A novel ELISA system for selective measurement of human adiponectin multimers by using proteases

BACKGROUND: Adiponectin, an antiatherogenic adipocyte-derived protein exists in human blood as multiple isoforms--trimeric low molecular weight (LMW), albumin-binding LMW (Alb-LMW), hexameric middle molecular weight (MMW), and high molecular weight (HMW) forms. We developed a novel ELISA system to detect total human adiponectin and the selective level of each adiponectin multimer for investigating the distribution of these levels in human blood. METHODS: Two monoclonal antibodies that were raised against human adiponectin were used to construct a sandwich ELISA to measure adiponectin levels. Adiponectin multimers were selectively measured after sample pretreatment with two proteases that specifically digested the trimeric forms or both the hexameric and trimeric forms. RESULTS: The ELISA had a dynamic range of 0.075-4.8 ng/ml. Intraassay variations (CV) were 5.3% (total adiponectin), 4.1% (MMW+HMW), and 3.3% (HMW). Comparison of the results of ELISA and quantitative western blot analysis of multimeric adiponectin in serum samples revealed good correlation (LMW+Alb-LMW, r=0.873; MMW, r=0.907; HMW, r=0.950). Each of the three forms of adiponectin multimer levels closely correlated with total adiponectin levels in healthy subjects. CONCLUSIONS: This ELISA system can be used to further investigate the physiological roles of human adiponectin multimers.     

Gas chromatographic measurement of myo-inositol in human blood,cerebrospinal fluid and seminal fluid

The concentration of myo-inositol in samples of human blood serum and plasma, cerebrospinal fluid, and seminal fluid, were determined by gas chromatography of the trimethylsilyl derivative.The average inositol level in blood serum was 6.9 μg/ml. Higher values were found in pools of serum obtained from a mixed patient population from a general hospital. High inositol values were found in pooled plasma from uremic patients. Patients with depression did not demonstrate significant differences from normal controls, whether or not they were treated with lithium carbonate.A survey of cerebrospinal fluids from a patient population revealed inositol values from 4–163 μg/ml, with the majority of samples falling between 6 and 35 μg/ml. In a few samples extremely high values were found. Clinical findings and discussion of these cases are included.     

A sensitive assay of transthyretin (prealbumin) in human cerebrospinal fluid in nanogram amounts by ELISA

A sensitive ELISA method for determining transthyretin (prealbumin) in human cerebrospinal fluid (CSF) is described. The method utilizes goat antihuman transthyretin antibody (IgG fraction) for capture and peroxidase conjugated antibody for color development. The assay has a linear range of 1-4 ng transthyretin added per well. The within-day and between-day coefficients of variation are 5.1 and 6.1%, respectively. The concentration of transthyretin in CSF (ranging from 5 to 20 mg per L) correlated significantly with the corresponding serum concentrations (range 170-420 mg/l). This suggests that synthesis of transthyretin in the brain and peripheral tissues is under similar biological control in normal subjects. The transthyretin concentrations in CSF did not correlate with total CSF protein concentration or age of the subject.     

Alpha-fetoprotein in human fetal cerebrospinal fluid

Alpha-fetoprotein (AFP) is a fetal glycoprotein. It has been ascribed a regulatory function of growth factor responses and immune functions. The concentrations of AFP and albumin (ALB) are highly variable in fetal serum and CSF and change with gestational age. The AFP index=[AFP(CSF)/AFP(SERUM)]/[ALB(CSF)/ALB(SERUM)] was determined in six normal fetuses at gestational age 17-23 weeks and found to be independent of gestational age and close to unity, mean 0.90+/-0.11 (S.D.). The ratio of CSF-serum concentrations of AFP and ALB both decreased significantly (p<0.05) with gestational age. The mean fraction of AFP being non-reactive with concanavalin A was 1.7% in serum and 1.9% in CSF, suggesting a common hepatic origin of AFP in both compartments. In conclusion, the concentration of AFP in CSF seems to be determined largely by the serum-CSF concentration gradient in normal fetuses. This finding, combined with the remarkable constancy of the AFP index compared to the highly variable absolute concentrations of AFP in both serum and CSF should make the AFP index the marker of choice when analyzing for intrathecal AFP synthesis during development and in pathological conditions.     

