Development of EIA systems for active-form MAP kinase.

Quantitative sandwich enzyme immunoassay (EIA) systems, that can distinguish between active-form subtypes of mitogen-activated protein kinases (p44 and p42 MAP kinase, also called ERK1 and ERK2), were developed employing subtype-specific antibodies as a solid phase and an antibody specific for the phosphorylated region of MAP kinases as the detector. Using these systems, we investigated the dynamic changes in the activity of ERK1 and ERK2 in platelet-derived growth factor (PDGF)-treated rat mesangial cells and nerve growth factor (NGF)-treated PC12. Both ERK1 and ERK2 were activated immediately after stimulation, and the activity reached a maximum at 5-10 min. The total activity of both subtypes correlated well with that obtained using the conventional method. Compared with the usual methods, these systems should have a higher specificity and be more convenient and suitable for experiments with multiple samples. Moreover, as these EIA systems can be applied not only to rat MAP kinases but also to human, mouse and rabbit MAP kinases, they are potentially very useful for a range of investigations.

Analysis of reactive oxygen species generated by neutrophils using a chemiluminescence probe L-012.

Reactive oxygen species (ROS) play important roles in the defense mechanism against infection and in the pathogenesis of various diseases. Although chemical properties of ROS generated by leukocytes have been studied extensively, methods available for their analysis are not sufficiently sensitive. We found that 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione (L-012) reacted with various types of ROS generated by activated neutrophils in human blood and oral cavity, and from peritoneal cavity of the rat, and developed strong chemiluminescence (CHL). Under physiological conditions, opsonized zymosan-dependent CHL intensity of L-012 in human blood and rat peritoneal neutrophils was about 100 and 10 times higher than that of luminol and luciferin analog MCLA, respectively. Phorbol ester-activated CHL of oral neutrophils was also higher with L-012 than that with luminol and MCLA. The presence of either superoxide dismutase, catalase, uric acid, deferoxamine, or azide decreased CHL intensity of L-012 by 52, 57, 57, 63, and 91%, respectively. Kinetic analysis revealed that L-012 developed CHL predominantly by reacting with hydroxyl radical and hypochlorite. Thus, highly sensitive L-012 permits studies on ROS generation by complex biological systems, such as leukocytes, and on the role of ROS in the pathogenesis of various diseases.

beta-Amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity.

beta-Amyloid protein (A beta) is the major component of the senile plaques in Alzheimer's disease (AD), and microglial cells have been shown to be closely associated with these plaques. However, the roles of A beta and microglial cells in pathogenesis of AD remain unclear. Incubation of rat microglial cells with A beta(1-40) caused a significant increase in nitrite, a stable metabolite of nitric oxide (NO), in culture media, while there was no detectable increase in nitrite in astrocyte-rich glial cells or cortical neurons after incubation with A beta(1-40). Nitrite production by microglial cells was also induced by A beta(1-42), but not A beta(25-35). An inhibitor of NO synthase, NG-monomethyl-L-arginine (NMMA), as well as dexamethasone and actinomycin D, dose-dependently inhibited this nitrite production. Among the various cytokines investigated such as interleukin-1, interleukin-6, tumor necrosis factor-alpha and interferon-gamma (IFN-gamma), only IFN-gamma markedly enhanced A beta-dependent nitrite production. Cultured cortical neurons were injured by microglial cells stimulated with A beta in a dose-dependent manner in the presence of IFN-gamma. Neurotoxicity caused by the A beta plus IFN-gamma-stimulated microglial cells was significantly attenuated by NMMA. Thus, although further investigations into the effect of A beta on human microglial cells are needed, it is likely that A beta-induced NO production by microglial cells is one mechanism of the neuronal death in AD.

Basic fibroblast growth factor as a candidate tumor marker for renal cell carcinoma.

