Epitope mapping of mAbs to denatured human testicular ACE (CD143).

Angiotensin I-converting enzyme (ACE; CD143) has two homologous enzymatically active domains (N and C) and plays a crucial role in blood pressure regulation and vascular remodeling. A wide spectrum of monoclonal antibodies (mAbs) to different epitopes on the N and C domains of human ACE have been used to study different aspects of ACE biology. In this study, we characterized a set of nine mAbs, developed against the C domain of human ACE, which recognize the denatured forms of ACE and thus are suitable for the detection and quantification of somatic ACE (sACE) and testicular ACE (tACE) using Western blotting and immunohistochemistry on paraffin-embedded human tissues. The epitopes for these mAbs were defined using species cross-reactivity, phage display library screening, Western blotting and ACE mutagenesis. Most of the mAbs recognized common/overlapping region(s) on both somatic and testicular forms of human ACE, whereas mAb 4E10 was relatively specific for the testicular isoform and mAb 5B9 mainly recognized the glycan attached to Asn 731. This set of mAbs is useful for identifying even subtle changes in human ACE conformation because of denaturation. These mAbs are also sensitive tools for the detection of human sACE and tACE in biological fluids and tissues using proteomic approaches. Their high reactivity in paraffin-embedded tissues provides opportunities to study changes in the pattern of ACE expression and glycosylation (particularly with mAb 5B9) in different tissues and cells.

MAP1D, a novel methionine aminopeptidase family member is overexpressed in colon cancer.

N-terminal methionine removal is an important cellular process required for proper biological activity, subcellular localization, and eventual degradation of many proteins. The enzymes that catalyze this reaction are called Methionine Aminopeptidases (MAPs). To date, only two MAP family members, MAP1A and MAP2, have been well characterized and studied in mammals. In our studies, we have cloned a full length MAP1D gene. Expression and purification of full length recombinant protein shows that the sequence encodes an enzyme with MAP activity. MAP1D is overexpressed in colon cancer cell lines and in colon tumors as compared to matched normal tissue samples. Downregulation of MAP1D expression by shRNA in HCT-116 colon carcinoma cells reduces anchorage-independant growth in soft agar. These data suggest that MAP1D is a potentially oncogenic, novel member of the MAP gene family that may play an important role in colon tumorigenesis.

An immunoblotting method for high-resolution isoelectric focusing of protein isoforms on immobilized pH gradients.

Post-translational modifications such as phosphorylation and acetylation are important elements for regulating the activity of enzymes or structural proteins. These modifications give rise to isoforms that are often not resolved by separation methods relying on the size of proteins. Here, we optimized an isoelectric focusing (IEF)-immunoblotting method suitable for analyzing protein isoforms in total cell extracts. The separations were carried out in parallel on commercially available immobilized pH gradient slab gels (IPG). The buffer used for separation contained urea, thiourea, dithiothreitol, as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), and was designed to match those used in two-dimensional polyacrylamide gel electrophoresis (PAGE) separations where efficient solubilization is required. Proteins were transferred to membranes by passive diffusion in the presence of 4 M guanidinium chloride using protocols optimized for several protein classes (tubulin, stathmin, 14-3-3 proteins) some of which required removal of CHAPS prior to transfer. In conjunction with narrow-range pH gradient gels, excellent resolution of isoforms differing by phosphorylation or acetylation was achieved. The usefulness of pI and titration curve calculations for predicting the pI shifts expected for post-translational modifications of proteins with known amino acid composition was demonstrated. Using stathmin--which contains four phosphorylation sites--as an example, the effects on the pI-shifts were well predicted. This sensitive and widely applicable IEF-blotting technology is expected to be especially suited for analyzing protein isoforms first detected by two-dimensional electrophoresis.

Phosphorylation disrupts the central helix in Op18/stathmin and suppresses binding to tubulin.

Protein phosphorylation represents a ubiquitous control mechanism in living cells. The structural prerequisites and consequences of this important post-translational modification, however, are poorly understood. Oncoprotein 18/stathmin (Op18) is a globally disordered phosphoprotein that is involved in the regulation of the microtubule (MT) filament system. Here we document that phosphorylation of Ser63, which is located within a helix initiation site in Op18, disrupts the transiently formed amphipathic helix. The phosphoryl group reduces tubulin binding 10-fold and suppresses the MT polymerization inhibition activity of Op18's C-terminal domain. Op18 represents an example where phosphorylation occurs within a regular secondary structural element. Together, our findings have implications for the prediction of phosphorylation sites and give insights into the molecular behavior of a globally disordered protein.

