Chemiluminescent Substrate Research Articles

Intraparticle Energy Level Alignment of Semiconducting Polymer Nanoparticles to Amplify Chemiluminescence for Ultrasensitive In Vivo Imaging of Reactive Oxygen Species.

Detection of reactive oxygen species (ROS), a hallmark of many pathological processes, is imperative to understanding, detection and treatment of many life-threatening diseases. However, methods capable of real-time in situ imaging of ROS in living animals are still very limited. We herein report the development and optimization of chemiluminescent semiconducting polymer nanoparticles (SPNs) for ultrasensitive in vivo imaging of hydrogen peroxide (H2O2). The chemiluminescence is amplified by adjusting the energy levels between the luminescence reporter and the chemiluminescence substrate to facilitate intermolecular electron transfer in the process of H2O2-activated luminescence. The optimized SPN can emit chemiluminescence with the quantum yield up to 2.30 × 10(-2) einsteins/mol and detect H2O2 down to 5 nM, which substantially outperforms the previous probes. Further doping of this SPN with a naphthalocyanine dye creates intraparticle chemiluminescence resonance energy transfer (CRET), leading to the near-infrared (NIR) luminescence responding to H2O2. By virtue of high brightness and ideal NIR optical window, SPN-NIR permits ultrasensitive imaging of H2O2 in the mouse models of peritonitis and neuroinflammation with the minute administration quantity. Thus, this study not only provides a category of optical probes that eliminates the need of external light excitation for imaging of H2O2, but also reveals the underlying principle to enhance the brightness of chemiluminescence systems.

Biological implications of somatic DDX41 p.R525H mutation in acute myeloid leukemia.

The DDX41 gene, encoding a DEAD-box type ATP-dependent RNA helicase, is rarely but reproducibly mutated in myeloid diseases. The acquired mutation in DDX41 is highly concentrated at c.G1574A (p.R525H) in the conserved motif VI located at the C-terminus of the helicase core domain where ATP interacts and is hydrolyzed. Therefore, it is likely that the p.R525H mutation perturbs ATPase activity in a dominant-negative manner. In this study, we screened for the DDX41 mutation of CD34-positive tumor cells based on mRNA sequencing and identified the p.R525H mutation in three cases among 23 patients. Intriguingly, these patients commonly exhibited acute myeloid leukemia (AML) with peripheral blood cytopenias and low blast counts, suggesting that the mutation inhibits the growth and differentiation of hematopoietic cells. Data from cord blood cells and leukemia cell lines suggest a role for DDX41 in preribosomal RNA processing, in which the expression of the p.R525H mutant causes a certain ribosomopathy phenotype in hematopoietic cells by suppressing MDM2-mediated RB degradation, thus triggering the inhibition of E2F activity. This study uncovered a pathogenic role of p.R525H DDX41 in the slow growth rate of tumor cells. Age-dependent epigenetic alterations or other somatic changes might collaborate with the mutation to causeAML.

Smart CuS Nanoparticles as Peroxidase Mimetics for the Design of Novel Label-Free Chemiluminescent Immunoassay.

In the present work, a novel label-free chemiluminescent (CL) immunoassay method was designed by employing smart CuS nanoparticles (CuSNPs) as peroxidase mimetics. The CuSNPs were synthesized through a simple coprecipitation method, and showed high catalytic activity and stability. This efficient label-free CL immunoassay could be easily achieved through a simple strategy. First, CuSNPs dispersed in chitosan were modified on the epoxy-functionalized glass slide to form a solid CL signal interface. Streptavidin was then used to functionalize CuSNPs to capture the biotinylated antibody, further producing a sensing interface. After online incubation with antigen molecules, the formed antibody-antigen complex on the biosensing substrate could prevent the diffusion channel of CL substrate toward the signal interface, and restrained the mimic enzyme-catalyzed CL reaction, finally resulting in the decrease of CL signals of the assay system. Compared to the label-based CL immunoassay, the proposed label-free assay mode is more simple, cheap and fast. Using a model analyte alpha-fetoprotein, the label-free CL immunoassay method had a linear range of 0.1-60 ng/mL and a low detection limit of 0.07 ng/mL. Moreover, the peroxidase mimetic-based label-free CL immunoassay system showed good specificity, acceptable repeatability, and good accuracy. The study provided a promising strategy for the development of highly efficient label-free CL immunoassay system.

