Conjugated To Horseradish Peroxidase Research Articles

Novel electrochemical aptamer biosensor based on an enzyme–gold nanoparticle dual label for the ultrasensitive detection of epithelial tumour marker MUC1

A novel platform based on a hairpin oligonucleotide (HO) switch, gold nanoparticles (AuNPs), and enzyme signal amplification for the ultrasensitive detection of mucin 1 protein (MUC1) was developed in this assay. This HO aptamers and horseradish peroxidase (HRP) were immobilised on the AuNPs to yield HO–AuNP–HRP conjugates. AuNPs were used as labels and bridges between the HO and HRP. HRP was also used as label for catalysing the oxidation of o-phenylenediamine by H2O2. The reaction product was 2,3-diaminophenazine (DAP), which was reduced and could be detected at surface of modified electrode. The reduction signal of DAP was used as a probe for the sensitive detection. After the recognition between oligonucleotide and MUC1, biotin was exposed. Biotin, along with the conjugate, was captured by streptavidin onto the surface of modified electrode. Therefore, the detection of target MUC1 which was a membrane-associated glycoprotein of the mucin family could be sensitively transduced via detection of the electrochemical reduction signal of DAP. Compared to other aptasensors, this biosensor has a good linear correlation ranges from 8.8nM to 353.3nM and a lower detection limit of 2.2nM for MUC1. The proposed method provided a new electrochemical approach for the detection of MUC1.

Detection of immunoglobulin E using an aptamer based dot-blot assay

A novel aptamer based dot-blot assay for the detection of immunoglobulin E (IgE) was developed. A biotinylated aptamer was employed as the bio-recognition element to specifically interact with the target protein immobilized onto a nitrocellulose membrane substrate. Avidin conjugated horseradish peroxidase was introduced onto the membrane through the biotin-avidin system to catalyze the hydrogen peroxide mediated oxidation of 3,3′,5,5′-tetramethylbenzidine, thereby producing the blue-colored insoluble product. The intensity of the dots increased as the concentration of IgE increased. The spot intensity was quantified using a simple portable instrument. A linear response relationship between the spot intensity and the concentration of IgE over the range of 50 nmol/L to 1 μmol/L was obtained. The detection limit for IgE using the aptamer-based assay was 2.89 nmol/L. This assay was found to discriminate IgE from non-target proteins such as thrombin, bovine serum albumin and immunoglobulin G.

Rapid monitoring of dicyclohexyl phthalate in foods using the direct competitive ELISA

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) has been developed for the quantitative detection of the dicyclohexyl phthalate (DCHP) in some liquid food. Specific polyclonal antisera to DCHP was raised, using the hapten-bovine serum albumin conjugates as the immunogen. The conjugate of horseradish peroxidase (HRP) with antibody was used as the detectable probe to provide the direct measurement of the antigen and analyte, which was synthesised by a modified glutaraldehyde method. Under the optimised assay, the quantitative working range was from 0.1 to 100 ng mL−1 (R2=0.9989), with a limit of detection (LOD) of 0.03 ng mL−1, and a recovery of 96.6–112.4%. The specificity and accuracy of developed method were evaluated. The cross-reactivities of antibody with structurally related phthalate esters were less than 10%. Results obtained indicated that the dc-ELISA was a sensitive, low expense method to improve the routine monitoring of trace constituents in some liquid food.

Development of ultrasensitive direct chemiluminescent enzyme immunoassay for determination of aflatoxin B1 in food products