Isotope-tagged cross-linking reagents. A new tool in mass spectrometric protein interaction analysis.

In protein interaction analysis, one promising method to identify the involved proteins and to characterize interacting sites at the same time is the mass spectrometric analysis of enzymatic hydrolysates of covalently cross-linked complexes. While protein identification can be accomplished by the methodology developed for proteome analysis, the unequivocal detection and characterization of cross-linked sites remained involved without selection criteria for linked peptides in addition to mass. To provide such criteria, we incorporated cross-links with a distinct isotope pattern into the microtubule-destabilizing protein Op18/stathmin (Op18) and into complexes formed by Op18 with tubulin. The deuterium-labeled cross-linking reagents bis(sulfosuccinimidyl)-glutarate-d4, -pimelate-d4, and -sebacate-d4 were prepared together with their undeuterated counterparts and applied as a 1:1 mixture of the respective d0 and d4 isotopomers. The resulting d0/d4 isotope tags allowed a straightforward mass spectrometric detection of peptides carrying the linker even in complex enzymatic protein hydrolysates. In the structure elucidation of the linked peptides by MS/MS, the assignment of the linked amino acids was again greatly facilitated by the d0/d4 tag. By applying two cross-linkers with similar reactivity but different spacer length in parallel, even doublets with very low intensity could be assigned with high confidence in MS and MS/MS spectra. Since in the Op18-tubulin complexes only a limited number of peptides carried the linker, the identification of the involved proteins per se was not impeded, thus accomplishing both protein identification and characterization of interacting sites in the same experiment. This novel methodology allowed us to significantly refine the current view of the complex between Op18 and tubulin corroborating the tubulin "capping" activity of the N-terminal domain of Op18.

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha. PTK787/ZK 222584 inhibits VEGF-induced autophosphorylation of kinase insert domain-containing receptor (KDR), endothelial cell proliferation, migration, and survival in the nanomolar range in cell-based assays. In concentrations up to 1 microM, PTK787/ZK 222584 does not have any cytotoxic or antiproliferative effect on cells that do not express VEGF receptors. After oral dosing (50 mg/kg) to mice, plasma concentrations of PTK787/ZK 222584 remain above 1 microM for more than 8 h. PTK787/ZK 222584 induces dose-dependent inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, as well as a tumor cell-driven angiogenesis model after once-daily oral dosing (25-100 mg/kg). In the same dose range, it also inhibits the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal carcinoma and its metastases in a syngeneic, orthotopic model. Histological examination of tumors revealed inhibition of microvessel formation in the interior of the tumor. PTK787/ZK 222584 is very well tolerated and does not impair wound healing. It also does not have any significant effects on circulating blood cells or bone marrow leukocytes as a single agent or impair hematopoetic recovery after concomitant cytotoxic anti-cancer agent challenge. This novel compound has therapeutic potential for the treatment of solid tumors and other diseases where angiogenesis plays an important role.

In vitro modulation of cytokine, cytokine inhibitor, and prostaglandin E release from blood mononuclear cells and synovial fibroblasts by antirheumatic drugs.

OBJECTIVE: To assess the effect of various antirheumatic drugs on cytokine, cytokine inhibitor, and prostaglandin E (PGE) production by normal blood mononuclear cells (MNC) and rheumatoid arthritis (RA) synovial fibroblasts in vitro. METHODS: MNC from healthy donors and RA synovial fibroblasts were preincubated with or without prostaglandin E2 (PGE2), indomethacin, dexamethasone, gold sodium thiomalate (GSTM), methotrexate (MTX), and cyclosporin A (CyA), and then cultured in the absence or presence of interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) for 48 h. We characterized cytokines such as IL-1 beta, IL-8, monocyte chemoattractant protein-1 (MCP-1), and cytokine inhibitors such as IL-1 receptor antagonist (IL-1ra) and soluble TNF receptors (sTNFR p55 + p75) as well as PGE in the cell-free culture supernatants. RESULTS: In MNC and synovial fibroblast cultures dexamethasone, GSTM, and PGE2 most markedly downregulated spontaneous and/or cytokine stimulated production of IL-1 beta, IL-14a, IL-8, and MCP-1, whereas sTNFR shedding was not affected. In contrast, MTX and CyA had only marginal or no effects on mediator release, whereas indomethacin inhibited only PGE production. CONCLUSION: Among several antirheumatic drugs examined, dexamethasone and GSTM exhibited the most potent inhibitory effects on inflammatory cytokine and cytokine inhibitor production by blood mononuclear cells and synovial fibroblasts. These drugs may exert their antiinflammatory actions by unspecific suppression of monocyte and fibroblast secretory function.

