Development and characterization of the NanoOrange protein quantitation assay: a fluorescence-based assay of proteins in solution

We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 micrograms/mL protein with a standard fluorometer, offering a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The protein-to-protein variability of the NanoOrange assay was comparable to those of standard assays, including Lowry, bicinchoninic acid, and Bradford procedures. We also found that the NanoOrange assay is useful for detecting relatively small proteins or large peptides, such as aprotinin and insulin. The assay was somewhat sensitive to the presence of several common contaminants found in protein preparations such as salts and detergents; however, it was insensitive to the presence of reducing agents, nucleic acids, and free amino acids. The simple assay protocol is suitable for automation. Samples are briefly heated in the presence of dye in a detergent-containing diluent, allowed to cool to room temperature, and fluorescence is measured using 485-nm excitation and 590-nm emission wavelengths. Therefore, the NanoOrange assay is well suited for use with standard fluorescence microplate readers, fluorometers, and some laser scanners.     

检测HSA—GLP-1融合蛋白双抗体夹心ELISA方法的建立

目的：为检测血清中HSA-GLP-1融合蛋白整体分子的浓度,建立一种特异、灵敏的定量检测食蟹猴体内HSA-GLP-1融合蛋白浓度的双抗体夹心ELISA的方法。方法：采用双抗体夹心ELISA方法,以GLP-1单克隆抗体为包被抗体、HSA-GLP-1融合蛋白为夹心抗原、anti-HSA-Biotin为检测抗体,用Streptavidin-HRP进行免疫放大,TMB显色。结果：建立了检测HSA-GLP-1融合蛋白的ELISA方法,其线性范围为15.6~1000 ng/mL,最低检测限为15.6 ng/mL,与GLP-1、HSA、IL2-HSA均无交叉反应,板内和板间精密度均小于15%,准确度为±15%,冻融稳定性和稀释稳定性良好。结论：建立的HSA-GLP-1蛋白检测方法符合新生物制品临床前药代动力学研究指导原则要求,可用于HSA-GLP-1融合蛋白在临床前药代动力学试验的定量检测。     

Enzyme-linked assay of cellulose-binding domain functions from Cellulomonas fimi on multi-well microtiter plate

Cellulose-binding domain (CBD) enriches cellulolytic enzymes on cellulosic surfaces and contributes to the catalytic efficiency by increasing enzyme-substrate complex formations. Thus, high affinity CBDs are essential for the development of efficient cellulose-degrading enzymes. Here, we present a microtiter plate-based assay system to measure the binding affinity of CBDs to cellulose. The assay uses a periplasmic alkaline phosphatase (AP) as a fusion reporter and its activity is detected using a fluorogenic substrate, 4-methylumbelliferyl phosphate. Lignocellulose discs of 6 mm in diameter were used as substrates in 96-well plate. As a result, the enzyme-linked assay detected the binding of CBDs on the cellulosic discs in a highly sensitive manner, detecting from 0.05 to 1.0 μg/mL of APCBD proteins, which is several hundred times more sensitive than conventional protein measurements. The proposed method was applied to compare the binding affinity of different CBDs from Cellulomonas fimi to lignocellulose discs.     

Establishment of ELISA for murrel vitellogenin and choriogenin, as biomarkers of potential endocrine disruption

Vitellogenin (Vg) and choriogenin (Chg) are sensitive biomarkers for testing endocrine disruption in fish. Therefore, we have developed immunoassays for Vg and Chg in the Indian freshwater murrel, Channa punctatus. Vg is a known precursor of egg-yolk proteins, whereas Chg contributes to the formation of egg-envelope. Vg and Chg were induced in male murrel by administration of estradiol-17beta. Chg had an apparent native molecular mass of 180 kDa. It consisted of a single peptide with a molecular mass of 110 kDa, whereas native Vg protein (530 kDa) contained 175 kDa peptide. Highly specific polyclonal antibodies against purified plasma proteins, Vg and Chg, were employed for developing competitive enzyme linked immunosorbent assays (ELISAs). The sensitivity of Vg assay was 3.9 ng/mL (working range 15-500 ng/mL) and of Chg assay was 1.56 ng/mL (working range 6-200 ng/mL). The inter- and intra-assay variations were well within acceptable limits. The two antisera did not cross-react with male plasma proteins. Antiserum to Vg did not cross-react with Chg. Similarly, antiserum to Chg showed no correlation with Vg. Further, immunofluorescence and Western blotting confirmed the specificity of Vg and Chg antisera.     

