Serum NSE, CEA, CT, CA 15-3 levels in human lung cancer

The significance of neuron specific enolase (NSE) was investigated in comparison with other tumor markers (CEA, CT, CA 15-3) used in the diagnosis and treatment monitoring of lung cancer. As previously described, the calcitonin assay proved to have very low sensitivity for small cell lung cancer (SCLC). The serum NSE assay was, however, shown to be a useful diagnostic aid for discrimination between histologically different lung cancers, and therefore this assay may be a valuable tool for treatment monitoring in SCLC patients. CA 15-3, also an unspecific marker, showed similar sensitivity to the NSE assay in SCLC patients, the sensitivity being higher than CEA in non small cell lung cancer (NSCLC).     

Release of neuron specific enolase (NSE) in cerebrospinal fluid following experimental lesions of the rat brain

Neuron-specific enolase (NSE) levels were measured by sandwich enzymo-immunoassay as well as by enzymatic assay in rat cerebrospinal fluid (CSF), following mechanical lesions of the brain tissue. Significant increases of NSE were observed in CSF, with a peak 2 h following lesions located near the lateral ventricle. Values returned to normal around 48 h later. In another experimental group, lesions were realized further away from the lateral ventricle; the elevation of NSE in CSF reached the maximal value 11 h later. In addition, measurements which were performed following lesions at the same location but of various sizes, indicated that the quantity of NSE released is proportional to the extent of brain damage. The possible factors which govern the time course and amount of NSE release in CSF are discussed. These results suggest that NSE could be a useful and easily detected marker of neuronal damage.     

Comparison of two rapid cortisol radioimmunoassays for use in the fetal sheep.

Cortisol radioimmunoassays (RIA's) utilizing highly specific antisera combined with a simple ethanol protein precipitation procedure (ETOH-PPT) are widely utilized to measure cortisol in human plasma. This same type of RIA has been assumed specific for measurement of cortisol in the plasma of several different species of experimental animals. In order to test this assumption as applied to fetal ovine plasma, we compared an ETOH-PPT cortisol RIA with another rapid cortisol assay which utilizes a dichloromethane extraction (DM-E) step. The DM-E assay in turn was compared with a chromatographic assay previously shown to be highly specific for measurement of fetal plasma cortisol in this species. Fetal ovine plasma cortisol concentrations determined by the DM-E method were nearly identical to the concentrations obtained by the specific chromatographic RIA procedure. On the other hand, the ETOH-PPT RIA grossly overestimated cortisol concentrations when compared with the DM-E RIA. While the rapid DM-E RIA appears to be suitable for use in fetal ovine plasma, the widely used ETOH-PPT RIA yields spuriously high and unpredictable values and must be considered unreliable. These comparisons demonstrate the need for careful reassessment of steroid assays prior to their application in experimental animals even though they have been previously documented as specific in human plasma.     

MEASUREMENT OF NEURON-SPECIFIC (NSE) AND NON-NEURONAL (NNE) ISOENZYMES OF ENOLASE IN RAT, MONKEY AND HUMAN NERVOUS TISSUE

Four double antibody solid-phase radioimmunoassay systems are described for the measurement of neuron-specific enolase (NSE) and non-neuronal enolase (NNE) from rat, monkey and human brain tissue. NSE and NNE are antigenically distinct, making their respective assays specific. The levels of neuronal and non-neuronal enolase (an enolase recently shown to be localized in glial cells) are determined in various regions of rat, monkey and human nervous system. Both neuronal and glial enolases are major proteins of brain tissue with each representing about 1.5% of total brain soluble protein. NSE levels are highest and NNE levels lowest in brain areas having a high proportion of grey matter, such as the cerebral cortex. The reverse is true for areas high in white matter, such as the pyramidal tract and the corpus callosum. Peripheral nervous system levels of NSE are much lower than those of brain with the spinal cord intermediate between the two. Radioimmunological and immunocytochemical data show that neuron-specific enolase is also present in neuroendocrine cells located in non-nervous tissue, which include pinealocytes, parafollicular cells of the thyroid, adrenal medullary chromaffin cells, glandular cells of the pituitary and Islet of Langerhans cells in the pancreas. Unlike neurons, these cells also contain non-neuronal enolase in high amounts.     

