Effects of chromium on the immune system

The performance of integral membrane antigens (IMAs) of Mycobacterium habana TMC 5135 in detecting antimycobacterial antibodies in serum and body fluids of patients mainly of extrapulmonary tuberculosis was evaluated. The IMAs were recovered from the detergent phase during Triton X-114 treatment of the plasma membrane of M. habana. Antimycobacterial antibodies were detected by ELISA using IMAs in serum and body fluids of 42 patients and 62 control subjects. As authentic adjunct Mycobacterium tuberculosis antigens were also detected (by ELISA) in body fluids and circulating immune complexes using anti-M. tuberculosis H37Ra antibodies. Anti-M. habana IMA antibody detection increased the positivity rate from 26.% (11/42) and 10% (4/42) obtained by culture and smear microscopy, respectively, to 86% (36/42). M. tuberculosis antigens were also found in 29 out of 36 anti-M. habana IMA antibody-positive cases. Interestingly, all 11 culture-positive cases were also positive for anti-M. habana IMA antibodies. The mean antigen titres in 23 cases, positive for antigens in body fluids, were 2.34 times higher in those who were also positive for anti-IMA antibodies in serum than in those negative for these antibodies. M. habana IMAs may be promising non-tubercular candidate antigens in ELISA-based serodiagnosis of extrapulmonary tuberculosis with substantial sensitivity, specificity and safety.     

Enzymatic recycling-based amperometric immunosensor for the ultrasensitive detection of okadaic acid in shellfish

Electrochemical immunosensors based on a competitive indirect enzyme-linked immunosorbent assay (ciELISA) and an enzymatic recycling system were developed for the detection of okadaic acid (OA). OA-ovalbumin (OA-OVA) conjugate was immobilised on screen-printed electrodes (SPEs) and competition of a newly generated monoclonal antibody (MAb) for free and immobilised OA was subsequently performed. Secondary antibodies labelled with alkaline phosphatase (ALP) or horseradish peroxidase (HRP) were used for signal generation. Experimental parameters were firstly optimised by colorimetric ELISA on microtiter wells and on SPEs. The ELISA system was then tested by amperometry at +300 mV vs. Ag/AgCl (detection of p-aminophenol produced by the reaction of p-aminophenyl phosphate with ALP) or -200 mV vs. Ag/AgCl (detection of 5-methyl-phenazinium methyl sulfate, redox mediator in the HRP bioelectrocatalysis). The limits of detection (LODs) with standard solutions were 1.07 and 1.98 microgL(-1) when using ALP and HRP labels, respectively. An electrochemical signal amplification system based on diaphorase (DI) recycling was integrated into the ALP-based immunosensor, decreasing the LOD to 0.03 microgL(-1) and enlarging the working range by two orders of magnitude. Preliminary results with mussel and oyster extracts were obtained and compared with the colorimetric immunoassay, the colorimetric protein phosphatase inhibition assay (PPIA) and LC-MS/MS.     

The use of streptavidin-biotin interaction for preparation of reagents for complement-dependent liposome immunoassay of proteins: detection of latrotoxin.

We have developed liposome sensitization by a protein, latrotoxin (LT), using immobilization of biotinylated LT via streptavidin with biotinylated phosphatidylethanolamine contained in liposomes. The use of such liposomes in the complement-dependent homogeneous liposome immune lysis assay (LILA) has allowed us to detect in the test sample as little as 2 micrograms/ml of polyclonal and 50-100 ng/ml of monoclonal IgG and IgM antibodies to LT. LT concentration in solution was determined by inhibition of immune lysis by free LT. The sensitivity of the LT assay varied from 1 x 10(-9) to 5-50 x 10(-9) M when antiserum (polyclonal antibodies) and monoclonal antibodies to LT were correspondingly used. The results show that a streptavidin-biotin spacer can be used to immobilize protein antigens on liposomes for a subsequent application in LILA. The suggested technique greatly simplifies the sensitization procedure and extends the applicability of the LILA.     

