A proteomic approach based on multiple parallel separation for the unambiguous identification of an antibody cognate antigen

Autoantibodies obtained from cancer patients have been identified as useful tools for cancer diagnostics, prognostics, and as potential targets for immunotherapy. Serological proteome analysis in combination with 2‐DE is a classic strategy for identification of tumor‐associated antigens in the serum of cancer patients. However, serological proteome analysis cannot always indicate the true antigen out of a complex proteome identified from a single protein spot because the most abundant protein is not always the most antigenic. To address this problem, we utilized multiple parallel separation (MPS) for proteome separation. The common identities present in the fractions obtained using different separation methods were regarded as the true antigens. The merit of our MPS technique was validated using anti‐ARPC2 and anti‐PTEN antibodies. Next, we applied the MPS technique for the identification of glycyl‐tRNA synthetase as the cognate antigen for an autoantibody that was overexpressed in the plasma of breast cancer patients. These results reveal that MPS can unambiguously identify an antibody cognate antigen by reducing false‐positives. Therefore, MPS could be used for the characterization of diagnostic antibodies raised in laboratory animals as well as autoantibodies isolated from diseased patients.     

Prostate-specific antigen immunoassays on the BioCD

The BioCD is a spinning-disc interferometric biosensor on which antibodies are immobilized to capture target antigens from biological samples. In this work, BioCDs measured the interferometric response to prostate-specific antigen (PSA). The ideal detection limit for PSA was determined using a BioCD with 12,500 printed target antibody spots with a corresponding number of reference protein spots. Statistical analysis projects the detection limit of PSA as a function of the number of spots included in the average. When approximately 10,000 spot pairs were averaged, the 3σ detection limit was 60 pg/ml in a 2 mg/ml simple protein background. A standard format for BioCD immunoassays uses 96 wells with 32 target spots paired with reference spots. In serum, the detection limit for this format was 1 ng/ml in 3:1 diluted female human serum using a sandwich assay with a nonfluorescent mass tag.     

Novel electrochemical sensor for the selective recognition of chlorogenic acid

In this article, we demonstrate the fabrication and simultaneous fluorescent detection of two biomarkers related to lung cancer. Polystyrene microspheres (PSM) were introduced as biomolecular immobilizing carriers and a 96-well filter plate was used as the separation platform. The whole experiment could be effectively carried out in a homogeneous system, as exemplified by the detection of carcinoembryonic antigen (CEA) and neuron specific enolase (NSE). First, two capture antibodies for CEA and NSE were immobilized on the PSM surface. Next, they reacted successively with two antigens and two modified detection antibodies. Finally, these two biomarkers could be recognized by streptavidin-conjugated quantum dots (QD) and goat-anti-FITC conjugated QD with a detection limit of 0.625 ng mL(-1), which was lower than the clinical cut-off level. The protocol showed good precision within 6.36% and good recovery in the range of 90.86-105.02%. Compared with several other assay formats reported previously, our new technique is competitive or even better. Furthermore, the immunosensor was successfully illustrated in 20 serum samples. Overall, this new immunoassay offers a promising alternative for the detection of biomarkers related to cancer diseases, taking advantage of simplicity, specificity, sensitivity and cost-efficiency.     

ITO pattern fabrication of glass platforms for electropolymerization of light sensitive polymer for its conjugation to bioreceptors on a micro-array

We present herein an effective and versatile method to fabricate a micro-patterned structure of conductive polymer, poly(pyrrole-benzophenone), on Indium Tin Oxide (ITO) glass chips for the subsequent photo-immobilization of various bioreceptor, antigens. Such methodologies are based on photolithography of ITO pattern fabrication on non-conductive surfaces, glass slides, and on a photo-active electrogenerated polymer films. The photo-active polymer serves as a substrate platform for the photo-immobilization of the bioreceptor reagents used for subsequent immunoreactions. We were able to show the resolution of electropolymerization on an ITO pattern as well as immobilization of more than one bioreceptor for the simultaneous detection of several analytes. The antigen micro-arrays were tested for sensitivity, specificity, and overall practicality for the simultaneous detection of analyte anti-Cholera Toxin B, anti-Hepatitis B virus surface and core protein antibodies. In addition we used our pattern ITO-poly(pyrrole-benzophenone) micro-array for the detection of serum samples of Hepatitis B virus patients previously screened by a standard hospital detection method.     

