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The development of robust and sensitive point-of-care testing platforms is necessary to improve patient care and outcomes. Surface-enhanced Raman scattering (SERS)-based immunosensors are especially suited for this purpose. Here, we present a highly sensitive and selective SERS immunoassay, demonstrating for example the detection of horseradish peroxidase (HRP), in a sandwich format. The strength of our biosensor lies in merging: (i) SERS-immunotags based on gold nanostars, allowing exceptional intense SERS from attached Raman probes, covalent attachment of anti-HRP antibodies by a simple chemical method providing exceptional antigen binding activity; (ii) the ease of preparation of the capture platform from a regenerated cellulose-based hydrogel, a transparent material, ideal for microfluidics applications, with low background fluorescence and Raman signal, particularly suited for preserving high activity of the covalently bound anti-HRP antibodies. The sandwich complexes formed were characterised by atomic force microscopy, and by scanning electron microscopy coupled with electron diffraction spectroscopy; and (iii) the robustness of the simple Classical Least Squares method for SERS data analysis, resulting in superior discrimination of SERS signals from the background and much better data fitting, compared to the commonly used peak integral method. Our SERS immunoassay greatly improves the detection limits of traditional enzyme-linked immunosorbent assay approaches, and its performance is better or comparable to those of existing SERS-based immunosensors. Our approach successfully overcomes the main challenges of application at point-of-care, including increasing reproducibility, sensitivity, and specificity, associated with an environmentally friendly and robust design. Also, the proposed design withstands several cycles of regeneration, a feature absent in paper-SERS immunoassays and this opens the way for sensitive multiplexing applications on a microfluidic platform.

Many emerging nanobiotechnologies rely on the proper function of proteins immobilized on gold nanoparticles. Often, the surface chemistry of the AuNP is engineered to control the orientation, surface coverage, and structure of the adsorbed protein to maximize conjugate function. Here, we chemically modified antibody to investigate the effect of protein surface chemistries on adsorption to AuNPs. A monoclonal anti-horseradish peroxidase IgG antibody (anti-HRP) was reacted with N-succinimidyl acrylate (NSA) or reduced dithiobissuccinimidyl propionate (DSP) to modify lysine residues. Zeta potential measurements confirmed that both chemical modifications reduced the localized regions of positive charge on the protein surface, while the DSP modification incorporated additional free thiols. Dynamic light scattering confirmed that native and chemically modified antibodies adsorbed onto AuNPs to form bioconjugates; however, adsorption kinetics revealed that the NSA-modified antibody required significantly more time to allow for the formation of a hard corona. Moreover, conjugates formed with the NSA-modified antibody lost antigen-binding function, whereas unmodified and DSP-modified antibodies adsorbed onto AuNPs to form functional conjugates. These results indicate that high-affinity functional groups are required to prevent protein unfolding and loss of function when adsorbed on the AuNP surface. The reduced protein charge and high-affinity thiol groups on the DSP-modified antibody enabled pH-dependent control of protein orientation and the formation of highly active conjugates at solution pHs (<7.5) that are inaccessible with unmodified antibody due to conjugate aggregation. This study establishes parameters for protein modification to facilitate the formation of highly functional and stable protein-AuNP conjugates.

Amino groups on the antibody surface (amino terminus and Lys) are very interesting conjugation targets due to their substantial quantities and selectivity toward various reactive groups. Oriented immobilization of antibodies via amino moieties on the Fc region instead of the antigen-binding fragment (Fab) is highly appreciated to conserve antigen-binding capacity. In this paper, targeting amino moieties on distinct regions, three antibody immobilization strategies were compared with the recognition ability of corresponding adsorbents. Our results demonstrate that oriented immobilization of antibodies onto heterofunctional chelate-epoxy support selectively involving Lys residues placed at the bottom of the Fc region, thus preserved the highest antigen recognition capacity (over 75% functionality). For homofunctional aldehyde support, immobilization at pH 10 demonstrates 50% remaining functionality due to the random orientation of tethered antibodies; while only 10% functionality remained when N-terminus were specifically conjugated at pH 8.5. With the rationalization of moieties density onto heterofunctional support, 2-fold recognition capacity was exhibited over randomly immobilization for antigens with higher size (β-galactosidase, 425 kDa vs. horseradish peroxidase, 40 kDa). Meanwhile, at least 97% of antigens with a varied concentration in diluted human serum were efficiently captured by the optimized chelate-epoxy support. Therefore, our antibody immobilization protocol proved the potential to be utilized as a promising candidate to capture voluminous antigens (large proteins and cells) in real samples., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2018. Published by Elsevier B.V.)

Antidoping testing for recombinant human erythropoietin (EPO) is routinely performed by gel electrophoresis followed by western blot analysis with primary and secondary antibodies. The two antibody steps add more than 24 h to the testing time of a purified sample. The aim of this study was to test the concept of using directly horseradish-peroxidase (HRP)-conjugated anti-EPO primary antibody, without the need for a secondary antibody, to reduce the analysis time and eliminate non-specific cross-reactivity with secondary antibodies. An in-house, periodate coupling (R&D systems, clone AE7A5) and three commercially available anti-human EPO-HRP conjugates from Genetex, Novus Biologicals and Santa Cruz were evaluated for specificity and sensitivity, using recombinant human EPO standards, negative human urine samples and urine samples from an EPO excretion study. The in-house anti-EPO-HRP conjugate was performed as well as the current two-step application of unconjugated primary and secondary antibodies used in routine analysis, with comparable specificity and sensitivity. The analysis time was markedly reduced for purified samples from 25 h with the routine method down to 7 h with the in-house HRP conjugate. Of the three commercially available conjugates tested, only the Santa Cruz anti-EPO-HRP conjugate showed comparable specificity but had lower sensitivity to both the in-house and the antibody combination currently applied routinely. The other two commercially available conjugates (Genetex and Novus Biologicals) did not show any visible bands with the EPO standards. The results clearly demonstrate the potential utility of a directly HRP-conjugated anti-EPO antibody to reduce analysis time for EPO in doping control., (© 2020 John Wiley & Sons, Ltd.)

Background: The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)‑horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum., Results: Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15‑HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti‑PCV2 antibodies. The cut‑off value of the cELISA is 20.72 %. Three hundreds and sixty porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68 % and 95.92 %, respectively. The coincidence rate of the two methods was 99.17 %. When detecting 620 clinical porcine serum samples, a good consistent (kappa value = 0.954) was found between the results of the cELISA and those of commercial kits., Conclusions: In brief, the newly developed cELISA based PCV2-Nb15‑HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccine efficacy and the indirect diagnosis of PCV2 infection.

Antibodies conjugated with horseradish peroxidase (HRP) are one of the most widely used bioreagents in the biological sciences. This protocol is a basic method for adding HRP to a thiolated antibody and can be adapted for use with different cross-linkers. Conjugation methods usually focus on linking through the lysines on HRP because there are only six of them and their modification does not adversely affect enzyme activity., (© 2019 Cold Spring Harbor Laboratory Press.)