The present investigation was conducted to determine serum levels of basic fibroblast growth factor (FGF) by enzyme immunoassay in patients with various urogenital tumors. Renal cell carcinoma had a higher tendency (28 of 52, 53.8%) toward increased serum levels of basic FGF than any of the other urogenital tumors, and increased serum basic FGF was detected more frequently in patients with advanced renal cell carcinoma. Analysis of histological pattern indicated that renal cell carcinoma with a solid or tubular component is more likely to produce basic FGF. However, no significant difference was seen between the percentage of clear cell type tumor patients with increased serum basic FGF (50.0%) and the percentage of granular cell type tumor patients with increased serum basic FGF (66.7%). Five of 8 patients with renal cell carcinoma who underwent selective renal venous sampling before nephrectomy showed increased serum basic FGF in the renal vein from the affected kidney. After resection of the affected kidney to remove the cancer, serum basic FGF disappeared within 2 weeks. However, residual huge tumor or postoperative disease prolonged the increased levels of basic FGF in 2 patients, indicating that basic FGF is produced from and secreted by tumor tissue itself. These findings suggest that serum basic FGF can be useful in the diagnosis, and in evaluating the prognosis, of patients with renal cell carcinoma.

Immunohistochemical expression of inducible nitric oxide synthase (iNOS) in reversible endotoxic shock studied by a novel monoclonal antibody against rat iNOS.

An antirat monoclonal antibody (mAb) against inducible nitric oxide synthase (iNOS), ANOS11, was used for immunohistochemistry to examine the expression of iNOS in various organs and tissues of adult rats in experimental endotoxic shock induced by lipopolysaccharide (LPS) injection. The phenotype of iNOS-expressed cells was also examined immunohistochemically using various mAbs. In control rats, very few cells were positive for ANOS11 except in the thymus. After intravenous injection of LPS, the number of iNOS-positive cells increased rapidly in almost all organs, except the thymus and brain, peaked 6 h after the injection, and decreased slowly. Of the numerous inflammatory cells that infiltrated the lungs, liver, and spleen after LPS injection, many were positive for ANOS11. Besides inflammatory cells, hepatocytes and endothelial cells of the aorta were also positive for ANOS11 but only around 6 h after injection. The cellular composition of iNOS-positive infiltrated cells changed along with the progression of endotoxic shock. At 4 to 6 h after injection, most iNOS-positive cells were considered polymorphonuclear leukocytes judging by their positive reactivity to OX42 and their nuclear morphology. The population of iNOS-positive macrophages positive for ED1 or ED2 increased with time. After 24 h, many iNOS-positive macrophages were found around the focal necrosis in the liver and spleen. These results indicate that the expression of iNOS in neutrophils, endothelial cells, and hepatocytes precedes that of macrophages in experimental endotoxic shock. The expression of iNOS in various cells and organs is closely associated with the progress and pathological changes of endotoxic shock.

Enhancers of the non-peroxidative metal porphyrin chemiluminescence reaction.

Metal porphyrins catalyse luminol chemiluminescence at pH13 without added peroxide. The effects of 22 different surface active compounds on this reaction were studied using six metal porphyrins and one metal porphyrin conjugate. The most active catalyst was Mn-meso-tetra(4-sulphonatophenyl)porphine. Tween-20 enhanced the activity of this catalyst best at a Tween-20 to luminol ratio of 74:1. However, lauryl sulphate enhanced best at an optimum lauryl sulphate to luminol ratio of over 1000:1 and both detergents enhanced the reaction when present below their critical micelle concentrations. Negatively charged aliphatic compounds such as fatty acids enhanced the reaction but positive-charged aliphatic compounds inhibited it. Small differences in enhancer structure resulted in differing enhancement. For example, linoleic acid enhanced Mn-meso-tetraphenyl porphine more than 10-fold, yet linolenic acid inhibited this catalyst. Conjugation of a metal porphyrin to antibody did not influence its enhancement by detergents. The results indicate that the enhancement mechanism does not require formation of pure detergent micelles but that direct association between enhancer and catalyst may be important.