Microfluidic Chip-Based Immunoassay for Reliable Detection of Cloxacillin in Poultry

By combining a chemiluminescence immunoassay with microfluidic chip technology, a simple assay was established for the sensitive detection of cloxacillin in poultry samples. The chip used in this approach was composed of an upper microchannel layer and a lower flat base layer. Each of the formed microchannels has a volume of 18 μL, thus enabling a considerable reduction of reagent consumption as well as sample size needed for analysis by the indirect competitive immunoassay. To obtain labeled antigens for coating, the active ester method was employed to couple cloxacillin to glucose oxidase (GlcOx). The immobilized GlcOx-cloxacillin conjugate on the base layer competed with residues of cloxacillin in samples for binding sites of the monoclonal antibodies (mAb) against cloxacillin. Horseradish peroxidase-labeled anti-mouse IgG antibodies and chemiluminescent substrate were used for signal generation. Under optimized conditions, the IC50 and the limit of detection (LOD) of the assay were at 96.5 ± 9.37 and 0.92 ± 0.07 ng/mL, respectively, and thus well below the maximum residue level of 300 ng/mL set by the European Commission. Recoveries for spiked chicken and duck samples were in the range from 94 to 118 % with relative standard deviations lower than 11 %. The results demonstrate that the microfluidic chip-based immunoassay can be used as a rapid and reliable platform for determination of cloxacillin in poultry.

Augmented Expression of Piezo1 in the Uterine Vasculature During Pregnancy

The maternal uterine circulation undergoes expansive remodeling during gestation to facilitate the many‐fold increase in uteroplacental blood flow required for normal pregnancy outcome. Although we and others have shown that endothelial nitric oxide (NO) is a key mediator of this process, the primary molecular sensor responsible for detecting shear stress and transducing it to eNOS activation has yet to be identified. The recent discovery of Piezo1 ‐ a large, mechanically‐activated cation channel that has been implicated in mechanosensing of shear stress in various cell types, including the endothelium, inspired this study. Notably, transfection of Piezo1 into HEK293 cells, which normally cannot sense shear stress, conferred a shear stress responsiveness evidenced by increased cytosolic calcium and eNOS activation. We hypothesized that Piezo1 may be present in uterine arteries, and that its expression would be augmented during pregnancy and linked to increased eNOS.Main uterine arteries (MUA) were collected from adult (12–14 week old) late pregnant (LP: gestational day 20, n=4) Sprague‐Dawley rats, and from age‐matched nonpregnant (NP, n=4) controls. Following euthanization, MUAs were dissected free of surrounding connective and adipose tissues, and cut open to remove any blood present in the lumen. Segments of MUA were either fixed and permeabilized for immunohistochemistry (IHC), or homogenized for Western Blotting (WB). Tissues were incubated with rabbit polyclonal Piezo1 antibody (Ab), eNOS Ab, β‐tubulin Ab; goat anti‐rabbit IgG‐HRP Ab (and/or Alexa Fluor® 488 Ab). Immunolabeled vessels were imaged using a Zeiss LSM 510 META confocal microscope @ 100x; for WB, proteins were detected with chemiluminescent substrate and analyzed using ImageJ software.Results(1) Piezo1 was detected in the MUA of both NP and LP rats (using either method), and localized to the arterial endothelium (IHC). (2) Both Piezo1 and eNOS expression were augmented in MUAs from LP vs. NP rats. Based on this preliminary information, we suggest that Piezo1 may play a role in transducing shear stress into the activation of eNOS in the uterine circulation during pregnancy, and thereby contributes to the expansive arterial remodeling required to maintain sufficient uteroplacental perfusion. This is the first study to identify this channel in uterine vessels, and to show a gestational effect on its expression. As both shear stress and uterine arterial remodeling are attenuated in preeclampsia (a serious gestational disease that afflicts ten million pregnant women worldwide each year), understanding the underlying mechanosensing/transducing mechanisms may lead to new therapeutic strategies for augmenting uteroplacental blood flow.Support or Funding InformationNIH R21 HD080156

Immunosorbent analysis of toxin contamination in milk and ground beef using IgY-based ELISA

