High Background Signal Research Articles

ToF-SIMS characterisation of diterpenoic acids after chromatographic separation

Microcolumn liquid chromatography (μHPLC) coupled on-line with time of flight secondary ion mass spectrometry (ToF-SIMS) was applied for mixture of diterpenoic acids (abietic, gibberellic and kaurenoic) analysis. Chromatographic effluent, with analytes separated, was carried out directly onto different, ToF-SIMS compatible surface substrates, for further ToF-SIMS analysis. Silica gel Si 60, aluminium backplate modified Si 60, monolithic silica gel and Raman spectroscopy chromatographic thin layers were used as the deposition substrates in this experiment. By ToF-SIMS surface imaging the deposition trace picture has been obtained. Effluent deposition surface area was scanned for diterpenoic acid fragment mass values based on mass spectrometric library. Measured ToF-SIMS dataset of fragment abundance and intensities were used for preliminary fragmentation schemes construction. The lowest substrate background activity has been established for monolithic silica gel thin layer and aluminium backplate modified Si 60 thin layer. In the case of Raman spectroscopy pre-treated thin layer or conventional chromatographic thin layer Si 60, the both, high background signal intensity and impossibility to construct negative ions surface image, were observed. Diterpenoic acids studied serve the similar mass spectrum but ToF-SIMS coupled with liquid chromatographic separation brings new impact to the positive identification of analytes studied.

Sentinel Lymph Node Mapping of the Gastrointestinal Tract by Using Invisible Light

Because many gastrointestinal (GI) tumors spread by way of lymphatics, histological assessment of the first draining lymph nodes has both prognostic and therapeutic significance. However, sentinel lymph node mapping of the GI tract by using available techniques is limited by unpredictable drainage patterns, high background signal, and the inability to image lymphatic tracers relative to surgical anatomy in real time. Our goal was to develop a method for patient-specific intraoperative sentinel lymph node mapping of the GI tract by using invisible near-infrared light. We developed an intraoperative near-infrared fluorescence imaging system that simultaneously displays surgical anatomy and otherwise invisible near-infrared fluorescence images of the surgical field. Near-infrared fluorescent quantum dots were injected intraparenchymally into the stomach, small bowel, and colon, and draining lymphatic channels and sentinel lymph nodes were visualized. Dissection was performed under real-time image guidance. In 10 adult pigs, we demonstrated that 200 pmol of quantum dots quickly and accurately map lymphatic drainage and sentinel lymph nodes. Injection into the mid jejunum and colon results in fluorescence of a single lymph node at the root of the bowel mesentery. Injection into the stomach resulted in identification of a retrogastric node. Histological analysis in all cases confirmed the presence of nodal tissue. We report the use of invisible near-infrared light for intraoperative sentinel lymph node mapping of the GI tract. This technology overcomes the limitations of currently available methods, permits patient-specific imaging of lymphatic flow and sentinel nodes, and provides highly sensitive, real-time image-guided dissection.

