On-line immunoaffinity extraction followed by high-performance liquid chromatography and radioimmunoassay for a novel retinobenzoic acid, AM-80, in human plasma

On-line coupled immunoaffinity chromatography and reversed-phase high performance liquid chromatography (on-line IAC-HPLC) with detection by radioimmunoassay (RIA) was developed for 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-carbamoyl] benzoic acid (Am-80) in human plasma. A 0.05-mL sample was directly loaded onto an immunoaffinity pre-column packed with immobilized polyclonal antibodies against Am-80. The immunoaffinity extract was then automatically introduced to reversed-phase     

A solid phase, direct competition, radioimmunoassay for quantitation of secretory IgA in human serum

A solid phase radioimmunoassay is described for measurement of secretory IgA (sIgA) in human serum. The assay is based on direct competition between sIgA in serum and purified labelled milk sIgA for anti-secretory-component antibodies coated on disposable plastic cups. The assay is relatively rapid, reproducible and of adequate sensitivity. A wide range of sIgA concentrations (5–120 μg/ml) can be tested with the same dilution of human serum. The arithmetic mean ± S.D. of the serum sIgA levels of 120 random blood donors (both sexes) was 10.9 ± 4.6 μg/ml. Compared to previously published methods and results of quantitation of sIgA in human sera, the present assay is an improvement which should yield interesting data in human hepatic physiopathology.     

高度敏感与特异的微量固相放射免疫分析法检测人IgE

应用两个针对不同抗原决定簇的抗人IgE单克隆抗体分别作为固相抗体和标记~(125)I,建立了一步法微量固相放射免疫分析系统。该系统灵敏度为12.5-15.0pg/ml,特异性强,与100μg/ml人IgG,IgA,IgM和IgD均无反应;标准曲线的线性范围宽,为25-51200pg/ml。批内变异系数为2.4-17.1%。全部材料均可采用国产品,可用于定量测定人淋巴细胞体外培养上清及外分泌液中的微量IgE。     

Isolation of the human myelin basic protein by immunoaffinity chromatography with a monoclonal antibody

Immunoaffinity chromatography has been developed for the isolation of the human myelin basic protein (MBP). The method is based on the use of a monoclonal antibody which was produced to bovine MBP, cross-reacting with human MBP. The protein isolated from acidic extracts of the brain proteins was shown to be native MBP by its immunochemical reactivity, by its ability to elicit experimental allergic encephalomyelitis and by its mol. wt (18,600 ± 400). It represented a single-band purity after hypersensitive silver staining. The MBP isolated by the method described represents a higher purity than that of the MBP purified by conventional multistep biochemical separation techniques.     

Glutaminase-like immunoreactivity in the lower brainstem and cerebellum of the adult rat

Distribution of putative glutamatergic neurons in the lower brainstem and cerebellum of the rat was examined immunocytochemically by using a monoclonal antibody against phosphate-activated glutaminase, which has been proposed to be a major synthetic enzyme of transmitter glutamate and so may serve as a marker for glutamatergic neurons in the central nervous system. Intensely-immunolabeled neuronal cell bodies were densely distributed in the main precerebellar nuclei sending mossy fibers to the cerebellum; in the pontine nuclei, pontine tegmental reticular nucleus of Bechterew, external cuneate nucleus, and lateral reticular nucleus of the medulla oblongata. Phosphate-activated glutaminase-immunoreactive granular deposits were densely seen in the brachium pontis and restiform body, suggesting the immunolabeling of mossy fibers of passage. In the cerebellum, neuropil within the granule cell layer of the cerebellar cortex displayed intense phosphate-activated glutaminase-immunoreactivity, and that within the deep cerebellar nuclei showed moderate immunoreactivity. These results indicate that many mossy fiber terminals originate from phosphate-activated glutaminase-containing neurons and utilize phosphate-activated glutaminase for the synthesis of transmitter glutamate. Intensely-immunostained neuronal cell bodies were further observed in other regions which have been reported to contain neurons sending mossy fibers to the cerebellum; in the dorsal part of the principal sensory trigeminal nucleus, dorsomedial part of the oral subnucleus of the spinal trigeminal nucleus, interpolar subnucleus of the spinal trigeminal nucleus, paratrigeminal nucleus, supragenual nucleus, regions dorsal to the abducens nucleus and genu of the facial nerve, superior and medial vestibular nuclei, cell groups f, x and y, hypoglossal prepositus nucleus, intercalated nucleus, nucleus of Roller, reticular regions intercalated between the motor trigeminal and principal sensory trigeminal nuclei, linear nucleus, and gigantocellular and paramedian reticular formation. Neuronal cell bodies with intense phosphate-activated glutaminase-immunoreactivity were also found in other brainstem regions, such as the paracochlear glial substance, posterior ventral cochlear nucleus, and cell group e. Although it is still controversial whether all glutamatergic neurons use phosphate-activated glutaminase in a transmitter-related process and whether phosphate-activated glutaminase is involved in other metabolism-related processes, the neurons showing intense phosphate-activated glutaminase-immunoreactivity in the present study were suggested to be putative glutamatergic neurons.     