Development and application of competitive ELISA assays for rat LH and FSH

Rat LH (rLH) and FSH (rFSH) were measured by sensitive and specific competition ELISAs. The rat LH ELISA used rLH-I-9 coated plates, an antiserum against rLH and an antibody against rabbit IgG labeled with peroxidase. Using rLH-RP-3 as a standard, rat LH was determined by binding of the anti-LH antibody to rLH-I-9 coated plates. The sensitivity of the assay was 0.8 ng/mL. Similarly, the rat FSH-ELISA used rFSH-I-8 coated plates, an antiserum against rFSH and an antibody against rabbit IgG labeled with peroxidase. Using rFSH-RP-3 as a standard, the FSH-ELISA was also determined by binding of the anti-FSH antibody to rFSH-I-8 coated plates. The sensitivity of this assay was 1.25 ng/mL. Both rat LH and FSH ELISA assays are highly specific and provide accurate determination of gonadotrophins in buffers, sera, cell culture media, and anterior pituitary extracts. These assays were used for monitoring the gonadotrophin surge-attenuating factor (GnSAF) and inhibin activities present in human follicular fluid (hFF). The 2 new ELISA procedures have practical advantages (safety, convenience, economy) over the RIA methods, and they perform as well as the RIA techniques at the same range of concentrations.     

A sensitive, robust high-throughput electrochemiluminescence assay for rat insulin

In diabetes research, mouse and rat models are used for in vivo experiments, and quantification of insulin in serum samples under different pathophysiological conditions and after treatment with compounds is essential. There are few commercial radioimmunoassay and enzyme-linked immunosorbent assay (ELISA) assay kits to determine the rat/mouse plasma levels of insulin. However, reliability in insulin measurements using the available biological assays is a great concern. The authors report a robust, extremely sensitive electrochemiluminescence (ECL) insulin assay using the Origen technology platform. The assay performance, as judged by the Z' value of 0.82+/-0.03 and the signal-to-background (S/B) ratio of 133, suggests that this is a robust and reliable assay. The intra-assay and interassay variation is less than 5%. The dynamic range of detection for insulin is 5 pg to 5 ng in the ECL assays. Recovery of insulin was about 100% when different volumes of serum were spiked with exogenous insulin. These results suggest that the ECL insulin assay is an extremely sensitive, robust, nonradioactive homogeneous assay and can be used successfully to determine the insulin levels in rodent serum samples.     

A sensitive and rapid gel retention assay for nuclear factor I and other DNA-binding proteins in crude nuclear extracts.

The paper describes a rapid and sensitive assay for DNA binding proteins which interact with specific and defined binding sites. It exploits the observation that complexes of proteins and small synthetic DNA fragments (40 bp) containing the protein/DNA binding site can enter native polyacrylamide gels and remain stably associated during electrophoresis under non-denaturing conditions. The assay was applied to nuclear factor I, to its identification and purification from porcine liver, to an analysis of its binding site on adenovirus type 5 DNA and to an exploration of other potential binding sites for DNA binding proteins within the inverted terminal repetition of adenovirus DNA. The extreme sensitivity of the assay which surpasses that of conventional footprint assays by at least two orders of magnitude permitted the identification of nuclear factor I-like activities in Saccharomyces cerevisiae.     

检测脂肪酸化胰岛素放射免疫分析方法的建立

目的:建立一种高灵敏度、高特异性、操作简单快捷的放射免疫分析法(RIA),用于检测比格犬血浆中的脂肪酸化胰岛素。方法:所用试剂盒主要以豚抗胰岛素抗体为主要特异性试剂,利用均相竞争抑制原理,采用平衡竞争法对比格犬血浆进行直接测定。在该系统中加入未标记的标准品或样品,则标记抗原和非标记抗原将竞争有限且定量的抗体结合位点。结果:建立了检测脂肪酸化胰岛素的RIA法并进行了确证,方法的线性范围为3.125~800μU/mL,最低检测限为3.125μU/mL,批内和批间精密度分别为4.5%~7.0%和3.9%~7.6%,冻融稳定性和稀释稳定性等良好。结论:方法学确证结果表明,本研究建立的脂肪酸化胰岛素RIA检测方法符合新生物制品临床前药代动力学研究指导原则要求,可用于脂肪酸化胰岛素在临床前药代动力学试验的检测。     