Ozonolysis of model olefins—Efficiency of antiozonants

In this study, the efficiency of several potential long lasting antiozonants was studied by ozonolysis of model olefins. 2-Methyl-2-pentene was selected as a model for natural rubber (NR) and 5-phenyl-2-hexene as a model for styrene butadiene rubber (SBR). A comparison was made between the efficiency of conventional antiozonants like N-(1,3 dimethylbutyl)-N′-phenyl-p-phenylene diamine (6PPD), N-isopropyl-N′-phenyl-p-phenylene diamine (IPPD), and a mixture of diaryl p-phenylene diamines (Wingstay 100) and some newly synthesized antiozonants. The stearic acid salt of 6PPD (PPD-C18), 2,4,6-tris(4-(phenylamino)phenyl)-1,3–5-triazinane (ADPAT), and 4-pyrole diphenylamine (PDPA) showed a higher efficiency than the conventional antiozonants in both NR as well as SBR model system. Special attention was paid to the carboxylic acid salts of 6PPD such as PPD-C18, which has shown good long-term protection of passenger tire sidewall compounds. It was demonstrated that by varying the chain length, C7, C18, and C22, of the carboxylic acid part of the 6PPD salts, the ozone protection was not influenced under the selected test conditions. The 6PPD salts made from strong acids like succinic acid (SA) and methyl sulfonic acid (MSA) appeared to be less efficient than PPD-C18. It was also investigated whether the reactions between ozone and the double bonds of the model rubber could be measured online by a spectroscopic technique. It was demonstrated that near infrared spectroscopy is a suitable technique to study these reactions. FT Raman looked also a promising technique because of the high response factor of double bonds. However, the addition of p-phenylene diamines (PPDAs) to the sample solution resulted in a strong discoloration (dark brown) and therefore in a high fluorescence background signal. This technique can therefore not be used for the evaluation of staining antiozonants. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci, 100: 853–866, 2006

Prevention of Nonspecific Bacterial Cell Adhesion in Immunoassays by Use of Cranberry Juice

The ability of Vaccinum macrocarpon, the North American cranberry, to prevent bacterial adhesion has been used to advantage in the prevention of urinary tract infections and has recently been described for the prevention of adhesion of bacteria responsible for oral infections and stomach ulcers. This report documents the ability of cranberry juice to reduce nonspecific adhesion of bacteria to the borosilicate glass microscope slides used in an immunoarray biosensor format. Nonspecific binding of analytes in the array sensor leads to high background signals that cause increased detection limits and false positives. Reduction in background-to-signal ratios can be seen as the juice concentration is increased from 0 to 50% of the sample. This impact cannot be duplicated with grape, orange, apple, or white cranberry juice. Sugar content and pH have been eliminated as the agents in the juice responsible for the anti-adhesive activity.

Matrix effects during phosphorus determination with quadrupole inductively coupled plasma mass spectrometry

A quadrupole inductively coupled plasma mass spectrometer was evaluated for use in the detection of phosphorus. The influences of nitric acid and methanol (simulating the composition of a sample solution after nitric acid digestion) on phosphorus determination were studied using two different measuring methods at different plasma conditions: detection of phosphorus ions at m/z 31 and detection of phosphorus oxide ions at m/z 47. The existence of polyatomic interferences at m/z 31 and 47 was explored. Nitric acid and methanol are shown to be the sources of polyatomic ions and therefore cause poorer detection limits. Better detection limits were achieved in such matrices when phosphorus was detected as 31P+. The presence of methanol improves the system sensitivity towards phosphorus sevenfold; however, this positive effect is hindered by the high background signal due to carbon-based polyatomic ions. For samples with an organic matrix an appropriate mineralization procedure should be applied (high excess of nitric acid and high temperature) to quantitatively oxidize organic compounds to carbon dioxide, which is easily removed from the sample, in order to achieve correct results.

δ13C values of pyrolysis products from cellulose and lignin represent the isotope content of their precursors

We investigated the δ 13C values of pyrolysis products from cellulose and lignin, the main components of wood, to prove that in the pyrolysis process no isotope exchange between different pyrolysis products appears and that hence pyrolysis products represent the isotope content of their precursors. Two cellulose samples and a lignin sample with δ 13C values of −23, −12 and −28‰, respectively, were pyrolysed using Curie point pyrolysis simultaneously coupled to GC/MS and GC/IRMS. All samples were pyrolysed individually and in mixtures of lignin with both isotopically different celluloses in two mixing ratios, e.g. 1:1 and 4:1; the latter is representative for wood. No intermolecular isotope exchange between pyrolysis products from cellulose and lignin was observed. This proves that Curie point pyrolysis is suitable for investigating the isotope content of pyrolysis products in complex mixtures like wood. Pyrolysis products from both celluloses and lignin have δ 13C values similar to the bulk δ 13C value of the respective sample. In mixtures of cellulose and lignin δ 13C values of some pyrolysis products change slightly due to peak overlap and higher background signals. However, detected changes were independent from the isotopic difference between the mixing partners. Pyrolysis products such as 2,6-dimethoxyphenol, 4-methyl-2,6-dimethoxyphenol and trans-4-(2-propenyl)-2,6-dimethoxyphenol for lignin and 2-hydroxymethyl-5-hydroxy-2,3-dihydro-(4 H)-pyrane-4-one and levoglucosane for cellulose were found to represent the isotopic signature from cellulose and lignin in mixtures regardless of the composition of the mixture.