Monoclonal antibodies against hst-1 gene product.

Four monoclonal antibodies (MAbs) against the hst-1 gene product (hst-1 protein) were obtained by a somatic cell hybridization technique. The recognition sites of these MAbs designated HS-131, HS-210, HS-233 and HS-276 on the hst-1 protein were evaluated by competitive binding assay with synthetic polypeptides. HS-131 MAb and HS-276 MAb recognize the epitope located within the 59-73 and the 197-206 amino acid sequences, respectively. The epitopes recognized by HS-210 and HS-233 MAbs could not be determined, but these MAbs showed neutralizing activity against hst-1 protein. Using HS-131 and HS-233 MAbs, a sensitive sandwich enzyme immunoassay (sandwich EIA) has been developed. The assay sensitivity was 1.2-2.5 pg/well of hst-1 protein. Acidic and basic fibroblast growth factors were not cross-reactive up to a concentration of 1 microgram/ml.

Stabilization of inducible nitric oxide synthase by monoclonal antibodies.

We have produced 14 monoclonal antibodies to inducible nitric oxide synthase purified from rat peritoneal cytotoxic activated macrophages. None of the antibodies showed neutralizing activity, but some of them enhanced the enzyme activity through stabilization of the enzyme.

Establishment of monoclonal antibodies against human nerve growth factor.

We have generated and characterized a monoclonal antibody to human nerve growth factor (hNGF). The monoclonal antibody NGFA-133 neutralizes hNGF activity, as assayed by neurite-outgrowth of nerve cells from chick embryonal dorsal root ganglion. Using this antibody, we have developed a sensitive and specific two-site enzyme immunoassay (EIA) system for hNGF. The assay is based on a sandwiching of the antigen between NGFA-133 coated on a microtiter plate and the same monoclonal antibody (NGFA-133) conjugated with horseradish peroxidase (HRP). The two-site EIA was sensitive enough to detect 920 fg/well of hNGF and did not cross-react either human neurotrophin-3 (hNT-3) or sodium dodecyl sulfate (SDS) denatured hNGF.

Monoclonal antibodies against human neurotrophin-3.

Hybridomas producing monoclonal antibodies (MoAbs) against human neurotrophin-3 (hNT-3) were established using recombinant hNT-3 produced in CHO cells and E. coli as immunogens. Of the five MoAbs obtained, MoAb 3w3 showed the highest antibody titer and also best neutralized NT-3 activity as measured by the survival of chick embryonic day-8 dorsal root ganglia neurons. A sandwich enzyme immunoassay (EIA) for NT-3 was established with solid phase MoAb 3W3 and the Fab' fragment of MoAb 3W3 conjugated to horseradish peroxidase. The detection limit was 2.7 pg/well of NT-3 and no cross-reactivity with nerve growth factor up to 100 ng/well was observed. Using this EIA system we have screened a variety of cell lines for NT-3 production. Among these tested, only human Burkitt's lymphoma Namalwa cells were found to be producing NT-3.

Improved enzyme immunoassay for human basic fibroblast growth factor using a new enhanced chemiluminescence system.

An enhanced chemiluminescence reaction has been incorporated into an enzyme immunoassay (EIA) for human basic fibroblast growth factor (hbFGF). We developed a new luminol derivative, designated L-012 and a new enhancer, 4-(4-hydroxyphenyl)thiazole. Using these compounds, the detection limit of hbFGF was improved to 0.1 pg/assay, which was 10-20 and 2 times better than the o-phenylenediamine colorimetric and luminol chemiluminescence assays, respectively. The average concentration of bFGF in sera from 25 normal volunteers was 5.9 pg/ml. On the other hand, serum bFGF levels were elevated in renal, lung and brain tumor patients. The data presented here indicate that the serum bFGF level could be a useful diagnostic marker for these tumors. Furthermore, these new compounds could easily be applied to any other EIA that uses horse radish peroxidase to improve sensitivity.