Antibody-based analytical instruments are under development to detect signatures of life on planetary bodies. Antibodies are molecular recognition reagents able to detect their target at sub-nanomolar concentrations, with high affinity and specificity. Studying antibody binding performances under space conditions is mandatory to convince space agencies of the adequacy of this promising tool for planetary exploration. To complement previous ground-based experiments on antibody resistance to simulated irradiation, we evaluate in this paper the effects of antibody exposure to real space conditions during the EXPOSE-R2 mission outside the International Space Station. The absorbed dose of ionizing radiation recorded during the 588 days of this mission (220 mGy) corresponded to the absorbed dose expected during a mission to Mars. Moreover, samples faced, at the same time as irradiation, thermal cycles, launch constraints, and long-term storage. A model biochip was used in this study with antibodies in freeze-dried form and under two formats: free or covalently grafted to a solid surface. We found that antibody-binding performances were not significantly affected by cosmic radiation, and more than 40% of the exposed antibody, independent of its format, was still functional during all this experiment. We conclude that antibody-based instruments are well suited for in situ analysis on planetary bodies.

Enzymatic crosslinking chemistry using horseradish peroxidase (HRP) has been widely utilized as an effective approach to fabricating injectable hydrogels with high efficiency under mild reaction conditions. However, their clinical applications are limited by the immunogenicity of the plant-derived enzyme. Herein we report the design, synthesis and characterization of HRP-immobilized porous silica particles (HRP-particles) and their use for in situ formation of HRP-free hydrogels. HRP was immobilized on aminopropyl-modified porous silica particles of 70-140 µm in diameter via poly(ethylene glycol) spacers of different molecular weights by reductive amination reaction. Two different HRP-free hydrogels based on dextran-tyramine and gelatin-hydroxyphenylpropionic acid (GHPA) conjugates were produced by passing a solution containing the conjugates and H 2 O 2 through a syringe packed with HRP-particles. The storage modulus and gelation rate of both hydrogels were tunable by varying the contact time between the polymer solution and HRP-particles. Our in vitro study revealed that HRP-free GHPA hydrogel was less stimulatory to activated mouse macrophages than HRP-containing GHPA hydrogel with the same stiffness. Furthermore, HRP-free GHPA hydrogel exhibited remarkably lower levels of local and systemic inflammation than HRP-containing one upon subcutaneous injection in immunocompetent C57BL/6J mice. The attenuated immunogenicity of HRP-free GHPA hydrogels makes them an attractive platform for tissue engineering applications. STATEMENT OF SIGNIFICANCE: The immunogenicity of HRP is a significant issue for clinical application of HRP-catalyzed in situ forming hydrogels. HRP-particles are developed to overcome the safety concerns by fabricating HRP-free hydrogels. The porosity of silica particles and molecular weight of poly(ethylene glycol) spacers are discovered as important factors determining the catalytic ability of HRP-particles to induce the in situ crosslinking of polymer-phenol conjugates. Although several articles speculate the potential of HRP to trigger immune responses when administered as a part of hydrogel formulation, no literature has attempted to investigate the immunogenicity of HRP-containing hydrogels in comparison with HRP-free hydrogels. Our findings suggest that the immunogenicity issue should be carefully considered before clinical translation of HRP-containing hydrogels., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Antibodies against citrinin (CTN) were generated from rabbits, which were injected with CTN-keyhole limpet hemocyanin (KLH). This work involved the development of a sensitive competitive direct enzyme-linked immunosorbent assay (cdELISA) and a rapid gold nanoparticle immunochromatographic strip (immunostrip) method for analyzing CTN in Monascus -fermented food. CTN at a concentration of 5.0 ng/mL caused 50% inhibition (IC 50 ) of CTN-horseradish peroxidase (CTN-HRP) binding to the antibodies in the cdELISA. The capable on-site detection of CTN was accomplished by a rapid antibody-gold nanoparticle immunostrip with a detection limit of 20 ng/mL and that was completed within 15 min. A close inspection of 19 Monascus -fermented foods by cdELISA confirmed that 14 were contaminated with citrinin at levels from 28.6⁻9454 ng/g. Further analysis with the immunostrip is consistent with those results obtained using cdELISA. Both means are sensitive enough for the rapid examination of CTN in Monascus -fermented food products.

A rapid and sensitive electrochemical biosensor based on magnetic nanoparticles and screen-printed electrodes (MNPs-SPEs sensor) was developed for the detection of ochratoxin A (OTA) in cereal and feed samples. Different types of magnetic nanoparticles-based ELISA (MNPs-ELISA) were optimized, and the signal detection, as well as sensitivity, was enhanced by the combined use of screen-printed electrodes (SPEs). Under the optimized conditions, the calibration curve of the MNPs-SPEs sensor was y = 0.3372 x + 0.8324 ( R ² = 0.9805). The linear range of detection and the detection limit were 0.01⁻0.82 ng/mL and 0.007 ng/mL, respectively. In addition, 50% inhibition (IC50) was detectable at 0.10 ng/mL. The limit of detection (LOD) of this MNPs-SPEs sensor in cereal and feed samples was 0.28 μg/kg. The recovery rates in spiked samples were between 78.7% and 113.5%, and the relative standard deviations (RSDs) were 3.6⁻9.8%, with the coefficient of variation lower than 15%. Parallel analysis of commercial samples (corn, wheat, and feedstuff) showed a good correlation between MNPs-SPEs sensor and liquid chromatography tandem mass spectrometry (LC/MS-MS). This new method provides a rapid, highly sensitive, and less time-consuming method to determine levels of ochratoxin A in cereal and feedstuff samples.

