Development of a Progesterone-Receptor-Based Pseudo-immunoassay for Multi-detection of Progestins in Milk and Studying Its Recognition Mechanism.

The residues of progestins in milk are dangerous to consumers, but an immunoassay capable of multi-determining progestins in milk has not been reported thus far. In this study, the ligand binding domain of the human progesterone receptor was expressed and its intermolecular interactions with the commonly used steroid hormones were studied. The docking results showed that the receptor fragment only recognized progestins and did not recognize other steroid hormones. Then, it was used as recognition material to develop a pseudo-direct competitive enzyme-linked immunosorbent assay for multi-determination of five progestins in milk. Because biotinylated horseradish peroxidase was combined with streptavidinated horseradish peroxidase to enhance the signal, the sensitivities for the five progestins (IC50 of 0.029-0.097 ng/mL) were improved 96-143-fold in comparison to the use of the conventional horseradish peroxidase signal system (IC50 of 3.0-12.5 ng/mL). This method showed negligible cross-reactivities to other steroid hormones, consistent with the docking results. This was the first paper developing a progesterone-receptor-based method for detection of progestins, and this method exhibited generally better performance than all of the previously reported immunoassays for progestins.

Ultrasensitive and Multiplexed Protein Imaging with Cleavable Fluorescent Tyramide and Antibody Stripping.

Multiplexed single-cell analysis of proteins in their native cellular contexts holds great promise to reveal the composition, interaction and function of the distinct cell types in complex biological systems. However, the existing multiplexed protein imaging technologies are limited by their detection sensitivity or technical demands. To address these issues, here, we develop an ultrasensitive and multiplexed in situ protein profiling approach by reiterative staining with off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this approach, the protein targets are recognized by antibodies labeled with horseradish peroxidase, which catalyze the covalent deposition of CFT on or close to the protein targets. After imaging, the fluorophores are chemically cleaved, and the antibodies are stripped. Through continuous cycles of staining, imaging, fluorophore cleavage and antibody stripping, a large number of proteins can be quantified in individual cells in situ. Applying this method, we analyzed 20 different proteins in each of ~67,000 cells in a human formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Based on their unique protein expression profiles and microenvironment, these individual cells are partitioned into different cell clusters. We also explored the cell-cell interactions in the tissue by examining which specific cell clusters are selectively associating or avoiding each other.

Rationale of 3,3',5,5'-Tetramethylbenzidine as the Chromogenic Substrate in Colorimetric Analysis.

Horseradish peroxidase (HRP)-based assays feature particular interests because of the simple colorimetric readout. In these assays, 3,3',5,5'-tetramethylbenzidine (TMB) is the most widely used chromogenic substrates for HRP. The later research in nanozyme and DNAzyme also used TMB (the chosen substrate) because they are both HRP-mimics. It should be noted that the substrate of HRP is not just limited to TMB but, in fact, a broad range of benzidine derivatives. However, except decreased carcinogenicity due to tetrasubstitution of benzidine, the rationale for the chosen substrate TMB is not clear yet. Here, we addressed such a fundamental issue from the chemistry point of view. Nine benzidine derivatives featuring varied properties (different substitution groups and varied number of substitutions) were selected and investigated with four typical TMB-involved chromogenic systems. Among the existing benzidine substrates that are used for peroxidase-based assays, TMB exhibited the highest sensitivity, better color purity of colored products, and reasonable stability of oxidation products. Moreover, two tetrasubstituted benzidine derivatives other than TMB (4OCH3 and 2OCH32CH3) were synthesized for comparison. It turned out that the performances (sensitivity, color purity, and stability of the colored products) of TMB are still superior, thus chemically confirming its status of "the chosen substrate" in colorimetric assays.

Visualization of Latent Fingermarks by Enhanced Chemiluminescence Immunoassay and Pattern Recognition.

Herein we report the combination of enzyme-linked immunoassay and pattern recognition analysis for extracting both chemical and spatial information from latent fingermarks (LFMs). The development approach basically involves two steps, namely, specific recognition of protein and polypeptide secretions present in the ridge residues of LFMs by horseradish peroxidase (HRP)-labeled antibodies and the HRP-catalyzed chemiluminescent (CL) reaction between luminol and H2O2. The emitted light can spatially resolve the ridges, generating a bright image against the dark object surface for visualization of an LFM. Meanwhile, thanks to the molecular specificity of the immunoassay step, the emission also provides us additional information on the existence of specific substances in LFMs. The developed LFMs are further processed by a set of digital image processing procedures. Quantitative analysis based on minutia features shows that even poorly developed fingermarks can be matched successfully. This work offers the promise of facilitating cross-disciplinary studies between data-processing approaches and fingermark development techniques, such as the extraction of more information from LFM evidence, as well as the establishment of evaluation criteria for an enhancement technique.

