title: Development of monoclonal antibodies and immunochromatographic lateral flow device for rapid test of alanine aminotransferase isoenzyme 1.
abstract: 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 Immunochromatographic 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(+). ALT1 recombinant protein was routinely prepared by E. coli BL21 (DE3) expression and Ni(2+) 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
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: The Blood Transfusion Department, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, PR China.
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Hu
authors.affiliation: Clinical Laboratory, Shandong Jiaotong Hospital, No.11 Wuyinshan Road, Tianqiao District, Ji'nan, Shandong Province, PR China.
authors.forename: Shiliang
authors.initials: S
authors.lastname: Cheng
authors.affiliation: Clinical Laboratory, Shandong Jiaotong Hospital, No.11 Wuyinshan Road, Tianqiao District, Ji'nan, Shandong Province, PR China.
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Liu
authors.affiliation: The Blood Transfusion Department, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, PR China.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: Clinical Laboratory, Shandong Jiaotong Hospital, No.11 Wuyinshan Road, Tianqiao District, Ji'nan, Shandong Province, PR China.
authors.forename: Wen
authors.initials: W
authors.lastname: Zheng
authors.affiliation: Artron BioResearch Inc., 3938 North Fraser Way, Burnaby, British Columbia, V5J 5H6, Canada.
authors.forename: Gang
authors.initials: G
authors.lastname: Lu
authors.affiliation: Artron BioResearch Inc., 3938 North Fraser Way, Burnaby, British Columbia, V5J 5H6, Canada.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Molecular Biology on Infectious Diseases, Chongqing Medical University, No.1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zheng
authors.affiliation: The Blood Transfusion Department, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, PR China. Electronic address: juanmcq@163.com.
authors.forename: Juan
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000410:Alanine Transaminase / Q000032:analysis* / Q000097:blood / Q000276:immunology; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis* / Q000737:chemistry; D002846:Chromatography, Affinity; D004926:Escherichia coli; D005260:Female; D056945:Hep G2 Cells; D006801:Humans; D006825:Hybridomas; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000032:analysis / Q000097:blood / Q000276:immunology
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D011994:Recombinant Proteins; D000410:Alanine Transaminase
keywords: Alanine aminotransferase; Alanine aminotransferase isoenzyme 1; ELISA; Lateral flow device; Monoclonal antibody; Rapid test; Western-blot