PHAGE ANTIBODY LIBRARY FROM CELIAC DISEASE PATIENT (1999)

96 Introduction In phage display, an antibody fragment (scFv) is displayed on the surface of a filamentous phage and the phage harbors the encoding gene. Antibody libraries have been made from patients suffering from diseases in which antibodies are known to play a role, the antibodies selected from such libraries having similar specificities to those found in patients' serum. Coeliac disease (CD) is associated with a strong Ab response to certain dietary cereals and autoantigens and is a good model to be studied with phage display. Methods Creation of combinatorial library Peripheral blood was obtained from a undiagnosed 30 year old celiac patient, with strong AGA and EMA responses. Total RNA was extracted from 107 PBL, V regions were amplified, Igκ and Igλ libraries were then constructed. Antibody selection Purified A-gliadin was obtained with alcohol extraction. α/β lactoglobulin and guinea pig tTG were purchased from Sigma. Human tTG (HutTG) was cloned from intestinal biopsy RNA. All the antigens were immobilized on immunotubes (Nunc) by coating at 10 µg/ml in PBS overnight. Results and discussion We have made and characterized an antibody (scFv) phage library from a patient with Celiac Disease. Many different Scfv against the toxic antigen (A-gliadin) and dietary antigens (α/β lactoglobulin) were isolated from this library after 2 rounds of selections. 93 % of the clones were positive for A-gliadin, 85% of these were polyreactive, only 8% were monoreactive. 12 anti A-gliadin clones were further characterized. Monoreactive Abs recognized antigens from wheat, barley and to a lesser extent rye in ELISA and this is probably due to the presence of a shared toxic antigenic determinant. All the clones were sequenced and the VH and VL families determined. 7 anti A-gliadin scFv out of 12 contained a VH chain matching the VH4 family. The same 7 Abs gave the highest OD in ELISA. This result may indicate a possible role of certain VH families in the onset of C.D. Recently the enzyme tissue-transglutaminase (tTG) was described as the possible endomysial autoantigen. Despite multiple attempt selecting on Guinea pig tTG gave no positive clones after 3 cycles. Considering that Guinea pig tTg is only 80% homologous to human tTG we cloned and expressed the human tTG. We showed that HutTG give higher specificity and sensitivity than guinea pig tTG. Also HuTtg gave negative results after 3 round of selection. More recently, preliminary results obtained with a phage library from intestinal lymphocytes revealed the presence of Abs reactive to HuTtg. This finding supports the hypothesis that anti-endomysium autoantibodies are originally synthesized at intestinal level. These Abs could be used for clear determination of the epitopes involved in the immune activation and response and in their pathogenic role, that actually is impossible with a polyreactive serum. Furthermore molecular analysis revealed a new VH family specific activation that could have a role in the disease. This analysis will be even more relevant for the anti tTG Abs. These could be used to further characterize the real role of transglutaminase which is still controversial.