ABSTRACTAnalytical methodology to detect ricin and Shiga toxins (Stx) in food matrices is important for food safety and biosecurity. Monoclonal antibodies (mAbs) that bind each toxin were used for capture in sandwich enzyme-linked immunosorbent assay, and IgY polyclonal antibodies were prepared as detection antibodies. The ricin assay systems, using colorimetric or chemiluminescent substrates, detected native, but not heat-denatured ricin. The lower limit of detection (LOD) was 0.13 ng mL−1 in milk and 0.8 ng g−1 in ground beef. The Stx2 assay systems detected native Stx2, but not heat-denatured Stx2 or Stx1. The LOD was 0.13 ng mL−1 in milk and 0.7 ng g−1 in ground beef. Using a standard 96-well-plate format, the assays can detect less than 1 × 10−4 of an estimated lethal oral dose of either toxin in a serving of milk. The IgY detection antibodies for ricin were more heat-stable than mouse polyclonal anti-ricin at 65°C.

糖类抗原125化学发光免疫定量检测试剂的研制与临床应用

为了研制肿瘤标志物糖类抗原125(CA125)化学发光免疫定量检测试剂，并分析其临床应用价值，将CA125单抗包被微孔板，以辣根过氧化物酶标记的CA125抗体为二抗，结合鲁米诺化学发光底物系统，建立CA125化学发光免疫定量检测方法。用CA125检测试剂国家参考品质控血清分析所建立方法的特异性、灵敏度、稳定性和精密性等试剂性能，并与进口的CA125定量检测试剂同时检测临床血清样本350例，比较检测结果。结果表明所研制的试剂性能符合CA125检测试剂国家参考品质量标准。批内变异系数2.8%~3.6%、批间变异系数3.7%；试剂盒置37℃孵育3d，其稳定性良好。临床样本比对检测结果表明，两种方法相关性良好(相关系数r = 0.9897，阴性符合率99.5%、阳性符合率99.3%，总符合率为99.4%，Kappa值为0.99，一致性强度为最强)。本文成功研制了快速、特异、敏感和稳定的肿瘤标记物CA125化学发光免疫定量检测试剂并建立其检测方法，适合临床检验推广应用。 To develop a chemiluminescence immunoassay (CLIA) kit for quantitative determination of tumor marker carbohydrate antigen 125 (CA125) and evaluate its value of clinical application, CLIA for quantitative determination of CA125 was established by using micro-plates coated with antibody to CA125, combined with HRP conjugated anti-CA125 antibody, as well as luminol chemiluminescence substrate system. The national reference control sera were used for evaluation of specificity, sensitivity, stability and accuracy of the established CLIA. CA125 levels in sera from 350 clinical samples were parallel determined by the established methods and reference kits produced by the Siemens. The results show that the efficiency of the CLIA reagents met the requirements of national reference standard. The intra coefficient variations (CV) were between 2.8%~3.6% and inter CV 3.7%. The kit showed good stability after being kept at 37˚C for 3d. The established CLIA showed good correlation with the reference kits (negative concordance rate, positive concordance rate and overall concordance rate were 99.5%, 99.3% and 99.4% respectively, and a correlation coefficient r = 0.9897. High degree of agreement was observed with a Kappa value of 0.99). A rapid, specific, sensitive and stable CLIA kit for quantitative determina-tion of CA125 was successfully established and that will be fit for widely application in clinical laboratory.

Probing Force with Mechanobase‐Induced Chemiluminescence

AbstractMechanophores capable of releasing N‐heterocyclic carbene (NHC), a strong base, are combined with triggerable chemiluminescent substrates to give a novel system for mechanically induced chemiluminescence. The mechanophores are palladium bis‐NHC complexes, centrally incorporated in poly(tetrahydrofuran) (pTHF). Chemiluminescence is induced from two substrates, adamantyl phenol dioxetane (APD) and a coumaranone derivative, upon sonication of dilute solutions of the polymer complex and either APD or the coumaranone. Control experiments with a low molecular weight Pd complex showed no significant activation and the molecular weight dependence of the coumaranone emission supports the mechanical origin of the activation. The development of this system is a first step towards mechanoluminescence at lower force thresholds and catalytic mechanoluminescence.

Probing Force with Mechanobase‐Induced Chemiluminescence

Mechanophores capable of releasing N-heterocyclic carbene (NHC), a strong base, are combined with triggerable chemiluminescent substrates to give a novel system for mechanically induced chemiluminescence. The mechanophores are palladium bis-NHC complexes, centrally incorporated in poly(tetrahydrofuran) (pTHF). Chemiluminescence is induced from two substrates, adamantyl phenol dioxetane (APD) and a coumaranone derivative, upon sonication of dilute solutions of the polymer complex and either APD or the coumaranone. Control experiments with a low molecular weight Pd complex showed no significant activation and the molecular weight dependence of the coumaranone emission supports the mechanical origin of the activation. The development of this system is a first step towards mechanoluminescence at lower force thresholds and catalytic mechanoluminescence.

