Proteinase inhibitory activities of antileukoprotease are represented by its second COOH-terminal domain.

Antileukoprotease or secretory leukocyte proteinase inhibitor is a potent serine proteinase inhibitor produced by exocrine glands of the human body. This monomeric protein (107 amino acids) comprises two homologous domains. It is generally thought that Leu19-Arg20-Tyr21 in the NH2-terminal domain represent the trypsin inhibitory activity, whereas Leu72-Met73-Leu74 in the COOH-domain represent the chymotrypsin and elastase inhibitory activity. Besides Met73, antileukoprotease contains three additional methionine residues all located in the COOH-terminal domain. Treatment of antileukoprotease with different amounts of methionine-selective reagents such as myeloperoxidase in the presence of H2O2 and Cl-, or cis-platinumdiammine dichloride resulted in a dose-dependent inactivation of all inhibitory activities, suggesting that methionine residues are involved in these activities. By using specific synthetic substrates, it was observed that elastase is able to displace trypsin from the inhibitor molecule, indicating that the trypsin and elastase inhibitory sites are located close to each other or at the same site. Incubation of antileukoprotease or its recombinant COOH-terminal domain with an antileukoprotease-specific monoclonal antibody (MoAb15) resulted in a strong selective increase of the trypsin inhibitory activity. The results presented reveal strong evidence that the inhibitory activities of antileukoprotease against trypsin, chymotrypsin and elastase are represented by its COOH-terminal domain, and that methionine residues are involved in interactions with these proteinases.

Fluobodies: green fluorescent single-chain Fv fusion proteins.

An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selected recombinant antibodies by flow cytometry or fluorescent cell staining. Two different single-chain variable fragment antibodies, both directed against the lipopolysaccharide of the bacterium Ralstonia solanacearum have been genetically fused to a red-shifted green fluorescent protein and the produced fusion protein tested for usefulness. These fluobodies can be produced in cultures of bacterial cells and purified using immobilized metal affinity chromatography. They function well in flow cytometry and immunofluorescent cell staining, are specific for their target antigens and, unlike FITC-conjugated antibodies, they do not fade upon illumination.

Development of Specific Recombinant Monoclonal Antibodies Against the Lipopolysaccharide of Ralstonia solanacearum Race 3.

ABSTRACT Recombinant single-chain antibodies (scFvs) against the lipopolysaccharide of Ralstonia solanacearum (biovar 2, race 3) were successfully selected by phage display from a large combinatorial antibody library. Characterization with regard to cross-reaction and use in routine immunoassays showed that the selected antibodies had improved characteristics when compared with the polyclonal antiserum that is currently used for brown rot diagnosis of potato in the Netherlands. The isolated monoclonal scFvs reacted in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence cell staining with all race 3 strains tested, but with only some strains belonging to other races. Furthermore, only a few cross-reactions with saprophytic bacteria, which also cross-reacted with polyclonal antisera, were observed. Using ELISA, one of the recombinant antibodies detected as few as 5 x 10(3) bacteria in potato tuber extracts. Therefore, this antibody is potentially useful for detection of R. solanacearum race 3.

Application of Phage Display in Selecting Tomato spotted wilt virus-Specific Single-Chain Antibodies (scFvs) for Sensitive Diagnosis in ELISA.

ABSTRACT A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S, which allowed the scFvs to be expressed as alkaline phosphatase fusion proteins, 17 different scFv antibodies were obtained. Of these, 12 scFvs were directed against the nucleoprotein (N) and 5, putatively, against the glycoproteins (G1 and G2). Five of the N-specific antibodies cross-reacted with two other tospoviruses (Tomato chlorotic spot virus and Groundnut ringspot virus), but none recognized the more distantly related tospoviruses Impatiens necrotic spot virus, Watermelon silverleaf mottle virus, Iris yellow spot virus, or Physalis severe mottle virus. The successful use of one of the antibodies as coating and detection reagent in a double-antibody sandwich enzyme-linked immunosorbent assay showed the potential of the phage display system in obtaining antibodies for routine TSWV diagnosis.

Simplification of a commercially available serum angiotensin-converting enzyme determination.

The spectrophotometric method for the determination of angiotensin-converting enzyme in serum using p-hydroxyhippuryl-L-histidyl-L-leucine as substrate is commercially available as a test kit. It shows excellent linearity over the whole range of catalytic concentration found in serum. We describe several modifications of this method to simplify and economize the procedure.

L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide, a highly specific substrate for granulocyte elastase.

