Validation of an ultrasensitive and specific immunofluorometric assay for mouse follicle-stimulating hormone

Sensitive and specific measurement of FSH is critical to research in reproductive biology, and the increasing availability of transgenic mouse models has created a need for a robust, sensitive, and specific mouse (m) FSH assay. The present study evaluated a time-resolved immunofluorometric assay (IFMA) for mFSH using monoclonal antibody to human (h) FSHbeta as a capture antibody and a biotinylated polyclonal antibody to rat alpha subunit as a detection probe, with signaling amplified by europium-labeled streptavidin. The mFSH IFMA lowered the detection limit 34-fold (5 vs. 170 pg/sample) compared with standard mFSH RIA. The mFSH IFMA demonstrated parallelism of response to dilutions of castrated mouse serum and rat FSH but no cross-reactivity with hFSH and mLH or hLH, whereas the RIA demonstrated nonparallel cross-reactivity with hFSH. The IFMA has a wide analytical range, with a good precision profile for within- and between-assay reproducibility. Because the IFMA is a sandwich-type assay with strict dimer-specificity by design, the lower readings and recovery obtained were compared with the RIA when both assays used a pituitary-purified mFSH assay standard that contained isolated or fragmented subunits as well as intact dimeric FSH. When used with mouse serum sample, the mFSH IFMA demonstrated the expected increases following orchidectomy as well as markedly enhanced sensitivity to very low levels of endogenous mFSH in gonadotropin-deficient mice. Furthermore, the IFMA measured mFSH with fidelity in both intact and orchidectomized male mice without any interference from transgenic hFSH. The greatly enhanced sensitivity, specificity, and technical convenience of this mFSH IFMA will allow wider application of FSH measurements to very small blood samples in immature and mature mice as well as transgenic models.     

The characterization of radioimmunoassay for rat pancreatic polypeptide in serum.

A radioimmunoassay for the measurement of rat pancreatic polypeptide (RPP) in serum or plasma has been developed and characterized using a new guinea-pig anti-rat-PP antibody. The assay provides a high degree of sensitivity and lacks cross-reactivity (CR less than 0.01%) to neuropeptide Y and peptide YY. It also does not interact with PPs of other species or peptide hormones namely, amylin, glucagon, human insulin, human-PP, human-proinsulin, rat C-peptide and rat insulin. The assay employs synthetic rat PP as standards from concentrations of 21-2100 pg/ml (i.e., 5-500 pM) and produces a sensitivity limit of 19 pg/ml (4.5 pM) PP at +/- 3 S.D. The intra- and interassay % coefficient of variations are 6.4% and 5.9%, respectively. The % recovery of RPP added to rat serum samples ranges from 98% to 103%. Assay of serum volumes ranging from 25 microliters to 100 microliters does not significantly alter the expected RPP level. The migration patterns of rat serum PP and that of a synthetic RPP are identical by Sephadex G-50 chromatographic analysis. The mean values of fasting and a 2 h post-feeding plasma RPP levels in normal rats are 40 +/- 2 and 80 +/- 10 pg/ml (9.5 pM and 19.0 pM), respectively. Rat-PP release during insulin induced hypoglycemia in conscious rats rises from 38 +/- 5 pg/ml to 261 +/- 34 pg/ml (9.0 to 62.1 pM, P less than 0.005) by 30 min. Additionally, the antibody used in this study cross-reacts well with mouse-PP as determined by linear serum dilution curves, thus making it useful in the measurement of murine-PP. In conclusion, we have developed and validated a sensitive and specific rat-PP assay. This assay provides a new tool for the reliable measurement of PP in physiologic studies using rat and mouse animal models.     

Development of Electrochemiluminescent Serology Assays to Measure the Humoral Response to Antigens of Respiratory Syncytial Virus

Sensitive and precise serology assays are needed to measure the humoral response to antigens of respiratory syncytial virus (RSV) following natural infection or vaccination. We developed and evaluated a collection of electrochemiluminescent (ECL) serology assays using four RSV antigens (F, N, Ga and Gb). To assess the merits of ECL technology, the four ECL serology assays were evaluated using a well-characterized “gold standard” panel of acute and convalescent serum samples from fifty-nine RSV-positive and thirty RSV-negative elderly subjects (≥65 years old). The combined results from the four ECL assays demonstrated good concordance to the “gold standard” diagnosis, reaching 95% diagnostic sensitivity and 100% diagnostic specificity. Additionally, a combination of ECL assays provided higher diagnostic sensitivity than a commercially available diagnostic ELISA or cell-based microneutralization assay. In summary, these data demonstrate the advantages of using ECL-based serology assays and highlight their use as a sensitive diagnostic approach to detect recent RSV infection in an elderly population.     

