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Production of recombinant human tissue transglutaminase using the baculovirus expression system, and its application for serological diagnosis of coeliac disease.

Abstract

BACKGROUND AND OBJECTIVES

Tissue transglutaminase was identified as the main autoantigen in coeliac disease (CD) but enzyme immunoassays applying the commercially available antigen from guinea pig liver show insufficient specificity and sensitivity for diagnosis as compared with endomysium antibodies (EmA). The aim of this present study was to develop a new method for the cloning and expression of human tissue transglutaminase (hu-tTG) and to test hu-tTG in the serological diagnosis of CD.

METHODS

Hu-tTG was cloned and expressed using a baculovirus system and SF9 insect cells. The enzyme carried a C-terminal His tag allowing efficient affinity purification from cell lysates. The recognition of hu-tTG by human sera was checked by using an enzyme linked immunosorbent assay (ELISA). For this, 35 patients with active CD were compared with 144 controls (18 patients with bioptically excluded CD, 89 blood donors, 30 patients with inflammatory bowel disease, and seven patients with cystic fibrosis).

RESULTS

The ELISA using hu-tTG showed a sensitivity of 100% and a specificity of 98.6%. Titres of antibodies against hu-tTG (anti-hu-tTG) were positively correlated with EmA titres. All results negative for EmA were also negative for anti-hu-tTG. There were, however, EmA positive results up to a titre of 1 : 80 below the cut-off for anti-hu-tTG. For comparison, antibodies against guinea pig tissue transglutaminase (anti-gp-tTG) were determined in parallel. All patients with anti-hu-tTG below the cut-off were also negative for anti-gp-tTG. However, there were eight patients positive for anti-hu-tTG but negative for anti-gp-tTG.

CONCLUSIONS

The new test reaches and even exceeds diagnostic efficiency of EmA for coeliac diagnosis.

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  • Authors+Show Affiliations

    ,

    Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University Hospital, Leipzig, Germany.

    , , , , , ,

    Source

    MeSH

    Adolescent
    Adult
    Aged
    Aged, 80 and over
    Baculoviridae
    Blotting, Western
    Celiac Disease
    Cells, Cultured
    Child
    Child, Preschool
    Cloning, Molecular
    Electrophoresis, Polyacrylamide Gel
    Enzyme-Linked Immunosorbent Assay
    Genetic Vectors
    Humans
    Middle Aged
    Recombinant Proteins
    Transfection
    Transglutaminases

    Pub Type(s)

    Journal Article
    Research Support, Non-U.S. Gov't

    Language

    eng

    PubMed ID

    12439116

    Citation

    Osman, Awad A., et al. "Production of Recombinant Human Tissue Transglutaminase Using the Baculovirus Expression System, and Its Application for Serological Diagnosis of Coeliac Disease." European Journal of Gastroenterology & Hepatology, vol. 14, no. 11, 2002, pp. 1217-23.
    Osman AA, Richter T, Stern M, et al. Production of recombinant human tissue transglutaminase using the baculovirus expression system, and its application for serological diagnosis of coeliac disease. Eur J Gastroenterol Hepatol. 2002;14(11):1217-23.
    Osman, A. A., Richter, T., Stern, M., Conrad, K., Henker, J., Brandsch, C., ... Mothes, T. (2002). Production of recombinant human tissue transglutaminase using the baculovirus expression system, and its application for serological diagnosis of coeliac disease. European Journal of Gastroenterology & Hepatology, 14(11), pp. 1217-23.
    Osman AA, et al. Production of Recombinant Human Tissue Transglutaminase Using the Baculovirus Expression System, and Its Application for Serological Diagnosis of Coeliac Disease. Eur J Gastroenterol Hepatol. 2002;14(11):1217-23. PubMed PMID: 12439116.
    * Article titles in AMA citation format should be in sentence-case
    TY - JOUR T1 - Production of recombinant human tissue transglutaminase using the baculovirus expression system, and its application for serological diagnosis of coeliac disease. AU - Osman,Awad A, AU - Richter,Thomas, AU - Stern,Martin, AU - Conrad,Karsten, AU - Henker,Jobst, AU - Brandsch,Corinna, AU - Zimmer,Klaus-Peter, AU - Mothes,Thomas, PY - 2002/11/20/pubmed PY - 2003/4/4/medline PY - 2002/11/20/entrez SP - 1217 EP - 23 JF - European journal of gastroenterology & hepatology JO - Eur J Gastroenterol Hepatol VL - 14 IS - 11 N2 - BACKGROUND AND OBJECTIVES: Tissue transglutaminase was identified as the main autoantigen in coeliac disease (CD) but enzyme immunoassays applying the commercially available antigen from guinea pig liver show insufficient specificity and sensitivity for diagnosis as compared with endomysium antibodies (EmA). The aim of this present study was to develop a new method for the cloning and expression of human tissue transglutaminase (hu-tTG) and to test hu-tTG in the serological diagnosis of CD. METHODS: Hu-tTG was cloned and expressed using a baculovirus system and SF9 insect cells. The enzyme carried a C-terminal His tag allowing efficient affinity purification from cell lysates. The recognition of hu-tTG by human sera was checked by using an enzyme linked immunosorbent assay (ELISA). For this, 35 patients with active CD were compared with 144 controls (18 patients with bioptically excluded CD, 89 blood donors, 30 patients with inflammatory bowel disease, and seven patients with cystic fibrosis). RESULTS: The ELISA using hu-tTG showed a sensitivity of 100% and a specificity of 98.6%. Titres of antibodies against hu-tTG (anti-hu-tTG) were positively correlated with EmA titres. All results negative for EmA were also negative for anti-hu-tTG. There were, however, EmA positive results up to a titre of 1 : 80 below the cut-off for anti-hu-tTG. For comparison, antibodies against guinea pig tissue transglutaminase (anti-gp-tTG) were determined in parallel. All patients with anti-hu-tTG below the cut-off were also negative for anti-gp-tTG. However, there were eight patients positive for anti-hu-tTG but negative for anti-gp-tTG. CONCLUSIONS: The new test reaches and even exceeds diagnostic efficiency of EmA for coeliac diagnosis. SN - 0954-691X UR - https://www.unboundmedicine.com/medline/citation/12439116/Production_of_recombinant_human_tissue_transglutaminase_using_the_baculovirus_expression_system_and_its_application_for_serological_diagnosis_of_coeliac_disease_ L2 - http://Insights.ovid.com/pubmed?pmid=12439116 DB - PRIME DP - Unbound Medicine ER -