L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide was found to be a highly specific substrate for human granulocyte elastase. At pH 8.3 and 37 degrees C, its Km = 0.55 mmol/l and the value for kcat was 6 sec-1, whereas with porcine pancreatic elastase these values were approximately 2 mmol/l and less than 0.001 sec-1, respectively. It is not cleaved by trypsin or chymotrypsin. With granulocyte elastase this new substrate is 50 times more sensitive compared to succinyltrialanyl-p-nitroanilide. L-Pyroglutamyl-L-prolyl-L-valine-p-nitroanilide can also be used for the assay of granulocyte elastase inhibitors.

Interactions among stimulated human polymorphonuclear leucocytes, released elastase and bronchial antileucoprotease.

1. We investigated the effect of stimulated human polymorphonuclear leucocytes (PMN's) on both the antitrypsin and anti-elastase activity of bronchial antileucoprotease (ALP). 2. Incubation of ALP with stimulated human PMN's resulted in a rapid loss of anti-elastase activity which paralleled that of the antitrypsin activity, suggesting that both inhibitor activities are represented by the same active site. 3. The myeloperoxidase-oxidizing system was found to be responsible for the inactivation of ALP. 4. The oxidized inhibitor was unable to form stable complexes with PMN elastase but was resistant to breakdown by proteolytic enzymes from stimulated PMN's. 5. It was observed that stimulated cells are capable of releasing elastase which shows full activity in the presence of a large molar excess of ALP. 6. We conclude from this study that stimulated PMN's are able to inactivate ALP by which released elastase is able to express enzymatic activity in spite of the presence of this low-molecular-weight inhibitor. Thus, inactivation of ALP by triggered PMN's may contribute to destructive processes in which elastase is thought to be a mediator.

Detection of extracellular neutrophil elastase in hamster lungs after intratracheal instillation of E. coli lipopolysaccharide using a fluorogenic, elastase-specific, synthetic substrate.

Repeated intratracheal instillations of E. coli lipopolysaccharide (LPS) in hamster lungs cause an influx of polymorphonuclear leukocytes (PMNs) into the alveolar walls, with concomitant development of severe emphysema. It has been suggested that elastase, released by these PMNs, is involved in the development of emphysema. This study demonstrates the release of elastase from recruited PMNs in cryostat sections of hamster lungs, after being treated once, twice, or thrice with LPS, intratracheally. Elastase activity was visualized using two elastase-specific synthetic substrates, to which a methoxynaphthylamine (MNA) group had been bound covalently. Liberated MNA, when made insoluble by coupling with 5-nitrosalicylaldehyde, fluoresces strongly. The authors observed that the interval between start of incubation and appearance of fluorescence and the intensity of fluorescence correlated with the number of LPS administrations. Fluorescence was observed to be located in or in close vicinity to alveolar walls. No fluorescence was observed in sections of untreated hamsters. Liberation of MNA from synthetic substrates was delayed strongly by the addition of a recombinant secretory leukocyte proteinase inhibitor or a substituted cephalosporin neutrophil elastase inhibitor. The authors conclude that LPS-mediated PMN influx into the lung is accompanied by release of elastase from these cells and speculate that this PMN-elastase is involved in the development of LPS-mediated emphysema.

pSKAP/S: An expression vector for the production of single-chain Fv alkaline phosphatase fusion proteins.

The vector pSKAP/S was constructed to enable overexpression of single-chain variable fragment antibody (scFv)-alkaline phosphatase fusion proteins. In pSKAP/S, the scFv were genetically fused to the mutated Escherichia coli PhoA/S gene that encodes an alkaline phosphatase with increased specific activity. The restriction sites incorporated into pSKAP/S allowed the scFv genes to be easily transferred from pUC119-derived phagemid vectors that are used frequently in phage display antibody library technology. Strong transcriptional control of expression was achieved using the tetracycline promoter, and induction of different individual clones with anhydrotetracycline resulted in secretion of most of the scFv-alkaline phosphatase fusion proteins into the culture medium. Although some of the clones secreted fusion proteins that were retained in the periplasm, these proteins could be isolated with a simple extraction procedure. Increased amounts of a scFv-alkaline phosphatase fusion protein were obtained when expressed in the pSKAP/S vector compared with expression in a vector incorporating the lac promoter. Testing for binding of the scFv-alkaline phosphatase fusion proteins to antigen was possible in an ELISA without the need for additional enzyme-conjugated antibodies. The pSKAP/S vector was successfully used to obtain scFv fragments from a preparation of phage-antibody clones after subcloning and expression of individual clones as scFv-alkaline phosphatase fusions, whereas fewer clones (and clones with different properties) were obtained from the same phage-antibody preparations when expressed as soluble scFv fragments. Therefore, the pSKAP/S vector was shown to be useful in extending the range of scFv obtained from phage display libraries.