Determination of phosphorescence lifetimes in the presence of high background signals using phase-shift measurements

Phosphorescence-based optical sensors are typically based on intensity or triplet-lifetime measurements. Lifetime determinations may be performed in the frequency-domain by exciting the luminophor with a sinusoidal modulated light source and measuring the phase shift between the excitation light and the phosphorescence emission. Modulated scattered light at the same frequency of the modulating signal must be removed in order to perform accurate measurements. A new and simple method based on phase-modulation measurements is presented to measure the triplet-lifetime of a long-lifetime phosphorescent luminophor, even if high levels of background short-lived fluorescence or scattered light are present. The method is based on the measurement of the phase shift between excitation and emission light at two different frequencies and it turned to provide reliable lifetime measurements if the background light lifetime is significantly shorter than that of the phosphorescent emission to be measured. Theoretical aspects of the proposed methodology, the design and construction of a fiber-optical measuring system and the selection of the optimal frequencies to perform the accurate lifetime measurements are discussed. Finally, the performance of the proposed measurement method has been assessed in the determination of the triplet-lifetime values of the metal chelate Al-ferron immobilized in an inorganic sol–gel support (an oxygen indicator which displays a strong fluorescence emission overlapping significantly with the measured phosphorescence emission).

Comparative detection of bacterial adhesion to Caco-2 cells with ELISA, radioactivity and plate count methods

Different methods are used to study bacterial adhesion to intestinal epithelial cells, which is an important step in pathogenic infection as well as in probiotic colonization of the intestinal tract. The aim of this study was to compare the ELISA-based method with more conventional plate count and radiolabeling methods for bacterial adhesion detection. An ELISA-based assay was optimized for the detection of Bifidobacterium longum and Escherichia coli O157:H7, which are low and highly adherent bacteria, respectively. In agreement with previous investigations, a percentage of adhesion below 1% was obtained for B. longum with ELISA. However, high nonspecific background and low positive signals were measured due to the use of polyclonal antibodies and the low adhesion capacity with this strain. In contrast, the ELISA-based method developed for E. coli adhesion detected a high adhesion percentage (15%). For this bacterium the three methods tested gave similar results for the highest bacterial concentrations (6.8 Log CFU added bacteria/well). However, differences among methods increased with the addition of decreased bacterial concentration due to different detection thresholds (5.9, 5.6 and 2.9 Log CFU adherent bacteria/well for radioactivity, ELISA and plate count methods, respectively). The ELISA-based method was shown to be a good predictor for bacterial adhesion compared to the radiolabeling method when good quality specific antibodies were used. This technique is convenient and allows handling of numerous samples.

Flow-through solid-phase based optical sensor for the multisyringe flow injection trace determination of orthophosphate in waters with chemiluminescence detection