A direct competitive chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for determination of aflatoxin B1 (AFB1) was developed. To improve the assay sensitivity, a mixture of 3-(10′-phenothiazinyl)-propane-1-sulfonate and 4-morpholinopyridine previously optimized by a factorial design was used as enhancer of horseradish peroxidase-induced chemiluminescence. Varying the concentrations of the coating anti-AFB1 antibody and conjugate of AFB1 and horseradish peroxidase the conditions of the chemiluminescent assay were optimized. The values of the detection limit value and dynamic working range of CL-ELISA of AFB1 were 0.0015ngmL−1 and 0.003–0.03ngmL−1, respectively. It was shown that a dilution of rice and mung beans extracts in 5 and 10 times, respectively, prevented a matrix effect of the food products in CL-ELISA. The recovery values from the spiked samples of rice and mung beans were in the range of 90–104% and 102–117%, respectively. Studying 8 rice and 8 mung beans samples purchased in commercial stores the developed CL-ELISA allowed to find 3 samples (1 rice and 2 mung beans) containing AFB1, the content of AFB1 in one sample being higher than the maximum acceptable level established in the European Community.

Elaboration of Immunoenzymatic Test-Kit for Total Human ΙgΕ Assay and Investigation of Its Analytical Properties

Enzyme-linked immunosorbent assay (ELISA) test-kit has been developed based on complex immuno-chemical, including epitop mapping, characteristics of monoclonal antibodies (MAbs) to human IgЕ. “Sandwich” type ELISA based on usage of different epitop directionality MAbs. It has been founded correlation between affinity of different MAb pairs and its sorbtion-detection ability. Optimal configuration of MAbs in “sandwich” ELISA was follow-ing: 164H10 - 165C12 and 164H10 - 166B7. Cooperative usage of horseradish peroxidase conjugates of MAbs directed to different epitops (165C12-HRP + 166B7-HRP) increased analytical sensitivity of assay and constituted 4.5 IU/ml. Analytical characteristics of developed test-kit were following: dynamic range – from 4.5 to 1600 IU/ml, variation coefficient value during one procedure – 4.2±2.1%, and between procedures – 4.8±2.3%. Presences of human IgG, IgA, IgМ, albumin (1 μg/ml) were not effect on the assay specificity (test concentration of IgE – 50 IU/ml).

Secretion-related structures of hypothalamo-hypophysial terminals in the rat posterior pituitary

Hypothalamic terminals were investigated in the rat posterior pituitary (PP). Injection of wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) and co-injection of WGA-HRP with Rab3A-siRNA were made into the hypothalamus, respectively. Additional injection of WGA-HRP was made into the hypothalamus in the animals exposed to ethanol. These injections resulted in heavy labeling of fibers exclusively confined to the PP. Ultrastructural observations showed terminals, fibers, pituicytes, capillaries and vascular spaces in the PP. Although the majority of terminals were observed to contain large dense core vesicles (LDCVs) and HRP-reaction products (HRP-RPs), exocytosis of LDCVs in close proximity to cell membrane was not found. Interestingly, a few terminals showed alteration of cell membrane called "apocrine-like structure" containing LDCV and RP. The narrow neck portion of the structure gave the appearance that it may have been in some stage of separating from terminals. Other remarkable feature was that terminals occasionally reveal the structure of "leakage" of RP discharged into vascular spaces crossing cell membrane. Such hormone-releasing mechanism might be involved in one of "diacrine-like secretion". In the present study secretion-related structures of hypothalamic terminals in the PP are quite different from normal vesicular exocytosis.

Aquaporins 6-12 in the human eye

Aquaporins (AQPs) are widely expressed and have diverse distribution patterns in the eye. AQPs 0-5 have been localized at the cellular level in human eyes. We investigated the presence of the more recently discovered AQPs 6-12 in the human eye. RT-PCR was performed on fresh tissue from two human eyes divided into the cornea, corneal limbus, ciliary body and iris, lens, choroid, optic nerve, retina and sclera. Each structure was examined to detect the mRNA of AQPs 6-12. Twenty-one human eyes were examined using immunohistochemical and immunofluorescence techniques to determine the topographical localization of AQPs 6-12. mRNA transcripts of AQP7, AQP9 and AQP11 were found in the ciliary body, corneo-limbal tissue, optic nerve, retina and sclera. AQP9 and AQP11 mRNA was also detected in the choroid. No mRNA of AQP6, AQP8, AQP10 or AQP12 was detected. Anti-AQP7 immunolabelling was detected in the corneal epithelium, corneal endothelium, trabecular meshwork endothelium, ciliary epithelia, lens epithelium, the inner and outer limiting membrane of the retina, the retinal pigment epithelium and the capillary endothelium of all parts of the eye. AQP9 immunolabelling was detected in the nonpigmented ciliary epithelium and retinal ganglion cells. AQP11 immunolabelling was detected in the corneo-limbal epithelium, nonpigmented ciliary epithelium and inner limiting membrane of the retina. Selective expression of AQP7, AQP9 and AQP11 was found within various structures of the human eye. The detection of these aquaporins in the eye implies a role that may be related not only to water transport but also to the transport of glycerol, lactate and ammonia, with importance for metabolism, especially in the retina.