A radioimmunoassay for human epidermal growth factor receptor

The development of a radioimmunoassay (RIA) for the human epidermal growth factor receptor solubilized with nonionic detergents which employs iodinated epidermal growth factor (125I-EGF) as the specific ligand is described. A monoclonal antibody (R1) that binds specifically to human EGF receptors [Waterfield, M. D., et al. (1982) J. Cell Biochem. 20, 149-161] was used to separate solubilized receptors saturated with 125I-EGF from free ligand by absorption to protein A-Sepharose, and the bound radioactivity was determined. The RIA was linear when increasing amounts of solubilized membrane protein were added and, when compared to the standard polyethylene glycol assay, was more reproducible. In addition, the background nonspecific binding obtained in the presence of a hundred-fold excess of unlabeled EGF was less in the RIA. Substitution of normal mouse serum for the monoclonal antibody gave very low nonspecific background ligand binding and avoided the use of large amounts of unlabeled EGF in the assay. Two major classes of binding sites for EGF were observed in membrane preparations from the cervical carcinoma cell line A431 or from normal human placental tissue. These were present in approximately equal amounts, with apparent dissociation constants of 4 X 10(-10) and 4 X 10(-9) M. Upon solubilization with the nonionic detergent Triton X-100, only one class of EGF binding sites was detected in both cases, with a dissociation constant of 3 X 10(-8) M. The RIA can be used to monitor receptor purification and for quantitation of receptor number and affinity in various cell types.     

Increased Nervous System-Specific Enolases in Rat Plasma and Cerebrospinal Fluid in Bilirubin Encephalopathy Detected by an Enzyme Immunoassay

Abstract: Three forms of enolase isozymes (αα, αγ, and γγ), including nervous system-specific forms, were measured in the cerebrospinal fluid and the blood plasma of jaundiced or nonjaundiced infant rats by means of enzyme immunoassay systems capable of detecting each form of enolase at the 1 amol (10⁻¹⁸ mol) level. Average enolase levels in cerebrospinal fluid in normal rat were 2.0, 0.2 and 0.1 pmol/ml for αα, αγ, and γγ forms, respectively. Levels of αγ and γγ forms (nervous system-specific enolases; NSE) in jaundiced rats, which suffer Purkinje cell degeneration due to the inborn hyperbilirubinemia, were three to four times as high as the normal values. When kernicterus was induced in jaundiced rats by an injection of bucolome, the NSE level in cerebrospinal fluid was elevated up to more than 30-fold the control, together with a significantly higher level of αγ form in blood plasma. These results suggest that assays of NSE in the cerebrospinal fluid or the blood plasma are helpful in detecting neuronal damage in the central nervous system.     

A dual radioimmunoassay and cytosol receptor binding assay for the measurement of estrogenic compounds applied to urine,fecal and plasma samples

A technique for co-chromatography using tritiated steroids, radioimmunoassay (RIA), and a cytosol receptor binding assay was developed to examine estrogen components following elution from a reverse phase high pressure liquid chromatography (HPLC) system. Urine, fecal or plasma samples from several species of animals were assayed by this method and the results demonstrated compounds previously unreported. Two of these, one labelled E_x in primates, and one labelled E_w in birds are shown to have both cytosol receptor binding activity as well as immunoreactivity.     

Characteristics of antibodies against 17 beta-oestradiol in homologous and heterologous systems of radioimmunoassay

Directly iodinated oestradiol-2(4)-iodo-[125I] and oestradiol-6-O-(CMO)-[125I]iodohistamine were prepared and used in conjunction with anti-oestradiol-2(4)-azo and anti-oestradiol-6-O-(CMO) sera for the development of various radioimmunoassay (RIA) systems which showed marked differences in sensitivity and relatively small differences in specificity. Whereas the heterologous combination of oestradiol-6-O-(CMO)-[125I]iodohistamine and anti-oestradiol-2(4)-azo-serum showed a sensitivity expressed in femtograms, the homologous combination using oestradiol-6-O-(CMO)-[125I]iodohistamine radioligand exhibited a sensitivity two orders lower. The specificity of both the heterologous and homologous system did not differ significantly from the RIA system using tritiated radioligand. The combination of directly iodinated oestradiol and anti-oestradiol-2(4)-azo-serum showed a higher sensitivity and a lower specificity as compared with tritiated radioligand.     

