Immunochemical Reaction Research Articles

Simplified calibration and analysis on screen-printed disposable platforms for electrochemical magnetic bead-based inmunosensing of zearalenone in baby food samples

An electrochemical immunosensor involving magnetic beads and disposable carbon screen-printed electrodes (SPEs) for zearalenone (Zea) sensing has been developed and evaluated using a certified reference material (CRM) and selected baby food samples. After the immunochemical reaction, the modified paramagnetic beads were confined by a magnet on surface of SPE platforms where electrochemical detection is simply achieved through the suitable substrate and mediator for the HRP enzyme. A remarkable detection limit of 0.007μgL−1 and excellent accuracy with recovery rate of 101–111% showed the proposed system to be a very suitable screening tool for the analysis of zearalenone in baby food samples.A new simple, fast and reliable strategy involving the sequential performing of calibration and analysis of target mycotoxin using just one disposable SPE each is additionally proposed. Excellent analytical performance in terms of accuracy and precision were again obtained with a remarkable low systematic error (less than 4%) and excellent reproducibility (RSD=6%). This strategy enhanced the analytical merits of immunosensor approach towards truly analytical disposable tools for food-safety scene.

Hemagglutinating activity of histones of the cell nucleus

Histone components of the cell nucleus having lectin activity are identified by differentiated determination of hemagglutinating activity and immunochemical reactions. It is shown that the hemagglutinating activity of deoxyribonucleoprotein is somewhat higher than the activity of total histones.

Identification and localization of the bilitranslocase homologue in white grape berries (Vitis vinifera L.) during ripening.

A homologue of the mammalian bilirubin transporter bilitranslocase (BTL) (TCDB 2.A.65.1.1), able to perform an apparent secondary active transport of flavonoids, has previously been found in carnation petals and red grape berries. In the present work, a BTL homologue was also shown in white berries from Vitis vinifera L. cv. Tocai/Friulano, using anti-sequence antibodies specific for rat liver BTL. This transporter, similarly to what found in red grape, was localized in the first layers of the epidermal tissue and in the vascular bundle cells of the mesocarp. In addition, a strong immunochemical reaction was detected in the placental tissue and particularly in peripheral integuments of the seed. The protein was expressed during the last maturation stages in both skin and pulp tissues and exhibited an apparent molecular mass of c. 31 kDa. Furthermore, the transport activity of such a carrier, measured as bromosulphophthalein (BSP) uptake, was detected in berry pulp microsomes, where it was inhibited by specific anti-BTL antibodies. The BTL homologue activity exhibited higher values, for both Km and Vmax, than those found in the red cultivar. Moreover, two non-pigmented flavonoids, such as quercetin (a flavonol) and eriodictyol (a flavanone), inhibited the uptake of BSP in an uncompetitive manner. Such results strengthen the hypothesis that this BTL homologue acts as a carrier involved also in the membrane transport of colourless flavonoids and demonstrate the presence of such a carrier in different organs and tissues.

The current status of urinary proteins as diagnostic and prognostic markers

Numerous proteins appear in urine under different pathophysiological conditions. Individual proteins are measured by immunoassays, while mass spectrometry is being introduced to evaluate proteins and peptides. The protein in urine is more or less unstable and heterogeneous due to degradation or modification by urine proteases, resulting in altered immunochemical reactions. A quality assurance system for each analyte by each is a key, by setting proper pre-analytical conditions and establishing reference measurement systems, especially by qualified reference material. Indication for urine protein measurement is to seek pathogenesis, localisation of the affected organ or tissue, evaluation of prognosis or response to therapy in systemic, renal and genitourinary diseases. Albumin, a marker for glomerular dysfunction, is essential not only for diabetic nephropathy, but a reliable risk and prognostic marker for cardiovascular diseases. Measurement of very low levels is underway for early detection and prevention of related diseases. α1-microglobulin is a well-established reliable tubular marker. Among post-renal markers IL-8 is a good inflammatory marker especially for urinary tract infection (UTI). The pre-renal marker may specifically detect systemic diseases; a specific antibody against microorganisms is in use by point of care testing (POCT). Clinical usefulness is being expanded by discovery of new proteins and peptides that may be incorporated in the expert system.

