特异性小鼠血清中瘦素放射免疫分析的建立和应用

目的　建立高特异性和高灵敏度的小鼠血清瘦素 (Leptin)放射免疫分析方法 ,用于测定小鼠血清中瘦素水平。方法　用人工重组的小鼠leptin多次免疫兔子获取高效价、高特异的小鼠leptin抗体。用氯氨T法制备1 2 5I标记的leptin ,经Sep hadexG 2 5纯化。抗原抗体反应采用平衡一步法 ,4℃培养 2 4h后经PR试剂分离结合和游离的标记抗原。该法测定范围为 1.1～ 30 0ng mL ,最低检出值为 0 .5ng mL ,批内和批间误差小于 4 .4 %和 6 .7%。结果　用该法测定正常小鼠 2 3例 ,血清leptin为 (39.6 5± 15 .72 )ng mL ,而人leptin放射免疫法为 (1.5 6± 0 .4 5 )ng mL ,具有显著的差异 ,2组数据有中等程度的相关性 (r=0 .6 2 18)。用该法测定正常人样本 30例 ,血清leptin为 (19.4 6± 7.0 9)ng mL ,而人leptin放射免疫法为 (10 .0 8± 5 .2 0 )ng mL ,具有显著差异 ,但不存在明显的相关性 (r =- 0 .0 95 9)。用该法发现烫伤大鼠血清leptin明显下降 (14 .33± 3.5 2 )ng mL与(6 .71± 3.14 )ng mL ,烫伤大鼠切痂后leptin于 96h内恢复正常水平 (12 .0 8± 4 .0 5 )ng mL。结论　该法稳定、简便、特异、灵敏 ,适于检出小鼠和大鼠血清中的leptin水平 ,而不适合于检测人血清内leptin水平     

Establishment of a sensitive radioimmunoassay for the detection of human IgE-binding factor (soluble CD23).

A monoclonal antibody (mAb) specific to low-affinity receptor for IgE (FceRII/CD23) was established by the fusion of spleen cells of BALB/c mice immunized with the FceRII+ human B lymphoblastoid cell line (RPMI 8866) with mouse myeloma P3U1. Four mAbs, 10/3 (IgG1), 11/4 (IgG1), 12/2 (IgG2b) and 15/6 (IgM), almost completely inhibited the IgE binding to FceRII+ cells but not to FceRII- cells. More directly, they were demonstrated to react only with 43-kD component/FceRII of the cell lysate of RPMI 8866 cells by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. Since they have a different epitope specificity, a solid-phase radioimmunoassay (RIA) for the measurement of IgE-binding factor (IgE-BF) was established. It was found that the RIA with the use of 10/3 and 125I-labeled 11/4 or 12/2 gave good results in the detection of IgE-BF derived from B cells and monocytes as well as of T-cell-derived IgE-BF. More importantly, serum IgE-BF was also quantitatively measured by this RIA. Although increased serum levels of IgE-BF were observed in atopic patients, serum IgE-BF was decreased rather than increased in patients with very high serum IgE. This phenomenon may be explained by the decreased ability of the patients' B cells to spontaneously release IgE-BF in vitro.     

A highly sensitive radioimmunoassay for human growth hormone using a monoclonal antibody

Biozzi-strain mice were immunized with a highly purified preparation of 20K variant of hGH. Spleen-cells were fused with SP2/0Ag14 myeloma cells. Clone productions were screened for specificity toward 20K and 22K hGH and for the affinity constant of antibody-antigen reaction. For the selected monoclonal antibody, Ka was 1.02.10(11) L/M using 22K hGH as both tracer and reference preparation. No cross reactivity was found with PRL and other pituitary hormones; hPL reactivity was 0.002 percent that of hGH. According to these antibody characteristics, a highly sensitive RIA system was developed and used for specific GH measurement in human serum. Using logit-log co-ordinates, the slope of the standard curve was -1.099 and the minimum detected dose was 0.5 uIU/ml. Excellent correlation (r = 0.9575) was found between assay data in this system and those of a conventional RIA method using specific polyclonal rabbit antiserum. The International Reference preparation (66/217) could adequately be used to calibrate the monoclonal antibody system since the in house internal 22K GH standard and international one were equally well recognized by the monoclonal antibody.     

