Development of monoclonal antibodies and immunochromatographic lateral flow device for rapid test of alanine aminotransferase isoenzyme 1

• Published in: Protein expression and purification Vol. 119; pp. 94 - 101
• Main Authors: Hu, Xiaomei, Cheng, Shiliang, Liu, Xinfeng, Li, Jie, Zheng, Wen, Lu, Gang, Zhang, Jun, Zheng, Jian, Zhang, Juan
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.03.2016
• Subjects: Alanine aminotransferase; Alanine aminotransferase isoenzyme 1; Alanine Transaminase - analysis; Alanine Transaminase - blood; Alanine Transaminase - immunology; Animals; Antibodies, Monoclonal, Murine-Derived - biosynthesis; Antibodies, Monoclonal, Murine-Derived - chemistry; ELISA; Escherichia coli; Female; Hep G2 Cells; Humans; Hybridomas; Immunochromatography; Lateral flow device; Mice, Inbred BALB C; Monoclonal antibody; Rapid test; Recombinant Proteins - analysis; Recombinant Proteins - blood; Recombinant Proteins - immunology; Western-blot; Alanine aminotransferase isoenzyme 1; Rapid test; Alanine aminotransferase; Monoclonal antibody; Western-blot; ELISA; Lateral flow device
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.11.016

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 Immunochromatographic lateral flow device (LFD) for rapid test of ALT1 in human serums. 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(+). ALT1 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. ALT1 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. The ALT1 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. •This is the first report on ALT1 MAb production and its LFD preparation.•The ALT1 LFD is valuable for testing ALT1 protein in human serums with high specificity and sensitivity.•The ALT1 MAb not only help understanding ALT1 distribution in human body but also paves the way for the future research on molecular basis of ALT elevation.