ENSAM: Europium Nanoparticles for Signal enhancement of Antibody Microarrays on nanoporous silicon

To improve the sensitivity of antibody microarray assays, we developed ENSAM (Europium Nanoparticles for Signal enhancement of Antibody Microarrays). ENSAM is based on two nanomaterials. The first is polystyrene nanoparticles incorporated with europium chelate (beta-diketone) and coated with streptavidin. The multiple fluorophores incorporated into each nanoparticle should increase signal obtained from a single binding event. The second nanomaterial is array surfaces of nanoporous silicon, which creates high capacity for antibody adsorption. Two antibody microarray assays were compared: ENSAM and use of streptavidin labeled with a nine-dentate europium chelate. Analyzing biotinylated prostate-specific antigen (PSA) spiked into human female serum, ENSAM yielded a 10-fold signal enhancement compared to the streptavidin-europium chelate. Similarly, we observed around 1 order of magnitude greater sensitivity for the ENSAM assay (limit of detection < or = 0.14 ng/mL, dynamic range > 10(5)) compared to the streptavidin-europium chelate assay (limit of detection < or = 0.7 ng/mL, dynamic range > 10(4)). Analysis of a titration series showed strong linearity of ENSAM ( R2 = 0.99 by linear regression). This work demonstrates the novel utility of nanoparticles with time-resolved fluorescence for signal enhancement of antibody microarrays, requiring as low as 100-200 zmol biotinylated PSA per microarray spot. In addition, proof of principle was shown for analyzing PSA in plasma obtained from patients undergoing clinical PSA-testing.     

Dye-binding protein assay using a long-wave-absorbing cyanine probe

A simple and fast protein assay that involves the binding of water-soluble sulfonate heptamethylene cyanine to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 778 to 904 nm, and the increase in absorption at 904 nm is monitored. This assay is very reproducible, of good color stability for at least 80 min, and sensitive at the 100 ng/mL level of human serum albumin (HSA) when a spectrophotometer with near-infrared wavelength is used to measure absorbance. Few chemicals except ionic surfactants such as cetyltrimethylammonium bromide and sodium dodecyl sulfonate interfere with the assay. Purified proteins have different capacities to interact with the dye; under the experimental conditions, the linear ranges of bovine serum albumin (BSA), HSA and gamma-IgG were 200-2000, 100-2400, and 200-3000 ng/mL, respectively. The relative standard deviation for the five replicate determinations of 1200 ng/mL BSA is 2.1%.     

Fluorescence protection assay: a novel homogeneous assay platform toward development of aptamer sensors for protein detection

Development of novel aptamer sensor strategies for rapid and selective assays of protein biomarkers plays crucial roles in proteomics and clinical diagnostics. Herein, we have developed a novel aptamer sensor strategy for homogeneous detection of protein targets based on fluorescence protection assay. This strategy is based on our reasoning that interaction of aptamer with its protein target may dramatically increase steric hindrance, which protects the fluorophore, fluorescein isothiocyannate (FITC), labeled at the binding pocket from accessing and quenching by the FITC antibody. The aptamer sensor strategy is demonstrated using a model protein target of immunoglobulin E (IgE), a known biomarker associated with atopic allergic diseases. The results reveal that the aptamer sensor shows substantial (>6-fold) fluorescence enhancement in response to the protein target, thereby verifying the mechanism of fluorescence protection. Moreover, the aptamer sensor displays improved specificity to other co-existing proteins and a desirable dynamic range within the IgE concentration from 0.1 to 50 nM with a readily achieved detection limit of 0.1 nM. Because of great robustness, easy operation and scalability for parallel assays, the developed homogeneous fluorescence protection assay strategy might create a new methodology for developing aptamer sensors in sensitive, selective detection of proteins.     

Monitoring protein-small molecule interactions by local pH modulation

We have developed a technique for sensing protein-small molecule and protein-ion interactions in bulk aqueous solution by utilizing a pH sensitive dye, 5-(and-6)-carboxyfluorescein, conjugated to free lysine residues on the surfaces of designated capture proteins. The fluorescein intensity was found to change by about 6% and 15% for small molecule and ion binding, respectively. The assay works by modulating the local electric fields around a pH sensitive dye. This, in turn, alters the dye's apparent pK(A) value. Such changes may result directly from the charge on the analyte, occur through allosteric effects related to the binding process, or result from a combination of both. The assay was used to follow the binding of Ca(2+) to calmodulin (CaM) and thiamine monophosphate (ThMP) to thiamine binding protein A (TbpA). The results demonstrate a binding constant of 1.1μM for the Ca(2+)/CaM pair and 3.2nM for ThMP/TbpA pair, which are in excellent agreement with literature values. These assays demonstrate the generality of this method for observing the interactions of small molecules and ions with capture proteins. In fact, the assay should work as a biosensor platform for most proteins containing a specific ligand binding site, which would be useful as a simple and rapid preliminary screen of protein-ligand interactions.     