Electrochemiluminescence assay for basic carboxypeptidases: inhibition of basic carboxypeptidases and activation of thrombin-activatable fibrinolysis inhibitor

Carboxypeptidases catalyze the removal of the C-terminal amino acid residues in peptides and proteins and exert important biological functions. Assays for carboxypeptidase activity that rely on change of absorbance generally suffer from low sensitivity and are difficult to adapt to high-throughput screening. We have developed a sensitive, robust assay for basic carboxypeptidase activity that makes use of electrochemiluminescent (ECL) detection of reaction product. In this assay, a peptide substrate contains the epitope for antibody (G2-10) binding which is masked by a C-terminal arginine. Carboxypeptidase activity exposes the epitope, allowing the binding of ruthenylated G2-10 which is then detected using ECL. High sensitivity allowed detection limits of 1-2 pM enzyme for carboxypeptidase B and activated thrombin-activatable fibrinolysis inhibitor (TAFIa). The inhibition of several basic carboxypeptidases by commercially available inhibitors was studied. This antibody-based method can be extended to other sensitive detection techniques such as amplified luminescent proximity homogeneous assay. The high sensitivity of the assay allowed the determination of the activatable levels of TAFI in human and other animal plasma in the presence of epsilon -aminocaproic acid, an active-site inhibitor that stabilizes TAFIa. A method to isolate in situ activated TAFIa from human serum in the presence of epsilon -aminocaproic acid was also developed.     

Immunomagnetic-electrochemiluminescent detection of Escherichia coli O157 and Salmonella typhimurium in foods and environmental water samples.

Hemorrhagic Escherichia coli O157:H7 strains and other virulent enteric pathogens can pose a serious health threat in tainted meats, poultry, and even drinking water. Traditional culture-based methods for assay of enteric pathogens in foods and water sources are relatively slow, and results can be ambiguous. Immunomagnetic separation (IMS) and detection methods have been investigated and appear promising for rapid bacterial assay of foods and environmental samples. In this work, a commercial sensor which combines IMS with electrochemiluminescence (ECL) detection is evaluated for detection of E. coli O157 and Salmonella typhimurium in foods and fomites. Results indicate that detection limits are in the range of 100 to 1,000 bacteria per ml in pristine buffer for E. coli O157 and S. typhimurium, respectively, or 1,000 to 2,000 bacteria per ml in food samples (depending on the sample) and that total processing and assay time is rapid (< 1 h) even in food samples. An immunologic "hook" or high-antigen-concentration prozone effect was observed above 10(4) and 10(5) bacteria per ml for E. coli O157 and S. typhimurium, respectively. IMS was accomplished in milk, juices, serum, supernatant fluids from ground beef, finely minced chicken, and fish suspensions as well as several freshwater sources and followed by ECL assay. Some samples, especially fish, gave unexpectedly high background ECL. Conversely, low ECL intensity was observed in nonfat and 2% fat milk samples, which appeared to be related to binding or entrapment of the antibody-coated magnetic beads by particulates in the milk, as revealed by microscopy. Results of this evaluation suggest the feasibility of immunomagnetic-ECL methodology for rapid, sensitive, and facile preliminary screening of various foods and fomites for the presence of virulent enteric pathogens.     

An antigen capture assay for the measurement of serum clusterin concentrations

We have developed and validated a robust antigen capture assay for the measurement of serum clusterin. Increased clusterin expression, and alterations in serum clusterin levels have been associated with a number of disease states. In particular, clusterin has been shown to be associated with tissue regression and apoptosis in the rat ventral prostate in response to androgen ablation or administration of anti-androgens. The object of this study was to determine if changes in human serum clusterin can be used as a diagnostic or prognostic marker to monitor the response to hormonal therapy in patients with prostate cancer, and to determine if clusterin concentrations increase with the progression towards androgen independence. The antigen capture assay was used for an extensive analysis of human serum clusterin concentration in fasting males, and to determine if there is any relationship between clusterin and age or cholesterol levels. The average clusterin level in serum is 101+/-42 microg/ml (n=96). There is no correlation to age or serum cholesterol levels. Analysis of serum clusterin levels in patients with newly diagnosed prostate cancer (n=5), hormone responsive tumors (n=5), and hormone refractory disease (n=5), demonstrates that no significant changes in serum clusterin levels accompany the progression of prostatic disease, or response to hormone therapy.     

A sensitive sandwich enzyme immunoassay for calcitonin gene-related peptide (CGRP): characterization and application.