Multiplex immunoassays of equine virus based on fluorescent encoded magnetic composite nanoparticles

A new detection format for multiplexed analysis based on fluorescent encoded magnetic composite nanoparticles is presented. Two kinds of virus were analyzed by this new method: equine influenza virus (EIV) and equine infectious anemia virus (EIAV). Firstly, EIV antigen and EIAV antigen were conjugated to two kinds of fluorescent encoded magnetic composite nanoparticles, while the green-emitting CdTe quantum dots (QDs) were attached to the antibody of EIV and EIAV. Then both green-emitting CdTe QD-labeled antibodies and antigens labeled with fluorescent encoded magnetic composite nanoparticles were used to form an immunoassay system for the detection of EIV and EIAV antigens. The method is time-saving and has higher sensitivity (1.3 ng mL⁻¹ for EIV antigens and 1.2 ng mL⁻¹ for EIAV antigens) than the conventional methods. A competitive immunoassay method based on this analysis system was used to detect EIV and EIAV antigens in spiked serum samples with satisfactory results.     

An optical tweezers-based immunosensor for detection of femtomoles-per-liter concentrations of antigens

We used optical tweezers—optical trapping with focused laser beams—to pull microspheres coated with antigens off of an antibody-coated surface. Using this technique, we could quantify the force required to separate antigen to antibody bonds. At very low surface density of antigen, we were able to detect the single antigen to antibody binding. The force required to break the antigen-antibody bonds and pull the microsphere off the surface was shown to increase monotonically with increasing surface density of antigens. Using the force determination as a transducer, we were able to detect concentrations of free antigens in solution as small as 10⁻¹⁵ mol/L in a competitive binding assay.     

Probe functionalization with a Rhop-3 antibody: toward a Rhop-3 antigen immunosensor for detection of malaria

The antibody specific for the malaria protein, Rhop-3, and FL-Rhop-3, were immobilized on the surface of a gold electrode modified with cysteamine. Colloidal gold was used to enhance the detection signal for Rhop-3 antigens. The Rhop-3 antibody was also immobilized on gold electrodes preactivated with dithiobis(succinimidyl proprionate) (DSP). Immobilization was performed at room temperature and at 37 °C. Cyclic voltammetry (CV) was used to monitor the interaction between the immobilized antibody and its cognate antigen in solution, using ferricyanide, K₃Fe(CN)₆, as reporting electroactive probe. Tests indicate recognition of Rhop-3 protein by the immobilized antibody. Antigen recognition was enhanced by incubation at 37 °C compared with room-temperature incubation. Our results suggest that an immunosensor can be developed and optimized to aid detection of Rhop-3 antigens in samples from malaria patients. As far as we are aware, this is the first amperometric immunosensor targeting Rhop-3 antigen as a malaria biomarker.     

Wall-less sample preparation of <Emphasis Type="Italic">μ</Emphasis>m-sized sample spots for femtomole detection limits of proteins from liquid based UV-MALDI matrices

Previously, we introduced wall-less sample preparation (WaSP), technology that involves the use of an electrodynamic balance (EDB) to prepare microm-sized sample spots for analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). In that work we demonstrated the detection of femtomole quantities of a low molecular weight peptide and a hydrophobic ester (both <600 Da). Here we use WaSP to test the hypothesis that the use of small sample spot sizes and an instrument equipped with delayed extraction would increase the analytical utility of liquid sample spots for peptide and protein (>2500 Da) analysis by UV-MALDI-TOF-MS (Sze et al.; J. Am. Soc. Mass Spectrom. 1998, 9, 166-174). To aid the optimization of preparing microm-sized sample spots by WaSP, optical microscopy and mass spectrometry were used to investigate nonvolatile solute concentration effects on droplet fission and sample spot size, modifications of the EDB electric field to control droplet ejection, and the use of multiple droplet deposition to increase sample loading. Also described is a rapid deposition mode of operation for WaSP that allows single droplets generated at 1 Hz to be levitated briefly ( approximately 500 ms) before being ejected autonomously and deposited as a concentrated sample spot with a spatial accuracy of +/-5 microm. To test the sensitivity of the method, one hundred glycerol droplets (270 pL each, 27 nL total) each containing 32 amol lysozyme were deposited on top of each other one-at-a-time to create a single sample spot. Using a mass spectrometer equipped with delayed extraction to analyze this sample spot, we verified the hypothesis of Sze et al. by achieving detection limits three orders of magnitude below that previously observed for the detection of a protein by UV-MALDI-TOF-MS with a chemical-doped liquid matrix sample preparation.     