The ELISA is recognized as an efficient diagnostic tool for antibody detection, but there is no standard ELISA assay for detection of antibodies against hemorrhagic septicemia (HS) in cattle. The present study reports on an indirect ELISA assay for antibody detection of HS in dairy cows, and evaluates the sensitivity (Se) and specificity (Sp) of the method using a Bayesian approach. An indirect ELISA was developed with two types of heat extract antigens, Pasteurella multocida strains P-1256 and M-1404, as coating antigens. A checkerboard titration was employed using dairy cow sera immunized with P. multocida bacterin and colostrum-deprived calf sera. The concentrations of heat extract antigen (160μg/mL), sample serum (1:100) and goat anti-bovine immunoglobulin G labeled with horseradish peroxidase (1:2000) were optimal for the assay. The cut-off values were 0.147 and 0.128 for P-1256 and M-1404 coating antigens, and there were no differences in the results of tests with positive and negative sera (p<0.05). The characteristics of three diagnostic tests were evaluated using a one-population Bayesian model, assuming conditional dependence between two types of coating antigen-based ELISAs and indirect hemagglutination assay (IHA). A total of 415 sera samples from dairy cows without HS vaccination and no history of disease were tested. The Se and Sp of the P-1256 and M-1404 ELISAs were higher than those of the IHA. The Se and Sp of the P-1256 ELISA were 90.3% and 90.1%, while the Se and Sp of the M-1404 ELISA were 92.1% and 71.9%. The median values of Se and Sp from the IHA were 36.0% and 58.2%., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Antibody-coated surfaces (Ab surfaces) play a key role in bioanalytical tools developed for biosensors and diagnostics. Therefore, a high and well-defined functional activity of the Ab surface is required. The functional activity of the Ab surface depends on its available binding sites i.e. "the active sites" that are able to capture antigen (Ag). The number of active binding sites strongly depends on the immobilization strategy used to fix the Ab on the solid surface. Determination of layer thickness or surface topology are often used to characterize the Ab surfaces but there is no gold standard method for the study of the functionality of the Ab surfaces. For that purpose, we aim at developing an assay allowing to determine the performances of Ab surfaces. In the present study, anti-horseradish peroxidase antibody (anti-HRP Ab) is used as capture Ab covalently bound to the surface and enzyme HRP as Ag. This direct assay permits, in one-step, to generate a signal utilizing the catalytic properties of HRP. The signal is directly proportional to the amount of HRP bound on the Ab surface, and therefore to the active binding sites of immobilized Ab. The HRP/anti-HRP Ab interactions may be a useful indicator to construct accurate and reproducible active Ab surfaces and also to improve their performances in term of stability and sensitivity. Optimization of the assay parameters and quality of the results are presented. A good repeatability and an acceptable inter-day precision (RSD < 10%) are reported., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Two sisters with hirsutism presented with mild hirsutism and isolated, grossly elevated (>34.9nmol/L) serum concentrations of androstenedione measured by competitive, homogeneous immunoassay. The clinically discordant laboratory results prompted us to look for assay interference. In this immunoassay, horseradish peroxidase (HRP)-conjugated androstenedione competes with endogenous androstenedione for binding with the solid-phase polyclonal rabbit IgG antibodies. After a wash step, the amount of signal generated by the bound HRP conjugate is inversely proportional to the androstenedione concentration. Alternative analysis by tandem mass spectrometry (a good first line option for troubleshooting) and repeating the competitive immunoassay after polyethylene glycol treatment returned androstenedione concentrations within reference limits. These findings suggested that the original result was spuriously elevated due to assay interference. Additionally, the patient samples were pre-incubated with heterophile blocking reagents, normal rabbit IgG antibodies and HRP-conjugated normal goat IgG antibodies, followed by repeat measurement using the immunoassay. Only samples pre-incubated with HRP-conjugate returned significantly lower androstenedione (9.5 and 12.5nmol/L, respectively), implying neutralisation of the interfering antibodies. Androstenedione remained grossly elevated in the other experiments. This deductive exercise showed that the interference is due to autoantibodies against the HRP label used in the immunoassay. Another immunoassay using HRP label (5α-dihydrotestosterone) also produced gross elevation that was normal by tandem mass spectrometry analysis. Assay interferences, though not uncommon, are frequently overlooked. Laboratory results discordant with clinical features should prompt consideration of assay interference to avoid unnecessary investigations and treatment. This is the first report of autoantibodies against the HRP label used in immunoassay., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Bispecific antibodies (BsAbs), with the ability to recognize two different epitopes simultaneously, offer remarkable advantages in bioassays, cancer therapy, biosensors, and enzyme electrodes. Preparation and purification of BsAbs in adequate quantities remains a major hurdle in their use in various applications. Poor yield is also the principal limitation in the preparation of BsAbs by the redox procedure. IgG with reduced inter-heavy chain disulfides do not dissociate into half molecules at neutral pH. In this study, we report that the dissociation occurs in presence of sodium dodecyl sulphate (SDS) and inclusion of the detergent during the redox procedure results in remarkable increase in the formation of the BsAbs. Exposure of antibodies to 0.1% (w/v) SDS causes only minor loss in secondary/tertiary structure and the ability to bind the antigen. The BsAbs prepared using the modified redox procedure that recognize the antigens HRP and α-LA were prepared and successfully employed for detecting α-LA in milk/dairy products by ELISA and dot blot techniques. BsAbs were also prepared from partially purified immunoglobulin gamma (IgG). This work shows for the first time that SDS, by dissociating IgG with reduced inter-heavy chain disulfides into half molecules, markedly enhances the formation of BsAbs by the redox procedure.

Fucoconjugates are key mediators of protein-glycan interactions in prokaryotes and eukaryotes. As examples, N-glycans modified with the non-mammalian core α1,3-linked fucose have been detected in various organisms ranging from plants to insects and are immunogenic in mammals. The rabbit polyclonal antibody raised against plant horseradish peroxidase (anti-HRP) is able to recognize the α1,3-linked fucose epitope and is also known to specifically stain neural tissues in the fruit fly Drosophila melanogaster. In this study, we have detected and localized the anti-HRP cross-reactivity in another insect species, the malaria mosquito vector Anopheles gambiae. We were able to identify and structurally elucidate fucosylated N-glycans including core mono- and difucosylated structures (responsible for anti-HRP cross reactivity) as well as a Lewis-type antennal modification on mosquito anionic N-glycans by applying enzymatic and chemical treatments. The three mosquito fucosyltransferase open reading frames (FucT6, FucTA and FucTC) required for the in vivo biosynthesis of the fucosylated N-glycan epitopes were identified in the Anopheles gambiae genome, cloned and recombinantly expressed in Pichia pastoris. Using a robust MALDI-TOF MS approach, we characterised the activity of the three recombinant fucosyltransferases in vitro and demonstrate that they share similar enzymatic properties as compared to their homologues from D. melanogaster and Apis mellifera. Thus, not only do we confirm the neural reactivity of anti-HRP in a mosquito species, but also demonstrate enzymatic activity for all its α1,3- and α1,6-fucosyltransferase homologues, whose specificity matches the results of glycomic analyses., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Thioglycolic acid modified superparamagnetic iron oxide nanoparticles (TG-APTS-SPION) were synthesized by using (3-aminopropyl) triethoxysilane (APTS) and thioglycolic acid (TG). Actin was immobilized on the nanoparticle surfaces. Binding amount of the actin (Act) on TG-APTS-SPIONs was determined by using a calibration curve equation that was drawn using fluorescence spectra at 280 and 342 nm of excitation and emission wavelengths. Anti-Actin (anti-Act) was interacted with the actin immobilized TG-APTS-SPIONs as primary antibody. Horse radish peroxidase (HRP) was also interacted with antibody conjugated nanoparticles as secondary antibody. The binding capacity of primary and secondary antibodies was also estimated by fluorescence spectroscopy. Scanning electron microscopy (SEM), Infrared spectroscopy (FTIR) and energy dispersive X-ray (EDX) analysis were also clarified binding of the protein and antibodies to the nanoparticles' surfaces. Western blot analysis was also done for actin conjunction with anti Act antibody to confirm binding of the antibody to the protein., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The multikinase inhibitor sorafenib has been used in the treatment of hepatocellular carcinoma, renal cell carcinoma, and differentiated thyroid carcinoma. Here we have demonstrated the production of the first specific antibody against sorafenib. Anti-sorafenib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and carboxylic modified 4-(4-aminophenoxy)-N-methyl-2-pyridinecarboxamide (AMPC) using the N-succinimidyl ester method. Enzyme labeling of sorafenib with horseradish peroxidase was similarly performed using carboxylic modified AMPC. A simple competitive enzyme-linked immunosorbent assay (ELISA) for sorafenib was developed using the principle of direct competition between sorafenib and the enzyme marker for anti-sorafenib antibody, which had been adsorbed by the plastic surface of a microtiter plate. Serum sorafenib concentrations lower than 0.04 µg/mL were reproducibly measurable using the ELISA. This ELISA was specific to sorafenib and showed very slight cross-reactivity (2.5%) with a major metabolite, sorafenib N-oxide. The values of serum sorafenib levels from 32 patients measured by this ELISA were comparable with those measured by HPLC, and there was a strong correlation between the values determined by the two methods (Y=1.016X-0.137, r=0.979). The specificity and sensitivity of the ELISA for sorafenib should provide a valuable new tool for use in therapeutic drug monitoring and pharmacokinetic studies of sorafenib.