A specific and highly sensitive radioimmunoassay of human brain natriuretic peptide

We established specific and highly sensitive radioimmunoassay (RIA) for the detection of human brain natriuretic peptide (BNP), and confirmed the presence of BNP-like immunoreactivity (BNP-LI) in human circulating blood and the heart. The antibody used in this RIA did not react any known human atrial natriuretic peptide (ANP). Gel permeation chromatography coupled with the RIA revealed that BNP-LI in atrium is composed with high molecular BNP and low molecular BNP. Furthermore, dominant form of BNP-LI in human plasma was thought to be low molecular BNP. Mean concentration of BNP-LI in plasma from normal subjects was 5.9 pg/ml, which was about 5 times lower than that of ANP-LI. Mean concentration of BNP-LI in human cardiac atrium was 45.6 ng/mg protein, which was about 40-fold lower than that of ANP-LI. Furthermore, concentration of BNP-LI in human cardiac ventricle was in a range of 0.1-4.8 ng/mg protein, being about 1/20 that of cardiac atrium. These data suggest that BNP synthesized in human heart and secreted into circulation.     

A sensitive radioimmunoassay for the detection of monoclonal anti-idiotope antibodies

A radioimmunoassay was developed in order to detect anti-idiotypic antibodies in the supernatants of hybrid cells. This assay is both sensitive and specific for anti-idiotypic (but not anti-allotypic) antibodies. Monoclonal antibodies present in test supernatants are bound by an anti-immunoglobulin coated solid phase. Subsequent incubation with a source of mouse immunoglobulin 'blocks' unreacted anti-immunoglobulin antibodies on the solid phase. Anti-idiotypic antibodies are then detected by their ability to bind 125I-labelled idiotype-bearing antibody. This paper describes the use of this assay to detect monoclonal anti-idiotypic antibodies in 2 systems: the cross-reactive idiotype of A/J anti-ABA antibodies, and the idiotype expressed by the myeloma protein HOPC 8. Similarly, 125I-labelled anti-idiotype antibodies may be used in this assay to detect monoclonal idiotype-bearing antibodies. Further modifications are described which would allow the detection of monoclonal anti-allotype antibodies.     

MICA-a highly sensitive and avidin-free immunohistochemical detection system

MICA is an acronym for mirror image complementary antibodies. The MICA system is a novel, avidin-free immunohistochemical detection system that provides a significant increase in sensitivity compared to traditional immunodetection systems. The principle of the system is the alternate and sequential application of mutually attractive polyclonal antibodies to form a large complex of antibodies which can cross-link tissue-bound primary antibodies. Peroxidase conjugated to the constituent antibodies permits signal generation. The formed peroxidase-containing complex is very stable and, by cross-linking tissue-bound primary antibodies, possesses high avidity for the target antigens, thereby producing high immunohistochemical sensitivity.     

Serum leptin levels in different types of hypertension during pregnancy

We determined the serum levels of leptin in 96 pregnant women with body mass index between 20 to 30, 30 normal (NP), 26 with mild preeclampsia (MPE), 27 with severe preeclampsia (SPE), 6 with chronic hypertension plus preeclampsia (CHT+PE) and 7 with chronic hypertension (CHT). A significant (p < 0.01) decrease in leptin levels was observed in the SPE group when compared with the NP group. On the contrary, significant (p < 0.05) increases were observed in the CHT and CHT+PE groups when compared with the NP group. Leptin levels were significantly higher in the MPE (p < 0.001), CHT (p < 0.01) and CHT+PE (p < 0.5) groups when compared with the SPE. No significant differences were observed in the CHT group when compared with CHT+PE. Moreover, a positive correlation was encountered (r = 0.6, p < 0.001) between platelet number and leptin levels for all the patients with preeclampsia. These results suggest that leptin levels may be useful metabolic parameter in different types of hypertension during pregnancy.     

A highly specific and sensitive radioimmunoassay of caerulein, an analogue of cholecystokinin-8

A highly specific and sensitive competitive radioimmunoassay was developed for caerulein (CLN), an analogue of cholecystokinin-8 (CCK-8), in plasma and brain. Antiserum was produced in rabbit by immunization with N delta-[CLN-(1-6)]-ornithine amide conjugated with bovine serum albumin by the glutaraldehyde method. N alpha-[CLN-(1-6)]-lysine amide was labelled with 125I-Bolton & Hunter reagent and used as a labelled antigen after purification by high-performance liquid chromatography. This assay was highly specific for CLN, and cross reactivities for other related peptides, CCK-4, CCK-8, gastrin-I, and gastrin-(14-17), were not observed (less than 0.01%). The limits of determination in biological specimens after CLN administration were 11 pg/ml in human plasma and rat plasma and 80 pg/g in rat brain. This study showed that the slight structure difference between hapten and 125I-labelled antigen is important to the assay performance.     