脑脊液蛋白质分析的临床意义

脑脊液充满于脑和脊髓的生理腔隙中 ,可视为中枢神经系统的组织液。随着人们对神经系统疾病的不断发现与认识 ,相应地对这些疾病的有价值的实验室诊断的研究也越来越多。现对近几年来国内外研究较多的脑脊液蛋白质及其临床意义作一简要综述     

Atrial natriuretic peptide in human cerebrospinal fluid

We measured immunoreactive atrial natriuretic peptide (ANP) levels in cerebrospinal fluid (CSF) collected from patients with various neurologic disorders requiring diagnostic lumbar puncture. ANP was present in all of the CSF samples from 45 patients (1.7 +/- 0.6 pmol/L, mean +/- SD). CSF ANP levels were not related to the underlying central nervous diseases of the patients, to the presence or the absence of consciousness disturbance, or to CSF osmolalities in individual patients. In 35 patients, the mean ANP concentration in CSF corresponded to 27% of that in plasma, and there was no significant correlation between ANP concentrations in each paired sample. When ANP in pooled CSF was extracted by anti-ANP-agarose and analyzed by reverse-phase high performance liquid chromatography (HPLC), multiple peaks of ANP were found. One of the major ANP peaks was identified as ANP-(103-126) on the basis of its retention time on HPLC and the specificity of the antiserum used in the radioimmunoassay; however, none of other peaks coeluted with ANP-(99-126), ANP-(101-126), ANP-(102-126), ANP-(103-125), or ANP-(105-126). We conclude from these results that ANP is present in human CSF that is differently processed from ANP in the cardiac atrium.     

Demonstration of fibronectin in human cerebrospinal fluid.

Fibronectin is a glycoprotein found in plasma (cold-insoluble globulin), connective tissues, and cultures of fibroblasts and astroglial cells. This paper describes the identification of fibronectin in human CSF. Fibronectin in CSF was immunologically indistinguishable from the plasma form, as shown by double-diffusion analysis and by radioimmunoassay specific for fibronectin. Fibronectin was isolated from human CSF by affinity chromatography on Sepharose-coupled gelatin and was further analyzed by SDS-polyacrylamide gel electrophoresis. It showed a polypeptide band similar to that of plasma fibronectin. The fibronectin concentration in CSF of 17 neurological outpatients without demonstrable organic lesion in the CNS was 3.0 +/- 1.6 microgram/ml (mean +/- S.D.) which is about 0.6% of total CSF protein. In CSF of 11 MS patients, the concentration was significantly (p less than 0.005) lower (1.6 +/- 0.2 microgram/ml). Of patients with brain tumors, seven had very low levels, three were normal, and two had very high levels. The cause for the low levels in MS and tumor patients is not known.     

检测人中性粒细胞防御素ELISA法的建立及初步应用

目的建立检测人中性粒细胞防御素(HNP)的ELISA法,并探讨其临床应用价值。方法以纯化的HNP-1为抗原制备特异性抗-HNP单克隆抗体,建立ELISA夹心法,同时检测36名健康献血员血浆中HNP含量,并观察血库4℃保存的全血不同保存时间血浆中HNP含量的变化。结果ELISA法批内和批间变异系数分别为4.8%和9.3%,与进口试剂比较相关性良好(r=0.98,P<0.01),36名健康献血员血浆HNP含量为(46±48)ng/m l。库存全血随保存时间延长血浆HNP含量逐渐升高,保存25天的血浆HNP含量达到760 ng/m l。结论建立的ELISA定量检测法与进口试剂盒有良好的相关性,除血浆外,推测也能适用于细胞培养上清或各种体液中HNP的定量测定。     

