Immunoaffinity purification and reconstitution of human alpha(2)-adrenergic receptor subtype C2 into phospholipid vesicles.

Large quantities of correctly folded, pure alpha(2)-adrenergic receptor protein are needed for structural analysis. We report here the first efficient method to purify human alpha(2)-adrenergic receptor subtype C2 to homogeneity from recombinant yeast Saccharomyces cerevisiae by one-step purification using a monoclonal antibody column (specific for alpha(2)C2). We show that the adrenoceptor antagonist phentolamine stabilized the receptor during purification. We used a very effective chaotropic agent, NaSCN, to elute the receptor from the immunoaffinity column with an overall yield of 34% before reconstitution. Ligand binding of detergent-solubilized, immunoaffinity-purified receptors could not be demonstrated, but partial recovery of ligand binding activity was achieved when purified receptors were reconstituted into phospholipid vesicles. The reconstituted receptors still bound radioligand after storage on ice for 4 weeks. This purification procedure can be easily scaled-up and thus demonstrates the utility of a monoclonal antibody column and NaSCN elution to purify large quantities of G-protein-coupled receptors.

In vivo detection of vascular adhesion protein-1 in experimental inflammation.

Vascular adhesion protein-1 (VAP-1) is an inflammation-inducible endothelial glycoprotein which mediates leukocyte-endothelial cell interactions. To study the pathogenetic significance of VAP-1 in inflammatory disorders, an in vivo immunodetection method was used to detect the regulation of luminally expressed VAP-1 in experimental skin and joint inflammation in the pig and dog. Moreover, VAP-1 was studied as a potential target to localize inflammation by radioimmunoscintigraphy. Up-regulation of VAP-1 in experimental dermatitis and arthritis could be visualized by specifically targeted immunoscintigraphy. Moreover, the translocation of VAP-1 to the functional position on the endothelial surface was only seen in inflamed tissues. These results suggest that VAP-1 is both an optimal candidate for anti-adhesive therapy and a potential target molecule for imaging inflammation.

Production and characterisation of monoclonal antibodies against a very small hapten, 3-methylindole.

Monoclonal antibodies were produced against a very small (131.2 Da) hapten, 3-methylindole. Nine derivatives of 3-methylindole were synthesised with spacers ending in a carboxyl group, and coupled to immunogenic carriers and europium chelate labels. Almost all the antigens elicited an antihapten response, but the majority of the mAbs produced strongly recognised the spacer group and did not bind free 3-methylindole. However, specific antibodies were obtained with five immunogens. Specificity could be directed against the pyrrole ring by locating the bridging group to the aromatic moiety of the indole ring system. Any modification in the position 3 of the indole ring strongly hindered mAb binding to the compound, and the cross-reactivity of physiologically important compounds, such as tryptophan and tryptamine, was negligible for all of the mAbs. The developed hapten structures successfully focused antibody recognition to the important sub-determinants in the indole ring system. Similar constructs could also be useful in the development of antibodies against other indolic compounds.

Endothelial tie receptor antigen in maternal and cord blood of healthy and preeclamptic subjects.

OBJECTIVE: To measure the vascular endothelial receptor tyrosine kinase Tie, essential in the process of angiogenesis, in blood from healthy and preeclamptic pregnant women, in umbilical cord blood from both, and in blood from nonpregnant women. METHODS: A total of 143 women participated in four arms of the study. Blood samples were collected from 54 healthy nonlaboring pregnant women (gestational weeks 14-41). Samples were collected immediately prepartum and postpartum from another 40 healthy women (15 delivered vaginally and 25 by cesarean) and 15 preeclamptic women (all delivered by cesarean). Arterial and venous cord samples were collected, when possible, from infants born by cesarean. Single blood samples were drawn from 34 nonpregnant controls. Of these, weekly samples from 11 were drawn during one menstrual cycle. A time-resolved fluoroimmunoassay was developed for the detection of the soluble extracellular domain of Tie. RESULTS: Maternal serum Tie levels decreased with advancing gestational age after 26 weeks (r = .6, P < .001). They were significantly higher in healthy women at term (median 233 ng/mL, range 152-414 ng/mL) compared with nonpregnant controls (median 173 ng/mL, range 107-333 ng/mL, P < .001) or with preeclamptic women at term (median 152 ng/mL, range 90-372 ng/mL, P < .05). This difference between healthy and preeclamptic women persisted on the first postpartum day (median 221 ng/mL, range 128-343 and median 152 ng/mL, range 90-372 ng/mL, respectively, P < .05). The highest levels of serum Tie receptor were observed in umbilical arterial and venous blood (median 240 ng/mL, range 174-474 ng/mL and median 340 ng/mL, range 245-690 ng/mL, respectively). In nonpregnant women, serum Tie levels did not vary with menstrual cycle. CONCLUSION: The high levels of the extracellular domain of Tie in healthy term maternal and cord blood may indicate a role for Tie in the vascular development of human fetuses and placentas.