Thirty mouse monoclonal antibodies (mAbs) directed against rat calcitonin gene-related peptide-alpha (CGRP-alpha) have been obtained. These mAbs are classified in 2 groups, one recognizing the peptide N-terminus and the other binding the C-terminus. A two-site immunometric assay was developed using mAb CGRP-83 as capture antibody, whereas mAb CGRP-72 acts as tracer, covalently labeled with enzyme acetylcholinesterase. This assay appeared sensitive (limit of detection: 2 pg/ml) and precise, allowing quantitative measurement of all human and murine CGRP isoforms. The assay was used to determine specific concentrations of CGRP in different rat, mice and guinea pig samples. The validity of the test was demonstrated by HPLC fractionation experiments.     

Radioimmunoassay of metallothionein in rabbit,rat, mouse,Chinese hamster,and human cells

We describe a competitive, solid-phase radioimmunoassay for metallothionein, which employs a rabbit antiserum directed against rat MT-2 to detect metallothionein (MT) from several different species (rabbit, mouse, rat, Chinese hamster, and human). The lower limit of detection of the assay for rat MT-2 was 0.7 ng; for rabbit MT-2 it was 2 ng. The method is capable of measuring both isoforms of MT (MT-1 and MT-2). When MT levels in rat and mouse tissues were estimated with this RIA and the silver-saturation method, both assays gave the same pattern of MT induction in control and cadmium-treated animals. Both methods measured high levels of MT in human liver samples. Chinese hamster ovary cells induced with cadmium also showed elevated MT expression. The detectability of MTs from a broad range of species is facilitated by the use of solid-phase MT, which has an avidity for the antiserum similar to that of the MT in the tested sample.     

神经生长因子的发光免疫分析法

介绍了一种灵敏的神经生长因子化学发光免疫测定方法。小鼠神经生长因子（ｍＮＧＦ）的抗体ＩｇＧ用亲和层析纯化并用吖啶酯标记。该法可用于大鼠组织中ＮＧＦ含量的测定。方法的灵敏度为１０ｐｇ／ｍＬＮＧＦ;批内批间变异系数分别为８．７％及１３．２％;回收率平均为１０３％。ｍＮＧＦ抗体对人的ＮＧＦ也有极强的交叉反应,故本方法也可能用于病人血清或脑脊液中ＮＧＦ含量的测定。     

Concomitant up-regulation of insulin binding and elevated heat sensitivity in serum depleted CHO cells

When HA-1 CHO cells were cultured in media with a range of fetal calf serum (FCS) concentrations, they became increasingly heat sensitive at low (5%) serum levels. Heat sensitization occurred concomitantly with increased insulin binding capacity. Insulin binding capacity also became more heat sensitive. Thermotolerance induced by a mild treatment (10 min/45 degrees C) 12 hr prior to assay, caused marked heat resistance expressed both as cell survival or insulin binding and abolished differences in sensitivity between the serum adapted cell lines.     

A radioimmunoassay for rat ghrelin: evaluation of method and effects of nonylphenol on ghrelin secretion in force-fed young rats

Antiserum YJC 13-31 against the rat ghrelin conjugated to bovine serum albumin (BSA) was produced in the rabbit and a double antibody radioimmunoassay (RIA) for ghrelin has been developed. Characterization results of this antiserum revealed no cross-reaction with human growth hormone and somatostatin. Weak cross-reactions with insulin (0.1%), rat growth hormone (0.1%) and glucagon (0.3%) were observed, which scarcely interfered the assay system. The sensitivity of this RIA was 5 pg per assay tube. With the rat serum samples, the within-assay precision was 7.1% and the between-assay precision was 12.3%. The RIA was also available to detect the ghrelin in rat tissue extracts with good parallelism to the rat ghrelin standard. In application, the serum ghrelin and corticosterone levels in weaned rats were measured by RIA. Gavage of saline was sufficient to raise serum ghrelin from 2.6 +/- 0.18 to 6.7 +/- 0.7 ng/ml (P < 0.01). Gavage with nonylphenol (NP) suppressed the elevation of serum ghrelin levels in a dose-dependent manner. Besides, gavages of saline elevated the serum levels of corticosterone from 108.8 +/- 13.5 to 188.7 +/- 23.5 ng/ml (P < 0.01) but the elevation effects of corticosterone from gavages were overcome by NP in the low dose of 50 mg/kg. It can be speculated that ingestion of NP is harmful to young animals during growth and environmental adaptation.     