Immunopurification and insertion into liposomes of native and mutant H-2Kb: quantification by solid phase radioimmunoassay

To study the interaction between T cells and isolated H-2Kb, we developed protocols for the immunopurification of the molecule from monoclonal anti-H-2Kb immunoadsorbent columns and for its insertion in lipid vesicles. Patterns of reactivity of two anti-H-2Kb monoclonal antibodies (mAb) (20-8-4 and Y3) on H-2 recombinant and H-2Kb mutant mice indicated that mAb Y3 reacted with all six mutant forms of H-2Kb tested. Binding competition studies indicated that Y3 and 20-8-4 recognized distinct epitopes of H-2Kb. A solid phase radioimmunoassay was established using these two mAb to monitor H-2Kb activity in detergent containing cell lysates, after immunopurification, and after insertion into liposomes. About 70% of H-2Kb activity could be eluted from anti-H-2Kb-immunoadsorbents in the presence of 3 M NH4SCN and octyl glucoside (pH 7.4). A procedure of liposome formation combining gel dilution and dialysis yielded liposomes bearing H-2Kb molecules which could inhibit antibody plus complement cytolysis and could stimulate in vivo primed T cells to generate cytotoxic T lymphocytes in vitro. The present protocol can be extended to immunopurify and obtain H-2Kbm1 bearing liposomes.     

Detection of enterovirus specific IgG and IgM antibodies in humans by an indirect solid phase radioimmunoassay

The development of a solid phase radioimmunoassay which is able to detect virus-specific IgG and IgM antibodies in serum specimens from patients with enterovirus infections is described. Viral antigen partially purified from infected tissue culture fluid was adsorbed passively to individual polystyrene microtiter wells. Dilutions of sera were incubated on these antigens and bound anti-viral antibodies were monitored by the addition of 125-iodine labeled anti-human-IgG or anti-human-IgM antibody. Specificity of the assay to detect virus-specific IgM antibody was ensured by highly specific anti-IgM antibody which did not cross react with IgG and 2-mercaptoethanol sensitivity of IgM antibody titers. Changes of IgM antibody titers clearly indicated a current infection by that virus strain which was isolated as etiological agent. Advantages and restrictions of the introduced radioimmunoassay are discussed.This study was supported by the Bundesministerium für Forschung und Technologie (ZA/NT/SS 0313/6)     

人肺癌相关单克隆抗体的制备及其抗原的纯化

目的：研制抗人肺癌细胞（A549）单克隆抗体，并对其所识别的抗原进行免疫亲和层析纯化。方法：以人肺癌细胞系A549免疫BALB／c小鼠，常规融合，以间接ELISA筛选，免疫组化研究单克隆抗体的特性，通过免疫亲和层析纯化所识别相关的抗原。结果：成功获得了一株能够稳定分泌抗人肺癌相关抗原单克隆抗体的杂交瘤细胞株289，并提取了其识别的相关抗原。结论：2139单克隆抗体及其所识别抗原有可能进一步用于肺癌的实验室诊断。     

The segmented regional volumes of the cerebrum and cerebellum in boys with Tourette syndrome

Neuropathological deficits are an etiological factor in Tourette syndrome (TS), and implicate a network linking the basal ganglia and the cerebrum, not a particular single brain region. In this study, the volumes of 20 cerebral and cerebellar regions and their symmetries were measured in normal boys and TS boys by brain magnetic resonance imaging. Brain magnetic resonance images were obtained prospectively in 19 boys with TS and 17 age-matched normal control boys. Cerebral and cerebellar regions were segmented to gray and white fractions using algorithm for semi-automated fuzzy tissue segmentation. The frontal, parietal, temporal, and the occipital lobes and the cerebellum were defined using the semiautomated Talairach atlas-based parcellation method. Boys with TS had smaller total brain volumes than control subjects. In the gray matter, although the smaller brain volume was taken into account, TS boys had a smaller right frontal lobe and a larger left frontal lobe and increased normal asymmetry (left>right). In addition, TS boys had more frontal lobe white matter. There were no significant differences in regions of interest of the parietal, temporal, or the occipital lobes or the cerebellum. These findings suggest that boys with TS may have neuropathological abnormalities in the gray and the white matter of the frontal lobe.     

