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.

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.

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.

Polystyrene balls as the solid-phase of a double-antibody radioimmunoassay for human serum albumin.

Polystyrene balls have been incorporated as the solid-phase of a model double-antibody radioimmunoassay for human serum albumin. Purified IgG from the secondary antiserum is adsorbed on the 6.4 mm diameter balls. The solid-phase secondary antibody is then used to separate primary antibody bound iodinated antigen from unbound antigen. The secondary antibody coated polystyrene balls are easily prepared and manipulated; several hundred sample dilutions can readily be processed in a single assay. Assay background values of 1.5% or less are consistently obtained without extensive or special washing procedures.

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.

Radioimmunoassay of herpes-simplex and measles virus antibodies in serum and cerebrospinal fluid of patients without infectious or demyelinating diseases of the central nervous system.

A solid-phase radioimmunoassay was used to detect IgG antibodies against herpes-simplex virus antigens (capsid, envelope and excreted) and against measles virus antigen in serum and cerebrospinal fluid (CSF) specimens of 61 patients with no evidence of infectious or demyelinating disease of the central nervous system. Quantitative determinations of IgG and albumin in serum and CSF were also performed. Of the 61 serum and 61 CSF samples tested, 57 and 56 respectively contained antibodies against subunit antigens of herpes simplex virus. Antibody against measles virus was found in 59 serum and 47 CSF specimens. A positive correlation (P less than 0-001) was found between each of the four serum to CSF antibody ratios and the serum to CSF total IgG ratios. This indicated that the distribution of antiviral IgG antibodies in serum and CSF normally follows the distribution of total IgG. The ratios between viral antibody in serum and CSF were also correlated with albumin ratios (P less than 0-05). An inverse relation (P less than 0-001) was found between the age of the patients and their serum to CSF albumin ratios, but not their IgG ratios, suggesting that the albumin ratio is a useful indicator of a blood brain barrier lesion and that the IgG ratio should be used in evaluating disturbed antibody ratios.

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.

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 Chlamydia trachomatis antigen and antiserum: a review.

In this review of the production of Chlamydia trachomatis antigen, a procedure is described in which Chlamydia-infected cells are harvested from culture cells by mechanical disruption 48 to 72 h after infection. Released elementary bodies (EB) are purified by differential centrifugation, followed by centrifugation through Renografin gradient. The purified, formalinized EBs are suitable for use in such serological tests as microimmunofluorescence (MIF) or radioimmunoassay (RIA). The reticulate body (RB) stage of the chlamydiae are harvested from infected cells 27 to 28 h postinfection; when formalinized, these are useful as a serological screening antigen. Chlamydial inclusions in infected cells may be used as a single antigen in screening serology. Type-specific antisera may be produced in mice. Broadly reactive antisera are produced by immunization of guinea pigs or rabbits with multiple C. trachomatis serotypes. Among the potential applications for such hyperimmune sera are the staining of inclusion-containing cells and the direct demonstration of chlamydial antigen by solid-phase RIA.

Detection of Chlamydia trachomatis antigen by radioimmunoassay.

A sensitive indirect radioimmunoassay (RIA) was developed for detection of chlamydial antigens in infected, irradiated McCoy cell culture. Polystyrene beads were used as the solid phase, guinea pig antichlamydial immunoglobulins were used as the captive antibodies, rabbit antichlamydial immunoglobulins were used as the secondary antibodies, and 125I- labelled sheep antirabbit immunoglobulin was used as the indicator antibodies. The immunizations were done intracutaneously with purified genital and lymphogranuloma venereum Chlamydia trachomatis strains grown in the yolk sacs of embryonated eggs. The bound radioactivity was a function of the amount of chlamydial antigen and the method demonstrated the antigen approximately 20 hours post infection. Also noninfectious chlamydial antigen was detectable by RIA. The sensitivity of the assay was about 10 ng/ml for purified antigen and less than 10 inclusions in the cell culture. Each chlamydial serotype could be detected. The RIA was found to be more sensitive than iodine staining and as rapid and sensitive as immunofluorescence method to demonstrate chlamydial infection in cell culture.

Immunochemical analysis of antigenic determinants of Chlamydia trachomatis by monoclonal antibodies.

Monoclonal antibodies to outer membrane of Chlamydia trachomatis lymphogranuloma venereum (LGV) strain L1 (440-L) were used for antigen characterization. Two separate type-specific antigenic determinants (epitopes) and one species-specific epitope were represented in the major outer-membrane protein (MOMP, molecular weight 40 000) of homologous (440-L) and heterologous (IOL-1962, 810-B) L1 strains. One of the type-specific antibodies, the reactivity of which was not affected by oxidation or reduction of the antigenic sites, partially prevented the binding of species-specific antibody as determined by solid-phase radioimmunoassay. The binding of type- and species-specific antibody could be destroyed with proteolytic enzyme treatment. The reactivity of genus-specific monoclonal antibody originating from another fusion (L2, 434-Bu) was unaffected by proteolytic treatment but was sensitive to periodate, indicating the carbohydrate nature of the genus-specific epitope. Heating of antigens had no effect on the affinity of monoclonal antibodies studied.

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)

Determination of human immunoglobulin M rheumatoid factor by a solid-phase radioimmunoassay which uses human immunoglobulin G in antigen-antibody complexes.

A solid-phase radioimmunoassay for the rapid determination of human immunoglobulin M (IgM) rheumatoid factor (RF) has been developed. Preparation of the solid phase for the assay involved the formation of complexes between respiratory syncytial virus-specific human IgG antibodies and virus antigen on the surface of polystyrene balls. Binding of serum RF to IgG in the immune complex was subsequently detected by 125I-labeled mu-chain-specific antibodies to human IgM. The amount of radioactive indicator antibody bound was converted to units of RF by comparison to the standard curve for an RF reference-serum pool. This assay should prove useful in studies of the physiological role of RF, since it can effectively measure low levels of circulating RF.

IgA antibody response in acute rubella determined by solid-phase radioimmunoassay.

A solid-phase radioimmunoassay (RIA) for detecting rubella virus IgA serum antibodies was developed. Purified rubella virus grown in roller cultures of Vero cells was adsorbed onto polystyrene beads. The coated beads were then incubated with dilutions of serum, and rubella IgA antibodies which attached to the virus antigen on the solid-phase were subsequently detected with 125I-labelled anti-human-alpha antibodies. The specificity of the iodinated anti-human immunoglobulins was confirmed by RIA analysis of fractions obtained by chromatography of an early convalescent serum on an agarose column. A complete separation of IgM, IgA, and IgG was observed. A total of 144 serial serum specimens from 31 adult patients with an acute rubella infection were tested for rubella IgA antibodies, and the results were compared with the RIA IgG and IgM titres reported earlier from the same specimens. The RIA IgA response was detected in each of the 31 patients and the IgA antibodies appeared almost simultaneously with the IgG and IgM antibodies. The maximum titres, which were lower than the IgG and IgM titres, were reached in about 1 week after the onset of rash. In 6 patients out of 31 the IgA antibody response was transient and persisted approximately two months, while in the remaining 25 patients the IgA antibodies persisted throughout the study period of more than 5 months. The results obtained indicate that the presence of rubella IgA antibodies in serum is not an indication for a recent rubella infection.

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.