Dextran as anti-nutritional factor is usually a result of bacteria activity and has associated serial problems during the process stream in the sugar industry and in medical therapy. A sensitive method is expected to detect dextran quantitatively. Here we generated four monoclonal antibodies (MAbs) against dextran using dextran T40 conjugated with bovine serum albumin (BSA) as immunogen in our lab following hybridoma protocol. Through pairwise, an MAb named D24 was determined to be conjugated with horseradish peroxidase (HRP) and was used in the establishment of a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) method for determination of dextran, in which MAb D9 was chosen as a capture antibody. The detection limit and working scope of the developed sandwich ELISA method were 3.9 ng/mL and 7.8-500 ng/mL with a correlation coefficient of 0.9909. In addition, the cross-reaction assay demonstrated that the method possessed high specificity with no significant cross-reaction with dextran-related substances, and the recovery rate ranged from 96.35 to 102.00%, with coefficient of variation ranging from 1.58 to 6.94%. These results indicated that we developed a detection system of MAb-based sandwich ELISA to measure dextran and this system should be a potential tool to determine dextran levels.

Spatially resolved electron energy loss spectroscopy (SR-EELS) using scanning transmission electron microscope (STEM) allows the identification and determination of the spatial distribution of the components/elements of immuno-functionalized core-shell superparamagnetic magnetite nanoparticles. Here, we report that SR-EELS measurements allow the direct identification and study of the biological moieties (protein G and anti-HRP antibody) in complex bionanocarriers of relevance for biomedical applications. Our findings show that the biomacromolecules are located on specific areas on the nanoparticles' surface. In addition, efficiency of this functionalization was evaluated by means of biochemical techniques.

In this study, a novel signal-amplified electrochemical immunosensor was proposed by using TiO(2) nanotube (TiNT) arrays as the platform. Due to the distinct tubular features-large surface area, high pore volume and good electrochemical conductivity, the TiNT based electrodes exhibited excellent signal-amplified effects. gold nanoparticle (AuNP) was further utilized to bind horseradish peroxidase (HRP) tagged antibodies as recognition elements. Compared to the immunosensor based on either flat electrode, the immunosensors using TiNT layer as electrode showed higher amplified electrochemical signals from the catalytic reaction of HRP relative to hydrogen peroxide (H(2)O(2)). Under optimal conditions, the proposed immunosensor exhibited a good electrochemical behavior to antigen in a concentration range from 0.1 ng mL(-1) to 10(5) ng mL(-1) with a detection limit of 0.01 ng mL(-1). The results showed that the TiNT-based electrochemical immunosensing platform could provide a great potential in clinical application for detection of low-abundant proteins., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A hen's egg is one of the most common causes of food allergy. The allergen quantitation in hot-processed food is always a difficult task, because the protein in these samples will be denatured, insoluble, and degraded. This article presents a competitive enzyme linked immunosorbent assay (ELISA) for the quantitation of ovalbumin in hot-processed food. Its recovery was improved nearly two times by the assay method as compared with previous sandwich ELISA. The heat and DL-Dithiothreitol treated ovalbumin was used as antigen for monoclonal antibody preparation. A smaller labeled antibody molecule, horseradish peroxidase (HRP) labeled Fab' fragment, was used to replace IgG in ELISA for improving sensitivity and analytical speed of the method. A binding site protection procedure was developed for Fab' fragment labeling with HRP, which prevented damage to the Fab' binding site. The combination and separation steps were efficiently completed in an affinity spin column. Based on the optimized ELISA condition, the IC50 was 1.2 µg/mL and the coefficients of variation were less than 10%.

Multiple antibody immobilization methodologies have been developed for several applications including affinity chromatography, immunosensing, and drug delivery. Most of them have been carried out without considering the orientation of the antigen binding site of the antibody, or after the chemical modification of the antibody. An efficient immobilization to improve the biological activity of the antibody is one of the key fundamental issues to pursue. A simple and effective methodology for well-oriented covalently immobilization of antibodies on nanoparticles is reported in this chapter.

Modification of protein and other biopolymers by labeling them with small or macromolecules has become a very powerful research tool in biochemistry, molecular biology, diagnostics, and therapeutics. However, current methodologies available for their preparations are not straightforward and take several hours of incubation time. In this paper, we describe a new filtration-assisted technique for covalent conjugation between the reactive functional groups of two different molecules (small or macromolecules). Compared to the current method, this new approach significantly reduces the total reaction time from several hours to just a few minutes. The technique has been used for the preparation of conjugates of a small molecule to a protein such as biotin-BSA conjugate or small molecules to a small molecule such as biotin-tyramine conjugate or protein-protein conjugation such as antibody-horseradish peroxidase conjugate. The procedure consists of filtering the reaction mixture multiple times through membrane micropores with the help of two syringes, which make the cross filtration process less laborious. The method saves time, allows conjugation of less than 1mg protein and produces conjugates better than those obtained by the current methods. Although the present technique has been applied on some common conjugation methods, it provides a potentially general method, and may further be expanded for the synthesis of several other macromolecular conjugates., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this study, we successfully developed a novel lectin enzyme-linked immunosorbent assay (lectin ELISA) for the detection of glycosides linked to carcinoembryonic antigen (CEA) as a model antigen using a scFv-immobilized hydrophilic polystyrene (phi-PS) plate and 12 different HRP-labeled lectins. Anti-CEA scFv genetically fused with a PS-tag at its C-terminus (scFv-PS) was successfully over-expressed in an insoluble fraction by cultivation of recombinant E. coli. A solid-phase refolding method was adopted to immobilize and refold scFv-PS on the surface of the phi-PS plate. Consequently, in sandwich ELISA using phi-PS plates immobilized with scFv and scFv-PS as well as Maxisorp™ plate with whole antibody (whole Ab), the highest sensitivity and S/N ratio were obtained from the scFv-PS-immobilized phi-PS plate as it was the antigen-binding domain with the highest surface immobilization density and remaining activity displayed on the phi-PS surface. During the lectin ELISA, high background signals were detected from the whole-Ab-immobilized Maxisorp™ plate, indicating that HRP-labeled lectins, particularly PHA-E, Con A, LCA, PSA, DSA, and MAA, directly recognized the glyco-chains of a whole Ab. However, a considerable amount of low background signals were detected from the scFv-PS-immobilized plate since the ligand antibody, namely scFv-PS, was produced by E. coli cells that had no potential for glycosylation during the process of post-transcriptional modification. Signals for glyco-chains conjugated with CEA were detectable using 6 kinds of HRP-labeled lectins, namely PHA-E, PHA-L, SBA, Con A, LCA, and DSA, with much higher sensitivities and S/N ratios. Thus, the lectin ELISA using scFv-PS as a ligand was considerably useful for detecting the glyco-chains of glycoproteins, including glyco-biomarkers, with much higher sensitivities and S/N ratios compared with a conventional whole Ab. By preparation of a phi-PS plate immobilized with different species of scFvs, this method is capable of the glyco-chain analysis of a number of glyco-biomarkers in the fields of clinical diagnosis and biochemical research., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Nanospheres lithographic (NSL) method has been used to fabricate nano-structured arrays (NAs) of hexagonally close-packed gold (Au) using polystyrene beads [PS, diameter ∼300 nm] as mask. The developed NA was incorporated with a customized and cheap microfluidics system to demonstrate its applicability as an alternative easy and efficient platform for multiplex analysis and Lab-on-a-Chip applications. The chip functionality was demonstrated with horseradish peroxidase (HRP) and anti-HRP antibody as model for recognition system. The enzyme-linked immunosorbent assay (ELISA) performed on fabricated protein biochip had a detection limit 100 pg/mL for HRP. The antibody chip was also checked for the shelf-life and it was found that these chips could be stored for 50 days when stored at 4°C without any significant loss of activity. Therefore, NAs based protein biochip with the correct microfluidics could find huge potential application in diagnostics and biosensing technology., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Influenza virus attachment to sialic acid-containing molecules on the cell surface initiates the infection. The spectrum of functional receptors on target cells and decoy receptors on cells and epithelial mucus varies substantially between animal species leading to variations in the receptor-binding specificity of viruses circulating in these species. Analysis of the receptor specificity of different animal and human influenza viruses can give insight into factors and mechanisms that determine viral host range, tissue and cell tropism, replication efficiency, and pathogenesis. Knowledge of viral receptor specificity may also be useful for the development of more efficient influenza vaccines and anti-influenza drugs.A majority of known receptor specificity assays measure influenza virus binding to sialic acid-containing natural and synthetic compounds (receptor analogues). Here, we describe protocols of two solid-phase enzyme-linked receptor-binding assays which are technically similar to standard ELISA. Each assay determines binding of the virus immobilized in the wells of 96-well plate to receptor analogues in solution. In the direct binding assay, the virus binds to either synthetic biotinylated sialylglycopolymers or to peroxidase-labeled sialylglycoprotein fetuin (Fet-HRP); the apparent association constants of the virus-receptor complexes are calculated from the Scatchard plots of the binding data. In the fetuin-binding inhibition assay, the virus is incubated with a mixture of unlabeled receptor analogue and standard preparation of Fet-HRP; the association constant for analogue is calculated based on the level of its competition with Fet-HRP.