A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells.

A sensitive chemiluminescence method for measuring the production of superoxide anion (O2-) by activated EoL-1 cells (human eosinophilic leukemia cell line) is described. Recently, we succeeded in synthesizing a new chemiluminescence probe, 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012). In the presence of L-012, activated EoL-1 cells which produce reactive oxygen species generated a marked chemiluminescence with negligible background. The L-012-dependent chemiluminescence was completely abolished by 100-300 U/ml superoxide dismutase, indicating that the main reactive oxygen species detected in this reaction was O2-. The light intensity and the sensitivity of L-012 to O2- were higher than those of other chemiluminescence probes such as luminol and Cypridina luciferin analog (MCLA). Thus, L-012 would provide an improved chemiluminescence method for measuring O2- from cells.

Establishment of an enzyme immunoassay using monoclonal antibody (HG1-219) and its application for the diagnosis of hepatocellular carcinoma.

A monoclonal antibody (MoAb HG1-219) against a human gastric cancer cell line (HuG-1) and its shedding antigen (HG1-219 Ag) was generated and a solid-phase sandwich enzyme immunoassay (EIA-219) was developed. The mean serum HG1-219 Ag concentration in normal individuals was 30.5 +/- 14.5 U/ml measured by EIA-219. When the mean +3 SD of the antigen concentration in normal individuals was used as a cut-off level, 4.3% (2/47) of patients with chronic hepatitis, 9.1% (4/44) of cirrhotic patients and 37.5% (18/48) of patients with hepatocellular carcinoma (HCC) had HG1-219 Ag above the cut-off value. The positive rates of a-fetoprotein (AFP) (> 400 ng/ml) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) for HCC were 26.7% (12/45) and 33.3% (12/36), respectively. There was no significant correlation between HG1-219 Ag and AFP or PIVKA-II in patients with HCC. The combination assay of EIA-219, AFP and PIVKA-II for HCC gave the positive rate of 75% (27/36). The effect of periodic acid on the HG1-219 Ag and the inhibition of EIA-219 by CA 19-9 suggest that the epitope of HG1-219 Ag is a suger chain similar to CA 19-9.

Interaction between endothelium-derived relaxing factors, S-nitrosothiols, and endothelin-1 on Ca2+ mobilization in rat vascular smooth muscle cells.

S-Nitrosothiols (S-nitrosocysteine, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine), which belong to the group of endothelium-derived relaxing factors (EDRFs), caused decreases of cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMCs). The endothelin-1 (ET-1)-induced sustained increase of [Ca2+]i in rat VSMCs was completely abolished by preaddition of at least an equal molar quantity of S-nitrosocysteine (Cys-SNO). Also exposure of VSMCs to a mixture of Cys-SNO and ET-1 at the same time resulted in the transient increase only. These results suggest that S-nitrosothiols may have no significant effect on ET-1-induced Ca2+ release from intracellular stores via inositol 1,4,5-triphosphate production but do affect Ca2+ influx through Ca2+ channels in the plasma membrane.

Activation of Src-like protein-tyrosine kinase Lyn and its association with phosphatidylinositol 3-kinase upon B-cell antigen receptor-mediated signaling.

Crosslinking of membrane-bound immunoglobulins, which are B-cell antigen receptors, causes proliferation and differentiation of B cells or the inhibition of their growth. The receptor-mediated signaling involves tyrosine phosphorylation of cellular proteins. The Src-like protein-tyrosine kinase Lyn is expressed preferentially in B cells and is an intracytoplasmic constituent of the B-cell antigen receptor complex. Crosslinking of membrane-bound immunoglobulin M with antibody induced rapid increases in the kinase activities of Lyn and Lyn-associated phosphatidylinositol 3-kinase. Crosslinking of B-cell antigen receptor also induced association of Lyn with an 85-kDa noncatalytic subunit of phosphatidylinositol 3-kinase. Thus, Lyn is functionally associated with membrane-bound immunoglobulin M and seems likely to participate in B-cell antigen receptor-mediated signaling.