Lymphatic endothelium and Kaposi's sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3.

Lymphatic vessels have been difficult to study in detail in normal and tumor tissues because of the lack of molecular markers. Here, monoclonal antibodies against the extracellular domain of the vascular endothelial growth factor-C receptor that we have named VEGFR-3 were found to specifically stain endothelial cells of lymphatic vessels and vessels around tumors such as lymphoma and in situ breast carcinoma. Interestingly, the spindle cells of several cutaneous nodular AIDS-associated Kaposi's sarcomas and the endothelium around the nodules were also VEGFR-3 positive. The first specific molecular marker for the lymphatic endothelium should provide a useful tool for the analysis of lymphatic vessels in malignant tumors and their metastases and the cellular origin and differentiation of Kaposi's sarcomas.

Analysis of Tie receptor tyrosine kinase in haemopoietic progenitor and leukaemia cells.

We generated a panel of monoclonal antibodies against the extracellular domain of the Tie receptor tyrosine kinase and studied its expression in human haemopoietic and tumour cell lines and in samples from leukaemia patients. Most of the erythroblastic/megakaryoblastic (6/8), 2/7 myeloid and 3/6 B-lymphoblastic leukaemia cell lines were Tie-positive. The erythroblastic/megakaryoblastic leukaemia cell lines also expressed the related Tie-2/Tek gene and, surprisingly, its recently cloned ligand gene angiopoietin-1, which was located in chromosome 8q23.1. In addition, 16% of freshly isolated leukaemia samples were Tie positive. Peripheral blood mononuclear cells were Tie negative, but a few Tie positive cells were found in immunoperoxidase staining of mobilized peripheral blood stem cells. Long-term culture of isolated umbilical cord blood CD34+ Tie+ and CD34+ Tie- cells indicated that the Tie+ fraction contained a slightly higher frequency of cobblestone area forming cells (CAFC). Thus, Tie is expressed on haemopoietic progenitor cells and some leukaemic blasts. The coexpression of Tie-2 and angiopoietin-1 in megakaryoblastic leukaemia cell lines suggests the existence of an autocrine ligand/receptor signalling loop in these cells.

Subtype specific recognition of human alpha2C2 adrenergic receptor using monoclonal antibodies against the third intracellular loop.

Monoclonal antibodies (Mabs) against human alpha2C2-adrenergic receptor (alpha2C2-AR) were raised in mice and characterized. Bacterially expressed fusion protein consisting a sequence from the putative third intracellular loop (amino acids 213-343) of human alpha2C2 and glutathione-S-transferase (GST) was used as antigen. Results from mass spectrometry of purified thrombin cleaved alpha2C2 polypeptide suggested that the epitope region would lie near the aminoterminal end of the 3rd intracellular loop of human alpha2C2-AR. Elevation of Mabs was detected with Western blotting from mouse blood samples. Three alpha2C2 specific cell clones were expanded to in vitro production in hollow fiber systems. The specificity of the Mabs was further determined by immunoprecipitation and immunocytochemistry. Scatchard analysis of thrombin digested, purified, Europium-labelled antigen (amino acids 213-343 of alpha2C2) revealed binding affinity constants of 0.4 x 10(9), 0.7 x 10(9) and 1.6 x 10(9) M(-1) and Kds of 2.6, 1.4 and 0.6 nM for the three Mabs 2B1, 3G3 and 7G1, respectively.