Cell Condensation and 3-Dimensional Dynamic Environment in a Rotation Culture Upregulates Osteogenic Differentiation of Mesenchymal Stromal Cells

Objective: For regeneration of bone in a clinical setting, an adequate number of committed osteoblasts is required. Bone marrow mesenchymal stromal cells could provide a source of osteoblasts for bone regeneration. In addition, it is well known that mesenchymal cell condensation plays an important role in the early stages of membranous osteogenesis, and dynamic flow can positively influence and enhance osteodifferentiation of mesenchymal stromal cells. Therefore, to imitate bone development, we adopted a rotation culture method rather than a static culture method.Materials and Methods: Osteogenic differentiation was evaluated by histological, immunochemical and reverse transcriptase-polymerase chain reaction analyses.Results: Cell condensation and a 3-dimensional dynamic environment in a rotation culture may enhance osteoblastic phenotype development and mineralised matrix synthesis in vitro. The extracellular matrix of cell aggregates was endogenously synthesised in an entirely natural manner by the cells themselves. The cell aggregates by rotation culture showed mineral deposition and osteocalcin expression at 7 days, whereas these effects were not detected in the static culture until 14 days.Conclusion: These findings indicate that, compared with static culture in well plates, rotation culture provides a more suitable culture environment that is much closer to the actual cellular environment in vivo.

Label-free impedimetric immunosensor for sensitive detection of ochratoxin A

A novel label-free electrochemical impedimetric immunosensor for sensitive detection of ochratoxin A (OTA) was reported. A two-step reaction protocol was elaborated to modify the gold electrode. The electrode was first derivatized by electrochemical reduction of in situ generated 4-carboxyphenyl diazonium salt (4-CPDS) in acidic aqueous solution yielded stable 4-carboxyphenyl (4-CP) monolayer. The ochratoxin A antibody was then immobilized making use of the carbodiimide chemistry. The steps of the immunosensor elaboration and the immunochemical reaction between ochratoxin A and the surface-bound antibody were interrogated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The impedance change, due to the specific immuno-interaction at the immunosensor surface was utilized to detect ochratoxin A. The increase in electron-transfer resistance (ΔRet) values was linearly proportional to the concentration of OTA in the range of 1–20ngmL−1, with a detection limit of 0.5ngmL−1.

Three-dimensional interdigitated electrode array as a transducer for label-free biosensors

A new transducer for biosensor applications has been developed based on a three-dimensional interdigitated electrode array (IDEA) with electrode digits separated by an insulating barrier. Binding of molecules to a chemically modified surface of the transducer induces important changes in conductivity between the electrodes. Three-dimensional sensor shows considerable improvement compared with a standard planar IDEA design. The potential of the developed device as a sensor transducer to detect immunochemical and enzymatic reactions, as well as DNA hybridization events is demonstrated. The immunosensor allows direct detection of the antibiotic sulfapyridine and shows the IC 50 parameter value of 5.6 μg L −1 in a buffer. Immunochemical determination occurs under competitive configurations and without the use of any label. Each modified sensor is of a single use. Nevertheless, biochemical reagents can be easily cleaned off the sensor surface for its reuse. Layer-by-layer method of used to deposit polyethyleneimine and glucose oxidase showed that the sensor is also highly effective for detecting single and multilayered molecular assemblies.

Piezoquartz immunosensors for assessing the interactions between Yersinia enterocolitica lipopolysaccharides and antibodies to them

The utility of a piezoquartz immunosensor coated with lipopolysaccharides (LPS) for the quantification of antibody specificities was demonstrated. Immunochemical reactions were monitored according to the changes in the weight of sensor bioreceptor layer with high sensitivity (detection limit, 1.3 microg/ml) and assay rate (10 min) without any additional labels. The capabilities of this sensor were demonstrated by the example of quantifying the cross-reactivity of blood serum antibodies with the LPS of Yersinia enterocolitica serotypes O:3, O:5, O:5.27, O:6.30, and O:6.31. The proposed approach is promising for clinical diagnostics of yersiniosis, an infectious intestinal disease.