In this work, a novel flow-through solid-phase based chemiluminescence (CL) optical sensor is described for the trace determination of orthophosphate in waters exploiting the multisyringe flow injection analysis (MSFIA) concept with multicommutation. The proposed time-based injection flow system relies upon the in-line derivatisation of the analyte with ammonium molybdate in the presence of vanadate, and the transient immobilisation of the resulting heteropolyacid in a N-vinylpyrrolidone/divinylbenzene copolymer packed spiral shape flow-through cell located in front of the window of a photomultiplier tube. The simultaneous injection of well-defined slugs of luminol in alkaline medium and methanol solution towards the packed reactor is afterwards performed by proper switching of the solenoid valves. Then, the light emission from the luminol oxidation by the oxidant species retained onto the sorbent material is readily detected. At the same time, the generated molybdenum-blue compound is eluted by the minute amount of injected methanol, rendering the system prepared for a new measuring cycle. Therefore, the devised sensor enables the integration of the solid-phase CL reaction with elution and detection of the emitted light without the typical drawbacks of the molybdenum-blue based spectrophotometric procedures regarding the excess of molybdate anion, which causes high background signals due to its self-reduction. The noteworthy features of the developed CL–MSFIA system are the feasibility to accommodate reactions with different pH requirements and the ability to determine trace levels of orthophosphate in high silicate content samples (Si/P ratios up to 500). Under the optimised conditions, a dynamic linear range from 5 to 50 μg P l −1 for a 1.8 ml sample, repeatability better than 3.0% and a quantification limit of 4 μg P l −1 were attained. The flowing stream system handles 11 analysis h −1 and has been successfully applied to the determination of trace levels of orthophosphate in environmental samples such as mineral, ground, tap and pond waters as well as samples from a water-steam cycle of an incineration plant. The t-test comparison of the means for the developed optical sensor and the molybdenum-blue spectrophotometric APHA/AWWA/WPCF reference method revealed that there is no evidence of significant differences between the obtained results at the 95% confidence level.

Simultaneous BOLD/perfusion measurement using dual-echo FAIR and UNFAIR: sequence comparison at 1.5T and 3.0T

Functional MRI (fMRI) studies designed for simultaneously measuring Blood Oxygenation Level Dependent (BOLD) and Cerebral Blood Flow (CBF) signal often employ the standard Flow Alternating Inversion Recovery (FAIR) technique. However, some sensitivity is lost in the BOLD data due to inherent T1 relaxation. We sought to minimize the preceding problem by employing a modified UN-inverted FAIR (UNFAIR) technique, which (in theory) should provide identical CBF signal as FAIR with minimal degradation of the BOLD signal. UNFAIR BOLD maps acquired from human subjects ( n = 8) showed significantly higher mean z-score of ∼17% ( p < 0.001), and number of activated voxels at 1.5T. On the other hand, the corresponding FAIR perfusion maps were superior to the UNFAIR perfusion maps as reflected in a higher mean z-score of ∼8% ( p = 0.013), and number of activated voxels. The reduction in UNFAIR sensitivity for perfusion is attributed to increased motion sensitivity related to its higher background signal, and, T2 related losses from the use of an extra inversion pulse. Data acquired at 3.0T demonstrating similar trends are also presented.

Use of Detergent Extracts ofBrucella AbortusRB51 to Detect Serologic Responses in RB51-Vaccinated Cattle

Serologic responses to the newly introduced rough Brucella abortus vaccine strain RB51 have been determined in a dot-blot format using gamma-irradiated RB51 cells as the antigen. Because gamma-irradiated cells are not easily prepared and the signal from cells was not always reliable, an alternative antigen was sought. Detergent extracts of B. abortus RB51 were prepared using zwittergent 3-14, Triton X-100, and sodium dodecyl sulfate (SDS) and examined in a dot-blot format. Zwittergent 3-14 extracts and gamma-irradiated RB51 cells gave the same titers. Unlike gamma-irradiated RB51 cells, zwittergent 3-14 extracts produced signals consistently, and the signals were easily interpreted. Triton X-100 extracts interfered with signal development, and SDS extracts resulted in a high background signal. Western blot analyses revealed several outer membrane proteins in the zwittergent 3-14 extract. The major antigens in the extract had apparent molecular weights of <20,000.