A mouse molecular mimic of human vascular adhesion protein-1.

Human vascular adhesion protein-1 (VAP-1) is an endothelial sialoglycoprotein which exists in forms of Mr 90000 and 170000 and mediates lymphocyte binding to vessels under shear. VAP-1 is functionally defined by an inhibitory mouse mAb 1B2. A large-scale immunoaffinity purification of VAP-1 from human tonsil lysates was performed to determine the protein sequence for VAP-1 cDNA cloning. A dominant protein of molecular weight 90000 was obtained which yielded an N-terminal sequence of 20 amino acids which bore no significant identity to any protein sequence in the data banks. A mouse mAb (5B11) against a synthetic peptide from this sequence was raised and found to stain tissues in an identical manner to mAb 1B2, to inhibit lymphocyte adhesion to endothelial cells and to recognize VAP-1. Later, the N-terminal sequence obtained from the 1B2 immunoprecipitations was found to be identical to a mouse cyclophilin C associated protein (mCyCAP) subsequently published by others. We show here by several criteria at the protein and DNA level that VAP-1 is distinct from mCyCAP. Moreover, we elucidate the mechanism which results in binding of mCyCAP to mAb 1B2 during antibody synthesis in hybridoma cells and the sequelae of co-precipitation of mCyCAP during the immunoaffinity chromatography. Binding of mCyCAP to a mouse mAb has not been described before and suggests a new function for this molecule in immunoglobulin synthesis and/or secretion. Moreover, these data indicate that the N-terminal peptide of mCyCAP is a molecular mimic of a functionally important epitope of VAP-1.

Epitope mapping of nine monoclonal antibodies against osteocalcin: combinations into two-site assays affect both assay specificity and sample stability.

Nine monoclonal antibodies (Mabs) were raised against human recombinant osteocalcin fusion protein (rGST-hOC) or bovine osteocalcin (bOC) and selected to develop two-site immunoassays for human osteocalcin (hOC). The detection system was based on the time-resolved measurement of the fluorescence of europium chelates conjugated to the tracer Mabs. Based on the ability of the Mabs to recognize different forms of hOC (carboxypeptidase Y-digested, alkylated hOC, thermally decarboxylated hOC, recombinant forms of hOC, and tryptic peptides derived from hOC) and the information obtained from combinations of the Mabs in two-site assays, an epitope map was created. The epitope map was useful in understanding the behavior of the two-site combinations of the Mabs with serum samples. The two-site combinations could be divided into subgroups detecting either full-length hOC or full length+large NH2-terminal fragment as stimulated by the carboxypeptidase Y-digested form of hOC (it lacks four COOH-terminal residues), which with intact specific assays showed cross-reactivities ranging from 7 to 14% when compared with full-length hOC. In addition, differences were observed in the ability of the combinations to detect thermally decarboxylated hOC (lacks gamma-carboxylation at residues 17, 21, and 24) with cross-reactivities ranging from 8 to 85% when compared to gamma-carboxylated hOC. The analysis of human serum samples showed considerable differences in the concentration and stability of serum OC. This was attributed as the varying ability of the Mabs to detect different proteolytic fragments derived from hOC and/or differences in the degree of gamma-carboxylation of hOC. The in vitro generation of the large NH2-terminal fragment during incubation of the serum samples at room temperature (RT) and during prolonged storage at -20 degrees C in an undercooled state was detectable as loss of immunoreactivity (ranging from -42 +/- 17 to -50 +/- 15% in 16 h at RT, n = 3) with Mab combinations detecting only full-length hOC. Two-site combinations detecting full-length+large NH2-terminal fragment showed no loss of immunoreactivity after incubation of the serum samples at RT for 16 h. With all assays, an increase of serum OC ranging from 16 to 38% was found in postmenopausal samples (n = 24) when compared with premenopausal samples (n = 17), but the degree of statistical significance varied from not significant to p < 0.01.

Endothelial Tie growth factor receptor provides antigenic marker for assessment of breast cancer angiogenesis.