Microwave irradiation in label-detection for diagnostic DNA-in situ hybridization.

Summary A DNA-in situ hybridization protocol was adapted for application to sections of routinely processed paraffin embedded material. This protocol was developed previously for detecting DNA-virus infected cells in whole cell preparations and employs biotinylated DNA as probe. Three different biotin detection methods were optimized and applied. The first uses streptavidin and a biotinylated complex of alkaline phosphatase, the second consists of an immunogold-silver staining, and the third of a peroxidase technique using a silver amplification. The alkaline phosphatase method was the most rapid, and as sensitive as the immunogold-silver staining. The peroxidase method was the most sensitive. Microwave irradiation was applied to the different incubation steps of these three detection methods. Short incubations with microwave irradiation gave very poor results when peroxidase labelled antibodies were used. Short incubation with microwave irradiation gave results comparable to those obtained with conventional incubations, when streptavidin, antibiotin, complexed alkaline phosphatase, or gold labelled goat antirabbit were used. It was thus shown that microwave irradiation creates the possibility of a very rapid label-detection for nonradioactive DNA-in situ hybridization.     

Development of a screen-printed carbon electrochemical immunosensor for picomolar concentrations of estradiol in human serum extracts

Investigations into the development of a prototype electrochemical immunosensor for estradiol (E(2)) are described. After optimising reagent loadings in a 96-well enzyme-linked immunosorbent assay (ELISA), antibodies (rabbit anti-mouse IgG and monoclonal mouse anti-E(2)) were immobilised by passive adsorption onto the surface of screen-printed carbon electrodes (SPCEs). A competitive immunoassay was then performed using an alkaline-phosphatase (ALP)-labelled E(2) conjugate. Calibration plots for E(2) buffer standards, performed colorimetrically on the SPCEs using a para-nitrophenyl phosphate substrate solution, were in good agreement with ELISA calibration plots. Electrochemical measurements were then performed using differential pulse voltammetry (DPV) following the production of 1-naphthol from 1-naphthyl phosphate. The calibration plot of DPV peak current versus E(2) concentration showed a measurable range of 25-500 pg/ml with a detection limit of 50 pg/ml. A coefficient of variation of between 13.0 and 15.6% was obtained for repeat measurements. The immunosensor was applied to the determination of E(2) in spiked serum, following an extraction step with diethyl ether. A mean recovery for the method of 102.5% was obtained with a CV of 19.1%. The options available for further development of the sensor regarding precision, limit of detection and direct sample analysis are discussed.     

Colloidal gold as an electrochemical label of streptavidin-biotin interaction

A new electrochemical method to monitor biotin-streptavidin interaction, based on the use of colloidal gold as an electrochemical label, is investigated. Biotinylated albumin is adsorbed on the pretreated surface of a carbon paste electrode (CPE). This modified electrode is immersed in colloidal gold-streptavidin labelled solutions. Adsorptive voltammetry is used to monitor colloidal gold bound to streptavidin, obtaining a good reproducibility of the analytical signal (R.S.D. = 3.3%). A linear relationship between peak current and streptavidin concentration from 2.5 x 10(-9) to 2.5 x 10(-5) M is obtained when a sequential competitive assay between streptavidin and colloidal gold-labelled streptavidin is carried out. On the other hand, the adsorption of streptavidin on the electrode surface was performed, followed by the reaction with biotinylated albumin labelled with colloidal gold. In this way, a linear relationship between peak current and colloidal gold labelled biotinylated albumin concentration is achieved with a limit of detection of 7.3 x 10(9) gold particles per ml (5.29 x 10(-9) M in biotin).     