Increased serum levels of basic fibroblast growth factor in patients with renal cell carcinoma.

The serum level and urinary output of basic and acidic fibroblast growth factors (FGFs) were measured by sandwich enzyme immunoassay (EIA) in patients with renal cell carcinoma. In over fifty percent (16/31) of renal cell carcinoma patients, basic FGF was elevated (greater than 30 pg/ml) in their sera. There is relatively good correlation between serum levels of basic FGF and tumor stage or grade, while urinary daily output of basic FGF did not correlate with increased malignancy. The present results indicate that serum basic FGF level of patients with renal cell carcinoma is a useful diagnostic and prognostic marker for renal cell carcinoma. On the other hand, acidic FGF was not detectable in all sera and urine.

A sensitive enzyme immunoassay for human basic fibroblast growth factor.

A sensitive sandwich enzyme immunoassay for human basic fibroblast growth factor (HbFGF) was developed employing three monoclonal antibodies (MAb3H3, MAb98 and MAb52). The Fab' fragment of MAb3H3 which inhibits HbFGF biological activity was conjugated to horseradish peroxidase. A mixture of MAb52 and MAb98 was used in the solid phase. Neither human acidic fibroblast growth factor, hst-1/KS3 product nor acid denatured HbFGF was cross-reactive in this assay system. The detection limit of this assay system was 1 pg/well. Using this assay, some tumor cell lines were revealed to produce a higher level of bFGF than a normal one. Serum samples from normal volunteers were also assayed, and immuno-reactive HbFGF could be detected in 16 out of 57 samples at range 30 approximately 206 pg/ml.

Establishment of monoclonal antibodies against human acidic fibroblast growth factor.

Four kinds of hybridomas secreting monoclonal antibodies (MAbs) against human acidic fibroblast growth factor (haFGF) were established using recombinant haFGF as an immunogen. The recognition sites of four MAbs designated AF1-52, 81, 114 and 1C10 for the haFGF molecule were examined by binding studies with synthetic polypeptides and with amino-terminal truncated forms of haFGF. These experiments suggested that AF1-52, 114, and 1C10 MAbs recognize epitopes within the 1-5, 44-132 and 6-43 amino acid sequences, respectively. However, the epitope recognized by the AF1-81 MAb could not be determined. The sandwich EIA method constructed with these MAbs was sensitive to 1.5 pg/well of haFGF and had no cross-reactivity with human basic FGF, bovine aFGF or the hst-1 gene product.

Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas.

Two hybridoma systems, mouse.human-human (m.h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m.h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semi-continuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.

Effective production of anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies by serum-free culture of hybridomas.

Two hybridoma systems, mouse·human-human (m·h-h) heterohybridoma and human-human (h-h) hybridoma, have been established, and hybridomas secreting anti-tetanus toxoid and anti-HBsAg human monoclonal antibodies (MoAbs), both having a neutralizing activity have been obtained. Cell-line improvement was shown to be an efficient method for improving the productivity in a cell culture process. Two kinds of serum-free media, GFS (a serum substitute)-containing media and polyethylene glycol (PEG)-containing media, have been established to produce human MoAbs. m·h-h Heterohybridomas could be cultivated for a long period by perfusion culture in an agitation vessel, but h-h hybridomas could not. We found that h-h hybridomas show growth-associated antibody production kinetics and established two kinds of long-term cultivation systems: continuous perfusion culture and semicontinuous immobilized perfusion culture. We also scaled up batch culture and short-term perfusion culture to 200-L and 50-L fermentors, respectively. Processes for large-scale purification from the culture supernatants of both GFS- and PEG-containing serum-free media have also been developed.