NADPH Oxidase Activation and Endothelial Nitric Oxide Synthase Uncoupling Contribute to Endothelial Dysfunction in Antiphospholipid Syndrome

Introduction: Antiphospholipid syndrome (APS) is characterized by thrombosis and/or recurrent fetal loss in the presence of persistently elevated antiphospholipid antibodies (APLA). The majority of pathologic APLA are directed against β2-glycoprotein I (β2GPI). APLA cause endothelial dysfunction, though the underlying mechanisms have not been clearly delineated.Methods: Endothelial cells (EC) were incubated with β2GPI and either control antibodies or anti-β2GPI antibodies affinity-purified from sera of patients with APS, in the absence or presence of diapocynin, an NADPH oxidase (NOX) inhibitor, or siRNA against NOX1, NOX2, or NOX4. Generation of reactive oxygen species (ROS) in EC and conditioned medium were measured using fluorescent dyes (CM-H2DCFDA and CellROX Deep Red) or chemiluminescent substrate. NOX1, NOX2, NOX4, 3-nitrotyrosine, and thioredoxin reductase 1 (TrxR1) expression were analyzed by western blot. eNOS monomer and dimer were detected using cold (4°C) SDS-PAGE and immunoblot. Immunoprecipitation of TrxR1 was performed using protein A/G agarose. EC activation was assessed by measuring expression of E-selectin.Results: Incubation of EC with β2GPI and anti-β2GPI antibodies stimulated ROS generation in EC, as well as the release of ROS into conditioned medium. Expression of NOX2, but not NOX1 or NOX4, was significantly increased in EC exposed to anti-β2GPI antibodies, but not control IgG. Preteatment of endothelial cells with diapocynin, a NOX inhibitor, or siRNA against NOX2 and NOX4, but not NOX1, inhibited endothelial cell activation by anti-β2GPI antibodies. Furthermore, anti-β2GPI antibody-treated EC generated more peroxynitrite, as determined by 3-nitrotyrosine expression. Treatment of EC with anti-β2GPI antibodies increased eNOS monomer/dimer ratio, suggesting eNOS uncoupling. Compared to control human IgG, the TrxR1 immunoprecipitate from EC treated with β2GPI and anti-β2GPI antibodies contained more 3-nitrotyrosine level, suggesting TrxR1 tyrosine residue modification by nitration and possible loss of function.Conclusions: β2GPI and anti-β2GPI antibodies stimulate ROS generation in EC, with release into the conditioned medium. The impairment of EC activation by diapocynin, NOX2 siRNA, or NOX4 siRNA suggests that NOX mediate EC activation by anti-β2GPI antibodies. Anti-β2GPI antibodies induce nitrative stress in EC, which might be explained by eNOS uncoupling induced by these antibodies. The nitration of TrxR1, an enzyme that prevents eNOS uncoupling, may result in the inactivation of TrxR1 and contribute to eNOS uncoupling. Taken together, these studies provide preliminary evidence of the contribution of NOX activation and eNOS uncoupling in mediating oxidative and nitrative stress and induction of EC dysfunction by APLA. DisclosuresMcCrae:Janssen: Membership on an entity's Board of Directors or advisory committees; Syntimmune: Consultancy; Momenta: Consultancy; Halozyme: Membership on an entity's Board of Directors or advisory committees.

Clinical performance of hepatitis B surface antigen quantification with high sensitivity chemiluminescent enzyme immunoassay technology