Disposable amperometric immunosensor system for rabbit IgG using a conducting polymer modified screen-printed electrode

A disposable and mediatorless immunosensor based on a conducting polymer (5,2':5'2"-terthiophene-3'-carboxylic acid) coated screen-printed carbon electrode has been developed using a separation-free homogeneous technique for the detection of rabbit IgG as a model analyte. Horseradish peroxidase (HRP) and streptavidin were covalently bonded with the polymer on the electrode and biotinylated antibody was immobilized on the electrode surface using avidin-biotin coupling. This sensor was based on the competitive assay between free and labeled antigen for the available binding sites of antibody. Glucose oxidase was used as a label and in the presence of glucose, H(2)O(2) formed by the analyte-enzyme conjugate was reduced by the enzyme channeling via HRP bonded on the electrode. The catalytic current was monitored amperometrically at -0.35 V vs. Ag/AgCl and this method showed a linear range of RIgG concentrations from 0.5 to 2 microg/ml with standard deviation +/-0.0145 (n=4). Detection limit was determined to be 0.33 microg/ml.     

Celiac disease detection using a transglutaminase electrochemical immunosensor fabricated on nanohybrid screen-printed carbon electrodes

Celiac disease is a gluten-induced autoimmune enteropathy characterized by the presence of tissue tranglutaminase (tTG) autoantibodies. A disposable electrochemical immunosensor (EI) for the detection of IgA and IgG type anti-tTG autoantibodies in real patient's samples is presented. Screen-printed carbon electrodes (SPCE) nanostructurized with carbon nanotubes and gold nanoparticles were used as the transducer surface. This transducer exhibits the excellent characteristics of carbon-metal nanoparticle hybrid conjugation and led to the amplification of the immunological interaction. The immunosensing strategy consisted of the immobilization of tTG on the nanostructured electrode surface followed by the electrochemical detection of the autoantibodies present in the samples using an alkaline phosphatase (AP) labelled anti-human IgA or IgG antibody. The analytical signal was based on the anodic redissolution of enzymatically generated silver by cyclic voltammetry. The results obtained were corroborated with a commercial ELISA kit indicating that the electrochemical immunosensor is a trustful analytical screening tool.     

Oriented immobilisation of anti-pneumolysin Fab through a histidine tag for electrochemical immunosensors

Orientation of reagents is a key step in the construction of immunosensors. When the immunoreagent is a recombinant protein, this can be achieved by employing hexahistidine tags. The orientation of recombinant histidine-tagged Fab fragments of monoclonal anti-pneumolysin antibodies on gold films is evaluated. Using histidine as a chelator of Ni or employing an anti-polyhistidine antibody for capturing the His6 residue is considered. Measurements are based in the signal of indigo, which comes from the hydrolysis of 3-indoxylphosphate by alkaline phosphatase (AP). The attachment of the enzyme occurs through the interaction of biotin with AP-labelled streptavidin or employing AP-conjugated immunoreagents. In the case of the interaction Ni-histidine, for the study of the self-assembling process a His-tagged and biotinylated protein (His6-GST-B) was employed. General conditions were studied and non-specific adsorption was avoided with the use of 1-hexanethiol. Improvements of the signal compared with the direct adsorption were only achieved by the use of histidine capturing antibodies. With an optimised ratio anti-polyhis:His6-Fab the signal increases approximately a 100%. Precision is adequate and the response is linear with the concentration of pneumolysin between 0.1 and 10 ng/mL.     

Localization of human pituitary hormones by multiple immunoenzyme staining procedures using monoclonal and polyclonal antibodies

Mouse monoclonal and rabbit polyclonal antibodies to human pituitary hormones were applied together to sections of normal and neoplastic human pituitary tissue. Binding sites were revealed with species-specific immune reagents combined with various enzymes (peroxidase, alkaline phosphatase, and beta-D-galactosidase). The enzymes were developed separately to give differently colored end-products. Where two hormones were present in the same cell, a mixed color was produced. Up to four hormones could be immunostained in a single section. Multiple immunoenzymatic staining has great potential for the analysis of plural antigen production by single cells and relationships between cells producing different antigens.     