To overcome the sensitivity limit for small molecules (haptens) in immunoassays based on antigen-horseradish peroxidase (HRP) conjugates as labels, a novel approach was established that afforded very low detection limits. Biotinylated anti-HRP antibody was utilized in order to attach, via a streptavidin bridge, liposomaly entrapped HRP. Fentanyl, used as a model antigen, could be determined via the generation of a high-intensity and relatively stable chemiluminescence (CL) signal of a HRP-catalyzed luminol/H(2)O(2)/enhancer system, immediately after the addition of a substrate solution. 4-(1-Imidazolyl)phenol (4-IMP) was used as an enhancer, and the outcome of this combination was a very low detection limit (0.895 pg mL(-1)) in plasma samples. The respective detection limit with the use of just the classical HRP-antigen conjugate was > 5-times higher. Intra- and inter-assay RSDs of the novel assay were 6.8 - 9.9 and 11 - 17%, respectively. The proposed method could be utilized for a wide range of molecules without replacing existing antigen-HRP based kits.

Finding a general solution for optimizing the grafting of antibody on solid surfaces is difficult due to the variety of material, grafting principles and chemistries or surface formats available (beads, microplates, fibers, etc.). Pre-screening methods able to assess grafting efficiency (GE) and specific activity (SA) are required. In this context, we present here two colorimetric assays that can be used on a wide variety of surface format, chemistry, etc. The first one, ADECA (Amino Density Estimation by Colorimetric Assay) allows a rapid estimation of grafted antibodies and allows calculating the GE. The second one, A(2)HRP (Antibody Anti-HorseRadish Peroxidase) provides a measure of the amount of active antibody, which, combined to ADECA, is used to determine the SA of grafted antibody. Analytical parameters (limit of detection, repeatability, linearity, etc.) of these two colorimetric assays are presented. Using two commercially available microplates, we demonstrated that, when used in parallel, these rapid and sensitive methods are well adapted to pre-screening of antibody grafting performances., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Four biotinylated tri and tetrasaccharide fragments of plant and invertebrate N-glycans were synthesized using methyl tert-butyl phenyl (MBP) thioglycosides donors in order to evaluate their involvement in cross-allergies as cross-reactive carbohydrate determinants (CCDs). Various levels of reactivity to anti-bee and anti-HRP antibodies and with sera from allergic patients were observed when the conjugates were coated on streptavidin microplates. The results showed the potential utility of these xylosylated and fucosylated oligosaccharide fragments in determining CCD antibody epitopes., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Background: In hymenoptera venom allergy, about 75% of detected in vitro double positivity to yellow jacket and honeybee venom is ascribed to specific IgE (sIgE) directed against cross-reactive carbohydrate determinants (CCDs). To date, for the detection of CCD-sIgE, different carbohydrate antigens and methods are used. The most suitable one still has to be identified., Methods: Eighty-seven patients with confirmed hymenoptera venom allergy and venom sIgE values of ≥0.7 kU/l were investigated. Sixty-five patients showed sIgE reactivity to both yellow jacket and honeybee venom, 22 were venom mono positive and served as controls. Occurrence of CCD-sIgE was determined using bromelain, horseradish peroxidase (HRP) and MUXF(3) on system A, and ascorbic acid oxidase (AAO), bromelain and HRP on system B. Further, a reference standard for CCD-sIgE evaluation was created: CCD positivity was assumed when at least 4 of the 6 test results were positive., Results: According to the defined reference standard, 45/65 venom double positive patients exhibited CCD-sIgE. Using system A, comparison with the reference standard revealed sensitivity and specificity values of 96 and 97%, respectively, for MUXF(3), 100 and 100%, respectively, for bromelain, and 96 and 97%, respectively, for HRP. Using system B, sensitivity and specificity was 98 and 97%, respectively, for AAO, 62 and 95%, respectively, for bromelain, and 96 and 69%, respectively, for HRP. Results of the 3 test allergens obtained with system A showed strong correlations (r = 0.932-0.976), whereas results with system B showed lower correlations (r = 0.714-0.898)., Conclusions: All 3 test allergens used with system A are suitable for CCD-sIgE detection in hymenoptera venom allergy. With system B, only AAO seems to be a reliable tool., (Copyright © 2011 S. Karger AG, Basel.)

Background: The purpose of this study was to devise a novel electrochemical immunosensor for ultrasensitive detection of alfa-fetoprotein based on Fe(3)O(4)/Au nanoparticles as a carrier using a multienzyme amplification strategy., Methods and Results: Greatly enhanced sensitivity was achieved using bioconjugates containing horseradish peroxidase (HRP) and a secondary antibody (Ab(2)) linked to Fe(3)O(4)/Au nanoparticles (Fe(3)O(4)/Au-HRP-Ab(2)) at a high HRP/Ab(2) ratio. After a sandwich immunoreaction, the Fe(3)O(4)/Au-HRP-Ab(2) captured on the electrode surface produced an amplified electrocatalytic response by reduction of enzymatically oxidized hydroquinone in the presence of hydrogen peroxide. The high content of HRP in the Fe(3)O(4)/Au-HRP-Ab(2) could greatly amplify the electrochemical signal. Under optimal conditions, the reduction current increased with increasing alfa-fetoprotein concentration in the sample, and exhibited a dynamic range of 0.005-10 ng/mL with a detection limit of 3 pg/mL., Conclusion: The amplified immunoassay developed in this work shows good precision, acceptable stability, and reproducibility, and can be used for detection of alfa-fetoprotein in real samples, so provides a potential alternative tool for detection of protein in the laboratory. Furthermore, this immunosensor could be regenerated by simply using an external magnetic field.