Stage-dependent Localization of a Novel Gene Product of the Malaria Parasite, Plasmodium falciparum

A novel Plasmodium falciparum gene, MB2, was identified by screening a sporozoite cDNA library with the serum of a human volunteer protected experimentally by the bites of P. falciparum-infected and irradiated mosquitoes. The single-exon, single-copy MB2 gene is predicted to encode a protein with an M(r) of 187,000. The MB2 protein has an amino-terminal basic domain, a central acidic domain, and a carboxyl-terminal domain with similarity to the GTP-binding domain of the prokaryotic translation initiation factor 2. MB2 is expressed in sporozoites, the liver, and blood-stage parasites and gametocytes. The MB2 protein is distributed as a approximately 120-kDa moiety on the surface of sporozoites and is imported into the nucleus of blood-stage parasites as a approximately 66-kDa species. Proteolytic processing is favored as the mechanism regulating the distinct subcellular localization of the MB2 protein. This differential localization provides multiple opportunities to exploit the MB2 gene product as a vaccine or therapeutic target.

Sequential Immunohistochemical Double-Staining Using Envision Polymer Technology

The detection of more than 1 antigen within a single tissue specimen is sometimes required in immunohistochemical staining. Several methods have been described as how to achieve this. In this experiment, a sequential doublestaining was performed, using enhanced polymer technology. The use of the Envision polymer technology combines a less complicated protocol with a high sensitivity and low background signal. Staining was performed on neutral buffered, formalin fixed, paraffin embedded human tissues of spleen, lymph node, follicle center cell lymphoma, and plasmacell infiltrate of a jaw cyst using antibodies against CD20, CD43, kappa light chains, and lambda light chains. Two vizualization systems were used, based on horseradish peroxidase-DAB and alkaline phosphatase-Fast Red. Staining patterns, color distribution, and practical approach are described in this article. (The J Histotechnol 24:107, 2001)Submitted: March 5, 2001; Accepted with revisions: March 8, 2001

Sequential Immunohistochemical Double-Staining Using Envision Polymer Technology

The detection of more than 1 antigen within a single tissue specimen is sometimes required in immunohistochemical staining. Several methods have been described as how to achieve this. In this experiment, a sequential doublestaining was performed, using enhanced polymer technology. The use of the Envision polymer technology combines a less complicated protocol with a high sensitivity and low background signal. Staining was performed on neutral buffered, formalin fixed, paraffin embedded human tissues of spleen, lymph node, follicle center cell lymphoma, and plasmacell infiltrate of a jaw cyst using antibodies against CD20, CD43, kappa light chains, and lambda light chains. Two vizualization systems were used, based on horseradish peroxidase-DAB and alkaline phosphatase-Fast Red. Staining patterns, color distribution, and practical approach are described in this article. (The J Histotechnol 24:107, 2001)Submitted: March 5, 2001; Accepted with revisions: March 8, 2001

Antimony speciation at ultra trace levels using hydride generation atomic fluorescence spectrometry and 8-hydroxyquinoline as an efficient masking agent

Depending on the pH of the matrix and the concentration of Sb(V), we observed that the atomic fluorescence emission due to Sb(V) could contribute up to 40% of the emission signal during the determination of Sb(III). This can cause significant errors in Sb speciation analyses. Besides, interferences from heavy metals such as Mn(II), Cu(II), Fe(III), Co(II) and Ni(II) can cause severe depression of the signals as compared to the insignificant effects caused by Zn(II), Fe(II), Sn(II) and Pb(II). The compound 8-hydroxyquinoline was found to be an efficient agent to eliminate interference of transition metals in the determination of Sb by hydride generation-atomic fluorescence spectroscopy (HG-AFS). In addition, 8-hydroxyquinoline can effectively mask undesired fluorescence emission from Sb(V) during the determination of Sb(III) and make Sb speciation more accurate. The presence of 8-hydroxyquinoline does not modify the oxidation state of Sb in the sample and the possible metal complexes formed with 8-hydroxyquinoline were stable for more than 8 h. When l-cysteine was introduced as a pre-reductant, a high background signal was observed and it proved to be inefficient as a reductant when the concentrations of Sb(III) were low. A sensitive and interference-free procedure was then developed for the speciation of lake water samples and for the determination of Sb in sediments.

