degradation of macromolecules derived from the cell exterior by endocytosis, or from within the cell by autophagy. While our knowledge of the biosynthesis and targeting of lysosomal hydrolases is considerable, much less is known about the lysosomal membrane itself. To identify the lysosomal membrane proteins that mediate these functions, we have isolated lysosomes from amebae and injected them into mice to produce monoclonal antibodies (MAbs). We produced nine MAbs against Dictyostelium lysosomes from the batches of fused cells. Among them, three MAbs were specific to lysosomal membrane and gave a strong signal, and thus used in this study. The MAbs specifically reacted with a single protein band of 27 kd and stained a lysosome-like structure by immunofluorescence microscopy. To identify the antigen that the MAbs recognize, we processed differential centrifugation with whole-cell extract of Dictyostelium and traced p27 protein by activity assay of organelle marker enzyme. We showed that p27 is one component of the lysosomal system on the basis of comigration with a lysosomal marker enzyme. We also demonstrated that the 27-kd lysosomal protein is a tightly bound integral membrane protein by using a phase separation method of Triton X-114.