Cross-talk-free multiplexed immunoassay using a disposable electrochemiluminescent immunosensor array coupled with a non-array detector

A disposable electrochemiluminescent (ECL) immunosensor array was fabricated on a screen-printed carbon electrode (SPCE) substrate to perform multiplexed immunoassay (MIA) for the first time. The SPCE substrate was composed of an array of four carbon working electrodes, one common Ag/AgCl reference electrode, and one common carbon counter electrode. The immunosensor array was constructed by site-selectively immobilizing multiple antigens on different working electrodes of the SPCE substrate. With a competitive immunoassay format, the immobilized antigens competed with antigens in the sample to capture their corresponding tri(2,2'-bipyridyl)ruthenium(II)-labeled antibodies. The ECL signals from the immunosensors in this array were sequentially detected by a photomultiplier with the aid of a homemade single-pore-four-throw switch. Due to the ECL readout mechanism and the sequential detection mode, it could avoid the cross-talk between the adjacent immunosensors, which was common in other reported immunosensor array. Human, rabbit and mouse immunoglobulin Gs were near-simultaneously assayed as the model analytes. The linear ranges for them were 10-400, 20-400, and 20-400 ng/mL, with detection limits of 2.9, 6.1 and 6.5 ng/mL (S/N=3), respectively. This novel ECL strategy based on immunosensor array coupled with non-array detector provided a simple, sensitive, low-cost and time-saving approach for MIA. It showed great application potential in point-of-care test and field analysis of bio-agents, with mass production potential and high throughput.     

An electrochemical enzyme bioaffinity electrode based on biotin-streptavidin conjunction and bienzyme substrate recycling for amplification

A signal amplificatory electrochemical immunoassay with biotin-streptavidin conjunction and multienzymatic-based substrate recycling was developed in this work. Biotinylated secondary antibody (bio-IgG) was preliminarily assembled onto the immunosensor interface based on the sandwich format. Streptavidin was then loaded based on biotin-streptavidin conjunction. Owing to four identical binding sites of streptavidin to biotin, amounts of biotinylated alkaline phosphatase (bio-AP) were attached, and this improved the catalytic performance of the proposed immunosensor. Under the enzyme catalysis of AP, the electroinactive p-aminophenylphosphate (PAPP) substrate was rapidly hydrolyzed into the electroactive p-aminophenol (PAP) product, which next oxidized at the electrode surface into p-quinoneimine (PQI). In the presence of diaphorase (DI), PQI was reduced back to PAP, leading to a reversible cycle of PAP. Then the oxidized state of DI was regenerated into its reduced native state by its natural substrate, nicotinamide adenine dinucleotide (NADH). With the several amplification factors mentioned above, a wider linear ranged from 10⁻¹⁴ to 10⁻⁵ g ml⁻¹ was acquired with a relatively low detection limit of 3.5 × 10⁻⁵ g ml⁻¹ for human IgG. In addition, the nonspecific adsorption of proposed immunosensor was also investigated here.     

Fixation of cryo-sections under HIV-1 inactivating conditions: integrity of antigen binding sites and cell surface antigens