A rapid method for the detection of antibodies to cell surface antigens: a solid phase radioimmunoassay using cell membranes

Cell membranes isolated from murine lymphocytes or ascites tumors bind tightly to the surface of flexible plastic microtiter plates in the absence of additional proteins. This allows the detection of membrane associated molecules by specific antibodies and thus forms the basis for a rapid and sensitive radioimmunoassay for antibodies to membrane-bound components. The assay compares favorably with a variety of methods currently used to detect antibodies to cell surface antigens. The assay detects a variety of well characterized murine cell surface antigens (H-2, I-A, T-200, Thy-1.2, Ig). The level of antibody binding to membranes on plates correlates well with antigen density on intact cells. A modification of the assay involving competition between cross-reacting antibodies allows detection and resolution of closely spaced antigenic determinants.     

A modified, solid phase radioimmunoassay for the differential diagnosis of acute and convalescent phases of hepatitis A infection

The commercial assays for diagnosing the presence of hepatitis A antibodies (HAVAB; Abbott Laboratories, North Chicago, IL) or the presence of IgM class anti-hepatitis A virus antibodies (HAVAB-M; Abbott) do not provide precise information as to the timing of the acute infection. IgM class antibodies are detected as late as six months after the acute infection. In this study the authors describe a modified HAVAB test that inactivates the IgM class antibodies. It thus measures the proportion of IgG antibodies out of the total anti-hepatitis A virus antibodies. In a study of 139 patients with impaired liver function, the available and modified tests showed good agreement except for the convalescent phase of hepatitis A. During serial testing for three months after the acute infection, the commercial tests continuously detected IgM class antibodies. The modified test detected predominantly IgG class antibodies from four weeks on. By six weeks, 85% of the patients had predominantly IgG class antibodies. The modified test thus provides information on the timing of recent hepatitis A infection.     

Smoking during early pregnancy affects the expression pattern of both nicotinic and muscarinic acetylcholine receptors in human first trimester brainstem and cerebellum

Prenatal nicotine exposure is associated with an increased risk of complications during pregnancy and childhood. In this study the expression of nicotinic and muscarinic acetylcholine receptors in first trimester pons, medulla oblongata and cerebellum from abortus (5-12 weeks of gestation) of smoking and nonsmoking women was compared. A significant age-related increase in binding of nicotinic receptor subtype alpha4 was found in both pons and cerebellum only in fetal tissue from non-smoking women, while a similar increase was observed in medulla oblongata from fetuses exposed to smoking. A significant age-related increase in binding of muscarinic receptor subtype m2 was observed in pons from abortus of smoking compared with non-smoking women. The gene expression pattern of both alpha4 and alpha7 nicotinic receptor subunits was changed after smoking in all three regions investigated. Smoking also changed the expression of m1 and 2 muscarinic receptor mRNA in pons, m1 mRNA in cerebellum and the m3 mRNA in medulla oblongata. The findings indicate that early prenatal nicotine exposure affects the normal developmental pattern of the cholinergic system in human fetal brain.     

Dissociation of 'on-line' and 'off-line' visuomotor control of the arm by focal lesions in the cerebellum and brainstem.

Visuomotor control of the arm was assessed in a single case study of a subject with focal lesions in the cerebellum and brainstem. A dissociation between 'on-line' and 'off-line' visuomotor control was revealed: impairments in 'on-line' visuomotor control included inaccuracy of tracking velocity, increase in spatial pointing variability and a delay in simple reaction time; whereas the patient was able to adapt to a gain change in 'off-line' visual feedback during a pointing task, and his adaptation was less affected than that of control subjects by trial-to-trial random fluctuations in 'off-line' visual feedback. We conclude that focal damage in the cerebellar peduncles may be principally responsible for this dissociation.     

Survey of aflatoxin B1 and ochratoxin A in commercial green coffee beans by high‐performance liquid chromatography linked with immunoaffinity chromatography

The natural occurrence of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) has been surveyed in 47 samples of commercial green coffee beans. Samples were collected in Nagoya City, Japan, during 1988–93. Using a combination of immunoaffinity chromatography and high‐performance liquid chromatography analysis, AFB₁ and OTA were quantified with detection limits of 2 ng kg^‐1 and 0.1 μg kg^‐1 respectively. Positive rates and levels of AFB₁and OTA were 32% and 2.0–32.9 ng kg^‐1, and 30% and 0.1–17.4 μg kg^‐1 respectively. The samples contaminated with AFB₁ or OTA were mainly imported from African and Asian countries, and several samples were found to be positive for these two mycotoxins for the first time in Japan, although their levels were low.     

A method for the confirmation and identification of drugs of misuse in urine using solid phase extraction and gas-liquid chromatography with mass spectrometry.