Neoepitope immunoassay: an assay for human interleukin 1 beta based on an antibody induced conformational change.

A secondary monoclonal antibody (mAb2) was generated by immunization with immune complexes of human IL-1 beta and a primary monoclonal (mAb1). mAb2 bound to a neoepitope on the IL-1 beta/mAb1-complex with a dissociation constant (Kd) of 26 pM but not to uncomplexed IL-1 beta. As assessed by the binding of labeled IL-1 beta and neutralization of bioactivity, mAb2 enhanced the affinity of IL-1 beta to mAb1; Kd-values were 108 pM in absence and 5.4 pM in presence of mAb2. By analyzing a series of mutants of IL-1 where surface loops had been exchanged with the corresponding loops of human IL-1 receptor antagonist protein, a critical region responsible for mAb2 binding was localized to the C-terminal region. In addition to mAb1/IL-1 beta-complexes, mAb2 bound pro-IL-1 beta/mAb1 complexes as well as pro-IL-1 beta suggesting that mAb2 recognized a conformation of IL-1 beta resembling that of pro-IL-1 beta. Using this pair of mAbs, chemiluminescent and enzyme linked assays with detection limits of 2 pg/ml hIL-1 beta have been established.

Opposite effects of interleukin-13 and interleukin-12 on the release of inflammatory cytokines, cytokine inhibitors and prostaglandin E from synovial fibroblasts and blood mononuclear cells.

We examined the effects of interleukin-12 (IL-12) and interleukin-13 (IL-13) on cytokine, cytokine inhibitor and prostaglandin E (PGE) release from synovial fibroblasts and blood mononuclear cells (MNC). In resting synovial fibroblasts, we found that IL-13 is an inhibitor of IL-8 and PGE release. A significant decrease of PGE synthesis caused by IL-13 was also observed in tumor necrosis factor (TNF)-alpha-stimulated synovial fibroblasts, whereas IL-12 had no regulatory effects on these cells. In resting and cytokine-stimulated MNC, IL-13 markedly inhibited IL-1 beta, IL-8 and monocyte chemoattractant protein-1 (MCP-1) release and potently stimulated interleukin-1 receptor antagonist (IL-1ra) synthesis. In contrast, IL-12 stimulated the production of IL-1 beta and MCP-1 in TNF-alpha-stimulated MNC and inhibited IL-1ra synthesis in cytokine-stimulated cells. These findings identify novel biological actions of IL-12 and IL-13 on connective tissue and on blood mononuclear cells which indicate their regulatory functions as enhancer and suppressor of inflammatory processes, respectively.

High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies.

We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.

Interleukin 1 (IL-1) receptor antagonist, soluble tumor necrosis factor receptors, IL-1 beta, and IL-8--markers of remission in rheumatoid arthritis during treatment with methotrexate.

OBJECTIVE: To examine circulating levels of cytokines and cytokine inhibitors and their production by blood mononuclear cells (MNC) in patients with active rheumatoid arthritis (RA) before treatment with methotrexate (MTX) and inactive disease upon treatment as well as healthy control individuals. METHODS: Interleukin-1 receptor antagonist (IL-1ra), soluble tumor necrosis factor receptors p55 and p75 (sTNFr; p55 and p75), interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8), and monocyte chemoattractant protein 1 (MCP-1) were assessed by immunoassays in sera and MNC culture supernatants of 27 patients with RA with active disease before and 14 patients with inactive disease during MTX treatment, and 10 healthy controls. RESULTS: Levels of circulating IL-1ra, sTNFr p55 and p75 were higher in patients with active RA compared to those with inactive disease or controls. At the cellular level, resting MNC of patients with active RA released more IL-1 beta and IL-8, but less IL-1ra, and showed a lower ratio of IL-1ra:IL-1 beta than MNC of patients without inflammatory symptoms or healthy controls. In addition, unstimulated and in vitro lipopolysaccharide stimulated MNC cultures of patients with inactive RA released higher amounts of sTNFr p75 than MNC of patients with active RA. CONCLUSION: Circulating levels of IL-1ra and sTNFr as well as IL-1 beta, IL-8, and sTNFr p75 release from MNC and the ratio of IL-1ra:IL-1 beta production by these cells serve as markers to assess complete disease remission in patients with RA during MTX treatment.