Objective To assess the clinic application of a new-style automated immunoassay system HISCL (high sensitivity chemiluminescent enzyme immunoassay) with high sensitive chemiluminescence substrate CDP-Star®, bind-free separate technology and filter conversion technology. Methods The performance verification test evaluated HISCL's specification including reproducibility, functional sensitivity, linearity, accuracy, and sensitivity for HBsAg seroconversion. To evaluate the specificity, 1 007 HBsAg-negative specimens, 82 potentially interfering specimens were from conventional specimens in West China Hospital, Sichuan University between October and December of 2014. At the same time, with these results to determine the rate of the specimens that their results are in negative grey zone. 259 HBsAg-positive specimens and 27 weakly-positive specimens were tested for the comparison between the HISCL and ECLIA (electrochemiluminescence immunoassay) HBsAg quantification. A linear regression model, Pearson's correlation and Bland-Altman analysis were used as statistical methods. Results The between day and within-lot variable coefficient of two level samples are 1.55%, 2.02%, 0.34%, 1.34% separately. The functional sensitivity of the HISCL HBsAg assay reaches 0.007 IU /ml. There is a good linearity (y=3.262x+ 0.082, r=0.994; y=2 303.608x-33.006, r=0.999) within range of 0-2 300 IU/ml. HISCL obtain the accurate results for the CAP control material, the agreement rate is 100%. The sensitivity for the detection of HBsAg seroconversion is high. The specificity is 99.91%. The negative grey zone rate is 1.66%. The results of these two methods have good correlation and uniformity, r=0.995, LOA: -0.3-0.19 log10 IU/ml. Conclusions HISCL HBsAg detection system shows good detection performance. Be of the function of both qualitative and quantitative detection. And the negativegrey zonespecimen rate is low. It is wholly capable of wide linearity and quick assays for quantifying serum HBsAg levels. HISCL could contribute to improve HBsAg quantitative detection process for clinical laboratory.(Chin J Lab Med, 2015, 38: 537-542) Key words: Hepatitis B surface antigens; Luminescence; Immunoassay

Fiber-optic immunosensor for detection of Crimean-Congo hemorrhagic fever IgG antibodies in patients.

Crimean-Congo hemorrhagic fever (CCHF) is a severe viral disease with high fatality rate. CCHF virus is endemic in parts of Africa, Asia, the Middle East, and southeastern Europe. Rapid diagnostics of CCHF is vital for appropriate clinical management and prevention of secondary spread from human-to-human. Currently, diagnostics relies on real-time RT-PCR and antibody or antigen detection using ELISA. These methods require trained personnel and expensive equipment and are not appropriate for point-of-care (POC) diagnostics. Furthermore, there are no POC assays available for CCHF. We developed a fiber-optic biosensor for the detection of CCHF IgG antibodies. In order to improve sensitivity, we optimized both the bioreceptor immobilization protocol and the chemiluminescence substrate formulation. The resulting protocol showed a 100-fold greater sensitivity for detection of CCHF antibodies. Finally, we evaluated the fiber-optic biosensor with two CCHF patient sera. We showed that the fiber-optic biosensor is 10-times more sensitive than colorimetric ELISA and is able to detect both patients with high and low levels of IgG antibodies. We believe that the fiber-optic biosensor is a suitable alternative to ELISA as it is much more sensitive and makes it possible to detect a small amount of antibodies at an early stage of infection and can be integrated as a point-of-care diagnostic system of CCHF.

Apelin Enhances Brown Adipogenesis and Browning of White Adipocytes

Brown adipose tissue expends energy in the form of heat via the mitochondrial uncoupling protein UCP1. Recent studies showed that brown adipose tissue is present in adult humans and may be exploited for its anti-obesity and anti-diabetes actions. Apelin is an adipocyte-derived hormone that plays important roles in energy metabolism. Here, we report that apelin-APJ signaling promotes brown adipocyte differentiation by increasing the expressions of brown adipogenic and thermogenic transcriptional factors via the PI3K/Akt and AMPK signaling pathways. It is also found that apelin relieves the TNFα inhibition on brown adipogenesis. In addition, apelin increases the basal activity of brown adipocytes, as evidenced by the increased PGC1α and UCP1 expressions, mitochondrial biogenesis, and oxygen consumption. Finally, we provide both in vitro and in vivo evidence that apelin is able to increase the brown-like characteristics in white adipocytes. This study, for the first time, reveals the brown adipogenic and browning effects of apelin and suggests a potential therapeutic route to combat obesity and related metabolic disorders.