A potentiometric DNA sensor for determining autoimmune antibodies to DNA

A new technique is proposed for detecting interactions between DNA and DNA autoimmune antibodies using a potentiometric sensor based on a glassy-carbon electrode modified with polyaniline and native DNA from chicken erythrocytes. It is shown that the DNA-antibody interaction changes the rate of polyaniline doping in transferring the DNA sensor from an alkaline (pH 7.5) solution, which is optimum for the immunochemical reaction, to an acidic (pH 3.0) solution. The dynamics of the variation of the DNA sensor potential depends on the titer of antibodies and their origin. The dependence of the DNA sensor signal on the dilution of the blood sera from systemic lupus erythematosus and autoimmune thyroiditis patients shows that DNA antibodies can be diagnosed by the characteristic maximum in the dilution curve found in the range of serum dilutions from 1: 20 to 1: 50.

False-negative results for bromazepam use observed by the EMIT II Plus benzodiazepine assays

During our extensive routine analyses of drugs of abuse and other drugs such as benzodiazepines using both immunoassays and gas chromatography-mass spectrometry (GC-MS), we have noticed that the EMIT II benzodiazepine assays for urine samples sometimes gave false-negative results for bromazepam use or abuse. The negative benzodiazepine immunoassays and positive GC-MS results for bromazepam in urine could be explained by the absence (or below the detection limit) of 3-hydroxybromazepam (3HB) and the presence of 2-amino-3-hydroxy-5-bromobenzoylpyridine (AHBBP), which are the two major metabolites of bromazepam. 3HB cross-reacted with the antibody contained in the EMIT II, but AHBBP did not, due to their structural characteristics. To avoid such false-negative results for bromazepam, AHBBP screening by GC-MS is recommended. The false-negative results observed for bromazepam are probably not limited to the EMIT II kit; the same phenomena may be also true for other commercially available kits that are based on immunochemical reactions.

Analysis of Follicle-Stimulating Hormone by CE with Chemiluminescence Detection Based on Competitive Immunoassay

A competitive immunoassay and capillary electrophoresis with enhanced chemiluminescence detection have been used for determination of follicle-stimulating hormone (FSH) in human serum. The method is based on the competitive immunochemical reaction of FSH and horseradish peroxidase (HRP)-labeled FSH with anti-FSH, CE separation of antibody-bound and free HRP-labeled FSH, then chemiluminescence detection. The detection limit depends on the stability of the immune complex, which depends on analysis time and detector design, and on the chemiluminescence enhancer used. A unique chemiluminescence detector without dead volume or diluent effects was therefore used, and sodium tetraphenylboron was selected as the optimum enhancer. As a result sensitivity was substantially improved. Free HRP-labeled FSH and the immune complex could be separated within 15 min in alkaline borate buffer by use of a potential of 15 kV. Under the optimum conditions a calibration plot for FSH was established in the concentration range 0–100 mIU mL−1; the detection limit was 0.06 mIU mL−1. The concentration sensitivity achieved was 30 times better than that of ELISA.

Improvement of analytical performances of a disposable electrochemical immunosensor by using magnetic beads