Comparison of two radiolabeled quinuclidinyl benzilate ligands for the characterization of the human peripheral lung muscarinic receptor

Quinuclidinyl benzilate, a muscarinic antagonist, has previously been used in its tritiated form ([3H]-QNB) to study the lung muscarinic receptor. We investigated whether a newer iodinated form of QNB ([125I]-QNB) of higher specific activity would be an appropriate ligand to study the human peripheral lung muscarinic receptor. Both the tritiated and iodinated ligands bound specifically to human lung at 23 degrees C. At 37 degrees C the specific binding of [3H]-QNB increased slightly, but no specific binding of [125I]-QNB was found. The data from multiple equilibrium binding experiments covering a wide range of radiolabeled QNB concentrations were combined and analyzed using the computer modeling program, LIGAND. The tritiated QNB identified a single affinity human lung binding site with a Kd of 46 +/- 9 pM and a receptor concentration of 34 +/- 3 fmol/mg protein. The iodinated QNB identified a single higher affinity human lung binding site (Kd = 0.27 +/- 0.32 pM) of much smaller quantity (0.62 +/- 0.06 fmol/mg protein). Competition studies comparing the binding of unlabeled QNB relative to labeled QNB indicated that unlabeled QNB had the same Kd as that measured for [3H]-QNB, but a 5 log greater Kd than that measured for [125I]-QNB. Other muscarinic receptor agonists and antagonists competed with [3H]-QNB, but not [125I]-QNB for binding to muscarinic receptors with the expected magnitude and rank order of potency. We conclude that of the 2 radiolabeled forms of QNB available, only the tritiated form should be used to study the human peripheral lung muscarinic receptor.     

人脑神经元特异性烯醇化酶的纯化方法

采用改良的Grace层析方法,经一次DEAE-Sephadex A50柱层析即从人脑中纯化了神经元特异性烯醇化酶,比活力为92.1U/mg,纯化倍数为59.4.该酶纯化后,经SDS-聚丙烯酰胺凝胶电泳鉴定为单一蛋白质谱带.此外,还测定了其部分理化性质,其亚单位分子量为45000,等电点pI为4.7,氨基酸组成分析表明其为一种酸性蛋白质;对2-磷酸甘油酸的K_m值为5.6×10^-4mol/L.     

alpha-Neo-endorphin: receptor binding properties of the tritiated ligand

Tritiated porcine alpha-neo-endorphin has been prepared from its corresponding iodinated analog. The iodinated analog (diiodotyrosine at position 1) was synthesized, along with its non-iodinated counterpart, by the solid-phase method. Catalytic exchange of this iodinated analog in the presence of tritium yielded tritiated porcine alpha-neo-endorphin having a specific activity of 45.5 Ci/mmole. Both the native, iodinated and tritiated alpha-neo-endorphin analogs were shown to be homogenous by chromatography on carboxymethylcellulose, paper chromatography, paper electrophoresis, high performance liquid chromatography and amino acid analysis. For the first time binding of alpha-neo-endorphin to rat membrane preparations is described using [3H2-Tyr1]alpha-neo-endorphin as the ligand. The binding is time-dependent and saturable with respect to alpha-neo-endorphin. Scatchard analysis was bi-phasic with KDs of 0.20 and 3.75 nM. Displacement binding studies indicate that the receptor for alpha-neo-endorphin has "kappa" and possibly "epsilon" binding characteristics.     

Discrimination of a colony stimulating factor subclass by a specific receptor on a macrophage cell line

Utilizing the high affinity interactions between pure ¹²⁵I-L cell colony stimulating factor and its receptor(s) on the murine macrophage cell line J774, a murine radioreceptor assay (RRA) has been developed. The murine RRA selectively detects a colony stimulating factor (CSF) subclass (CSF-1) previously defined by murine radioimmunoassay (RIA) (E.R. Stanley, Proc. Nat. Acad. Sci., USA, 76:2969–2973 ('79)). CSF-1 stimulates macrophage production exclusively, and the occurrence of the CSF-1 receptor(s) appears to be restricted to cells of the mononuclear phagocytic system (L.J. Guilbert and E.R. Stanley, J. Cell Biol. 85:153–160 ('80)). The murine CSF-1 RRA failed to detect a variety of other CSF subclasses, growth factors, and hormones. In contrast to data obtained with the murine CSF-1 RIA, human CSF-1 (e.g., human urinary CSF) is detected by the mouse CSF-1 RRA almost as sensitively as murine CSF-1. In addition, there was an absolute correlation between CSF-1 levels determined by murine CSF-1 RRA and those determined by a human CSF-1 RIA for a variety of human CSF-1 sources. The murine CSF-1 RRA is a sensitive (sensitivity 5 units or 1.0 femtomole of CSF-1 protein), rapid, and highly specific assay for CSF-1 in both murine and human sources.     