Introduction of spacers in enzyme conjugates is known to exert an influence on the assay parameters of steroid enzyme immunoassays. We have introduced 3 to 10 atomic length linkers between enzyme and steroid moieties and studied their effects on sensitivity and specificity of dehydroepiandrosterone enzyme immunoassays. Dehydroepiandrosterone-17-carboxymethyloxime-bovine serum albumin (DHEA-17-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using DHEA-7-CMO as carboxylic derivative of DHEA and horseradish peroxidase (HRP) as label. These were DHEA-7-CMO-HRP, DHEA-7-CMO-urea-HRP (DHEA-7-CMO-U-HRP), DHEA-7-CMO-ehylenediamine-HRP (DHEA-7-CMO-EDA-HRP), DHEA-7-CMO-carbohydrazide-HRP (DHEA-7-CMO-CH-HRP), and DHEA-7-CMO-adipic acid dihydrazide-HRP (DHEA-7-CMO-ADH-HRP). The influence of different atomic length linkers on sensitivity and specificity were studied with reference to label without linker. The results of the present investigation revealed that with incorporation of linkers, the sensitivity improves, whereas specificity only marginally improves. These differential behaviors of various linkers toward the sensitivity and specificity of assays might be due to the difference in the magnitude of overall forces of attraction between the antibody and the enzyme conjugates.

Norethisterone is one kind of widely used anabolic steroid hormones which can help to promote livestock growth and sometime has been illegally used for livestock breeding. The residues of norethisterone in animal food will harm people's health, therefore, it has been banned to use for the growth promotion purposes in livestock. In this study, amino-group functionalized mesoporous silica nanoparticles (MSN) were prepared and used to immobilize Au nanoparticles, which was further utilized for the adsorption of horseradish peroxidase (HRP) and the secondary antibody (Ab2). The resulting nanoparticles, Au-MSN-HRP-Ab2 were used as labels for immunosensors to detect norethisterone antigen. A sandwich-type protocol was used to prepare the immunosensor with the primary antibody (Ab1) immobilized onto thionine (TH) and graphene sheet (GS) modified glassy carbon electrode surface. The sensitivity of the sandwich-type immunosensor using Au-MSN-HRP-Ab2 as labels for norethisterone antigen detection was much higher than that using either Au-MSN-Ab2 or MSN-HRP-Ab2 as labels. Within norethisterone concentration range (0.01-10 ng/mL), a linear calibration plot (Y=0.55671+8.27101X, r=0.9993) was obtained with a detection limit of 3.58 pg/mL under optimal conditions. The proposed immunosensor shows good reproducibility, selectivity, and acceptable stability. This new type of labels for immunosensors may provide many potential applications for the detection of growth hormone in animal derived food., (Copyright © 2010 Elsevier B.V. All rights reserved.)

A new electrochemical immunoassay protocol for the determination of carcinoembryonic antigen (CEA) in human serum was developed by means of immobilizing horseradish peroxidase-labeled anti-CEA antibodies (HRP-anti-CEA) on the nanogold/nano-Fe(3)O(4) particles-functionalized polyion complex (PIC) membrane. With a one-step immunoassay format, the formed immunocomplex inhibited partly the bioactive center of the immobilized HRP, and decreased the HRP toward the reduction of H(2)O(2). Nanogold-actuated nano-Fe(3)O(4) mimetic peroxidase was used for the sensitivity improvement of the electrochemical immunosensor with Prussian blue (PB) as mediators. Under optimal conditions, the electrochemical immunosensor displayed a wide dynamic range of 0.1-220 ng mL(-1) with a relative low detection limit (LOD) of 10 pg mL(-1) CEA (at 3σ). Intra- and inter-assay show good precisions with a coefficient of variation (CV) lower than 7.8% and 7.3%, respectively. No significant difference at the 95% confidence level was encountered in the analysis of 17 human serum specimens between the electrochemical immunosensor and automatic electrochemiluminescent (ECL) analyzer for the determination of CEA., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Few common carbohydrate epitopes consisting of terminal beta-(1,2)-xylose and/or alpha-(1,3)-fucose residues are shared by a variety of glycoproteins from plants, insects and parasitic worms, termed cross-reactive carbohydrate determinant (CCD), and frequently recognized by IgE antibodies of patients with food and/or respiratory allergy, though clinical relevancy of such CCD-specific IgE is still controversial. Attention has also been focused on CCDs from the undesired post-translational modification of recombinant therapeutic proteins produced by transgenic plants and insects. In the present study, to clarify immunogenic potentials of CCD-bearing glycoproteins, the antibody response to a model plant glycoprotein, horseradish peroxidase (HRP) was investigated in a mouse model. C3H/He mice were immunized with HRP plus Al(OH)(3) or Freund's adjuvant, and IgG and IgE responses to CCDs in addition to HRP were analyzed by ELISA using some distinct glycoproteins with known N-glycan structures. IgE response to HRP was induced remarkably, whereas that to CCD was weaker and delayed. Moreover, apparent ratio of the CCD-specific antibodies to HRP-specific ones tended to be higher in IgG2a and IgG2b isotypes than IgG1, IgG3 and IgE. In contrast to rabbit antibodies, the CCD-specific antibodies from the mice gave poor reactivity with bromelain and honeybee phospholipase A2, suggesting the critical role of both beta-(1,2)-xylose and alpha-(1,3)-mannose in the CCD-recognition by the mouse antibodies. Moreover, the mouse antibodies showed weaker cross-reactivity to pollen- and insect-derived glycoproteins than the rabbit ones. Thus, in this mouse model, not only IgE but also IgG2 antibody responses to CCDs were induced by immunizing with a CCD-bearing glycoprotein, suggesting that CCDs affected not only Th2-type but also Th1-type antibody response at least in C3H/He mice., (Copyright 2010 Elsevier B.V. All rights reserved.)

Various types of microplates made of polystyrene have been widely used for immunoassays. A new microplate suitable for the covalent immobilization of proteins and peptides was developed by thermal deposition of amino-modified parylene (parylene-A) on the microplate. The primary amine groups of the parylene-A was exploited for the covalent coupling of proteins and peptides. The optical transmittance at the wavelength of 400-500 nm was estimated to be suitable for the application to immunoassays. The immobilization efficiency of the parylene-A coated microplate was demonstrated to be far improved in comparison to the conventional microplate by using horseradish peroxidase (HRP), anti-HRP antibody and a peptide with 9-residues as model biomolecules., (2009 Elsevier B.V. All rights reserved.)

A highly sensitive protein detection method based on a novel enzyme-labeled gold nanoparticle (AuNP) probe has been developed. In this method, we firstly prepared the enzyme-labeled AuNP probe by coating AuNP with antibody, single-stranded DNA (ssDNA), and horseradish peroxidase (HRP). Magnetic microparticle (MMP) functionalized with another antibody was used as capture probe. Then, target protein was sandwiched by the enzyme-labeled AuNP probe and the capture probe through immunoreaction. The target immunoreaction event could be sensitively transduced via the enzymatically amplified optical signal. By using this strategy, carcinoembryonic antigen (CEA), as a model protein, was detected with high sensitivity and good specificity. The detection limit for this approach was 12 ng L(-1), which was approximately 130-fold more sensitive than the conventional enzyme-linked immunosorbent assay (ELISA). The practical application of the proposed immunoassay was carried out for determination of CEA in serum samples. The demonstrated capability of the proposed method shows potentially applications for early diagnoses of diseases.