Assessment of cerebral gliomas by a new dark fluid sequence, high intensity REduction (HIRE): a preliminary study.

The purpose of this study was to assess the diagnostic potential of a new dark fluid sequence, high intensity reduction (HIRE) in the diagnostic workup of patients with cerebral gliomas. The HIRE sequence utilizes a very long T(2) value of the cerebrospinal fluid (CSF) to suppress its high signal contribution in T(2)-weighted imaging by a image subtraction technique. Fifteen patients with histologically confirmed cerebral gliomas were examined with T(2)-weighted fast spin-echo (FSE), T(1)-weighted SE, fast fluid-attenuated inversion recovery (FLAIR), and HIRE imaging using identical scan parameters. In patients with enhancing lesions, fast FLAIR and HIRE were added to the contrast-enhanced T(1)-weighted SE images. Images were analyzed in a qualitative and quantitative evaluation. In the qualitative analysis, lesion detection, lesion delineation, and differentiation between enhancing and non-enhancing tumor tissue were assessed in a two-reader study. For the quantitative analysis, lesion-to-background and lesion-to-CSF contrast and contrast-to-noise ratios were determined in a region of interest analysis. HIRE achieved a significant reduction of the CSF signal without losing the high gray-to-white matter contrast of T(2)-weighted sequences. In the quantitative analysis, the contrast ratios of the HIRE images were lower compared with the FLAIR images due to a relatively high background and CSF signal. After administration of contrast media, HIRE images presented a significant signal increase in enhancing lesions, which subsequently increased the contrast and contrast-to-noise ratios. In the qualitative analysis, both readers found all tumors clearly delineated on HIRE imaging. Compared with T(2)-weighted FSE, the tumor delineation with HIRE was better in nine patients, equal in four patients, and less in one patient. Compared with the FLAIR images, HIRE was rated superior in three patients, equal in nine patients, and inferior in another three patients. Delineation of the enhancing tumor parts was possible with HIRE in all patients. HIRE images had significantly fewer image artifacts than FLAIR images due to reduced inflow effects. The T(2)-based HIRE sequence presented is an alternative to the T(1)-based FLAIR sequence, with the advantage of better gray-to-white matter contrast and shorter measurement time. Due to the subtraction technique, signal intensities from tissues with relaxation times in the range T(2 WM) < < T(2) < T(2 CSF) are also gradually affected, corresponding to their T(2) values. With respect to this unwanted effect, an improvement in HIRE imaging will be possible by using a self-weighted subtraction algorithm. In a forthcoming study this concept will first be tested on appropriate phantom fluids.

Selenium Determination in Biological Fluids Using Zeeman Background Correction Electrothermal Atomic Absorption Spectrometry

Procedures for the direct determination of total selenium in urine, serum, and blood using electrothermal atomic absorption spectrometry are presented. In the selected experimental conditions, Zeeman correction is mandatory to compensate for the high background signals. The sample diluted and containing 0.1% (w/v) Triton X-100 is introduced directly into the electrothermal atomizer. A solution containing 15% (w/v) hydrogen peroxide, 0.65% (w/v) nitric acid, and 0.5% (w/v) nickel is injected separately into the atomizer. Calibration is carried out using the standard additions method. The detection limit is 30 pg selenium. If palladium, instead of nickel, is used as the chemical modifier, calibration can be carried out against aqueous standards, and the detection limit is 45 pg. In this case, three separate injections are required to prevent precipitation problems in the automatic injector. The reliability of the procedures is checked by analyzing three certified reference materials and by recovery studies. Mean recoveries are 99.7% for serum, 99.4% for urine, and 100.8% for blood samples. Relative standard deviation values are ±4.0% for serum, ±3.9% for urine, and ±4.5% for blood.