A thin layer-based amperometric enzyme immunoassay for the rapid and sensitive diagnosis of respiratory syncytial virus infections

A simple electrochemical sandwich immunoassay involving a polystyrene microarray slide coated with monoclonal capture antibodies and carbon screen-printed sensors (SPS) was designed for the rapid diagnosis of respiratory syncytial virus (RSV). The detection of the antibody-antigen complex formation relied on the use of a horseradish peroxidase conjugate. Its chronoamperometric measurement detection was performed by confining a droplet of H2O2/3,3',5,5'-tetramethylbenzidine enzyme substrate/mediator solution within a thin layer between one spot of the microarray and the surface of one screen-printed electrochemical cell. The accumulation of the enzyme product in the thin film of liquid enhanced the electrochemical response which allowed the development of a rapid (25min) and sensitive thin layer-based amperometric (TLA) enzyme immunoassay. The method was successfully compared to commercially-available immunofluorescent and real-time PCR assays for RSV testing in respiratory secretion clinical samples. This suggests that owing to its rapidity, convenience, low-cost, portability and ability to provide quantified results, the reported concept could be a promising point-of-care diagnostic tool to screen patients with suspected respiratory infection or other types of infectious diseases.

A Perioculomotor Nitridergic Population in the Macaque and Cat

We determined the distribution of cells containing synthetic enzymes for the unconventional neurotransmitter, nitric oxide, with respect to the known populations within the oculomotor complex. The oculomotor complex was investigated in monkeys and cats by use of histochemistry to demonstrate nicotinamide adenine dinucleotide phosphate diaphorase positive (NADPHd(+)) cells and antibodies to localize neuronal nitric oxide synthase positive (NOS(+)) cells. In some cases, wheat germ agglutinin conjugated horseradish peroxidase (WGA-HRP) was injected into extraocular muscles to allow comparison of retrogradely labeled and NADPHd(+) cell distributions. The distribution of the NADPHd(+) and NOS(+) neurons did not coincide with that of preganglionic and extraocular motoneurons in the oculomotor complex. However, labeled perioculomotor neurons were observed. Specifically, in monkeys, they lay in an arc that extended from between the oculomotor nuclei into the supraoculomotor area (SOA). Comparison of WGA-HRP-labeled medial and superior rectus motoneurons with NADPHd staining confirmed that the distributions overlapped, but showed that the C- and S-group cells were not NADPHd(+). This suggested that NADPHd(+) cells are part of the centrally projecting Edinger-Westphal population (EWcp). Examination of the NADPHd(+) cell distribution in the cat showed that these cells were indeed found primarily within its well-defined EWcp. Based on their similar distributions, it appears that the peptidergic EWcp neurons, which project widely in the brain, also may be nitridergic. While the preganglionic and C- and S-group motoneuron populations do not use this nonsynaptic neurotransmitter, nitric oxide produced by surrounding NADPHd(+) cells may modulate the activity of these motoneurons.