Versatile protein recognition by the encoded display of multiple chemical elements on a constant macrocyclic scaffold.

In nature, specific antibodies can be generated as a result of an adaptive selection and expansion of lymphocytes with suitable protein binding properties. We attempted to mimic antibody-antigen recognition by displaying multiple chemical diversity elements on a defined macrocyclic scaffold. Encoding of the displayed combinations was achieved using distinctive DNA tags, resulting in a library size of 35,393,112. Specific binders could be isolated against a variety of proteins, including carbonic anhydrase IX, horseradish peroxidase, tankyrase 1, human serum albumin, alpha-1 acid glycoprotein, calmodulin, prostate-specific antigen and tumour necrosis factor. Similar to antibodies, the encoded display of multiple chemical elements on a constant scaffold enabled practical applications, such as fluorescence microscopy procedures or the selective in vivo delivery of payloads to tumours. Furthermore, the versatile structure of the scaffold facilitated the generation of protein-specific chemical probes, as illustrated by photo-crosslinking.

Inner Filter Effect-Based Sensor for Horseradish Peroxidase and Its Application to Fluorescence Immunoassay.

Being an important model peroxidase, horseradish peroxidase (HRP) has been thoroughly understood, and the detection of HRP is not only directly related to peroxidase-triggered catalytic process, but also linked to the development of HRP-based enzyme-linked immunosorbent assay (ELISA). Herein, we have reported an unconventional fluorescent sensor for convenient assay of HRP activity based on the HRP-catalyzed specific conversion of p-phenylenediamine (PPD) into chromogenic PPDox with H2O2 as the oxidizing agent, accompanied by the fluorescence quenching effect on fluorescein. By combining UV-vis absorption spectrum, isothermal titration calorimetry, and fluorescence lifetime analysis, we have confirmed the inner filter effect as a main quenching mechanism in our proposed fluorescent assay. According to the intrinsic sensitivity of fluorescent sensor and high selectivity, our PPD/fluorescein-based sensing system can be utilized for real-time monitoring of the HRP activity in real biological samples. Furthermore, the unambiguous response mechanism and excellent sensing performance encourage us to extend such HRP assay into the HRP-based fluorescent ELISA, which has a broad prospect of application in fluorescent diagnosis of hepatocellular carcinoma (HCC) by sensing alpha-fetoprotein, the well-known serologic HCC marker.

A Boronic Acid Assay for the Detection of Mucin-1 Glycoprotein from Cancer Cells.

Cell surface glycoproteins are commonly aberrant in disease and act as biomarkers that facilitate diagnostics. Mucin-1 (MUC1) is a prominent example, exhibiting truncated glycosylation in cancer. We present herein a boronic acid microplate assay for sensitive and high-throughput detection of such glycoproteins. The immobilization of biotin-boronic acid 1 onto streptavidin plates generated a multivalent surface for glycoprotein recruitment and detection. We first validated the binding properties of 1 in solution through titrations with alizarin dye. Next, the microplate assay was explored through horseradish peroxidase (HRP) analysis as a proof-of-concept glycoprotein with chemiluminescence detection. Finally, this platform was applied for the detection of MUC1 directly from MCF-7 human breast cancer cell lysates by using an HRP-tagged antibody that targets the cancerous form of this glycoprotein. Sensitive, dose-dependent detection of MUC1 was observed, showcasing the efficacy of this platform for detecting disease-associated glycoproteins.

A homogeneous chemiluminescent immunoassay method.

A new homogeneous chemiluminescent immunoassay method featuring the use of specific binding members separately labeled with an acridan-based chemiluminescent compound and a peroxidase is reported. Formation of an immunocomplex brings the chemiluminescent compound and the peroxidase into close proximity. Without any separation steps, a chemiluminescent signal is generated upon addition of a trigger solution, and the intensity is directly correlated to the quantity of the analyte.

Digoxin immune fab protects endothelial cells from ouabain-induced barrier injury.