A method is described for the confirmation/identification of a range of commonly misused drugs in urine samples. The method has been used for two years without problems for a range of purposes including hospital/clinic drugs of misuse screening and for toxicology in coroner's cases. Urine samples which have given a positive result on immunochemical screening for a particular drug group or groups (for example, opiates) can be processed with identification of the drugs present using a single procedure. Bond ElutCertify columns are used for the extraction of drugs from the samples followed by propionylation and gas chromatography with mass selective detection.     

Mapping of alpha-neo-endorphin- and neurokinin B-immunoreactivity in the human brainstem

We have studied the distribution of alpha-neo-endorphin- or neurokinin B-immunoreactive fibres and cell bodies in the adult human brainstem with no prior history of neurological or psychiatric disease. A low density of alpha-neo-endorphin-immunoreactive cell bodies was only observed in the medullary central gray matter and in the spinal trigeminal nucleus (gelatinosa part). Alpha-neo-endorphin-immunoreactive fibres were moderately distributed throughout the human brainstem. A high density of alpha-neo-endorphin-immunoreactive fibres was found only in the solitary nucleus (caudal part), in the spinal trigeminal nucleus (caudal part), and in the gelatinosa part of the latter nucleus. Neurokinin B-immunoreactive cell bodies (low density) were found in the periventricular central gray matter, the reticular formation of the pons and in the superior colliculus. The distribution of the neurokinin-immunoreactive fibres was restricted. In general, for both neuropeptides the density of the immunoreactive fibres was low. In the human brainstem, the proenkephalin system was more widely distributed than the prodynorphin system, and the preprotachykinin A system (neurokinin A) was more widely distributed than the preprotachykinin B system (neurokinin B).     

Expression of brain-derived neurotrophic factor and TrkB receptor in the sudden infant death syndrome brainstem

This study compared the expression of BDNF (proBDNF and rhBDNF forms) and its receptor TrkB, in the medulla of sudden infant death syndrome (SIDS) infants and infants who died from known causes (non-SIDS). This study also evaluated these markers in association with SIDS clinical risk factors including, sleep position, cigarette smoke exposure and gender. Brainstem tissue was immunohistochemically stained and quantitative analyses were made for eight nuclei of the caudal and rostral medulla. Compared to non-SIDS, SIDS infants had lower rhBDNF in the caudal nucleus of the solitary tract and higher TrkB in the caudal dorsal motor nucleus of the vagus. Within the SIDS cohort, prone sleep position was associated with lower rhBDNF in the caudal arcuate nucleus, and cigarette smoke exposure was associated with lower rhBDNF and TrkB in the inferior olivary nucleus. Abnormal expression of BDNF and TrkB suggests that neuroprotective functions of the BDNF/TrkB system may be reduced in respiratory-related nuclei of SIDS infants.     

Corticotropic-releasing hormone and serotonin interact in the human brainstem: behavioral implications.

The objective of this human post mortem study was to determine whether neurons which synthesize corticotropic-releasing hormone and serotonin form circuits implicated in the pathophysiology of major depression and suicide. For the first time, a sensitive, dual immunocytochemical procedure was used to identify circuits formed by corticotropic-releasing hormone-synthesizing and serotonergic cell groups. Corticotropic-releasing hormone-immunoreactive varicose fibers and puncta with morphological characteristics of terminals were labeled in the midline raphe, periventricular gray and pontine parabrachial complex, on single-labeled tissues processed immunocytochemically with a rabbit antibody to rat/human corticotropic-releasing hormone. Presumptive synaptic interactions with monoaminergic neurons were demonstrated with dual labeling techniques. Corticotropic-releasing hormone-immunoreactive terminals apposed neuronal somata and primary dendrites of serotonergic neurons in the pontine raphe. Serotonergic neurons were immunolabeled with a mouse antibody to phenylalanine hydroxylase, an enzyme with substantial sequence homology to tryptophan hydroxylase. Interactions in the lateral parabrachial nucleus were suggested by precise overlap of corticotropic-releasing hormone and serotonergic terminal fields. Corticotropic-releasing hormone projections were confirmed to noradrenergic neurons containing neuromelanin in the locus ceruleus. Maps of corticotropic-releasing hormone fiber trajectories suggest that these pathways may derive from the forebrain and, locally, from the human homologue of Barrington's nucleus--a neurochemically specialized division of the laterodorsal tegmental complex. Chemosensory functions were predicted by novel evidence for corticotropic-releasing hormone- and monoaminergic neurovascular and subependymal fiber plexuses. In conclusion, corticotropic-releasing hormone may influence the activity of two major monoaminergic cell systems implicated in the stress-diathesis model of mental illness, through neural and humoral mechanisms.