A miniaturized cell-based fluorescence resonance energy transfer assay for insulin-receptor activation

This report describes the development, optimization, and implementation of a miniaturized cell-based assay for the identification of small-molecule insulin mimetics and potentiators. Cell-based assays are attractive formats for compound screening because they present the molecular targets in their cellular environment. A fluorescence resonance energy transfer (FRET) cell-based assay that measures the insulin-dependent colocalization of Akt2 fused with either cyan fluorescent protein or yellow fluorescent protein to the cellular membrane was developed. This ratiometric FRET assay was miniaturized into a robust, yet sensitive 3456-well nanoplate assay with Z' factors of approximately 0.6 despite a very small assay window (less than twofold full activation with insulin). The FRET assay was used for primary screening of a large compound collection for insulin-receptor agonists and potentiators. To prioritize compounds for further development, primary hits were tested in two additional assays, a biochemical time-resolved fluorescence resonance energy transfer assay to measure insulin-receptor phosphorylation and a translocation-based imaging assay. Results from the three assays were combined to yield 11 compounds as potential leads for the development of insulin mimetics or potentiators.     

HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues

We report robust HPLC/UV methods for quantifying retinyl esters (RE), retinol (ROL), and retinal (RAL) applicable to diverse biological samples with lower limits of detection of 0.7, 0.2, and 0.2 pmol, respectively, and linear ranges greater than 3 orders of magnitude. These assays function well with small, complex biological samples (10-20 mg tissue). Coefficients of variation range from 5.9 to 10.0% (intraday) and from 5.9 to 11.0% (interday). Quantification of endogenous RE, ROL, and RAL in mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, skin, brain, and brain regions) reveals utility. Ability to discriminate spatial concentrations of ROL and RE is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum). We also developed a method to distinguish isomeric forms of ROL to investigate precursors of retinoic acid. The ROL isomer assay has limits of detection between 3.5 and 4.5 pmol and has a linear range and coefficient of variation similar to those of the ROL/RE and RAL assays. The assays described here provide for sensitive and rigorous quantification of endogenous RE, ROL, and RAL to elucidate retinoid homeostasis in disease states such as Alzheimer’s disease, type 2 diabetes, obesity, and cancer.     

Anti-bilirubin monoclonal antibody. II. Enzyme-linked immunosorbent assay for bilirubin fractions by combination of two monoclonal antibodies

Among 16 monoclonal antibodies raised against covalently coupled bilirubin-bovine serum albumin, we selected two antibodies: one (designated 24G7) reacted with unconjugated and conjugated bilirubin and the other (designated 25H17) reacted only with unconjugated bilirubin. Combination of these two antibodies enabled us to determine extremely low concentrations of unconjugated and conjugated bilirubin independently by enzyme-linked immunosorbent assay (ELISA). In the assay, samples were incubated with each anti-bilirubin IgG, and then free remaining IgG was allowed to bind to the immunotiter plates coated with bilirubin-bovine serum albumin. The bound fraction of the IgG was visualized with horseradish peroxidase-conjugated rabbit anti-mouse IgG and substrate. Bilirubin concentration was determined from the absorbance at 425 nm. In this system, we could measure 10(-7)-10(-5) M unconjugated and conjugated bilirubin in samples, which is 100-fold more sensitive than Micha?lsson's diazocoupling method. The assay results gave a good correlation coefficient (0.86) compared with those determined with high performance liquid chromatography.     

Histamine monoclonal antibody for brain immunocytochemistry.

Among five monoclonal antibodies (AHA-1 to 5 mAbs) prepared against glutaraldehyde (GA)-conjugated histamine (HA) in our previous study, only mAb AHA-2 was found to detect HA specifically in rat brain neurons by an immunocytochemistry method (ICC) using GA as a tissue fixative. All the other mAbs, except for AHA-5, reacted with HA in the enterochromaffin-like cells (ECL cells) of rat stomach [Fujiwara et al. (1997) Histochem. Cell Biol. 107, 39-45]. Enzyme-linked immunosorbent assay (ELISA) binding and inhibition tests demonstrated that AHA-2 is specific for HA, with almost no detectable cross-reaction with any other established or putative amino acid neurotransmitters, LH-RH, TRH, or peptides with N-terminal histidines. ELISA assays also suggested that the AHA-2 mAb recognizes a HA epitope structure different from the one recognized by the AHA-1 mAb. The immunostaining patterns with AHA-2 mAb, as seen in the five subgroups of the tuberomammillary nuclei in the rat posterior hypothalamus, were very similar to those described by Inagaki et al. [(1988) Brain Res. 439, 402-405; (1990) Exp. Brain Res. 80, 374-380] and Panula et al. [(1984) Proc. Natl. Acad. Sci. USA 81, 2572-2576; (1988) J. Histochem. Cytochem. 36, 259-269] using polyclonal anti-HA serum. However, it was also noted that moderate numbers of immunoreactive nerve fibers projected into the median eminence. The present HA ICC method using AHA-2 mAb allows highly sensitive HA detection in brain, and thus might permit detailed studies of HA localization hitherto impossible using previously available anti-HA polyclonal antibodies produced against carbodiimide-conjugated HA.     

