Background: Alanine aminotransferase (ALT) has been used as a sensitive marker for liver injury in people and in preclinical toxicity studies. But measurement of ALT isoenzymes, ALT1 and ALT2, was reported to be of more diagnostic value. The aim of this study is to develop an ideal pair of anti-ALT1 monoclonal antibodies (MAbs) of high specificity and affinity, and subsequently prepare a Immuno-chromatographic lateral flow device (LFD) for rapid test of ALT1 in human serums. Methods: The complete coding sequence of ALT1 gene (1500 bp) was cloned from human hepatoma G2 cells (HepG2) and inserted into the expression vector pET-32a(+). ALTI recombinant protein was routinely prepared by E. coli BL21 (DE3) expression and Ni2+ affinity purification. Balb/c mice were immunized with purified ALT1 and the splenocytes were fused with Sp2/0 myeloma cells. The positive clones, verified by indirect enzyme-linked immunosorbent assay (ELISA) using purified ALT1, were subcloned to single clones by limiting dilution process. A MAb pair was selected from the obtained MAbs according the sandwich ELISA pairing results and then used for lateral flow device (LFD) production. After evaluation of the sensitivity and specificity, the LFD strips were employed to test human serum samples with known ALT activity levels. Results: ALTI recombinant protein was expectedly prepared by expression and purification. A total of 8 stable clones that produced antibodies specifically recognizing ALT1 protein were developed. After sandwich ELISA pairing, an ideal pair of anti-ALT1 MAbs, designated as BD7 and DG3, were selected and proved to be of high specificity, titer and affinity. Based on the MAb pair, LFD strips specifically for ALT1 rapid test were subsequently prepared. The detection threshold of the LFD strips was 12 U/L. No cross reaction was found. Conclusions: The ALTI LFD with high sensitivity and specificity was successfully developed. It is valuable for testing ALT1 protein in human sera and can be a beneficial complement for traditional ALT test. (C) 2015 Elsevier Inc. All rights reserved.