Rapid and cost effective immunoassay development and fit-for-purpose validation of a biomarker assay using Spatial Proximity Analyte Reagent Capture Luminescence (SPARCL) (TECH2P.760)

Abstract A novel detection technology was used for rapid immunoassay development and validation. SPARCL (Spatial Proximity Analyte Reagent Capture Luminescence) is a proximity dependent and homogeneous chemiluminescent detection method. In a SPARCL assay, a chemiluminescent substrate (acridan) is brought into the proximity of an oxidative enzyme (horseradish peroxidase) through the specific antigen/antibody interaction. A flash of light is generated and is proportional to the amount of IL-8 bound. Assay development was completed in less than 3 days. The validated assay features a wide dynamic range (2 - 4,000 pg/mL), low MRD, short assay run time (60 min.), and sensitivity (2 pg/mL). Quality control samples (QC’s) run in plasma resulted in statistics that show the assay is precise and accurate, with a total error of all QC levels of under 12%. Matrix spiked with IL-8 diluted in a linear manor. The SPARCL technology enables rapid assay development, delivers an assay with desirable performance characteristics and allows for considerable savings in labor, disposables and capital equipment compared to popular platforms. Existing software and luminometers may be used for a SPARCL data acquisition. As a result, there may not be a need to validate new software or plate readers already running in existing Laboratory Information Management Systems or in a regulated environment. The SPARCL assay is microtiter plate-based and is suitable for use with robotics and in HTS applications.

New chemiluminescent substrates of paraoxonase 1 with improved specificity: synthesis and properties.

Paraoxonase 1 (PON1) is an important hydrolase, and the enzyme activity decreases in patients with liver disease, diabetes, coronary heart disease, etc. Phenyl acetate and organophosphates are usually employed as substrates for serum PON1 activity assay. However, phenyl acetate for arylesterase activity assay exhibits disadvantage of high background. According to properties of PON1, four new chemiluminescent acridinium esters were designed, prepared through three steps, and characterized with (1)H NMR and mass spectrometry (MS) data, and their properties as PON1 substrates were investigated. The hydrolyses of the four compounds catalyzed by recombinant human PON1 (rhPON1) (or serum) followed first-order kinetics within 22 min. The PON1 activator (NaCl, 0.10 mol L(-1)) could boost the rhPON1-mediated and serum-mediated hydrolyses of the acridinium esters to 2.01 ~ 2.26 folds, but 1.0 mol L(-1) NaCl decreased the serum arylesterase activity. RhPON1 showed selectivity over other serum esterases such as lipase, acetylcholinesterase, and esterase D more than 300 folds. By using ethylene diamine tetraacetic acid (EDTA) inhibitor, the specificities of the four substrates toward serum PON1 were determined as 78.3 ~ 92.9%, which is improved than that of the model compound 9-(4-chloro-phenoxycarbonyl)-10-methylacridinium ester triflate. Due to low toxicity, high specificity, and sensitivity of the substrates, they are useful for serum PON1 activity assay.

Nonstripping "Rainbow" and Multiple Antigen Detection (MAD) Western Blotting.

A variation of immunoblotting method (the "Rainbow Western"), permits sequential detection of multiple antigens (MAD) on a single protein blot without stripping off prior antibodies. Because no stripping is involved, immobilized proteins are not lost from the membrane, thus allowing for multiple reprobings of the same membrane with different primary antibodies (≥12), retaining strong signal intensities for all sequential antibody probings. The procedure utilizes horseradish peroxidase (HRPase)-based detection with both a chemiluminescent and colorimetric substrate. Initial incubation of the blot with secondary antibody followed by colorimetric development prior to probing the blot with primary antibodies markedly reduces background in ECL-based detection procedures and permits sequential use of antibodies derived from a single species. In the "Rainbow Western," four different HRPase-colorimetric substrates that produce black, brown, red, and green colors are employed sequentially for detection and simultaneous display of four different antigens on the same membrane. By allowing large amounts of data to be obtained from a single blot, the MAD-immunoblotting and Rainbow Western methods have the potential for researchers to compare the expression of several proteins within a single biological sample. Both techniques could be particularly valuable for analysis of cellular populations that are difficult to isolate in large numbers or of clinical specimens where the amounts of protein samples is minute or only available on a one-time basis.

Improvements and Variants of the Multiple Antigen Blot Assay-MABA: An Immunoenzymatic Technique for Simultaneous Antigen and Antibody Screening.