Breast cancer prognosis has previously been linked to the degree of tumour vascularisation. In order to establish additional markers for tumour angiogenesis, we have used monoclonal antibodies against the endothelial Tie receptor tyrosine kinase to study the degree of vascularisation of breast carcinomas and the regulation of Tie expression in the vascular endothelial cells. Antibodies were used for Tie detection and the results were correlated with other prognostic markers. Of four monoclonal antibodies directed against different epitopes of the Tie extracellular domain, two reacted against Tie in unfixed histopathological sections of breast carcinomas. One of these antibodies (clone 7e8) was specific for the endothelial cells whereas the other (clone 10f11) also reacted with basement membranes and occasional carcinoma cells. When Tie expression was studied with the antibody clone 7e8, all 27 carcinomas, two in situ carcinomas, samples of histologically normal breast tissue (n = 16) or normal skin or lymph node tissue (n = 5) showed staining. Microvessel counts were higher in carcinomas (median 14; range 3-27) than in fibrodenomas (median 10; range 5-18) or histologically normal breast tissue (median 7; range 3-15, P = 0.0006). A similar result was obtained using antibodies against the CD31 (PECAM) antigen. Microvessel counts in 7e8 staining were not significantly associated with primary tumour size, axillary nodal status, histological grade or staining for oestrogen receptor, progesterone receptor, Ki-67 proliferation marker or p53 oncoprotein.

The Tie receptor tyrosine kinase is expressed by human hematopoietic progenitor cells and by a subset of megakaryocytic cells.

Growth factor receptors in human hematopoietic progenitor cells have become the focus of intense interest, because they may provide tools for the monitoring, enrichment, and expansion of stem cells. We have shown earlier that the Tie receptor tyrosine kinase is expressed in erythroid and megakaryoblastic human leukemia cell lines, in the blood islands of the yolk sac, and in endothelial cells starting from day 8.0 of mouse development. Here, the expression of Tie was studied in human hematopoietic cells of various sources. Peripheral blood mononuclear cells were Tie-. However, a large fraction of CD34+ cells from umbilical cord blood (UCB) and bone marrow (BM) expressed tie protein and mRNA. On average, 64% of the fluorescence-activated cell sorting-gated UCB CD34+ cells including CD38- cells and a fraction of cells expressing low levels of c-Kit were Tie+. Also, 30% to 60% of BM CD34+ cells were Tie+, including most of the BM CD34+CD38-, CD34+Thy-1+, and CD34+HLA-DR- cells. Under culture conditions allowing myeloid, erythroid, and/or megakaryocytic differentiation, purified UCB CD34+ cells lost Tie mRNA and protein expression concomitantly with that of CD34; however, a significant fraction of cells expressed Tie during megakaryocytic differentiation. These data suggest that, in humans, the Tie receptor and presumably its ligand may function at an early stage of hematopoietic cell differentiation.

Free and complexed prostate-specific antigen (PSA): in vitro stability, epitope map, and development of immunofluorometric assays for specific and sensitive detection of free PSA and PSA-alpha 1-antichymotrypsin complex.

Generation of 15 monoclonal antibodies (MAbs) allowed construction of epitope maps and specific two-site immunofluorometric assays for free prostate-specific antigen (PSA) and PSA complexed with alpha 1-antichymotrypsin (ACT). Close correlation of PSA concentrations obtained with the use of different assays of free PSA suggested extensive similarity in immunodetection of free PSA in serum. Assays of the PSA-ACT complex overestimated the concentration of PSA-ACT in serum because of nonspecific adsorbance of ACT or cathepsin G-complexed ACT to the solid phase. This interference was substantially decreased in the presence of heparin. In studying the stability of purified PSA and PSA-ACT complexes formed in vitro, we found that the free PSA was stable during storage for 4 weeks at 35 degrees C, whereas PSA-ACT complexes largely dissociated in these conditions. The instability of PSA-ACT complexes was counteracted by storage at low temperatures, by adjusting the pH of the storage buffer between 6.8 and 7.4, and through addition of 100-1000-fold molar excess of native ACT. The ease of calibration and the accuracy of free PSA assays in comparison with assays of the PSA-ACT complex suggest that measurements of free to total PSA most accurately reflect the inverse of the proportion of PSA complexed to ACT in serum.

