Specific monoclonal antibodies against the active sites of two genetically engineered pancreatic secretory trypsin inhibitor (PSTI) variants (PSTI 0 and PSTI 4) were produced. The protease inhibitors PSTI 0 and PSTI 4 differ only by three amino acid substitution at their active sites. PSTI 0 inhibits trypsin, whereas PSTI 4 inhibits human granulocyte elastase and chymotrypsin. Immunization was performed in vitro with a synthetic heptapeptide that covers the mutated region of the protein. For this purpose in vitro culture conditions for the production of specific monoclonal antibodies against synthetic peptides were improved. The monoclonal antibodies obtained react specifically with the corresponding protease inhibitor variant. Competition experiments with trypsin and human elastase demonstrate that the protease displace the monoclonal antibody from the active site of PSTI 0 and PSTI 4 respectively.The genes of human pancreatic secretory trypsin inhibitor (PSTI 0, [l]) and a variant (PSTI 4) of this wild-type sequence were chemically synthesized, cloned and expressed by a secretion vector in Escherichia coli [2]. It was shown that the substitution of Lys-18, Ile-19 and Asn-21 in PSTI 0 by Leu-18, Glu-19 and Arg-21 in PSTI 4 (Fig. 1) led to a distinct alteration of the specificity of the protease inhibitor. While PSTI 0 inhibits human trypsin (Ki = 1.5 x M, PSTI 4 inhibits human granulocyte elastase (Ki = 6 x lo-" M) and chymotrypsin (Ki < 5 x lo-" M).For clinical diagnostic purposes we aimed at producing specific monoclonal antibodies against the active sites of PSTI 0 and PSTI 4. These should allow the determination of the amount of free protease inhibitor in the presence of bound inhibitor in serum. To produce monoclonal antibodies that are directed against the active sites of PSTI 0 and PSTI 4 immunization was carried out with synthetic heptapeptides, which comprise these target sites.Immunizations for the production of monoclonal antibodies can be performed both in vivo and in vitro. The advantages of the in vitro over in vivo immunization are (a) it requires only 5 -9 days, (b) the yield of positive stable hybridoma clones is considerably larger, (c) picomolar amounts of antigen are sufficient to produce monoclonal antibodies and (d) the probability of producing antibodies against self-antigens is greater, since suppression or tolerance mechanisms are less able to function. Nevertheless the poor survival capacity of