Differential effects of prednisolone and indomethacin on zymosan-induced inflammation in a modified murine tissue-chamber model.

A tissue-chamber model of inflammation in mice has been modified and used to investigate the kinetics of zymosan-induced inflammatory mediators such as tumour necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta) and prostaglandin E2 (PGE2). In addition, the influx of polymorphonuclear leukocytes (PMN) into the chamber fluid and the granuloma surrounding the chamber was measured by myeloperoxidase (MPO) activity using a new microtitre plate assay. TNF alpha and IL-1 beta reached a peak concentrations at 3 and 6 h respectively after zymosan injection. Intermediate high concentrations of IL-1 beta were observed until the end of the experiment at 72 h, but TNF alpha concentrations decreased from 24 h to biologically insignificant values. In contrast, exudate PGE2 and MPO activity increased up to 24 h after zymosan injection and remained high until 72 h. At 6 h after zymosan challenge, oral pre-treatment with prednisolone (3 to 30 mg/kg) dose-dependently reduced TNF alpha, IL-1 beta and PGE2 concentrations while indomethacin (0.3 to 3 mg/kg) significantly attenuated PGE2, slightly enhanced TNF alpha and had no effect on IL-1 beta concentrations in the exudate. Both drugs had similar potencies against exudate and tissue MPO activities. Prednisolone inhibited IL-1 beta at 72 h post-zymosan. Indomethacin was more potent than prednisolone against PGE2 (ID50 of< 0.3 versus 0.6 mg/kg). The data obtained confirm the usefulness and reliability of this model in evaluating the effects of anti-inflammatory agents on inflammatory mediators induced by zymosan.

Methotrexate action in rheumatoid arthritis: stimulation of cytokine inhibitor and inhibition of chemokine production by peripheral blood mononuclear cells.

This open label study examines whether methotrexate (MTX) treatment modulates ex vivo synthesis of interleukin-1 receptor antagonist (IL-1ra), soluble tumour necrosis factor receptors (sTNFR p55 and p75), interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha), interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) by peripheral blood mononuclear cells (PBMC) and whether changes reflect clinical response. Significant stimulation of IL-1ra and sTNFR p75 as well as inhibition of IL-8 production of PBMC were associated with clinical improvement observed in patients treated with MTX. When defining the characteristics of patients at study entry retrospectively in responders and non-responders, a significantly lower ratio of IL-1ra:IL-1 beta production before and its increase upon treatment was associated with clinical response in 13 patients compared to five patients not responding to MTX. In addition, clinical improvement was associated with decreased synthesis of IL-1 beta, TNF-alpha and IL-8 induced by bacterial lipopolysaccharide, IL-1 alpha and IL-1 beta in PBMC in vitro. These findings suggest that MTX therapy reverses the inflammatory type of rheumatoid arthritis (RA) blood mononuclear cells by stimulating cytokine inhibitor production while inhibiting inflammatory cytokine release at the same time. This may explain the powerful anti-inflammatory properties of low-dose MTX as observed in most RA patients. Pretreatment determination of the IL-1ra:IL-1 beta ratio in PBMC may be predictive with regard to a favourable therapeutic response and therefore may be useful for the selection of RA patients to be treated with MTX.

Cytokine immunotrapping: an assay to study the kinetics of production and consumption or degradation of human interferon-gamma.