A comparison of two electrochemical immunosensing strategies for PCBs detection, based on the use of two different solid phases, is here discussed. In both cases, carbon-based screen-printed electrodes (SPEs) are used as transducers in a direct competitive immunoassay scheme, where PCBs in solution compete with the tracer PCB28-alkaline phosphatase (AP) labeled for antibodies immobilized onto the solid-phase. In the standard format (called EI strategy), SPEs are both the solid-phase for immunoassay and electrochemical transducers: in this case the immunochemical reaction occurs onto the working electrode. Finally, the enzymatic substrate is added and an electroactive product is generated and detected by electrochemical measurement. In order to improve the performances of the system, a new approach (called EMI strategy) is developed by using functionalized magnetic beads as solid phase for the competitive assay; only after the immunosensing step they are captured by a magnet onto the working surface of the SPE for the electrochemical detection. Experimental results evidenced that the configuration based on the use of separate surfaces for immunoassay and for electrochemical detection gave the best results in terms of sensitivity and speed of the analysis. The improvement of analytical performances of the immunosensor based on EMI strategy was also demonstrated by the analysis of some spiked samples.

Electrochemical magneto immunosensing of antibiotic residues in milk

A novel electrochemical immunosensing strategy for the detection of sulfonamide antibiotics in milk based on magnetic beads is presented. Among the different strategies for immobilizing the class-specific anti-sulfonamide antibody to the magnetic beads – such as those based on the use of Protein A or carboxylate modified magnetic beads – ,the best strategy was found to be the covalent bonding on tosyl-activated magnetic beads. The immunological reaction for the detection of sulfonamide antibiotics performed on the magnetic bead is based on a direct competitive assay using a tracer with HRP peroxidase for the enzymatic labelling. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magneto sensor made of graphite–epoxy composite (m-GEC), which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate for the enzyme HRP and an electrochemical mediator. The electrochemical approach is also compared with a novel magneto-ELISA with optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked milk samples, and the detection limit was found to be 1.44 μg L −1 (5.92 nmol L −1) for raw full cream milk. This strategy offers great promise for rapid, simple, cost-effective and on-site analysis of biological, food and environmental samples.

Histological evaluation of buccal penetration enhancement properties of chitosan and trimethyl chitosan

The aim of the present work was to compare the penetration enhancement properties of chitosan hydrochloride (HCS) both as a polymeric solution and as a nanoparticulate system with that of trimethyl chitosan hydrochloride (TMC) on buccal mucosa. The hydrophilic high molecular weight fluorescein isothiocyanate dextran (FD4; 4400 Da) was used as a macromolecule model. The mechanism involved in the HCS (solution and nanoparticles) and TMC solution penetration enhancement was investigated on pig buccal mucosa, characterized by having stratified epithelium and lacking in tight junctions. The permeation/penetration of FD4 and the change in morphology and histology of the mucosa after contact with the polymers were assessed: the experiments were performed ex-vivo by applying the formulations on excised porcine buccal tissue. For the morphology and the histology studies, the epithelial cell layers from freshly excised pig buccal mucosa were analysed with light microscopy by means of routine histopathology analysis (haematoxylin and eosin staining and Toluidine blue staining) and immunohistochemistry reactions. The organization of desmosomal junctions was assessed by means of an immunochemical reaction on desmosomes and transmission electron microscopy. Confocal laser scanning microscopy (CLSM) was used to find evidence of the location of FD4 in the tissue. Furthermore, the increase of the FD4 apparent permeability coefficient was quantified by means of Franz diffusion cells using isolated buccal epithelium to demonstrate the penetration enhancement properties of the polymer systems. Morphological analysis, performed by light microscopy, transmission electron microscopy and CLSM, suggests a similar mechanism of penetration enhancement for both HCS and TMC solutions and for HCS nanoparticles. Such a mechanism probably involves a repackaging of the epithelial cells up to the basal membrane and a partial disarrangement of desmosomes. The cell viability and the nuclear integrity indicated on the semi-thin section stained with Toluidine blue and by CLSM analysis, respectively, suggest that HCS as a polymer solution and a nanoparticulate system, and TMC polymer solution, do not cause cell damage. Trimethyl chitosan and chitosan nanoparticulate systems were able to increase FD4 permeation across buccal epithelium to a greater extent than the chitosan solution.