The Synaptic Accumulation of Hyperphosphorylated Tau Oligomers in Alzheimer Disease Is Associated With Dysfunction of the Ubiquitin-Proteasome System

In Alzheimer disease (AD), deposition of neurofibrillary tangles and loss of synapses in the neocortex and limbic system each correlate strongly with cognitive impairment. Tangles are composed of misfolded hyperphosphorylated tau proteins; however, the link between tau abnormalities and synaptic dysfunction remains unclear. We examined the location of tau in control and AD cortices using biochemical and morphologic methods. We found that, in addition to its well-described axonal localization, normal tau is present at both presynaptic and postsynaptic terminals in control human brains. In AD, tau becomes hyperphosphorylated and misfolded at both presynaptic and postsynaptic terminals, and this abnormally posttranslationally modified tau is enriched in synaptoneurosomal fractions. Synaptic tau seems to be hyperphosphorylated and ubiquitinated, and forms stable oligomers resistant to SDS denaturation. The accumulation of hyperphosphorylated tau oligomers at human AD synapses is associated with increased ubiquitinated substrates and increased proteasome components, consistent with dysfunction of the ubiquitin-proteasome system. Our findings suggest that synaptic hyperphosphorylated tau oligomers may be an important mediator of the proteotoxicity that disrupts synapses in AD.

HMGN1 Protein Regulates Poly(ADP-ribose) Polymerase-1 (PARP-1) Self-PARylation in Mouse Fibroblasts

In mammalian cells, the nucleosome-binding protein HMGN1 (high mobility group N1) affects the structure and function of chromatin and plays a role in repair of damaged DNA. HMGN1 affects the interaction of DNA repair factors with chromatin and their access to damaged DNA; however, not all of the repair factors affected have been identified. Here, we report that HMGN1 affects the self-poly(ADP-ribosyl)ation (i.e., PARylation) of poly(ADP-ribose) polymerase-1 (PARP-1), a multifunctional and abundant nuclear enzyme known to recognize DNA lesions and promote chromatin remodeling, DNA repair, and other nucleic acid transactions. The catalytic activity of PARP-1 is activated by DNA with a strand break, and this results in self-PARylation and PARylation of other chromatin proteins. Using cells obtained from Hmgn1(-/-) and Hmgn1(+/+) littermate mice, we find that in untreated cells, loss of HMGN1 protein reduces PARP-1 self-PARylation. A similar result was obtained after MMS treatment of these cells. In imaging experiments after low energy laser-induced DNA damage, less PARylation at lesion sites was observed in Hmgn1(-/-) than in Hmgn1(+/+) cells. The HMGN1 regulation of PARP-1 activity could be mediated by direct protein-protein interaction as HMGN1 and PARP-1 were found to interact in binding assays. Purified HMGN1 was able to stimulate self-PARylation of purified PARP-1, and in experiments with cell extracts, self-PARylation was greater in Hmgn1(+/+) than in Hmgn1(-/-) extract. The results suggest a regulatory role for HMGN1 in PARP-1 activation.

Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

Congenital defects in the Na/H exchanger regulatory factor-1 (NHERF1) are linked to disordered phosphate homeostasis and skeletal abnormalities in humans. In the kidney, these mutations interrupt parathyroid hormone (PTH)-responsive sequestration of the renal phosphate transporter, Npt2a, with ensuing urinary phosphate wasting. We now report that NHERF1, a modular PDZ domain scaffolding protein, coordinates the assembly of an obligate ternary complex with Npt2a and the PKA-anchoring protein ezrin to facilitate PTH-responsive cAMP signaling events. Activation of ezrin-anchored PKA initiates NHERF1 phosphorylation to disassemble the ternary complex, release Npt2a, and thereby inhibit phosphate transport. Loss-of-function mutations stabilize an inactive NHERF1 conformation that we show is refractory to PKA phosphorylation and impairs assembly of the ternary complex. Compensatory mutations introduced in mutant NHERF1 re-establish the integrity of the ternary complex to permit phosphorylation of NHERF1 and rescue PTH action. These findings offer new insights into a novel macromolecular mechanism for the physiological action of a critical ternary complex, where anchored PKA coordinates the assembly and turnover of the Npt2a-NHERF1-ezrin complex.