Determination of myrosinase (thioglucoside glucohydrolase) activity by a spectrophotometric coupled enzyme assay

The hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system was used to assay for plant thioglucoside glucohydrolase (myrosinase, EC 3.2.3.1) by measuring the rate of glucose released during hydrolysis of glucosinolates. This coupled assay was compared with two other assays for myrosinase: a pH-stat assay that measures the rate of acid released during glucosinolate hydrolysis, and a spectrophotometric assay in which the decrease in the absorbance at 227.5 nm is used to measure the disappearance of the substrate, 2-propenylglucosinolate (DSA assay). The coupled and pH-stat assays were found to give comparable activities and were linear with enzyme concentration over the range 0 to 30 micrograms. The DSA assay gave lower myrosinase activity in comparison to the coupled and pH-stat assays. This is due to the lower concentrations of substrate and activator (ascorbate) which must be used in the assay. The DSA assay was found to give a nonlinear relationship with enzyme concentration over the range 2 to 30 micrograms. For these reasons this assay was found to be unsatisfactory. The coupled assay was found to be more sensitive and more widely applicable than the pH-stat assay as a routine continuous assay for myrosinase activity.     

肠出血性大肠杆菌(O157：H7)的特异性荧光探针检测方法的建立

目的:建立一种real-time PCR,快速准确检测肠出血性大肠杆菌O157:H7。方法:以肠出血性大肠杆菌0157:H7 rfbE为待检靶基因,设计一对引物和一条Taqman探针,探针5’端用FAM基团标记,3’端用TAMRA标记。通过重组质粒的构建,建立并优化了大肠杆菌0157:H7的荧光定量PCR检测方法。结果:在人工污染样本无需富集的情况下,检测的最低DNA浓度是10拷贝/反应(3CFU/mL);特异性检测实验中,0157菌株检测结果均为rfbE阳性,而非0157:H7菌株检测结果均为阴性;重复性实验中,批内、批间变异系数均小于3%。结论:实验结果显示此荧光定量PCR方法特异性、灵敏度高,重复性好,可对分离的可疑大肠杆菌0157:H7菌株进行快速鉴定。     

Development of a sensitive ELISA for the quantification of human tumour necrosis factor-alpha using 4 polyclonal antibodies

Despite the availability of many assays to measure concentrations of tumour necrosis factor alpha (TNF-alpha) in body fluids, these assays often lack specificity or sensitivity and are often of questionable reliability, resulting in inconsistent results. Therefore, we have developed an ELISA that is sensitive, reliable and not susceptible to disturbances by interfering substances such as heterophilic antibodies. The assay involves a combination of four polyclonal antibodies. The antibodies, which capture the analyte, were raised in chicken and the trapping anti-analyte antibodies were raised in rabbit. The immobilization of capture antibodies was achieved via a coating antibody raised in a duck against chicken IgY and the recognition of trapping antibodies was achieved by a detection antibody raised in a goat against rabbit IgG and labelled with HRP. The analytical and functional sensitivities of the ELISA are 8 pg/mL and 13 pg/mL, respectively. The assay showed good precision and, in contrast to our in-house RIA, excellent parallelism in serial dilutions. The recovery of TNF-alpha spiked to plasma samples ranged from 97% to 119%. Comparison of the newly developed, sensitive ELISA with our in-house RIA showed that the median TNF-alpha value obtained by RIA (range: 0.095-10.0, median 0.578 ng/mL) was found to be 1.5-2 times higher than that obtained with the ELISA (range 0.008-5.84, median 0.213 ng/mL). Spearman correlation was 0.755 (p < 0.0001). In addition, analysis of the TNF-alpha concentrations in blood from healthy individuals and from patients suffering from tuberculosis, with RIA and ELISA, showed the same differences although TNF-alpha levels obtained with ELISA were lower. We feel that this ELISA is a major improvement compared to the currently available assays for TNF.     