Evaluation of the Residual Antigenicity of Dairy Whey Hydrolysates Obtained by Combination of Enzymatic Hydrolysis and High-Pressure Treatment

Dairy whey was hydrolyzed for 15 min with five food-grade enzymes (Alcalase, Neutrase, Corolase 7089, Corolase PNL, and Papain) at atmospheric pressure (0.1 MPa) and in combination with high pressure (HP) at 100, 200, and 300 MPa, applied prior to or during enzymatic digestion. The peptide profile of the hydrolysates obtained was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and their residual antigenicity was assessed by immuno-blotting with anti–β-lactoglobulin monoclonal antibodies and the sera from pediatric patients allergic to cow's milk proteins. Moreover, to evaluate the presence of residual trace amounts of casein in bovine whey hydrolysates, immunoblotting with anti–cow's milk protein polyclonal antibodies was performed. SDS-PAGE analysis showed that HP treatment increased hydrolysis by the proteases assayed, especially when it was applied during the enzymatic digestion. Positive reactions at the band corresponding to β-lactoglobulin were detected for Corolase PN-L and Corolase 7089 hydrolysates, except for those obtained under 300 MPa by the last protease. However, the immunochemical reaction was lower in the hydrolysis products obtained under HP than in those obtained at atmospheric pressure and after the HP treatment. On the contrary, no residual immunochemical reactivity associated with β-lactoglobulin was observed in the hydrolysates obtained by Alcalase and Neutrase under HP, and none was observed in any of the hydrolysis products obtained by Papain. The presence of traces of casein was not significant. These results suggest that HP combined with selected food-grade proteases is a treatment that can remove the antigenicity of whey protein hydrolysates for their use as ingredients of hypoallergenic infant formulae.

Blocking agents for ELISA quantification of compounds coming from bovine muscle crude extracts

Non-specific binding (NSB) of compounds other than the capture antigen or antibody to the solid support is one of the most important error sources occurring in ELISA. Undesirable NSB may be minimized by saturating the remaining binding sites of the plastic surface with different protein compounds (blocking agents) that must not interfere with the immunoassay. This work had as main objective the evaluation of different blocking agents for their ability to avoid unwanted NSB in ELISA using bovine muscle crude extracts and a polyclonal antibody raised against purified cathepsin L. Results obtained showed that skimmed milk, casein, BSA, goat and rabbit serum proved to be efficient in suppressing NSB. In the case of skimmed milk and casein, an effective blocking effect was observed even in absence of the surcoating step. However, these two compounds and BSA interfered considerably the immunochemical reaction, making their use inadequate. Of the two assayed serums, rabbit serum would be a more suitable blocking agent than goat serum when working with bovine crude extracts because it did not interfere in great manner with the antigen–antibody reaction and, at the same time, did not increase background.

Biomarker discovery in breast cancer serum using 2‐D differential gel electrophoresis/ MALDI‐TOF/TOF and data validation by routine clinical assays

In the present study, we used 2-D differential gel electrophoresis (2-D DIGE) and MS to screen biomarker candidates in serum samples obtained from 39 patients with breast cancer and 35 controls. First, we pooled the serum samples matched with age and menopausal status. Then, we depleted the two most abundant proteins albumin and IgG by immunoaffinity chromatography under partly denaturing conditions in order to enrich low-abundance proteins and proteins with low molecular weight. Concentrated and desalted samples were labeled with three different CyDyes including one internal standard, pooled from all the samples, and separated with 2-D DIGE in triplicate experiments. Biological variations of the protein expression level were analyzed with DeCyder software and evaluated for reproducibility and statistical significance. The profile of differentially expressed protein spots between patients and controls revealed proapolipoprotein A-I, transferrin, and hemoglobin as up-regulated and three spots, apolipoprotein A-I, apolipoprotein C-III, and haptoglobin alpha2 as down-regulated in patients. Finally, routine clinical immunochemical reactions were used to validate selected candidate biomarkers by quantitative determination of specific proteins in all individual serum samples. The serum level of transferrin correlated well with the 2-D-DIGE results. However, the serum levels of apolipoprotein A-I and haptoglobin could not be detected with the clinical routine diagnostic tests. This demonstrated an advantage 2-D DIGE still has over other techniques. 2-D DIGE can distinguish between isoforms of proteins, where the overall immunochemical quantification does fail due to a lack of isoform-special antibodies.