Use of a hollow fiber bioreactor for large-scale production of alpha 2-adrenoceptors in mammalian cells.

Gene cloning has revealed the existence of receptors, which are structurally similar but pharmacologically distinct. One recent example is the alpha 2-adrenergic receptor (alpha 2AR) family with three members. Preparation of membrane-embedded G-protein coupled receptor subtypes in pure form is practically impossible from natural sources and only recombinant techniques have provided possibilities to study these receptors in great detail. In this respect, both yeast and insect cell hosts have been applied successfully but no good mammalian alternative has been described for large-scale production. We describe in this report the use of S115 mouse mammary tumor cells as an effective host for large-scale production of alpha 2-adrenoceptors. These cells can be easily adapted to grow in a hollow fiber bioreactor, with up to 2.8 g of total cellular protein produced in one 0.8 m2 casette. We also show that each recombinant alpha 2-subtype exhibits their expected ligand binding properties, and suggest therefore that this system could be generally applicable to other eukaryotic plasma membrane proteins.

Production of alpha-1-antichymotrypsin by PSA-containing cells of human prostate epithelium.

Prostate-specific antigen (PSA) is a chymotrypsin-like serine protease exclusively produced by the prostate epithelium, and abundant in seminal fluid. In serum, PSA is predominantly complexed to a liver-derived serine protease inhibitor, alpha-1-antichymotrypsin (ACT). A higher proportion of serum PSA is complexed to ACT in prostate cancer than in benign prostate hyperplasia. Since the molecular basis for this is unclear, we have investigated whether or not ACT may be produced in the prostate gland. Immunocytochemistry, using either monoclonal or polyclonal IgGs, demonstrated specific immunostaining for ACT in normal PSA-containing prostate epithelium. Production of ACT in the normal PSA-producing prostate epithelium was demonstrated by means of nonradioactive in situ hybridization using 30-mer anti-sense DNA probes for ACT and for PSA. The ACT and PSA coding transcripts, as detected by in situ hybridization, were distributed perinuclearly, in contrast to the specific immunostaining for ACT and PSA which was most intense in the apical portion of the secretory cells. The results strongly suggest local production and release of ACT by the normal prostate epithelium that may be important for interaction between PSA and ACT in extracellular compartments.

Serum prostate specific antigen complexed to alpha 1-antichymotrypsin as an indicator of prostate cancer.

Prostate specific antigen (PSA) in serum has recently been shown to occur in complex with alpha 1-antichymotrypsin and as an approximately 30 kDa. noncomplexed molecular form. We characterized PSA by 3 different assays in samples from 144 patients with benign prostatic hyperplasia (BPH) and 121 with carcinoma of the prostate. One of these noncompetitive assays measured total PSA by detecting PSA complexed to serine proteinase inhibitors and the noncomplexed molecular form, a second measured only PSA in complex with alpha 1-antichymotrypsin, whereas a third detected the noncomplexed form. PSA in complex with alpha 1-antichymotrypsin was the predominant form in all patient sera. Noncomplexed PSA constituted a minor fraction that was significantly smaller in patients with untreated prostate cancer than in those with BPH (p < 0.0001). The proportion of noncomplexed PSA does not correlate to the serum concentration of PSA or that of alpha 1-antichymotrypsin. In men with a serum PSA concentration of less than 10 micrograms./l. the combination of assays measuring total PSA immunoreactivity, the noncomplexed molecular form and PSA in complex with alpha 1-antichymotrypsin may facilitate discrimination between prostate cancer and BPH.

Prostate-specific antigen in serum occurs predominantly in complex with alpha 1-antichymotrypsin.