CE-based simultaneous liquid-phase noncompetitive enzyme immunoassay for three tumor markers in human serum using electrochemical detection

A method for indirectly detecting horseradish peroxidase (HRP) was described by CE with electrochemical detection. Details of selection for optimum conditions were presented. The detection limit of free HRP was 1.09 x 10(-12) M or 0.94 zmol (S/N = 3). A novel CE-based liquid-phase binding noncompetitive enzyme immunoassay (CE-EIA) was developed. In this method, after the noncompetitive immunoreaction in liquid phase, the free enzyme (HRP)-labeled antibody (Ab*) and the bound enzyme-labeled complex (Ag-Ab*) were separated and then the system of HRP catalyzing H(2)O(2)/o-aminophenol (OAP) reaction was adopted. Prostate specific antigen (PSA), carcinoembryonic antigen (CEA), and human chorionic gonadotropin (HCG) in human serum samples were detected without any sample preparation, with the detection limits (S/N = 3) of 0.22, 0.17 and 0.30 ng/mL, respectively. This technique has been successfully applied to detect simultaneously PSA, CEA, and HCG in 12 min, upon adding these three antigens into human serum to simulate patient serum. It proves that the CE-EIA technique proposed could be developed into a sensitive and new method for simultaneous clinical assay of multianalytes.     

Matrix‐free laser desorption/ionization mass spectrometry using silicon glancing angle deposition (GLAD) films

Glancing angle deposition (GLAD) was used to fabricate nanostructured silicon (Si) thin films with highly controlled morphology for use in laser desorption/ionization mass spectrometry (DIOS‐MS). Peptides, drugs and metabolites in the mass range of 150–2500 Da were readily analyzed. The best performance was obtained with 500 nm thick films deposited at a deposition angle of 85°. Low background mass spectra and attomole detection limits were observed with DIOS‐MS for various peptides. Films used after three months of dry storage in ambient conditions produced mass spectra with negligible low‐mass noise following a 15 min UV‐ozone treatment. The performance of the Si GLAD films was as good as or better than that reported for electrochemically etched porous silicon and related materials, and was superior to matrix‐assisted laser desorption/ionization (MALDI)‐MS for analysis of mixtures of small molecules between 150–2500 Da in terms of background chemical noise, detection limits and spot‐to‐spot reproducibility. The spot‐to‐spot reproducibility of signal intensities (100 shots/spectrum) from 21 different Si GLAD film targets was ±13% relative standard deviation (RSD). The single shot‐to‐shot reproducibility of signals on a single target was ±19% RSD (n = 7), with no indication of sweet spots or mute spots. Copyright © 2010 John Wiley & Sons, Ltd.     

Multiplexed detection of protein cancer markers on Au/Ag-barcoded nanorods using fluorescent-conjugated polymers

Integration of fluorescent-conjugated polymers as detection moiety with metallic striped nanorods for multiplexed detection of clinically important cancer marker proteins in an immunoassay format was demonstrated in this report. Specifically, cationic conjugated polymers were introduced to protein complexes through electrostatic binding to negatively charged double-stranded DNA, which was tagged on detection antibodies prior to antigen recognition. The intense fluorescence emission of conjugated polymers resulted in highly sensitive detection of cancer marker proteins wherein an undiluted bovine serum sample as low as ∼25 target molecules captured on each particle was detectable. Meanwhile, the use of polymer molecules as the detection probe did not obscure the optical pattern of underlying nanorods, i.e., the encoding capability of barcoded nanorods was preserved, which allowed simultaneous detection of three cancer marker proteins with good specificity.     

A microarray enzyme-linked immunosorbent assay for autoimmune diagnostics

In order to quantify autoantibodies in the sera of patients with autoimmune disease, we have created a microarray-based immunoassay that allows the simultaneous analysis of 18 known autoantigens. The microarrays contain serial dilutions of the various antigens, thereby allowing accurate determination of autoantibody titer using minimal amounts of serum. The assay is very sensitive and highly specific: as little as 40 fg of a known protein standard can be detected with little or no cross-reactivity to nonspecific proteins. The signal intensities observed from serial dilutions of immobilized antigen correlate well with serial dilutions of autoimmune sera. Miniaturized and highly parallelized immunoassays like these will reduce costs by decreasing reagent consumption and improve efficiency by greatly increasing the number of assays that can be performed with a single serum sample. This system will significantly facilitate and accelerate the diagnostics of autoimmune diseases and can be adapted easily to any other kind of immunoassay.     

Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain

The characteristics of protein detection and quantitation with SYPRO Ruby protein gel stain in one- and two-dimensional polyacrylamide gels were evaluated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of three different purified recombinant proteins showed that the limits of detection were comparable to the limits of detection with ammoniacal silver staining and were protein-specific, ranging from 0.5 to 5 ng. The linearity of the relationship between protein level and SYPRO Ruby staining intensity also depended on the individual protein, with observed linear dynamic ranges of 200-, 500-, and, 1000-fold for proteins analyzed by SDS-PAGE. SYPRO Ruby protein gel stain was also evaluated in two-dimensional electrophoretic (2-DE) analysis of Escherichia coli proteins. The experiment involved analysis of replicates of the same sample as well as dilution of the sample from 0.5 to 50 nug total protein across gels. In addition to validating the 2-DE system itself, the experiment was used to evaluate three different image analysis programs: Z3 (Compugen), Progenesis (Nonlinear Dynamics), and PDQuest (Bio-Rad). In each program, we analyzed the 2-DE images with respect to sensitivity and reproducibility of overall protein spot detection, as well as linearity of response for 20 representative proteins of different molecular weights and pI. Across all three programs, coefficients of variation (CV) in total number of spots detected among replicate gels ranged from 4 to 11%. For the 20 representative proteins, spot quantitation was also comparable with CVs for gel-to-gel reproducibility ranging from 3 to 33%. Using Progenesis and PDQuest, a 1000-fold linear dynamic range of SYPRO Ruby was demonstrated with a single known protein. These two programs were more suitable than Z3 for examining individual protein spot quantity across a series of gels and gave comparable results.     

Generation and characterization of a specific polyclonal antibody against the mouse serotonin receptor 1A: a state-of-the-art recommendation on how to characterize antibody specificity

A series of different antibodies against serotonin receptor 1A (5HT1A_R) have been reported although only limited information on the specificity of these antibodies and the antigens recognized is available. Herein, we characterized reactivity of an antibody by a gel-based proteomics method that should represent a model how antibodies may be defined in the future. An antibody against the 5HT1A_R was generated, used for immunoprecipitation and immunoblotting on blue-native gels containing a 5HT1A_R complex. The 5HT1A_R was isolated from tissue and was defined by nano-LC-ESI-MS/MS. A single band on the native gel and a single spot representing the denatured receptor in the 3rd dimensional step of gel electrophoresis was detected. Immunoprecipitation revealed a single band for the denatured 5HT1A_R. Herein, a procedure is proposed to characterize an antibody by the use of a robust method unambiguously identifying and characterizing the antigen, 5HT1A_R, from mouse whole brain.     

Electrochemical Immunoassay for Tumor Marker CA19-9 Detection Based on Self-Assembled Monolayer

A CA19-9 electrochemical immunosensor was constructed using a hybrid self-assembled membrane modified with a gold electrode and applied to detect real samples. Hybrid self-assembled membranes were selected for electrode modification and used to detect antigens. First, the pretreated working electrodes were placed in a 3-mercaptopropionic acid (MPA)/β-mercaptoethanol (ME) mixture for 24 h for self-assembly. The electrodes were then placed in an EDC/NHS mixture for 1 h. Layer modification was performed by stepwise dropwise addition of CA19-9 antibody, BSA, and antigen. Differential pulse voltammetry was used to characterize this immunosensor preparation process. The assembled electrochemical immunosensor enables linear detection in the concentration range of 0.05–500 U/mL of CA19-9, and the detection limit was calculated as 0.01 U/mL. The results of the specificity measurement test showed that the signal change of the interfering substance was much lower than the response value of the detected antigen, indicating that the sensor has good specificity and strong anti-interference ability. The repeatability test results showed that the relative standard deviations were less than 5%, showing good accuracy and precision. The CA19-9 electrochemical immunosensor was used for the actual sample detection, and the experimental results of the standard serum addition method showed that the RSD values of the test concentrations were all less than 10%. The recoveries were 102.4–115.0%, indicating that the assay has high precision, good accuracy, and high potential application value.     

Comparative study of natural autoantibodies in the serum and cerebrospinal fluid of normal individuals and patients with multiple sclerosis and other neurological diseases

Using a panel of antigens (actin, myosin, tubulin, albumin, transferrin, peroxidase, thyroglobulin, DNA, prolactin, TNP and myelin basic protein (MBP)), we have tested the antibody activity of serum and cerebrospinal fluid (CSF) from healthy individuals, patients with multiple sclerosis (MS) and individuals with other neurological diseases. No differences in the concentrations and specificities of the serum antibodies were observed among the 3 groups. In contrast, we found that MS patients often had elevated CSF antibody levels against many antigens of the panel. The MS patients with local immunoglobulin production in the central nervous system (CNS) had the highest antibody levels. Restricted antibody activity against a given antigen of the panel was not observed. Compared to the two other groups, the MS group had equivalent titres of anti-MBP antibodies in the CSF.These results suggest that, in MS, a general immune dysregulation exists which leads to a local expansion of B lymphocytes producing autoantibodies with reactivities similar to those of serum natural autoantibodies.     