Electrochemical Magnetoimmunosensing Strategy for the Detection of Pesticides Residues

A novel electrochemical immunosensing strategy for the detection of atrazine based on magnetic beads is presented. Different coupling strategies for the modification of the magnetic beads with the specific anti-atrazine antibody have been developed. The immunological reaction for the detection of atrazine performed on the magnetic bead is based on a direct competitive assay using a peroxidase (HRP) tracer as the enzymatic label. After the immunochemical reactions, the modified magnetic beads can be easily captured by a magnetosensor made of graphite-epoxy composite, which is also used as the transducer for the electrochemical immunosensing. The electrochemical detection is thus achieved through a suitable substrate and mediator for the enzyme HRP. The electrochemical approach is also compared with a novel magneto-ELISA based on optical detection. The performance of the electrochemical immunosensing strategy based on magnetic beads was successfully evaluated using spiked real orange juice samples. The detection limit for atrazine using the competitive electrochemical magnetoimmunosensing strategy with anti-atrazine-specific antibody covalent coupled with tosyl-activated magnetic beads was found to be 6 x 10(-3) microg L(-1) (0.027 nmol L(-1)). This strategy offers great promise for rapid, simple, cost-effective, and on-site analysis of biological, food, and environmental samples.

Implementation of immunohistochemical methods in diagnostics of bovine sarcocystosis

Bovine sarcocystosis was examined in three age categories of cattle using direct and immunohistochemical methods. The direct methods of compression, trypsin digestion and a histological method were applied, and the applied immunohistochemical methods included peroxidase antiperoxidase (PAP), the highly sensitive streptavidin biotin immunoperoxidase complex technique (LSAB+) and indirect immunofluorescence (IF). Primary rabbit antiserum obtained for the zoites Sarcocystis cruzi syn. S. bovicanis was used for all immunochemical and immunohistochemical examinations. Cross reactivity was determined using primary rabbit antiserum to antigens of S. ovifelis zoites. Both primary antiserums were produced in the laboratory, as no commercial preparations were available on the market. The evaluation of the sensitivity and efficacy of the immunohistochemical methods was performed on the grounds of comparisons with the direct methods: compression, trypsin digestion and a histological method. The applied immunohistochemical analyses are very specific in proving sarcocystosis in cattle, especially the LSAB+ technique, and, according to their sensitivity in detecting this infection, they are similar to the method of trypsin digestion. The obtained rabbit antiserum against antigens of S. bovicanis zoites yielded good results in the diagnosis of the homologous specie. Proven cross reactivity of the homologous and heterologous antiserums and antigens, as an immunochemical reaction of complete identity in one part to the antigen structure, was established in primary rabbit antiserum to S. ovifelis zoites, which indicates that heterologous antiserums can also be used in immunohistochemical reactions in the diagnostics of bovine sarcocystosis. .

The Rise of EIA/ELISA

Elsewhere in this issue of Clinical Chemistry , Lequin (1) tells the story of how, in the late 1960s, enzyme immunoassay (EIA)/ELISA was conceived and how the technology subsequently found wide application in biomedical analysis. When we started the project at Organon, we certainly did not have the vision that it would lay the foundation for billions of future laboratory analyses. The main reason for starting the project was that Organon’s management liked the success of the immunochemical pregnancy tests that had been introduced some years before (based on inhibition of hemagglutination or latex agglutination), but felt that the cumbersome and time-consuming test procedures formed a threat to continued success. A simple dip-and-read strip giving a color change seemed a more secure basis for future success. Thus, Anton Schuurs’ proposal to explore the potential of enzymes linked to antigens or antibodies for immunochemical reactions with a colorimetric endpoint found approval. Of course, we used the pregnancy hormone human chorionic gonadotropin …