Immunologic measurements of the serum concentration of prostate-specific antigen (PSA), an abundant prostatic-secreted serine proteinase, are frequently used to monitor patients with prostate cancer, though it has not been ascertained whether this immunoreactivity represents a PSA zymogen, the active proteinase, or PSA complexed to extracellular proteinase inhibitors. To characterize the PSA immunoreactivity in serum, we used monoclonal antibodies produced against PSA and a polyclonal rabbit IgG against alpha 1-antichymotrypsin in the design of three noncompetitive PSA assays: assay T, which detected PSA both when present as the active proteinase and when complexed to alpha 1-antichymotrypsin; assay F, which recognized the active proteinase but most poorly detected PSA complexed to alpha 1-antichymotrypsin; and assay C, which was specific for PSA complexed to alpha 1-antichymotrypsin. We used the three assays to measure PSA immunoreactivity in 64 patients' sera and in the effluent after gel chromatography of sera from four patients. This identified an 80- to 90-kDa complex between PSA and alpha 1-antichymotrypsin as the predominant fraction of the PSA immunoreactivity in blood plasma; an immunoreactive 25- to 40-kDa compound was the minor fraction.

Serum fructosamine in the assessment of glycaemic control in diabetes mellitus.

Serum fructosamine determination was evaluated in the assessment of glycaemic control in diabetes mellitus. Intra- and inter-assay variation of the method was 0.5-0.8 and 1.5-2.9%, respectively. The fructosamine concentration in serum was found to be stable for at least 10 days independent of prevailing serum glucose concentration is stored at +4 degrees C or colder. Stability of serum fructosamine with respect to rapid fluctuations of blood glucose was of the same order as that of HbA1c. The reference interval (mean +/- 2 SD) for 92 non-diabetic individuals was 1.9-2.7 mmol/l. Good correlation was found between HbA1c and serum fructosamine (r = 0.79). Serum fructosamine and HbA1c correlated well with the mean blood glucose values of the preceding week (r = 0.88 and 0.75, respectively, p less than 0.001). Significant correlations of fructosamine and HbA1c with fasting blood glucose were also found (r = 0.53 and 0.55, respectively, p less than 0.001). Fructosamine determined simultaneously with fasting blood glucose in 65 oral glucose tolerance tests (OGTT) did not separate normal subjects from those with impaired glucose tolerance. Two of the three subjects with diabetic response in the OGTT had, however, elevated fructosamine concentrations. Determination of serum fructosamine is a technically simple, reproducible and moderately inexpensive method for the assessment of glycaemic control in diabetes mellitus. Standardization of the method is, however, not without problems. Uniformity of the calibration and assay protocol is essential for reliable interlaboratory comparison of results. Physiological states altering the rate of synthesis or elimination of serum proteins should be considered in the interpretation of fructosamine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of pH on reactivity of monoclonal antibodies to Chlamydia.

The influence of pH on the reactivity of anti-Chlamydia monoclonal antibodies with genus-, species- and type-specific activity to chlamydial elementary body (EB) antigens was studied by solid-phase enzyme immunoassay. The antibodies bound readily to EB antigen from pH 4 to 9, but had a reduced binding efficiency at more extreme pH conditions. Antibodies although having a wide pH range of optimal binding, bound non-specifically at acidic pH (4-6). This non-specific binding was not seen with polyclonal guinea pig or rabbit anti-Chlamydia antibodies. Thus it seems to be a specific property of mouse immunoglobulins when tested against cell antigen.

Relation between avidity and specificity of monoclonal anti-chlamydial antibodies in culture supernatants and ascitic fluids determined by enzyme immunoassay.

The relation between avidity and specificity of monoclonal antibodies to Chlamydia trachomatis elementary body antigens was studied by enzyme immunoassay. Avidity was estimated by performing the assay in 6 sample dilutions and using a curve-fitting procedure to extrapolate antibody binding in the presence of large antibody excess. Specificity was judged by the difference between end-point titers against homologous and heterologous antigens. By this method antibodies are divided into 2 groups, high specificity (Hs) and low specificity (Ls). Usually, antibodies from ascitic fluids belonged to the Ls group. However, the Ls group also included a few antibodies in supernatants. There was an inverse relationship between avidity and specificity in the Ls group. Immunoglobulin M (IgM) and IgG antibodies of the Hs group had different relative avidities. Avid IgM antibodies from ascitic fluid are not necessarily highly specific.