This simple, versatile, reliable, reproducible, multipurpose, and inexpensive technique is based on the adhesion of different antigens to a single nitrocellulose strip using, as template, an acrylic device containing 28 parallel channels. The inclusion of channels containing normal human serum improves the quality control of this assay. Antigen-sensitized nitrocellulose strips are cut perpendicularly to the antigen-rows, exposed to immune sera followed by the appropriate conjugate. Positive signals are recorded using chemiluminescent or precipitable colorimetric substrates. This assay allows the simultaneous qualitative demonstration of antigenicity and immunogenicity of antigens obtained as synthetic peptides, recombinant molecules, or crude preparations, with high sensitivity and specificity. Its major value is based on the rapid and simultaneous comparative evaluation of various antigenic preparations allowing the diagnosis of a variety of infectious, allergic, and autoimmune diseases. It can in general be used to detect any type of antibody or circulating antigen. Some improvements and variants of the original technique are included.

Direct ELISA.

First described by Engvall and Perlmann, the enzyme-linked immunosorbent assay (ELISA) is a rapid and sensitive method for detection and quantitation of an antigen using an enzyme-labeled antibody. Besides routine laboratory usage, ELISA has been utilized in medical field and food industry as diagnostic and quality control tools. Traditionally performed in 96-well or 384-well polystyrene plates, the technology has expanded to other platforms with increase in automation. Depending on the antigen epitope and availability of specific antibody, there are variations in ELISA setup. The four basic formats are direct, indirect, sandwich, and competitive ELISAs. Direct ELISA is the simplest format requiring an antigen and an enzyme-conjugated antibody specific to the antigen. This chapter describes the individual steps for detection of a plate-bound antigen using a horseradish peroxidase (HRP)-conjugated antibody and luminol-based enhanced chemiluminescence (ECL) substrate. The methodological approach to optimize the assay by chessboard titration is also provided.

High-throughput imaging assay of multiple proteins via target-induced DNA assembly and cleavage.

This work integrates target-induced DNA assembly and cleavage on a DNA chip to design a versatile imaging strategy as an assay for multiple proteins. The DNA assembly is achieved via immunological recognition to trigger the proximity hybridization for releasing a DNA sequence, which then hybridizes with FITC-DNA1 immobilized on the chip to induce the enzymatic cleavage of DNA1 and thus decrease the signals. The signal readout is performed with both fluorescent imaging of the left FITC and chemiluminescent (CL) imaging, by adding peroxidase labelled anti-FITC in assembly solution and CL substrates to produce CL emission. This one-step incubation can be completed in 30 min. The imaging method shows wide detection ranges and detection limits down to pg mL-1 for the simultaneous detection of 4 protein biomarkers. This high-throughput strategy with good practicability can be easily extended to other protein analytes, providing a powerful protocol for protein analysis and clinical diagnosis.

Peptide conjugated chitosan foam as a novel approach for capture-purification and rapid detection of hapten--example of ochratoxin A.

A novel bioassay for the detection and monitoring of Ochratoxin A (OTA), a natural carcinogenic mycotoxin produced by Aspergillus and Penicillium fungi, has been developed and applied for the screening of red wine. Here we report the immobilization and orientation of NOF4, a synthetic peptide, onto 3-D porous chitosan supports using a N-terminal histidine tag to allow binding to M(++) ions that were previously adsorbed onto the high surface area biopolymer. Three divalent cations (M(++)=Zn(++), Co(++), Ni(++)) were evaluated and were found to adsorb via a Langmuir model and to have binding capacities in the order Zn(++)>Co(++)>Ni(++). Following Zn(++) saturation and washing, C-terminus vs. the N-terminus His-tagged NOF4 was evaluated. At 1000 µg L(-1) OTA the N-terminus immobilization was more efficient (2.5 times) in the capture of OTA. HRP labeled OTA was added to the antigen solutions (standards or samples) and together competitively incubated on biospecific chitosan foam. The chemiluminescence substrate luminol was then added and after 5 min of enzymatic reaction, light emission signals (λmax=425 nm) were analyzed. Calibration curves of %B/B0 vs. OTA concentration in PBS showed that half-inhibition occurred at 1.17 µg L(-1), allowing a range of discrimination of 0.25 and 25 µg L(-1). In red wine, the minimum concentration of OTA that the system can detect was 0.5 µg L(-1) and could detect up to 5 µg L(-1). Assay validation was performed against immunoaffinity column (IAC) tandem reversed-phase high pressure liquid chromatography with fluorescence detection (HPLC-FLD) and provided quite good agreement. The association of chitosan foam and specific peptide represents a new approach with potential for both purification-concentration and detection of small molecules. In the future this assay will be implemented in a solid-sate bioelectronic format.