PROBLEM Endogenous digitalis-like factors (EDLF) inhibit sodium pump Na(+) /K(+) ATPase activity, and maternal EDLF levels are elevated in preeclampsia (PE). This study determined whether digoxin immune Fab (DIF) could protect endothelial cells (ECs) from EDLF-induced endothelial barrier dysfunction. METHOD OF STUDY ECs were treated with escalating doses of ouabain (a known EDLF) in the presence or absence of DIF. EC barrier integrity was examined by junction protein VE-cadherin and occludin expressions. EC permeability was determined by horseradish-peroxidase (HRP) leakage and transendothelial electrical resistance (TEER). RESULTS EC junction protein VE-cadherin distribution was disrupted in cells treated with ouabain. DIF, but not control IgG Fab fragment, blocked ouabain-induced decreases in VE-cadherin and occludin expressions and prevented ouabain-induced HRP leakage and TEER changes. CONCLUSION DIF protects ECs from ouabain-induced barrier injury, providing evidence of beneficial effects of DIF on EC function and supporting that Na(+) /K(+) ATPase might be a therapeutic target to ameliorate endothelial dysfunction.

Assessment of permeability in barrier type of endothelium in brain using tracers: Evans blue, sodium fluorescein, and horseradish peroxidase.

Blood-brain barrier (BBB) constituted primarily by the capillary endothelial cells functions to maintain a constant environment for the brain, by preventing or slowing down the passage of a variety of blood-borne substances, such as serum proteins, chemical compounds, ions, and hormones from the circulation into the brain parenchyma. Various diseases such as brain tumors, epilepsy, and sepsis disturb the BBB integrity leading to enhanced permeability of brain microvessels. In animal models, a variety of experimental insults targeted to the BBB integrity have been shown to increase BBB permeability causing enhanced passage of molecules into the brain paranchyma by transcellular and/or paracellular pathways. This alteration can be demonstrated by intravascular infusion of exogenous tracers and subsequent detection of extravasated molecules in the brain tissue. A number of exogenous BBB tracers are available, and they can be used for functional and structural analysis of BBB permeability. In this chapter, we aimed to highlight the basic knowledge on the use of three most commonly performed tracers, namely Evans blue dye, sodium fluorescein, and horseradish peroxidase. The experimental methodologies that we use in our laboratory for the detection of these tracers by macroscopy, spectrophotometry, spectrophotofluorometry, and electron microscopy are also discussed. While tracing studies at the morphological level are mainly aimed at the identification and characterization of the tracers both in the barrier related cells and brain parenchyma, spectrophotometric and spectrophotofluorometric assays enable quantification of BBB permeability. The results of our studies that we performed using the mentioned tracers indicate that barrier type of endothelial cells in brain play an important role in paracellular and/or transcytoplasmic trafficking of macromolecules across BBB under various experimental settings, which may provide new insights in both designing approaches for the management of diseases with BBB breakdown and developing novel trans-BBB drug delivery strategies.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics.

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 ± 0.2) × 107M⁻¹ s⁻¹) and peroxynitrite ((3.7 ± 0.4) × 105 M⁻¹ s⁻¹) at pH 7.4 and 25°C.

Comparison of luminescent immunoassays using biotinylated proteins of aequorin, alkaline phosphatase and horseradish peroxidase as reporters.

We compare aequorin, alkaline phosphatase and horseradish peroxidase as reporters for luminescent immunoassays using alpha-fetoprotein (AFP) as a model analyte. Biotinylated aequorin was prepared from mutated aequorin containing a reactive cysteine residue by chemical conjugation. The measurable range of AFP using this biotinylated aequorin was 0.02-200 ng/ml, with a lower background level than the other biotinylated enzymes tested.

A novel sensitive immunoassay method based on the Invader technique.

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-alpha (hTNF-alpha). Detection limits obtained were 0.1 pg/ml hTNF-alpha when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-alpha enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.

A tris(2,2'-bipyridyl)cobalt(III)-bovine serum albumin composite membrane for biosensors.

A concept based on a novel redox-biocompatible composite protein membrane fabrication, double enzyme membrane modification technique and antibody immobilization, was exploited to develop a highly sensitive amperometric enzyme immunosensor for detection of carcinoembryonic antigen (CEA). In this concept, a solution of bovine serum albumin (BSA) containing horseradish peroxidase (HRP) is coated on the gold electrode in such a way that the first enzyme membrane is achieved. Then tris(2,2'-bipyridyl) cobalt(III) (Co(bpy)(3)3+), as a mediator, was embedded in BSA-HRP composite membrane vis the electrostatic force and hydrophobe functions. Later a self-assembled conductive nano-Au monolayer was constructed onto the resultant electrode surface by electrostatic interaction between the negatively charged nano-Au and positively charged Co(bpy)3(3+). Protein A is used as a binding material to achieve an adjusted (but not random) orientation of the antibodies surface for efficient combination of antigens. Finally, the HRP, was employed to block the possible remaining active sites and avoid the non-specific adsorption, which acts not only as a blocking reagent instead of the commonly used BSA but also as the conventional enzyme-labeling to amplify the response of the antigen-antibody reaction. The immunosensor constructed with the double layer biocatalytic HRP membranes and the desirable Co(bpy)(3)3+/BSA redox-biocompatible composite membrane performed high sensitivity and a wide linear response to CEA in the range of 0.50-80.00 ng/mL with a limit of detection of 0.14 ng/mL, as well as good stability and long-term life.

