Production of Monoclonal Antibodies

Aims: To measure the content of immunoreactive human pancreatic secretory trypsin inhibitor (irPSTI) in colonic carcinoma and adjacent normal colonic mucosa. Methods: From a stable hybridoma cell line producing monoclonal antibodies specific for human PSTI, a specific enzyme linked immunosorbent assay (ELISA) for human PSTI was developed. In a precipitation assay system these antibodies bound human PSTI in a dosedependent manner. The specimens were obtained from resectional surgery. Results: The content of irPSTI was 199 ,ug/g protein (0.55 ug/g tissue wet weight) in colonic carcinoma. In adjacent normal colonic mucosa 43-6 ,ugIg protein (1.12 uglg tissue wet weight) was shown. Conclusions: The enzymatic degradation of surrounding tissue necessary for tumour cell invasion could be facilitated by this relative deficit of the inhibitor in infiltrative carcinoma. (J Clin Pathol 1992;45:1066-1069) Department of Surgery P Jonsson H Bohe M Bohe Department of Pathology C Lindstrom Department of Surgical Pathophysiology K Ohlsson University of Lund, Malmo General Hospital, S-214 01 Malmo, Sweden Correspondence to: Dr Hans Bohe Accepted for publication 3 June 1992 Pancreatic secretory trypsin inhibitor (PSTI) was first isolated from pancreatic tissue in 1948 by Kazal.' PSTI is thought to protect the pancreatic gland from damage by preactivated trypsin. The serum concentration of immunoreactive PSTI (irPSTI) is increased in severe diseases such as carcinoma ofpancreatic gland, breast, thyroid, stomach, oesophagus,2 and gall bladder.' irPSTI has also been shown in and isolated from different parts of the normal gastrointestinal mucosa.4 "' irPSTI has been found in the foveolar cells in the gastric mucosa. In duodenal and small intestinal mucosa irPSTI found in Paneth5 7 and goblet cells.58 Normal gall bladder mucosa did not contain irPSTI while well differentiated carcinoma and tissue resembling adenoma did." In colonic mucosa ir PSTI has been found in the goblet cells in the basal parts of the crypts.6 In colonic adenomas, however, irPSTI was also found in the upper parts of the polyps following the reverse mode of regeneration.'2 irPSTI has not been shown in most colonic carcinoma tissue. 1 3 Methods Electrophoresis reagents, dextran T500, protein A sepharose Cl-4B, and CNBr-activated sepharose CI-4B were produced by Pharmacia LKB Biotechnology, Uppsala, Sweden. Class specific goat anti-mouse immunoglobulins were obtained from Nordic Immunologic Laboratories, Tilburg, the Netherlands. Alkaline phosphatase conjugated avidin was obtained from Dakopotts AB, Hagersten, Sweden. Tissue culture medium and fetal calf serum were supplied by Gibco Laboratories, Grand Island, NewYork, USA. Pansorbin was obtained from Calciobiochem Corporation, San Diego, California, USA. Recombinant PSTI (rPSTI) was obtained from Synergen Inc, Boulder, Colorado, USA. All other chemicals and proteins were purchased from Sigma Chemical Co, St Louis, Missouri, USA. PRODUCTION OF MONOCLONAL ANTIBODIES BALB/c mice (Bommice, Bomholtgard Breeding and Research Center Ltd, Bomholtvej, Denmark) were immunised by a subcutaneous injection of 30 ,ug of rPSTI in complete Freund's adjuvant, repeated five times at three week intervals with 30 ,ug rPSTI in Freund's incomplete adjuvant. Hybridomas were produced by fusion of SP2/0, non-secreting BALB/c myeloma cells with spleen lymphocytes from two out of five immunised mice.'4 '5 SELCTION OF ANTIBODY-PRODUCING HYBRIDOMAS Hybridoma culture fluids were tested for antibody using solid phase ELISA technique. Microtitre plates were coated with 50 pl of rPSTI, 10 ,ug/ml. Hybridomas producing antibodies specific for PSTI were selected for cloning. The subclass type of the monoclonal antibodies produced was determined by the double immunodiffusion test.'6 Cells harvested from cultures of hybridomas producing monoclonal antibodies against PSTI were injected intraperitoneally into BALB/c mice treated with Pristane (Aldrich, Beerse, Belgium) for the production of ascites fluid. The ascites fluid was purified by affinity chromatography on a protein A sepharose CI-4B column equilibrated with 1-5 M glycineNaOH, 3 M NaCI, at pH 8 9. The column was washed with this buffer after application of the ascites fluid and the IgG was eluted stepwise with 0-1 M citric acid-NaOH at pH 1 066 group.bmj.com on June 20, 2017 Published by http://jcp.bmj.com/ Downloaded from