Experimental Brain Ischemia: Neuron-Specific Enolase Level in Cerebrospinal Fluid as an Index of Neuronal Damage

Levels of neuron-specific enolase (NSE) were measured in rat CSF following occlusion of the four major arteries to the brain for 10, 20, or 30 min. In the CSF of rats submitted to 30 min of total ischemia, an up to nine-fold increase of NSE level occurred within the first few hours and then slowly diminished. Significant levels were seen for as long as 8 days. Histological observations 3 days after ischemia showed neuronal loss as well as neuronal damage in several forebrain regions such as hippocampus, striatum, and thalamus. Ischemia was followed by transient decreases in exploration behavior and neurological states that were no longer visible 24 h later. After 10 or 20 min ischemia, NSE levels were increased to a lesser degree and fewer damaged neurons were observed. The positive correlation between duration of ischemia and amount of NSE release in CSF indicates that the measurement of NSE in the CSF is a sensitive and reliable index of neuronal lesions.     

Peptidylarginine deiminase modulates the physiological roles of enolase via citrullination: links between altered multifunction of enolase and neurodegenerative diseases

The citrullination of enolase by PAD (peptidylarginine deiminase) has emerged as an important post-translational modification in human disorders; however, the physiological function of citrullination remains unknown. In the present study, we report that citrullination diversely regulates the biological functions of ENO1 (α-enolase) and NSE (neuron-specific enolase). We developed three mouse IgG1 monoclonal antibodies with specificity to the following: (i) citrullination of Arg9 of ENO1 [ENO1Cit9; anti-CE1 (citrullinated enolase 1) antibody]; (ii) citrullination of Arg9 in ENO1 and NSE (ENO1Cit9/NSECit9; anti-CE1/2 antibody); and (iii) citrullination of Arg429 of NSE (NSECit429; anti-CE2 antibody). Regardless of the total protein expression level, the levels of ENO1Cit9 and NSECit429 were elevated, and their immunoreactivities were also increased in cortical neuronal cells or around blood vessels in the frontal cortex of patients with sporadic Creutzfeldt-Jakob disease and Alzheimer's disease compared with controls. In a time- and dose-dependent manner, PAD negatively regulated enolase activity via citrullination, and enolase in diseased patients was more inactive than in controls. Interestingly, the citrullination of enolase effectively promoted its proteolytic degradation by Ca2+-dependent calpain-1, and leupeptin (calpain inhibitor I) abrogated this degradation. Surprisingly, using an affinity assay, the citrullination of enolase enhanced its plasminogen-binding affinity, which was blocked by the lysine analogue ?-aminocaproic acid. These findings suggest that PAD-mediated citrullination regulates the diverse physiological activities of enolase and that CE may be a candidate diagnostic/prognostic factor for degenerative diseases.     

Production of colony stimulating factor in long-term bone marrow cultures

Previous studies have shown no detectable colony-stimulating factor (CSF) in media harvested from long-term bone marrow cultures. In the present experiments supernatants from long-term cultures established in three laboratories were assayed for CSF by colony assay and by radioimmunoassay (RIA). Most samples were devoid of biologic activity but all contained CSF as judged by RIA. Biologic activity was found in the majority of samples after diafiltration to remove low molecular weight inhibitors or 5-fold concentration by ultrafiltration. Samples that remained inactive in the colony assay were subjected to gel filtration on Sephadex G-150 to remove potential high molecular weight inhibitors. Biologic activity remained lower than that by RIA in two of three samples tested. Thus, most long-term cultures appear to contain biologically active CSF but this activity is masked by various types of inhibitors. In addition some media appear to contain material that is only detected by RIA.     

Pituitary autoantibodies in lymphocytic hypophysitis target both gamma- and alpha-Enolase - a link with pregnancy?