A dual-label time-resolved fluoroimmunoassay (TRFIA) for simultaneous quantification of aflatoxin B1 (AFB1) and ochratoxin A (OTA) is described. For this, microtitration wells were coated with AFB1-horse radish peroxidase (HRP) and OTA-bovine serum albumin. The standards and samples were loaded on the coated plates, and diluted antibodies and Eu3+- and Sm3+-labeled IgG were then added. Our results showed that the sensitivity of TRFIA for AFB1 was 0.02 microg/L (range 0.02-100 microg/L). The intra- and inter-batch coefficient of variation (CV) was 3.2 and 7.3%, respectively, and the average recovery rate was 88.1%. On the other hand, the sensitivity of OTA was 0.05 microg/L (range 0.05-50 microg/L), the intra- and inter-batch CV was 2.9 and 7.9%, respectively, and the average recovery rate was 89.9%. In the AFB1/OTA-TRFIA, AFB1 and OTA did not mutually interfere. The correlation coefficients between the dual-label AFB1/OTA-TRFIA and the single-label AFB1-TRFIA or OTA-TRFIA were 0.972 and 0.981, respectively, indicating that the results were consistent. Our study suggests that AFB1/OTA-TRFIA allows the simultaneous detection of AFB1 and OTA; is a simple, fast, and economic method for screening large quantities of samples, and has good prospects of application.

In this work, the biospecific recognition antigen-antibody reaction was implemented in gravitational field-flow fractionation (GrFFF), a flow-assisted separation technique for micron-sized particles, in order to realize a hybrid immunomodulated GrFFF system in which two different principles are combined to achieve highly versatile fractionation. Micron-sized polystyrene beads coated with horseradish peroxidase (HRP) were used as a model sample, and anti-HRP antibodies were immobilized on the accumulation wall of the GrFFF channel. Ultrasensitive chemiluminescence imaging was employed to visualize the beads during elution and to optimize experimental conditions. The same principle was then applied to real biological samples composed by Yersinia enterocolitica and Escherichia coli cells. Results show the possibility to modify the elution of selected sample components and even to retain them into the channel. The hybrid immunomodulated GrFFF system is a step towards the development of a module that could be integrated in a lab-on-a-chip-based point-of-care testing device which includes sample pre-analytical cleanup and analysis.

Antibodies are very often used as specific cell and/or tissue markers. An example of this is anti-horseradish peroxidase (HRP), an antibody raised against a plant glycoprotein, which was shown some twenty-five years ago to specifically stain neural tissue in an animal, Drosophila melanogaster. This peculiar finding was later expanded to other invertebrate species including Caenorhabditis elegans, which were also shown to bear anti-HRP epitopes. Initial experiments indicated that the epitopes recognised by anti-HRP in invertebrates are of carbohydrate nature. Indeed, more recent experiments have characterised relevant core alpha1-3-fucosylated N-glycan structures that act as epitopes in various model and parasitic organisms. Moreover, a number of enzymes required for the synthesis of such structures have been identified. Over the years, medically-relevant roles of these structures have become apparent as regards allergenicity and immunoregulation. Although major advances have been made in understanding of the underlying mechanisms and structures related to the anti-HRP epitope, the in vivo role of the relevant epitopes in neural and other tissues is yet to be resolved. Current understanding of the anti-HRP epitopes synthesis and their relevance is discussed and elaborated.

The beta(1,2)-xylose- and/or alpha(1,3)-fucose-containing cross-reactive carbohydrate determinants (CCDs) are present in various plant and insect N-glycans, and have been attracted as potential antigens in IgE-mediated allergies and immunologically undesired post-translational products on some recombinant therapeutic proteins. By using ELISA and immunoblotting, CCDs-specific IgG and IgE antibodies from some, but not all, of mice and humans were found to fully retain their binding activity after a typical periodate-treatment to CCDs, which did cause the CCDs' antigenic activity to those from the other mice and rabbits to disappear almost completely. Furthermore, the mouse IgE recognizing the periodate-resistant CCDs induced the CCDs/IgE-dependent degranulation of rat basophilic RBL-2H3 cells. These findings indicate that in some cases CCDs include those dependent of the core trisaccharide more strongly than the terminal xylose and fucose, which might have been screened out in the CCDs analyses based on the loss of antibody-binding by the periodate-treatment.

The enzyme-labeled antigen method is a histochemical technique that visualizes antigen-specific antibody-producing cells in tissue sections, originally documented in 1968. In this study, we attempted to reemerge this hidden but potentially useful method in rat models immunized with horseradish peroxidase (HRP), ovalbumin (OA), or keyhole limpet hemocyanin (KLH). After repeated immunization in footpads, popliteal, groin, and axillary lymph nodes and spleen were sampled. Paraformaldehyde-prefixed frozen sections were incubated with HRP, biotinylated OA, or biotinylated KLH. Proteinase K pretreatment and the secondary use of HPR-labeled streptavidin were applied in the latter two situations. Plasma cells producing antigen-specific antibodies were visualized. Proportions of antigen-specific antibody-producing cells in total plasma cells shown with the immunoperoxidase method for rat immunoglobulins were evaluated. The percentage of antigen-specific plasma cells reached approximately 50% of total plasma cells in the regional lymph nodes. The specificity was confirmed by (a) negativity in non-immune rat tissue, (b) negativity with indifferent antigen probes, and (c) abolishment of the reactivity with the corresponding rat serum. In buffered formalin-fixed, paraffin-embedded tissues, fewer plasma cells were labeled for HRP and KLH antibody reactivity after strong proteolysis and prolonged incubation. Expectedly, this method allows us to observe antigen-specific antibody-producing cells under varied pathological conditions.

Arrestins mediate G protein-coupled receptor desensitization, internalization, and signaling. Dopamine D(2) and D(3) receptors have similar structures but distinct characteristics of interaction with arrestins. The goals of this study were to compare arrestin-binding determinants in D(2) and D(3) receptors other than phosphorylation sites and to create a D(2) receptor that is deficient in arrestin binding. We first assessed the ability of purified arrestins to bind to glutathione transferase (GST) fusion proteins containing the receptor third intracellular loops (IC3). Arrestin3 bound to IC3 of both D(2) and D(3) receptors, with the affinity and localization of the binding site indistinguishable between the receptor subtypes. Mutagenesis of the GST-IC3 fusion proteins identified an important determinant of the binding of arrestin3 in the N-terminal region of IC3. Alanine mutations of this determinant (IYIV212-215) in the full-length D(2) receptor generated a signaling-biased receptor with intact ligand binding and G-protein coupling and activation, but deficient in receptor-mediated arrestin3 translocation to the membrane, agonist-induced receptor internalization, and agonist-induced desensitization in human embryonic kidney 293 cells. This mutation also decreased arrestin-dependent activation of extracellular signal-regulated kinases. The finding that nonphosphorylated D(2)-IC3 and D(3)-IC3 have similar affinity for arrestin is consistent with previous suggestions that the differential effects of D(2) and D(3) receptor activation on membrane translocation of arrestin and receptor internalization are due, at least in part, to differential phosphorylation of the receptors. In addition, these results imply that the sequence IYIV212-215 at the N terminus of IC3 of the D(2) receptor is a key element of the arrestin binding site.