Novel indirect enzyme-linked immunosorbent assay (ELISA) method to detect Total E. coli in water environment

In order to establish ELISA (enzyme-linked immunosorbent assay) method to detect Total E. coli in water environment, E. coli multi-characters antigens in water environment were prepared according to the characters of kinds of E. coli serotypes, including antigen of whole cell, antigen of disrupted whole cell, somatic antigen, flagellar antigen and fimbrial antigen. Total E. coli polyclonal antibodies were obtained from the New Zealand rabbits immunized with these five antigens, respectively. Antibodies generated in this research are with high titers and good purity, can conjugate with antigens, specifically, stably and strongly. Indirect ELISA shows the titers of antibody of whole cell and antibody of disrupted whole cell are both over 1 × 10⁵. The cross-reactivity of the antibody is from 12 to 30% which indicate the specificity of the antibody against Total E. coli. Based on these antibodies, we established indirect ELISA method to detect Total E. coli in water environment. The matrix effects were studied and the results show that there is no significant influence by all the factors. The ELISA result shows that the detection limitation could be 10⁴ CFU (colony forming units) L⁻¹. The indirect ELISA method developed in this study is well suited for Total E. coli analysis in real water samples as a rapid screen method.     

Liquid Chromatography with Electrochemical Detection of Phenol and NADH for Enzyme Immunoassay

Abstract

Enzyme immunoassays based on chromatographic separation and amperometric detection of an enzyme generated product have been investigated. These assays combine the selectivity of the antigen/antibody reaction with the high sensitivity of thin layer amperometry. The feasibility of utilizing LCEC as a detection scheme was demonstrated using the Syva EMIT® kit for phenytoin. NADH production by glucose-6-phosphate dehydrogenase was monitored following a homogeneous procedure. Heterogeneous assays were developed for alkaline phosphatase labeled species which were based upon LCEC determination of phenol. Assays were designed for a common serum glycoprotein (orosomucoid) and a clinically important drug (digoxin). Detection limits approach the pg/mL level and as such may prove fruitful in the quantitation of numerous antigens of clinical interest.     

A new method for non-labeling attomolar detection of diseases based on an individual gold nanorod immunosensor

Herein, we present the use of a single gold nanorod sensor for detection of diseases on an antibody-functionalized surface, based on antibody-antigen interaction and the localized surface plasmon resonance (LSPR) λ(max) shifts of the resonant Rayleigh light scattering spectra. By replacing the cetyltrimethylammonium bromide (CTAB), a tightly packed self-assembled monolayer of HS(CH(2))(11)(OCH(2)CH(2))(6)OCH(2)COOH(OEG(6)) has been successfully formed on the gold nanorod surface prior to the LSPR sensing, leading to the successful fabrication of individual gold nanorod immunosensors. Using prostate specific antigen (PSA) as a protein biomarker, the lowest concentration experimentally detected was as low as 111 aM, corresponding to a 2.79 nm LSPR λ(max) shift. These results indicate that the detection platform is very sensitive and outperforms detection limits of commercial tests for PSA so far. Correlatively, its detection limit can be equally compared to the assays based on DNA biobarcodes. This study shows that a gold nanorod has been used as a single nanobiosensor to detect antigens for the first time; and the detection method based on the resonant Rayleigh scattering spectrum of individual gold nanorods enables a simple, label-free detection with ultrahigh sensitivity.     

Magnetic core-shell Fe3O4@Ag nanoparticles coated carbon paste interface for studies of carcinoembryonic antigen in clinical immunoassay

This study demonstrates a novel approach toward development of advanced immunosensors based on chemically functionalized core-shell Fe3O4@Ag magnetic nanoparticles, and the preparation, characterization, and measurement of relevant properties of the immunosensor useful for the detection of carcinoembryonic antigen (CEA) in clinical immunoassay. The immunosensor based on the combination of a magnetic nanocore and an Ag metallic shell shows good adsorption properties for the attachment of the CEA antibody selective to CEA. The core-shell nanostructure presents good magnetic properties to facilitate and modulate the way it was integrated into a carbon paste. Under optimal conditions, the resulting composite presents good electrochemical response for the detection of CEA, and allows detection of CEA at a concentration as low as 0.5 ng.mL(-1). Importantly, the proposed methodology could be extended to the detection of other antigens or biocompounds.