Creatine kinase isoenzymes in human cerebrospinal fluid and brain

Extracts of normal brains obtained at autopsy and cerebrospinal fluid (CSF) from patients with global brain ischemia were analyzed for creatine kinase (CK; EC 2.7.3.2) isoenzymes. We used both qualitative and quantitative assays (electrophoresis and immunoinhibition). Brain extracts contained CK-BB isoenzyme and mitochondrial CK. In 54 CSF samples free of blood contamination and with total activities ranging from 7 to 2010 U/L (mean 202 U/L), virtually all of the CK activity was due to CK-BB, and none to CK-MM or CK-MB. We conclude that brain contains CK-BB and mitochondrial CK, but lacks CK-MM and CK-MB. After cardiac arrest, CK-BB is released into the CSF. Any CK-MM in the CSF is probably from blood contamination, in which case immunoinhibition with anti-CK-M antibodies accurately quantifies CK-BB.     

ELISA-IgM applied to cerebrospinal fluid in human leptospirosis

Specific IgM antibodies in cerebrospinal fluid (CSF) from patients with leptospirosis were detected by enzyme-linked immunosorbent assay (ELISA). Thirty CSF samples from patients with the icterohaemorrhagic form of the disease (Weil's syndrome) with or without alterations in cell and protein contents were analysed. Thirty CSF samples obtained during anaesthesia from apparently healthy individuals with no clinical or epidemiological history of leptospirosis, six samples from patients with neurosyphilis and 109 samples from patients with meningitis of determined, non-leptospirotic aetiology were used as a control. The test showed 100% sensitivity, specificity and efficacy and showed advantages in terms of yield, rapidity and ease of execution, requiring only a single CSF sample and producing effective results in human leptospirosis.     

3-Methoxy-4-hydroxyphenylglycol in human cerebrospinal fluid

3-Methoxy-4-hydroxyphenylglycol (MHPG) free and sulphate conjugate concentrations were investigated in human lumbar cerebrospinal fluid from a group of 48 subjects of both sexes, using a sensitive and direct reversed-phase liquid chromatographic method with amperometric detection, improved with the adoption of 4-methoxy-3-hydroxyphenylglycol (iso-MHPG) as internal standard. The mean value of free MHPG was 7.30 ± 1.90 μg/1 (SD) and 8.10 ± 2.8 μl/1 in males (28) and females (20) respectively, and the MHPG sulphate conjugate represented about 5% of free metabolite in both sexes. In addition, the correlations between MHPG levels and patients' age and sex were also discussed, as were criteria for subject selection.     

Automated assay of gamma-aminobutyric acid in human cerebrospinal fluid

We describe an automated amino acid analyzer with fluorescence detection (o-phthalaldehyde) which permits sensitive and rapid determinations of gamma aminobutyric acid in human cerebrospinal fluid. Concentrations as low as 50 nmol/liter can be accurately determined in 100 mul samples at the rate of one sample per hour. Concentrations in untreated cerebrospinal fluid increase rapidly after sampling by lumbar puncture. The concentration in immediately deproteinized samples from 38 patients with intervertebral disc disorders was 220 +/- 81 nmol/liter (mean +/- SD).     

Radioreceptor assay of methionine-enkephalin-like substance in human cerebrospinal fluid

A sensitive and simple radioreceptor assay system for measuring methionine(met)-enkephalin-like substance in human cerebrospinal fluid (CSF) was developed using a particulate fraction of rat brain as a receptor preparation and [³H]dihydromorphine as a radiolabeled ligand in the presence of 1 mM ethylenediamine tetraacetate (EDTA) and 2 mM magnesium acetate. Metenkephalin-like substance was purified from CSF by the combination of Sephadex G-10 and SP-Sephadex (H⁺) column chromatographies to be free of sodium and large molecular weight substance such as β-endorphin. The assays were carried out on samples obtained from normal subjects and patients with the disease of the brain or pituitary by lumbar or ventricular puncture. Mean level in samples obtained from normal subjects by lumbar puncture was 2.6 ± 1.0 pmoles/ml.