Determination of arsenic in biological fluids by electrothermal atomic absorption spectrometry.

A procedure for the determination of the total content of arsenic in urine, serum and blood by electrothermal atomic absorption spectrometry (ETAAS) is described. Zeeman correction is used to compensate the high background signals. The samples are diluted (1 + 1 for urine and 1 + 3 for both serum and blood samples) in a medium containing 0.1% w/v Triton X-100 before being introduced directly into the furnace. A solution containing 15% w/v hydrogen peroxide, 0.65% w/v nitric acid and 0.5% w/v nickel is also introduced into the atomizer by means of a separate injection. Calibration is carried out against aqueous standards for blood and serum samples and using the standard additions method for urine samples. The detection limit is 20 pg (2 ng ml-1). The reliability of the procedure is checked by analyzing three certified reference materials and by recovery studies.

Differential gene expression between human neurons and neuronal progenitor cells in culture: an analysis of arrayed cDNA clones in NTera2 human embryonal carcinoma cell line as a model system

To elucidate the highly complex expression pattern of the genes involved in human neuronal differentiation, differential gene expression between human neurons and neuronal progenitor cells was investigated by analysis of a cDNA expression array in a pluripotent human embryonal carcinoma cell line NTera2 (NT2), a model system of human neuronal differentiation. Among 588 arrayed cDNA clones, 87 genes showed a differential expression pattern between undifferentiated neuronal progenitor cells (NT2-U) and NT2-derived differentiated neurons induced by treatment with retinoic acid (RA) (NT2-N), while 26 genes could not be analyzed due to high background signals. The levels of expression of 76 genes, including those encoding a group of transcription factors, intracellular signal-transducing proteins, cell death-regulatory proteins, and growth factors/cytokines/neurotransmitters and their receptors, were elevated after neuronal differentiation, while the levels of 11 genes, including those coding for cellular proliferation-related proteins, were decreased. Among the differentially expressed genes following induction of neuronal differentiation, significant up-regulation of the growth-associated protein (GAP-43), low-affinity nerve growth factor receptor p75 (LNGFR), and defender against apoptotic cell death (DAD1) mRNAs and substantial down-regulation of the proliferation-associated gene (PAG), fibroblast growth factor receptor-1 (FGFR-1), and cellular RA-binding protein-II (CRABP-II) mRNAs were verified by Northern blot analysis. These results indicate that the analysis of cDNA expression arrays provides a useful approach for screening and identification of a set of distinct genes that undergo highly complex regulation during human neuronal differentiation.

Immobilized chicken antibodies improve the detection of serum antigens with surface plasmon resonance (SPR)

Surface plasmon resonance (SPR) and other refractive index and mass sensitive methods are, due to complement activation by mouse monoclonal antibodies and with concomitant high background signal, only rarely used for the detection of antibody–antigen interactions in the blood serum milieu. In the present study chicken IgY and mouse IgG were immobilized to a sensor chip CM5 dextran matrix and compared for their background signal and detection of serum antigen. Ellipsometry with antibodies adsorbed to methylated silicon surfaces was used as a complementary detection method. As expected, fundamental differences in binding properties between the two kinds of antibodies were observed. Mouse antibodies bound large quantities of human serum. Human C1q was detected on mouse IgG and the complement system was activated, as seen from the rapid C3 and properdin depositions. Chicken antibodies bound low quantities of human serum and no human C1q. Moreover, C3 and properdin deposited only after prolonged serum incubations. Addition of EDTA to serum reduced the background signal modestly for both IgG and IgY. Serum samples with different concentrations of human C3 were injected over surfaces with immobilized chicken anti-C3, and the response was measured by SPR. Small concentration differences (<1.25 μg/ml) in a physiologically relevant range (1–40 μg/ml after 100 times dilution) could then be detected reproducibly. The SPR signal was totally obscured when a mouse monoclonal anti-C3 antibody was used for the detection.