With the aim of determining the rate of cytokine production, we have investigated immunoassay conditions which prevent consumption/degradation. These assays, termed cytokine immunotrapping assays (CITA), are based on early capturing of cytokines secreted during cell culture by immobilised or soluble mAbs and a recently described chemiluminescent immunoassay. Here we describe assay conditions using IFN-gamma as a prototype cytokine. For production of IFN-gamma, PBMC, purified CD4+ or CD8+ T cells, or diluted whole blood were cultured with different T cell stimulating agents. Polystyrene macrobeads precoated with an anti-IFN-gamma mAb were put in culture and after a defined incubation period, a dimethyl acridinium ester (DMAE)-labelled second anti-IFN-gamma mAb and sodium azide were added into the culture for additional 24 h. The beads were washed and chemiluminescence signals determined in a luminometer. Trapping experiments were also performed with the beads or the soluble mAbs alone. Irrespective of the configuration, IFN-gamma concentrations measured in trapping conditions were always higher (3-20-fold) than in conventional cultures. By using the best trapping combination, i.e. both bead-mAb1 and DMAE-mAb2 added at the start of culture (single step), it was possible to detect IFN-gamma production as early as 2 h. Also, IFN-gamma secreted by less than 500 PBMC or whole blood cells could be detected within 24 h. When purified CD4+ or CD8+ cells were used instead of PBMC, a reduction of the trapping effect was observed. Conversely, addition of monocytes to purified T cells increased the trapping factor suggesting that a substantial amount of IFN-gamma was consumed or degraded both by CD14+ cells as well as T cells in culture. Preliminary results show that this assay is also suitable for the early detection of IL-1 and IL-4 which are known to be more tightly regulated. Thus, the new principle described here is expected to be useful in clinical settings where both the time and amounts of material are limited to investigate the role of cytokines in particular disease.

Interleukin-10 differentially regulates cytokine inhibitor and chemokine release from blood mononuclear cells and fibroblasts.

In this study we have examined the effects of interleukin-10 (IL-10) on blood mononuclear cells (MNC) and on skin as well as on synovial fibroblasts. In unstimulated MNC, we found that IL-10 is a potent stimulator of interleukin-1 receptor antagonist (IL-1ra) and monocyte chemoattractant protein-1 (MCP-1) production and an inhibitor of IL-8 release. In cells exposed to IL-1 beta, it also moderately stimulated IL-1ra production and release of soluble tumor necrosis factor receptor p75 (sTNF-R p75) and inhibited IL-8 and MCP-1 production. In addition, we have evidence that the biological effects of IL-10 are not restricted to hematopoietic cells. IL-10 stimulated sTNF-R p55 dose-dependently and inhibited MCP-1 release from IL-1 beta-activated fibroblasts, whereas IL-8 production was not affected. Taken together, these findings identify novel biological actions of IL-10 on blood mononuclear and connective tissue cells which support its regulatory functions as a suppressor of inflammatory processes.

Sandwich immunoassay for the hapten angiotensin II. A novel assay principle based on antibodies against immune complexes.

Immunoassays for haptens such as short peptides or drugs are usually based on the principle of competition for a limited number of binding sites on antibody molecules. Owing to the small size of these antigens it has been thought that two specific antibodies cannot simultaneously bind a hapten. However, antisera containing so called anti-metatypic antibodies have been reported (Voss et al. (1988) Mol. Immunol. 25, 751-759) that bind to hapten-mAb complexes in a reaction where conformational changes on the primary antibody are important. Here, we report on monoclonal antibody pairs able to form ternary complexes with the octapeptide angiotensin II. The first mAb (mAb1) is conventional and binds angiotensin II with high affinity (Kd 10(-11) M). The secondary (anti-metatypic) mAbs (mAbs2s) recognize the immune complex consisting of angiotensin II bound to mAb1, but only poorly recognize mAb1 alone. An immunization technique involving tolerization with uncomplexed mAb1 was used to generate mAb2s. None of the mAbs2s were able to bind angiotensin II by themselves but all efficiently bound the complex of angiotensin II and mAb1. All mAb2s stabilized the angiotensin II-mAb1 complex and one mAb2 distinctly improved the specificity of the assay for angiotensin II. By either labelling mAb1 and immobilizing mAb2 (or vice versa) two-site immunometric assays with detection limits of 1 pg/ml angiotensin II have been established. The kinetics of the complex formation was investigated by fiber optic biospecific interaction analysis (FOBIA), a system allowing real time observation of binding events on the surface of a glass fiber. The association rate towards the liganded conformation of mAb1 was higher than towards the free mAb1. By contrast, the mAb2s dissociated at similar rates from complexed and uncomplexed mAb1.