Summary Cryostat-sections of biopsies from HIV-infected patients or HIV/SIV-infected experimental animals pose a biohazard risk to laboratory workers. The objective of this study was to select a procedure that appropriately fixes cryo-sections and reduces the risk of HIV-1 infectivity. This inactivation procedure should preserve antigen binding capacity of host-produced antibodies and the antigenic structure of epitopes present in these tissues, while retaining sufficient morphologic detail. We tested the effect of seven different established fixation-inactivation procedures for HIV-1 on the detection of specific antibodies and membrane markers, compared to acetone fixation as a reference. Frozen sections of spleens from mice immunized with trinitrophenyl (TNP)-Ficoll were incubated with TNP-alkaline phosphatase to detect specific antibody-forming cells and follicular immune complexes containing TNP-specific antibodies. In addition, sections were stained with monoclonal antibodies directed against IgM (187-1), T-cells (anti Thy-1), and marginal metallophilic macrophages (MOMA-1). Five procedures proved useful as they gave results similar to regular acetone fixation. In contrast, two procedures with a methanol-containing fixative obscured both antigen binding sites and membrane antigens. Subsequently, these five selected procedures were tested on glass slide preparations of HIV-1 infected cell lines, expressing HIV-1 determinants defined by monoclonal antibodies. Finally, the procedures were tested on sections of an HIV-1 infected human lymph node. for detection of HIV-specific B-cells. We show that fixation-inactivation in 0.37% (v/v) formaldehyde in PBS for 10 min at room temperature and 0.5% paraformaldehyde (w/v) in PBS for 10 min at room temperature are the methods of choice, combining preservation of antigen binding sites (Fab), membrane antigens, and HIV-1 determinants with good tissue morphology.Abbreviations AFC antibody forming cell - AP alkaline phosphatase - MAb monoclonal antibody - HIV-1 human immunodeficiency virus type 1 - HRP horseradish peroxidase - TNP trinitrophenyl     

Incorporation of antigens from Mannheimia haemolytica culture supernatant, and recombinant bovine C3d into ISCOM matrix using neutravidin-biotin interaction

The aim of this study was to incorporate antigens from Mannheimia haemolytica culture supernatant, and an immune modulatory molecule, recombinant bovine C3d (rBoC3d), into immune stimulating complexes (ISCOMs) using neutravidin-biotin interaction. Biotinylated ISCOM matrix was generated using a commercial kit. The biotinylated ISCOM matrix was incubated with neutravidin and then centrifuged in a sucrose density gradient. The rBoC3d was expressed as an in vivo biotinylated protein and with a c-Myc tag (EQKLISEEDL) engineered to facilitate detection. The neutravidin-coated ISCOM matrix was incubated with biotinylated antigens from M. haemolytica culture supernatants and rBoC3d. To test the association among the neutravidin-coated ISCOM matrix, biotinylated antigens and rBoC3d, an analytical sucrose density gradient (10-40%, w/w) was performed. The experimental formulations were run in SDS-PAGE gels under reducing conditions. For Western immunoblot analysis, polyclonal bovine antiavidin, monoclonal anti-c-Myc, monoclonal antileukotoxin, and anti-GS60 antibodies were used to detect the presence of neutravidin, rBoC3d, leukotoxin, and GS60 antigens, respectively. By taking advantage of the biotin-neutravidin interaction, not only leukotoxin but also the recombinant immunomodulatory molecule, rBoC3d, was incorporated into ISCOM particles.     

Differentiation of human adult and fetal intestinal alkaline phosphatases with monoclonal antibodies

Two forms of intestinal alkaline phosphatase have been recognized in humans. They are very similar in a number of biochemical and immunologic characteristics, but the exact genetic relationship between them remains unclear. To further study this problem, six monoclonal antibodies and a polyclonal rabbit antiserum to human fetal intestinal alkaline phosphatase have been produced. All of the monoclonal antibodies and the rabbit antiserum crossreact with adult intestinal alkaline phosphatase and with the intestinal-like alkaline phosphatase found in D98/AH-2 human tissue-culture cells. Four of the monoclonal antibodies and the rabbit antiserum crossreact with placental alkaline phosphatase, while none of the antibodies or the antiserum recognize liver or kidney alkaline phosphatase. Four of the monoclonal antibodies can distinguish between adult and fetal intestinal alkaline phosphatase in electrophoretic titration-binding studies, with the relative binding of adult enzyme being significantly greater than that of the fetal enzyme in each case. One of these antibodies, which also reacts with placental alkaline phosphatase, can distinguish the type 3 allelic variant of the placental enzyme from types 1 and 2. This indicates that the antibody detects a structural difference in the protein moiety of one of the allelic forms of the enzyme. These data suggest that adult and fetal intestinal alkaline phosphatases represent structurally distinct proteins, either encoded for by different genes or produced by differential processing of a common precursor molecule determined by a single gene.     