瘦素水平与妊娠高血压疾病的关系

目的探讨妊娠高血压疾病患者血清瘦素水平的变化及其与妊娠高血压疾病发病的关系。方法采用放射免疫分析法测定了30例妊娠高血压疾病患者产前、产后及脐血清瘦素水平,30例正常妊娠妇女产前、产后及脐血清瘦素水平。结果1.妊娠高血压疾病患者产前血清瘦素水平(16.01±4.01μg/L),高于正常妊娠妇女产前血清瘦素水平(12.27±5.17μg/L)。2.妊娠高血压疾病患者组,妊娠高血压组与轻度子痫前期无差异;重度子痫前期产前血清瘦素水平高于妊娠高血压组、轻度子痫前期。3.妊娠高血压疾病组重度患者产前与产后血清瘦素水平有差异,4.妊娠高血压组及轻度与正常妊娠组无差异,重度患者产后血清瘦素水平仍高于正常妊娠组。结论妊娠高血压疾病患者血清瘦素水平增高,瘦素参与了妊娠高血压疾病的发生和发展。     

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。     

Studies on the gonadal function in patients with anorexia nervosa using a highly sensitive radioimmunoassay kit for estradiol

We measured serum estradiol (E2) using a highly sensitive radioimmunoassay kit in patients with anorexia nervosa (AN). It is possible to determine the ovarian function with hypogonadism in patients with AN whose levels of E2 were below 10 pg/ml. In patients with extremely low levels of BMI (less than 15 kg/m2), basal levels of E2, LH, FSH and IGF-I increased significantly with gain of body weight. Recovery of the hypothalamic-pituitary-ovarian function in AN patients were correlated with weight gain and nutritional status. The levels of IGF-I and E2 raised in advance of gonadotropins. Using highly sensitive assay of E2, we recognized the clinical usefulness for evaluation of the ovarian function in patients with AN in the course of treatment.     

Development of a highly sensitive gold nanoparticle probe-based assay for bluetongue virus detection

A simple gold nanoparticle (GNP) probe based assay (GNPA) that was modified from a bio-barcode assay (BCA) technique, was developed for ultra-sensitive, rapid detection of the bluetongue virus (BTV) VP7 outer-core protein. This assay captures the VP7 target antigen using the GNP probe coated with anti-VP7 polyclonal antibodies and single-stranded signal DNA. Magnetic microparticle (MMP) probes coated with anti-VP7 monoclonal antibodies were then added to form a sandwich immuno-complex. The single-stranded signal DNA coated onto the GNP probe present in the immuno-complex could then be detected by PCR and real-time fluorescence PCR using a TaqMan probe. The assay has a purified VP7 detection limit of 10(-2)fg/ml which is 8 orders of magnitude greater than that of conventional antigen capture ELISAs and 1 order of magnitude more sensitive than that of a conventional BCA. These results indicate that the GNPA is a highly sensitive method for easy detection of BTV proteins and that it can be modified as needed to measure the presence of other proteins.     

Establishment of a highly sensitive enzyme-linked immunosorbent assay for interleukin-1 alpha employing a fluorogenic substrate

We have previously established a non-competitive solid-phase enzyme-linked immunosorbent assay (ELISA) specific for interleukin-1 alpha (IL-1 alpha) using a combination of polyclonal antibody as the immobilized antibody, biotinylated monoclonal antibody as the second antibody and avidin-peroxidase. The level of detection of that ELISA was 200-500 pg/ml. In order to improve its sensitivity, we have used streptavidin-beta-D-galactosidase and the fluorogenic substance 4-methylumbelliferyl-D-galactopyranoside as enzyme substrate. With this system IL-1 alpha could be detected at concentrations as low as 10-50 pg/ml, which was about 10-20 times more sensitive than conventional mouse thymocyte co-stimulator assays. Furthermore, the assay system was specific for IL-1 alpha in that neither IL-1 beta nor interleukin-2 (IL-2) interfered.     