Detection of ciguatoxins: advantages and drawbacks of different biological methods

Ciguatera is a seafood intoxication that results from ingestion of reef fish contaminated with ciguatoxins at levels orally toxic for humans. Precursors of those toxins, gambiertoxins, are produced by benthic dinoflagellates (genus Gambierdiscus), and then accumulated and biotransformed by herbivorous and carnivorous fishes into ciguatoxins, more toxic for humans. In the absence of specific treatment, that disease remains a health problem with otherwise adverse socio-economic impacts. Thus a cost-effective means of detecting ciguatoxins in fish has long been searched for. Many assays have been developed, including in vivo, in vitro, chemical or immunochemical approaches. This review focuses on some biological methods, from the well-standardised mouse assay to the specific radio-labelled ligand binding assay that is performed on rat brain synaptosomes. In addition to the mouse, the chick and the mongoose were still recently used, in particular for preliminary tests before ciguatoxin extraction from fish, since assays in these animals can directly assay the whole flesh. In contrast, various other in vivo methods, such as the kitten, mosquito and diptera larvae assays, were abandoned despite their interesting results. Finally, the mouse neuroblastoma and rat brain synaptosome assays, carried out in vitro as alternative approaches to animal-using assays, are highly sensitive and much more specific than the in vivo methods to detect ciguatoxins.     

Serodiagnosis of microbiosis in mice with a quantitative assay of immunoglobulins by a microcomputer-introduced ELISA system. 1. Anti-Sendai virus antibodies in naturally infected mouse sera

An enzyme-linked immunosorbent assay system combined with microcomputer data analysis was established as a quantitative assay method of immunoglobulins. The assay system was applied to measure IgG and IgM levels of anti-microbe antibodies in animals, especially mouse and rat. And now the measurement of IgG and IgM levels (ng/ml) of anti-Sendai virus (HVJ) antibodies in naturally infected mice is available. The assay system could improve serodiagnosis in the specificity and sensitivity and in the rapid treatment of many serum samples. The operation of this system was performed by a microcomputer, FM 8 connected Titertek Multiskan MC. The limited sensitivity of this assay for IgG and IgM was 10 ng/ml and 30 ng/ml, respectively. Ninety-one of serum samples were positive for IgG and/or IgM (45 samples for IgG and IgM, 44 samples for IgG, 2 samples for IgM) to Sendai virus in the tested 279 mouse sera, and serum titers were ranged from 1: 10 to 1: 12,800 in the IgG, and from 1: 20 to 1: 160 in the IgM. In these titers, serum IgG and IgM amounts were estimated to be 0.1 to 154 micrograms/ml and 0.5 to 4.8 micrograms/ml, respectively. Relationships of serum titers and antibody amounts were almost consisted, being judged like that approximately 10 micrograms/ml is 1: 400, 30 micrograms/ml is 1: 1,600 in IgG, and 2.4 micrograms/ml is 1: 80, 4 micrograms/ml is 1: 160 in IgM.     

Capillary electrophoresis coupled with electrochemiluminescence for determination of atomoxetine hydrochloride and the study on its interactions with three proteins

A simple, rapid and sensitive method for the determination of atomoxetine hydrochloride (AH) by capillary electrophoresis with electrochemiluminescence detection (CE‐ECL) using tris(2,2′‐bipyridyl) ruthenium (II) was developed. Under optimized conditions, the determinations of AH in capsules and rat plasmas and the study on its interactions with three plasma proteins, including bovine serum albumin, cytochrome c and myoglobin were performed successfully. Relative to some previous studies, in this paper the methodologies for the determination of AH in aqueous solution and spiked plasma systems were established, respectively. By comparing the difference between the two work curves of two systems, the matrix effect in plasma samples on the determination of AH by the CE‐ECL method was discussed in detail. The results indicated that the effect of the matrix in plasma samples should not be ignored even if no obvious interference was found in the electropherograms and the establishment of method validation in complex samples by the CE‐ECL method was necessary. Copyright © 2014 John Wiley & Sons, Ltd.