Two time-resolved fluorometric high-throughput assays for quantitation of GDP-L-fucose

Two rapid and simple procedures for the quantitative analysis of GDP-l-fucose (GDP-Fuc) are described. The methods are based on time-resolved fluorescence and microplate assay technology. The first assay relies on measuring the enzyme activity of alpha1, 3-fucosyltransferase. In this assay, transfer of fucose from GDP-Fuc converts sialyllactosamine to sialyl Lewis x tetrasaccharide, which is detected and quantified by relevant antibodies on a microplate. The formation of the reaction product is directly dependent on the presence of GDP-Fuc in the concentration range of 10-10,000 nM. In the second method GDP-Fuc inhibits the binding of fucose-specific Aleuria aurantia lectin to fucosylated glycan on a microwell. The lectin-based assay is less sensitive than the enzyme assay, but it is cheaper and faster. We used these assays in monitoring the amount of GDP-Fuc in crude lysates of transgenic yeast, which expresses the enzymes producing GDP-Fuc. The newly developed assays are versatile and applicable to measure also other nucleotide sugars or glycosyltransferase activities in a high-throughput manner.     

Comparison of colorimetric and chemiluminescent enzyme‐linked immunosorbent assay for the detection of endosulfan in food samples

Pesticides have become part of food protection since their inception. Endosulfan, an organochlorine insecticide, has been used against insect pests such as whiteflies, aphids, red spiders and mites. Methods of immunochemical assays have been devised for the determination and analysis of pesticides and commonly used for the analysis of contaminants in food, water, soil and body fluids. Chicken IgY antibodies raised against endosulfan haptens were used for the detection of endosulfan. We have compared colorimetric (CO) and chemiluminescence (CL) enzyme‐linked immunosorbent assay (ELISA) techniques for the detection of endosulfan isomers in a food matrix. CL ELISA assay was found to be more sensitive than CO assay. The mean recovery was 81.2–95.6% for α‐ and β‐endosulfan‐spiked food samples with 2.8–4.6% relative standard deviation. The detection of the endosulfan isomers was linear in the range 100 µg/mL–5 fg/mL, with a limit of detection at 100 µg/mL and 5 fg/mL for the CL ELISA method and 100 µg/mL and 1 ng/mL for the CO ELISA method respectively. These methods can be used for the rapid and reliable detection of organochlorine pesticide endosulfan. Copyright © 2015 John Wiley & Sons, Ltd.     

Amperometric immunosensor for the determination of IgA deficiency in human serum samples

An electrochemical immunosensor for the detection of human IgA deficiency in real human blood serum has been developed. The performance of the immunosensor presents a large but sensitive dynamic range that allows the determination of non-deficient IgA levels (>70 μg/mL) as well as of severe IgA deficiencies (0.5-5.0 μg/mL). The assay architecture involves the immobilisation of a coating antibody on an electrode surface using carboxylic-ended bipodal alkane-thiol self-assembled monolayers (SAMs). The long chain bipodal SAM presents intercalated poly(ethylenglycol) groups that confer the immunosensor the ability to retain its optimum performance in very complex matrices and serum with negligible non-specific adsorption phenomena. Amperometric optimisation of the assay resulted in limits of detection of 142 ng/mL in just 30 min total assay time. Real patients' serum samples were analysed using the developed electrochemical immunosensor demonstrating an excellent correlation in terms of sensitivity and reproducibility compared with standard enzyme linked immunosorbent assays (ELISA).     

An enzyme capture assay for analysis of active hyaluronan synthases

We describe a sensitive assay for detection of active hyaluronan synthases (HASs) capable of synthesizing hyaluronan (HA) without use of radioactive uridine 5'-diphosphate sugar precursors. The HAS capture assay is based on the binding of a biotinylated HA binding protein (bHABP) to HA chains that are associated with HAS and the subsequent capture of bHABP-HA-HAS complexes with streptavidin-agarose. Specific HAS proteins (e.g., HAS1, not HAS2 or HAS3) captured in this pull-down approach are readily immunodetected by Western blot analysis using appropriate antibodies. The assay was used to detect active HAS proteins in cell membranes, purified recombinant Streptococcus equisimilis HAS (SeHAS), and in vitro translated human HAS1 or SeHAS. The HAS capture assay was also used to assess the fraction of HAS molecules that were active, which cannot be done using standard assays for synthase activity. Assay sensitivity for detection of purified SeHAS is <1 pmol.