Establishment of a feline T-lymphoblastoid cell line highly sensitive for replication of feline immunodeficiency virus

Summary Interleukin-2 dependent feline T-lymphoblastoid cells designated as MYA-1 cells were established. The cells were free from exogenous retroviruses and sensitive for replication of feline immunodeficiency virus (FIV). FIV can grow more efficiently in MYA-1 cells than in feline primary peripheral blood mononuclear cells. This line of cells will be useful not only for isolation and propagation of FIV, but also for further investigation of properties of FIV.     

Quantitation of estradiol receptors employing a sensitive radioimmunoassay.

A method aimed at the identification and quantitation of estradiol receptors has been developed. The test is based on competitive binding reaction between solubilized cell receptors and anti-estradiol antibodies bound to acrylamide beads, for 3H-estradiol. The technique can be employed both for estradiol radioimmunoassay and quantitation of specific receptors in tissues and appears to be a sensitive, specific and reproducible method which can be applied to the evaluation of any hormone and its receptor.     

Establishment and primary application of a highly-sensitive orexin-A radioimmunoassay

Orexin-A was labeled by 125I using the chloramine-T method, and was purified with a Sephadex G-25 chromatographic column. The reaction between antigen and antibody was carried out by a one-step balance method and was incubated at 4 degrees C for 24 hours, then bonded and free antigen were separated by PR reagent. The detection range of this RIA is 21-2000 pg/mL; the lowest detection level is 21 pg/mL. The intra-assay and inter-assay variations were 7.8% and 9.7%, respectively. Plasma orexin-A levels of 30 normal individuals and 30 patients with hyperlipidemia (serum triglyceride > 1.7 mmol/L and serum total cholesterol > 5.7 mmol/L) were detected by this RIA, while orexin-A levels of plasma and hypothalamus in rat intestinal ischemia-reperfusion injury model were also measured. Plasma orexin-A levels of normal individuals was 338.48 +/- 20.24 pg/mL, while those of patients with hyperlipidemia were 343.51 +/- 15.49 pg/mL; there were no significant differences between these two groups t = -0.1976; P = 0.8441. We also found that orexin-A levels of rat plasma and hypothalamus did not express a significant change during the early stages of intestinal ischemia-reperfusion injury. These results have shown that this orexin-A radioimmunoassay is stable, simple, and specific, being sensitive enough to test orexin-A levels in human plasma, rat plasma, and hypothalamus.     

COLD PCR HRM: a highly sensitive detection method for IDH1 mutations

The p.Arg132His mutation of isocitrate dehydrogenase 1 (IDH1(R132H) ) is a frequent alteration and a major prognostic marker in gliomas. However, direct sequencing of highly contaminated tumor samples may fail to detect this mutation. Our objective was to evaluated the sensitivity of a newly described amplification method, coamplification at lower temperature-PCR (COLD PCR), combined with high-resolution melting (HRM) for the detection of the IDH1(R132H) mutation. To this end, we used serial dilutions of mutant DNA with wild-type DNA. PCR-HRM assay detects IDH1(R132H) at an abundance of 25%, similar to the detection limit of direct Sanger sequencing. Introducing a run of COLD PCR allows the detection of 2% mutant DNA. Using two consecutive runs of COLD PCR, we detected 0.25% mutant DNA in a background of wild-type DNA, that mimics a tumor sample highly contaminated by normal DNA. We then analyzed 10 biopsies of tumor edges, considered free of tumor cells by histological analysis, and showed that immunohistochemistry of IDH1(R132H) was positive in three cases (30%), whereas double COLD PCR HRM was positive in the 10 cases studied (100%). In summary, COLD PCR HRM analysis is 100-fold more sensitive than Sanger sequencing, rendering this rapid and powerful strategy particularly useful for samples highly contaminated with normal tissue.     

A sensitive radioimmunoassay for a component of mouse casein

Mouse casein (m.w. 22,000 daltons) has been purified by employing Sephadex G-100 and DEAE-cellulose column chromatographies. A sensitive radioimmunoassay method has been developed by using [125I]-labelled casein and antiserum elicited in rabbits after injection of glutaraldehyde-treated casein. The assay method is capable of detecting as little as 0.1 ng of casein. The use of the present radioimmunoassay method in detecting casein production in cultured mouse mammary explants has also been demonstrated.