Measurement of enzyme activity in single cells by voltammetry using a microcell with a positionable dual electrode.

The electrochemical single-cell analysis for enzyme activity was developed using microcells on a microcell array coupled with a positionable dual microelectrode. The microcell array with the nanoliter-scale microcells was constructed using simple chemical etching without photolithographic techniques. The positionable dual microelectrodes consisted of the nanometer-to-micrometer-radius Au disk working electrode and a approximately 80-microm-radius Ag/AgCl reference electrode. Peroxidase was chosen as the model enzyme. Factors that concern electrochemical single-cell analysis in microcells such as solution evaporation, interference of soluble oxygen, electrode size, solution volume, and electrode fouling were investigated and discussed. The 20 or 100 nL of detection volume was found to be suitable for peroxidase determination in single neutrophils or single acute promyelocytic leukemia cells without interference from intracellular macromolecules and electrode fouling, when the dual electrode with a 10-microm-radius Au disk working electrode was used. Cells were perforated with digitonin before transferring them into the microcells, to lyse cells easily. The perforated cells were transferred into the microcells by pushing a microscope slide on a drop of the cell suspension on the microcell array. After a single cell in the microcell was lysed using a freeze-thawing technique and allowed to dry, physiological buffer saline containing 2.0 x 10(-3) mol/L hydroquinone and 2.0 x 10(-3) mol/L H2O2 as the substrates of the enzyme-catalyzed reaction was added. The microcell array was positioned in a constant-humidity chamber to prevent evaporation. Then the dual electrode was inserted into the microcell by means of a scanning electrochemical microscope and the product benzoquinone of the enzyme-catalyzed reaction was voltammetrically detected. Peroxidase activity could be quantified using the steady-state current on the voltammogram after subtracting the blank and using the calibration curve.

Effect of double antigen bridging immunoassay format on antigen coating concentration dependence and implications for designing immunogenicity assays for monoclonal antibodies.

The double antigen bridging immunoassay has been used extensively for detection of immunogenicity responses to therapeutic monoclonal antibodies. We have analyzed parameters affecting performance of this type of immunoassay including microtiter plate antigen coating concentration, enzyme-labeled antigen conjugate dilution and assay format (one-step versus two-step). We present results demonstrating that the format of the assay has a significant impact on the optimal parameters to maximize assay performance. A one-step assay format achieves maximal sensitivity across a broad range of coating concentrations and at a lower concentration of conjugate than that in a two-step format. In contrast, a two-step format requires very low coating concentrations and higher conjugate concentrations to achieve maximal sensitivity and suffers from significantly reduced sensitivity at higher coating concentrations. Together, these findings indicate that a one-step assay format can greatly reduce the effect of coating concentration variation on assay performance.

Filter function and immune complex trapping in splenic ellipsoids.

The role of splenic ellipsoids in the trapping of particulate material and immune complexes was investigated in mink (Mustela vison). The ellipsoids were prominent, with typical features such as a permeable endothelium and a discontinuous basement membrane surrounded by a sheath of macrophages and reticular cells. Ellipsoidal trapping of circulating particles was demonstrated 10 min after intracardiac injection of colloidal carbon and fluorescent microspheres. Preformed peroxidase-antiperoxidase immune complexes were detected in ellipsoids 10 min and also 1 h after intracardiac injection. Erythrocytes were frequently observed in the ellipsoidal sheath, and many phagocytized fragments of erythrocytes were found in the ellipsoidal macrophages. It was concluded that mink ellipsoids are effective blood filters with a role in retention of circulating particulate material, and that mammalian splenic ellipsoids also have the ability to trap immune complexes.

Micro-analytical GO/HRP bioreactor for glucose determination and bioprocess monitoring.