Filtration, adsorption and immunodetection of virus using polyelectrolyte multilayer-modified paper

A new method for detection of viruses has been developed. The entire assay can be performed within 2h, and consists of a polyelectrolyte-multilayer-modified cellulosic filter paper combined with immunodetection. The M13 bacteriophage was used as a model virus. A visual colour-based detection system, anti-M13 horseradish peroxidase (HRP) conjugate and 3,3′,5,5′-tetramethylbenzidine (TMB), was selected to allow semi-quantitative assessment by human eye, or quantitative assessment using a digital scanner. By filtering a volume of 0.50ml, it was possible to visually detect a concentration of 106pfu/ml. The detection limit was improved to 5×104pfu/ml by increasing the volume of the sample to 100ml. For comparison, it was only possible to detect a concentration of 107pfu/ml using conventional sandwich enzyme-linked immunosorbent assay (ELISA) with the same detection system.

Endonuclease cleavage combined with horseradish peroxidase-assisted signal amplification for electrochemical monitoring of DNA

A novel and universal electrochemical strategy for monitoring DNA was developed based on specific cleavage of MspI endonuclease combining with streptavidin–horseradish peroxidase (SA–HRP) conjugate. By introducing the strategy to mycobacterium tuberculosis (MTB) DNA detection, the probe DNA was immobilized on Au electrode via AuS bond and SA–HRP conjugate was linked to probe DNA by biotin–SA specific interaction, which led to the first application in MTB DNA electrochemical detection. This proposed electrochemical method exhibited satisfactory performance which could detect MTB DNA linearly ranging from 10pM to 100nM with a detection limit of 2.3pM. The novel strategy of DNA analysis showed a promising application in clinic diagnostics.

A highly sensitive method for quantifying gallocatechin gallate and its epimer using a catechin-specific peptide

We have developed a method for quantifying gallocatechin gallate (GCg) and epigallocatechin gallate (EGCg) using a catechin-binding peptide (part of the 67-kDa laminin receptor). Using micro titer plates, we investigated various conditions, including the quantifiable range of EGCg concentrations, the optimal concentration of the catechin-binding peptide, and the optimal reaction conditions. In this microplate assay, after each well was coated with bovine serum albumin, sample containing GCg and EGCg was added at pH 8.0, and allowed to stand at 37 °C for 2 h. After washing, biotinylated-peptide solution was added at 1 μg mL−1 and allowed to react for 1 h at 37 °C. Each well was added with streptavidin–horseradish peroxidase conjugate, followed by chromogenic reaction for 25 min at room temperature. After the reaction, absorbance was measured at 405 nm. Our method is capable of quantifying EGCg in the range of approximately 0.1–2.0 mg L−1 with a high degree of sensitivity and a high correlation (R2 = 0.98) between EGCg concentration and absorbance. The method was specific to GCg and EGCg and seems capable of estimating GCg and EGCg contents in the presence of other catechin compounds. The method is simple and highly sensitive for quantitative GCg and EGCg measurement that requires no special equipment or operation and can measure multiple samples simultaneously.

Abstract 2443: MR imaging in human glioma tumor xenograft models using enzyme-conjugated anti-EGFRvIII-specific antibody fragments