The light output of the peroxidase-catalyzed luminol chemiluminescent oxidation reaction can be greatly increased by incorporating different enhancers. Such an increase is attributed to the preferential oxidation of the enhancer by peroxidase intermediates and the rapid formation of enhancer radicals that, in turn, quickly oxidize luminol to its radical anion. These enhancers, which include substituted phenols, substituted boronic acids, indophenols, and N-alkyl phenothiazines, behave as electron transfer mediators. A further, very significant increase in light output was also observed by the addition of nucleophilic acylation catalyst to the enhancer/luminol/oxidant substrate. The effect of the new component is general and applicable to many of the known enhancers but is much more remarkable in association with phenothiazine enhancers (up to 10-fold light output). The addition of a nucleophilic acylation catalyst to these substrates lowered the limit of detection for horseradish peroxidase from 50 to 8 amol. Similar improvements were observed in "sandwich" enzyme-linked immunosorbent assays and Western blot assays.

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.

Immobilization of antibodies by their oxidized sugar chain on aminated supports is a very efficient methodology to have a properly oriented antibody. However, these supports may behave as anionic exchangers, producing the unspecific adsorption of other proteins and reducing the selectivity of the system. To overcome this problem, we have proposed two solutions based in tailor-made support surfaces to immobilize antihorseradish peroxidase (HRP). The first solution was the use of supports having a very low amount of amino groups. These amino groups need to be very reactive with the aldehyde groups generated in the protein sugar chains to be efficient. Using supports having 7 micromol EDA/g (e.g., ethylenediamine modified glyoxyl-agarose), the antibody may be immobilized, keeping over 90% of the anti-HRP functionality. Second, by mixing amino groups and carboxylic groups, a neutral surface of the support has been generated. Again, this support has been unable to adsorb proteins while oxidized anti-HRP could be immobilized, giving functional anti-HRP antibodies. Both preparations retained 100% functionality after 2 months of storage at 4 degrees C. This way, the tailoring of the support surfaces has permitted solving some limitations of the immobilization of sugar-chain oxidized antibodies on primary amino supports.

Using a homologous combination of immunogen and enzyme conjugate, a highly specific and sensitive Enzyme Linked Immunosorbent Assay (ELISA) was developed to measure 17-alpha-hydroxy-progesterone (17-alpha-OH-P) in human serum. The antiserum was raised against 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime bovine serum albumin (17-alpha-OH-P-3-O-CMO-BSA) in New Zealand white rabbits. The enzyme conjugate was prepared by labeling 17-alpha-hydroxy-progesterone-3-O-carboxymethyloxime with horseradish peroxidase (HRP). Checkerboard assay was performed to determine the working dilutions of antiserum and enzyme conjugate. Dose-response studies were carried out by incubating 25 microL enzyme conjugate along with 50 microL of standards on the primary antibody coated wells for 1 hour. The bound enzyme activity was measured colorimetrically using Tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The enzyme substrate reaction was terminated with 100 microL of 0.5 M H2SO4 after 20 min and the intensity of the color was measured using Tecan ELISA reader at 450 nm. The assay was validated in terms of sensitivity, specificity, precision and recovery. The detection limit of the assay was 180 pg/mL. The assay was more specific as compared to most other reported immunoassays for 17-alpha-OH-P. Cross reaction with analogous C18, C19, and C21 steroids was less than 0.1% except for progesterone which showed 2.1% cross reaction. The intra- and inter-assay coefficients of variation ranges from 3.7-7.5% and 6.9-11.7%, respectively. The developed ELISA correlated well with established RIA, with a correlation coefficient of 0.9 (n=30).

Escherichia coli O157:H7 is a serious human pathogen that causes hemorrhagic colitis, and occasionally hemolytic uremic syndrome. Identification of the O157 antigen is an essential part of the detection and management of E. coli O157:H7. A quadroma P126 secreting a bispecific hybrid MAb (bsMAb), which recognizes both E. coli O157 and horseradish peroxidase in one molecule was produced by somatic hybridization of hybridomas specific for E. coli O157 and HRPO molecule. A bridge ELISA was used to select the quadromas obtained for bispecific monoclonal antibody purification and characterization. Benzhydroxamic-acid agarose (BHA) affinity co-chromatography was used as a convenient one-step method for purifying the HRPO-bsMAb complex for ultrasensitive diagnostic applications. Sandwich ELISA for detecting E. coli O157:H7 with HRPO-bsMAb allows quick one step detection of spiked E. coli O157:H7. The detection sensitivities were 100 CFU, 750 CFU and 500 CFU per 1 ml of tap water, lake water and apple juice respectively by microtiter assay. E. coli O157:H7 detection with immunofilter ELISA and immunomagnetic ELISA formats was approximately 1 CFU/ml and 10 CFU/ml respectively. BsMAbs avoid enzyme conjugation, has highest specific activity and molecular uniformity without aggregates and contribute to good signal to noise ratios. This new bispecific antibody can be generated and purified from quadroma cultures by affinity co-chromatography in one step and can be used to develop a new generation of assays for public health applications in water, food and human sample testing.

The multifunctional protein, beta-catenin, has essential roles in cell adhesion and, through the Wnt signaling pathway, in controlling cell differentiation, development, and generation of cancer. Could distinct molecular forms of beta-catenin underlie these two functions? Our single-molecule force spectroscopy of armadillo beta-catenin, with molecular dynamics (MD) simulation, suggests a model in which the cell generates various forms of beta-catenin, in equilibrium. We find beta-catenin and the transcriptional factor Tcf4 form two complexes with different affinities. Specific cellular response is achieved by the ligand binding to a particular matching preexisting conformer. Our MD simulation indicates that complexes derive from two conformers of the core region of the protein, whose preexisting molecular forms could arise from small variations in flexible regions of the beta-catenin main binding site. This mechanism for the generation of the various forms offers a route to tailoring future therapeutic strategies.

Recent studies have shown that, in mast cells, membrane microdomains rich in cholesterol and glycosphingolipids called lipid rafts play an important role in FcepsilonRI signaling. The present study demonstrates that, in RBL-2H3 cells following stimulation, the mast cell-specific gangliosides associated with FcepsilonRI are internalized from lipid rafts along with the receptor. When the cells are labeled with iodinated antibodies against the gangliosides or against FcepsilonRI and the cell components are then fractionated on Percoll density gradients, in stimulated cells the gangliosides are internalized with the same kinetics as FcepsilonRI and at 3 hr are present in the dense lysosome fraction. Using transmission electron microscopy, with antibody against the gangliosides conjugated to horseradish peroxidase and antibody against FcepsilonRI conjugated to colloidal gold, it was possible to demonstrate that the gangliosides and FcepsilonRI are internalized in the same coated vesicles. At 5 min, the gangliosides and FcepsilonRI can be identified in early endosomes and at 3 hr are found together in acid phosphatase-positive lysosomes. This study demonstrates that the mast cell-specific gangliosides are internalized from lipid rafts in the same vesicles and traffic intracellularly with the same kinetics as FcepsilonRI. This study contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.