Quantitation of interferon-induced Mx protein in whole blood lysates by an immunochemiluminescent assay: elimination of protease activity of cell lysates in toto.

Due to the rapidly expanding usage of interferons and its costliness of therapy, it is important to evaluate the clinical efficacy of the various interferons. Directly assaying circulating interferon is technically quite difficult. Here, we present an alternate method to evaluate interferon therapy by assaying a unique protein, called Mx protein, which is a 78 kDa cytoplasmic protein selectively induced by type-1 interferon in human leukocytes. The current assay is a two-site chemiluminescent immunoassay, designed to detect Mx protein in whole blood lysates. Since the Mx protein once solubilized, is highly susceptible to proteolysis in whole blood lysates, we have devised a new procedure both to maximize its solubility and virtually eliminate its proteolytic degradation. A mouse monoclonal antibody conjugated to the derivatized-paramagnetic particles and an acridinium ester-labeled antibody serve as the solid phase capture and detector antibodies, respectively. This assay is applicable to both manual and automated modes with a detection limit of Mx protein at 20 ng/ml whole blood. Availability of a reliable assay for Mx protein should facilitate the clinical evaluation of many of the newly constructed type-1 interferons.

Structure-function analysis of angiotensin I-converting enzyme using monoclonal antibodies. Selective inhibition of the amino-terminal active site.

Angiotensin I-converting enzyme (ACE; kininase II) contains two very similar domains (the NH2- and COOH-terminal domains (N and C domains, respectively)), each bearing an active site. These active sites hydrolyze the same peptides, but do not have the same catalytic properties and substrate specificities. In an attempt to develop domain-specific immunological probes, two series of monoclonal antibodies (mAbs), 19 clones in all, were produced and tested against human ACE. These mAbs recognized at least nine different epitopes within three antigenic regions of the ACE molecule. Testing on wild-type recombinant ACE and several mutants with only one intact domain showed that these epitopes were all located in the N domain. None of the mAbs recognized the C domain. This particular specificity and analysis of results obtained with several polyclonal antibodies to human ACE suggest that ACE immunogenicity is determined mainly by the N domain. Two mAbs (3A5 and i2H5) recognizing epitopes from different antigenic regions of ACE inhibited the enzymatic activity of the N (but not of the C) domain. mAb 3A5 had the same inhibitory potency toward hippuryl-His-Leu, benzyloxycarbonyl-Phe-His-Leu, and angiotensin I hydrolysis, with 50% inhibition achieved at a mAb/ACE molar ratio of 6. mAb i2H5 was roughly three times more effective than mAb 3A5 inhibiting the hydrolysis of benzyloxycarbonyl-Phe-His-Leu and the natural substrates angiotensin I and bradykinin (50% inhibition at a molar ratio of 1-2), but was less effective in inhibiting hippuryl-His-Leu cleavage (50% inhibition at a molar ratio of 22-25), indicating that this substrate interacts with a specific subsite. mAb i2H5 almost completely inhibited the hydrolysis of the luteinizing hormone-releasing hormone by the isolated N domain. Both the primary carboxyl- and amino-terminal cleavages of this peptide were suppressed. This antibody suppressed the primary amino-terminal cleavage of the luteinizing hormone-releasing hormone by wild-type ACE by > 90%, indicating that this particular ACE function is mediated mainly by the N domain active site. These data provide evidence for structural differences between the two homologous domains of ACE despite their high degree of sequence homology and show that monoclonal antibodies are able to distinguish between the two active sites in ACE.

Chemiluminescent and enzyme-linked immuno assays for sensitive detection of human IFN-gamma.

We have produced and characterized 4 mAbs to human IFN-gamma and established sensitive, non-radioactive immuno-assays. The first two assays use microtiter plates as the solid phase and enzymes or chemiluminescence (acridinium ester) for development. The use of chemiluminescence instead of peroxidase increased the sensitivity of the assay by a factor of about 75. The third and the fourth assays utilize polystyrene beads as the solid phase and enzymes or acridinium ester for development. The use of beads also increased the sensitivity of detection. The most sensitive IFN-gamma detection was achieved by the combination of bead with acridinium ester. In this configuration we were able to detect about 0.2 pg/ml IFN-gamma (1/250th of a unit). These chemiluminescent immunoassays (CLIA) appear to be more sensitive than existing ELISAs or radioimmunoassays and may find new application areas.