Abstract A truncated and constitutively active form of the EGF receptor variant III (EGFRvIII) is a major determinant of tumor growth and poor prognosis in glioblastoma multiforme [1]. To test a system for targeted imaging of EGFRvIII we investigated the retention of peroxidase-generated products of a paramagnetic substrate diTyr-DTPAGd in U87αEGFR orthotopic human glioma xenografts. F(ab’)2 fragments of EMD72000 mAb were conjugated to deglycosylated horseradish peroxidase (HRP) and glucose oxidase (GOX) and used as a self-complementing enzymatic signal amplification system [2] for EGFRvIII targeted imaging. We anticipated that receptor expression sites will exhibit a prolonged MR signal enhancement due to the formation of polymerized products of diTyr-DTPAGd oxidation by mAb conjugates [2]. F(ab’)2 fragments of mAb were linked to enzymes using bisaromatic hydrazone bonds and purified by HPLC. The conjugates were characterized in U87αEGFR cell culture and optimal ratios of HRP and GOX conjugates were determined to provide the maximum signal with low cytotoxicity. F(ab’)2-GOX and F(ab’)2-HRP were also modified with NHS-MAG3 for radiolabeling. U87αEGFR cells were stereotaxically implanted in the brains of RNU rats. Ten days after tumor implantation, each animal was imaged using T1-weighted spin-echo MRI (TR/TE=700ms/8.2ms) at 3T on two occasions: Day 1 - images were acquired after IV injection of 0.1 mmol/kg diTyr-DTPAGd over a 1.5h period; Day 2 - anti-EGFRvIII conjugates (100 µg mAb/animal) were injected IV, and then 4h later 0.1 mmol/kg diTyr-DTPAGd was injected IV followed by image acquisition for 2h. Both 99mTc-labeled conjugates showed specific binding to U87αEGFR cells in vitro and in vivo as demonstrated using SPECT/CT. Cell-binding and internalization studies showed that 80% conjugates were internalized at 37°C. MR T1-weighted images showed significantly higher enhancement and longer retention of the MR signal on Day 2 in rats pre-injected with conjugates compared to Day 1 over the same time period due to the conjugate co-localization at the EGFRvIII target sites. The washout of the contrast agent was best modeled using a biexponential decay as compared to Gli36αEGFR-bearing rats [4]. U87αEGFR tumors showed biexponential MR signal decay on both days. Administration of EGFRvIII-targeted mAb conjugates resulted in specific binding to U87αEGFR cells of which at least ∼20% remained on the surface enabling the reaction with the contrast agent. The biexponential decay constants on Day 2 for both Gli36αEGFR and U87αEGFR animals were not significantly different indicating a similarity of conjugate accumulation in both tumor models which resulted in a similar bimodal washout of the contrast agent.

Dual amplification strategy of highly sensitive thrombin amperometric aptasensor based on chitosan–Au nanocomposites

A highly sensitive and selective electrochemical aptasensor for thrombin was developed. By introducing chitosan-gold nanoparticles and horseradish peroxidase (CS-AuNPs-HRP) conjugates to the sensitive union, the thrombin detection signal was dual amplified. The capture probe was prepared by immobilizing an anti-thrombin aptamer on core-shell Fe(3)O(4)-Au magnetic nanoparticles (AuMNPs) and which was served as magnetic separation material as well. The detection probe was prepared from another anti-thrombin aptamer, horseradish peroxidase (HRP), thiolated CS nanoparticle and gold nanoparticle (CS-AuNPs-HRP-Apt2). In the presence of thrombin, the sandwich structure of AuMNPs-Apt1/thrombin/Apt2-CS-AuNPs-HRP was formed and abundant HRP was captured in it. The resultant conjugates are of magnetic characters and were captured onto the surface of a screen printed carbon electrode (SPCE) to prepare the modified electrode by a magnet located on the outer flank of the SPCE. It was demonstrated that the oxidation of hydroquinone (HQ) with H(2)O(2) was dramatically accelerated by the captured HRP. The electrochemical signal, which correlated to the reduction of BQ (the oxidation product of HQ), was amplified by the catalysis of HRP toward the reaction and the enrichment of HRP on the electrode surface. Under optimized conditions, ultrasensitive and high specific detection for thrombin was realized with the proposed assay strategy. The signal current was linearly correlated to the thrombin concentration in the range of 0.01-10 pM with a detection limit of 5.5 fM (S/N = 3). These results promise extensive applications of this newly proposed signal amplification strategy in protein detection and disease diagnosis.