The first target autoantigen to have been identified in lymphocytic hypophysitis is a 49 kDa protein, identified as alpha-enolase. Pituitary autoimmunity is strongly associated with pregnancy and we have shown that pituitary autoantibodies from patients with peripartum lymphocytic hypophysitis also recognise enolase in the placenta. Enolase exists in different forms as a number of isoenzymes, which are homo- or heterodimers of three subunits, alpha, beta and gamma. alphaalpha-enolase is ubiquitous, betabeta-enolase is muscle-specific and gammagamma-enolase, which is restricted to neuronal tissue and neuroendocrine cells, is known as neuron-specific enolase (NSE). NSE is expressed in normal human pituitary and pituitary neoplasms. The current study investigated which isoforms of enolase in pituitary and placenta reacted with the sera of patients with lymphocytic hypophysitis. Immunoblotting of two-dimensional gels of human pituitary cytosolic proteins showed that autoantibodies in patient sera react with both an acidic form, and more neutral forms of enolase. Immunoblotting with a monoclonal antibody to NSE confirmed the identity of the acidic enolase isoform as the gammagamma-isoform in both pituitary and placental samples. Gamma-enolase, i.e. NSE, was detected by immunohistochemistry in term placenta in decidua, syncytiotrophoblasts, anchoring villi and terminal villi. Our study is the first to describe the cellular localisation of NSE in normal human placenta, thus establishing a direct link between pituitary and placental autoantigens. This link provides a theoretical basis for the strong prediliction of lymphocytic hypophysitis to occur during or after pregnancy.     

A simple modification of the Salmonella liquid-incubation assay. Increased sensitivity for detecting mutagens in human urine

A simple modification of the Salmonella/microsome liquid-incubation procedure improves the sensitivity of the assay for detecting mutagens in human urine. Extracts from cigarette smokers' urine were used as a model complex mutagenic mixture for validation of the assay. The modification consists of adding increased numbers of bacterial cells (approximately 10(9] in a concentrated suspension to liver homogenate mix and urine extract, all in 0.2-ml volume. After 90 min incubation at 37 degrees C, the mixture is processed according to the standard Ames test protocol. This procedure is 20 times more sensitive than the standard plate-incorporation test and 13 times more sensitive than a previously reported liquid-incubation protocol. The number of spontaneous revertants did not increase under these conditions and, compared to the plate-incorporation test, 10-fold less liver homogenate and 5-fold less enzymatic cofactors were needed per plate. The procedure was approximately 14 times more sensitive in detecting the mutagenic activity of benzo[ a ]pyrene. We also used the modification to determine mutagenic activity in urine from a group of nonsmokers. The method may be generally useful for investigations of mutagenic activity in human urine samples.     

Content of neuron-specific and non-neuron-specific enolase isoenzymes in human brain structures

The distribution of neuron-specific (NSE) and non-neuronal (NNE) isoforms of glycolytic enzyme enolase (EC 4.2.I.II.) in the human brain was studied using immunoenzyme assay. The maximum NSE concentration was measured in the frontal and occipital brain cortex, hippocampus, limbic cortex and hypothalamus (12-14 micrograms/mg of water-soluble protein), the minimum level was observed in the brain stem structures (3-6 micrograms/mg). The maximum NNE content was determined in thalamus (34 micrograms/mg). The data can prove useful for the study of enolase isoform distribution in the brain of neurological and psychiatric patients.     

Immunoradiometric assay of chromogranin A in the diagnosis of small cell lung cancer: comparative evaluation with neuron-specific enolase

The aims of our work were 1) to determine the diagnostic performance of an immunoradiometric assay of chromogranin A (CgA) in small cell lung cancer and 2) to compare its discriminatory power with that of neuron-specific enolase (NSE), the marker currently used for SCLC. We selected 166 cases of small cell (64) and non-small cell (102) lung cancer and 106 cases of non-malignant lung diseases as controls. Both CgA and NSE were assayed by immunoradiometric methods and cutoff values were established on the basis of a pre-fixed specificity of 95% in non-malignant lung diseases. The CgA assay showed better diagnostic sensitivity than NSE in SCLC (61% versus 57%), especially in limited disease, and a low positivity rate in NSCLC with respect to NSE (14% versus 22%). By contrast, NSE reflected disease extent more accurately than CgA (U test: CgA p<0.05, NSE p<0.001). Finally, we found that the CgA assay was not affected by hemolysis whereas NSE serum levels greatly increased in hemolyzed sera. In conclusion, CgA assaying by an IRMA method is a reliable procedure in the diagnosis of SCLC. NSE remains the marker of choice in staging and monitoring of the disease. Further studies are needed to evaluate the prognostic significance of the marker and its role in therapy monitoring and patient follow-up.