Immunoelectron microscopic analysis of the binding of monoclonal antibodies to molecular variants of human placental alkaline phosphatase

Three monoclonal antibodies with distinct antigenic specificities were examined by electron microscopy for their binding to three common genetic variants (SS, FS, and FF) of human placental alkaline phosphatase. In the reaction with the monoclonal antibody H5, all three variants of human placental alkaline phosphatase preferentially formed circular immune complexes composed of two antibodies and two enzyme molecules. In separate reactions with the F11 and B2 monoclonal antibodies, the SS variant formed circular complexes and the FS variant formed Y-shaped complexes composed of one antibody and two enzyme molecules, whereas the FF variant scarcely reacted. These results confirm immunochemical data showing that H5 binds to both S and F subunits with similar affinities, whereas F11 and B2 bind the S subunit with markedly higher affinity than they do the F subunit. Furthermore, the formation of circular complexes in the reaction of the mixture of the two antibodies, F11 and B2, with FS molecules suggests that these two antibodies bind to different sites on the S subunit. Therefore, the F and S subunits differ from one another at more than one site. This is the first indication that alleles of human placental alkaline phosphatase may result from more than just single point mutations in the gene encoding them.     

A magnetoelastic bioaffinity-based sensor for avidin

A magnetoelastic bioaffinity sensor coupled with biocatalytic precipitation is described for avidin detection. The non-specific adsorption characteristics of streptavidin on different functionalized sensor surfaces are examined. It is found that a biotinylated poly(ethylene glycol) (PEG) interface can effectively block non-specific adsorption of proteins. Coupled with the PEG immobilized sensor surface, alkaline phosphatase (AP) labeled streptavidin is used to track specific binding on the sensor. This mass-change-based signal is amplified by the accumulation on the sensor of insoluble products of 5-bromo-4-chloro-3-indolyl phosphate catalyzed by AP. The resulting mass loading on the sensor surface in turn shifts the resonance frequency of the magnetoelastic sensors, with an avidin detection limit of approximately 200 ng/ml.     

An immunosensor with potential for the detection of viral antigens in body fluids, based on surface second harmonic generation

Field methods of assessing the immune status of animals are required to optimise vaccination programmes to control bovine viral diarrhoea (BVD) virus. An optoelectronic immunosensor was evaluated for the detection of viral antigens in a crude cell lysate in a pilot study. Binding of (BVD) virus antigen by two monoclonal antibodies immobilised on two different media (ELISA plate wells, and glass coverslips) was detected and quantified using the laser induced surface second harmonic generation (SSHG) technique. The results for both assays were correlated with an enzyme-linked immunoassay (ELISA) used for the diagnosis of BVD virus infection in cattle (ELISA plate; R(2)=0.86, coverslips; Exp. 1; R(2)=0.75, Exp. 2; R(2)=0.67). The method will allow rapid detection of antigens in the body fluids of farm animals.     

Electrochemical immunosensor with aptamer-based enzymatic amplification

An electrochemical immunosensor is reported by using aptamer-based enzymatic amplification with immunoglobulin E (IgE) as the model analyte. In this method, the IgE antibody is covalently immobilized as the capture probe on the gold electrode via a self-assembled monolayer of cysteamine. After the target is captured, the biotinylated anti-IgE aptamer is used as the detection probe. The specific interaction of streptavidin-conjugated alkaline phosphatase to the surface-bound biotinylated detection probe mediates a catalytic reaction of ascorbic acid 2-phosphate substrate to produce a reducing agent ascorbic acid. Then silver ions in the solution can be reduced, leading to the deposition of metallic silver on the electrode surface. The amount of deposited silver, which is determined by the amount of IgE target bound on the electrode surface, can be quantified using the stripping voltammetry. The results obtained demonstrated that the electrochemical immunosensor possesses high specificity and a wide dynamic range with a low detection limit that possibly arises from the combination of the highly specific aptamer and the highly sensitive stripping determination of enzymatically deposited silver.