Rapid immunochemical tests for qualitative and quantitative determination of T-2 and HT-2 toxins

Rapid immunotests were developed for qualitative and quantitative detection of T-2 and HT-2 toxins in cereal samples. Specific antibodies were immobilized either onto polyethylene filters or sepharose gel inside the transparent column. The tests combined the basic functions of purification, concentration and immunodetection of the target analytes through the enzymatic reaction of horseradish peroxidase (HRP). To make the test suitable for detection of both T-2 and HT-2 toxins, anti-T-2 toxin antibody was combined with HT-2 toxin–HRP conjugate. Matrix interference was eliminated by appropriate dilution of sample extracts with polyethylenglycol solution followed by filtration through a solid sorbent in a separate clean-up column. For the qualitative assay (visual detection) the cut-off level was 2 ng mL−1 of T-2 and HT-2 toxins, while the quantitative assay enabled the detection of 0.55 ng g−1 T-2 toxin and 1.7 ng g−1 HT-2 toxin in spiked wheat samples. Analysis of naturally contaminated wheat samples resulted in a good agreement between the developed immunotest and confirmatory LC-MS/MS. The proposed immunotest is fast, easy-to-use and suitable for rapid screening of T-2 and HT-2 toxins in wheat.

591: Rapid test for growth and determination of antibiotic sensitivity of Group B Streptococcus (GBS) in antepartum women

antibiotic sensitivity of Group B Streptococcus (GBS) in antepartum women Jonathan Faro, Allan Katz, Mildred Ramirez, Mark Turrentine, Karen Bishop, Gerald Riddle, Sebastian Faro UT Health Science Center at Houston, Obstetrics, Gynecology and Reproductive Sciences, Houston, TX, Kelsey Seybold, Obstetrics and Gynecology, Houston, TX OBJECTIVE: We determined if a new method of rapidly growing GBS in 6 hours could also determine antibiotic sensitivities in a shorter period compared to standard cultures. STUDY DESIGN: Women were screened between 35 to 37 weeks gestation. Vaginal-rectal swabs were collected in Stuarts transport medium. Swabs were then inoculated on a nitrocellulose membrane (NCM) that had been coated previously with polyclonal rabbit antibody against GBS in the presence or absence of clindamycin. At six hours, the NCM was removed from the sheep blood agar medium, and horseradish-peroxidase conjugate polyclonal antibody against GBS was added. An additional swab from each patient was cultured by traditional methods for 48-72 hours and those positive for GBS were then tested for resistance/sensitivity to clindamycin. A final control consisted of sending a separate swab from each patient to a commercial lab. RESULTS: 124 patient samples were screened, of which 33 were positive for GBS by the rapid test (97.0% concordance with traditional culture). Of these 33 positive samples, 10 were resistant to clindamycin, which agreed 100% with traditional culture. Results of the both the rapid and traditional culture showed 100% concordance with sensitivity or resistance to clindamycin when compared with commercial lab results. CONCLUSION: This new NCM assay offers a rapid and unique method for both detection and determination of antibiotic sensitivities for GBS; thus improving the targeting of antibiotic prophylaxis for GBS colonization.

Simple Chemiluminescence Aptasensors Based on Resonance Energy Transfer

In the present work, two aptamer-based probes and related sensor systems were developed with chemiluminescence signaling. The detection was based on "turning-on" chemiluminescence with switching "off" of the resonance energy transfer after the aptamer's recognition of the target molecule. In this design, a DNA/aptamer duplex linked a chemiluminescence group and a gold nanoparticle together. Only low-intensity chemiluminescence was obtained due to the highly efficient resonance energy transfer. After introducting the target molecule, structure-switching took place with turning off the energy transfer; thus, a restoration and turning on of the chemiluminescence was obtained. The two designs differed in the chemiluminescence groups, since one was a covalently linked luminol molecule, while the other was a conjugated horseradish peroxidase for the catalysis of further chemiluminescence reactions. These schemes provided simple and effective sensing toward a model analyte, adenosine.