Production of recombinant human tissue transglutaminase using the baculovirus expression system, and its application for serological diagnosis of coeliac disease.
OBJECTIVE
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.
Osman
Awad A
AA
Institute of Laboratory Medicine, Clinical Chemistry, and Molecular Diagnostics, University Hospital, Leipzig, Germany.
Richter
Thomas
T
Stern
Martin
M
Conrad
Karsten
K
Henker
Jobst
J
Brandsch
Corinna
C
Zimmer
Klaus-Peter
KP
Mothes
Thomas
T
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016367:Baculoviridae / Q000276:immunology*; D015153:Blotting, Western; D002446:Celiac Disease / Q000175:diagnosis* / Q000276:immunology; D002478:Cells, Cultured; D002648:Child; D002675:Child, Preschool; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D008875:Middle Aged; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection; D011503:Transglutaminases / Q000096:biosynthesis*
D011994:Recombinant Proteins; D011503:Transglutaminases