A bi-enzymatic micro-analytical bioreactor integrated in a FIA system for glucose measurements is described. Its robustness and small dimensions (working volume of about 70 microl containing approximately 1.2 mg GO and 0.26 mg HRP) make it easy to operate. The column is based on immobilisation of glucose oxidase (GO) and horseradish peroxidase (HRP) on alkylamine controlled pore glass (CPG) beads. The column has excellent shelf life (no significant loss of activity after 1 year if kept at 4 degrees C), and a very high operational stability that was demonstrated through extensive usage for glucose determinations over 1 year period during which the column retained almost all of its activity. More importantly, this operational stability allows glucose monitoring in the culture media without a decay of signal over the experiment time and consequently no signal correction or re-calibration is needed. This high operational stability was also confirmed by continuous glucose conversion with 30% activity loss after converting quantity of glucose equivalent to 21600 FIA injections of 20 microl with 1.7 mM glucose. Such good performance is a result of an optimised immobilisation method and moreover of the implementation of in situ enzyme stabilisation strategy which consisted on promoting the instantaneous H2O2 consumption produced by the GO. This strategy has the additional advantage of allowing concomitant assay of the H2O2 based on the HAP catalysed co-oxidation of phenol-4-sulphonic acid (PSA) in the presence of 4-aminoantipyrine (4-AAP). The glucose measurements are reproducible with high precision against the standard HPLC method. Linear range and sensitivity depend on sample injection volume; the upper limit is about 1.1 g/l. Lower detection limit is 10mg/l. The column performance has been validated for E. coli and S. cerevisiae fermentation monitoring, and glucose measurements in an animal cell culture (rat Langerhans islets).

Microfluidic enzyme immunosensors with immobilised protein A and G using chemiluminescence detection.

Affinity proteins were covalently immobilised on silicon microchips with overall dimensions of 13.1 x 3.2 mm, comprising 42 porous flow channels of 235 microm depth and 25 microm width, and used to develop microfluidic immunosensors based on horseradish peroxidase (HRP), catalysing the chemiluminescent oxidation of luminol/p-iodophenol (PIP). Different hydrophilic polymers with long flexible chains (polyethylenimine (PEI), dextran (DEX), polyvinyl alcohol, aminodextran) and 3-aminopropyltriethoxysilane (APTS) were employed for modification of the silica surfaces followed by attachment of protein A or G. The resulting immunosensors were compared in an affinity capture assay format, where the competition between the labelled antigen and the analyte for antibody-binding sites took place in the bulk of the solution. The formed immunocomplexes were then trapped by the microchip affinity capture support and the amount of bound tracer was monitored by injection of luminol, PIP and H2O2. All immunosensors were capable of detecting atrazine at the sub-microg l(-1) level. The most sensitive assays were obtained with PEI and DEX polymer modified supports and immobilised protein G, with limits of detection of 0.006 and 0.010 microg l(-1), and IC50 values of 0.096 and 0.130 microg l(-1), respectively. The protein G based immunosensors were regenerated with 0.4 M glycine-HCl buffer pH 2.2, with no loss of activity observed for a storage and operating period of over 8 months. To estimate the applicability of the immunosensors to the analysis of real samples, PEI and DEX based protein G microchips were used to detect atrazine in surface water and fruit juice, spiked with known amounts of the atrazine, giving recovery values of 87-102 and 88-124% at atrazine fortification levels of 0.5-3 and 80-240 microg l(-1), respectively.

Contribution of functional voltage-gated Na+ channel expression to cell behaviors involved in the metastatic cascade in rat prostate cancer: II. Secretory membrane activity.

The secretory membrane activities of two rat prostate cancer cell lines of markedly different metastatic potential, and corresponding electrophysiological characteristics, were studied in a comparative approach. In particular, voltage-gated Na(+) channels (VGSCs) were expressed in the strongly metastatic MAT-LyLu but not in the closely related, but weakly metastatic, AT-2 cells. Uptake and release of the non-cytotoxic marker horseradish peroxidase (HRP) were used as indices of general endocytotic and exocytotic membrane activity, respectively. The amount of tracer present in a given experimental condition was quantified by light microscopic digital imaging. The uptake of HRP was an active process, abolished completely by incubating the cells at low temperature (5 degrees C) and suppressed by disrupting the cytoskeleton. Interestingly, the extent of HRP uptake into the strongly metastatic MAT-LyLu cells was almost twice that into the weakly metastatic AT-2 cells. Vesicular uptake of HRP occurred in a fast followed by a slow phase; these appeared to correspond to cytoplasmic and perinuclear pools, respectively. Importantly, the overall quantitative difference in the uptake disappeared in the presence of 1 microM tetrodotoxin which significantly reduced the uptake of HRP into the MAT-LyLu cells. There was no effect on the AT-2 cells, consistent with functional VGSC expression occurring selectively in the former. A similar effect was observed in Na(+)-free medium. The uptake was partially dependent upon extracellular Ca(2+) but was not affected by raising the extracellular K(+) concentration. We suggest that functional VGSC expression could potentiate prostate cancer cells' metastatic ability by enhancing their secretory membrane activity.