Microfluidic electrochemical immunoarray for ultrasensitive detection of two cancer biomarker proteins in serum

A microfluidic electrochemical immunoassay system for multiplexed detection of protein cancer biomarkers was fabricated using a molded polydimethylsiloxane channel and routine machined parts interfaced with a pump and sample injector. Using off-line capture of analytes by heavily-enzyme-labeled 1 μm superparamagnetic particle (MP)-antibody bioconjugates and capture antibodies attached to an 8-electrode measuring chip, simultaneous detection of cancer biomarker proteins prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum was achieved at sub-pg mL?1 levels. MPs were conjugated with ～90,000 antibodies and ～200,000 horseradish peroxidase (HRP) labels to provide efficient off-line capture and high sensitivity. Measuring electrodes feature a layer of 5 nm glutathione-decorated gold nanoparticles to attach antibodies that capture MP-analyte bioconjugates. Detection limits of 0.23 pg mL?1 for PSA and 0.30 pg mL?1 for IL-6 were obtained in diluted serum mixtures. PSA and IL-6 biomarkers were measured in serum of prostate cancer patients in total assay time 1.15 h and sensor array results gave excellent correlation with standard enzyme-linked immunosorbent assays (ELISA). These microfluidic immunosensors employing nanostructured surfaces and off-line analyte capture with heavily labeled paramagnetic particles hold great promise for accurate, sensitive multiplexed detection of diagnostic cancer biomarkers.     

Carbon nanotube-based ultrasensitive multiplexing electrochemical immunosensor for cancer biomarkers

A multiplexing electrochemical immunosensor was developed for ultrasensitive detection of cancer related protein biomarkers. We employed disposable screen-printed carbon electrode (SPCE) array as the detection platform. A universal multi-labeled nanoprobe was developed by loading HRP and goat-anti-rabbit IgG (secondary antibody, Ab₂) onto multiwalled carbon nanotube (MWNT). This universal nanoprobe was available for virtually any sandwich-based antigen detection and showed superiority in several areas. By using the SPCE array and the universal nanoprobe, we could detect as low as 5 pg mL⁻¹ of prostate specific antigen (PSA) and 8 pg mL⁻¹ of Interleukin 8 (IL-8) with the electrochemical immunosensor. We also demonstrated simultaneous detection of two protein biomarkers with this platform. With these attracted features, our immunoassay system shows promising applications for in-field and point-of-care test in clinical diagnostics.     

Levels of plasma glycan-binding auto-IgG biomarkers improve the accuracy of prostate cancer diagnosis

Strategies to improve the early diagnosis of prostate cancer will provide opportunities for earlier intervention. The blood-based prostate-specific antigen (PSA) assay is widely used for prostate cancer diagnosis but specificity of the assay is not satisfactory. An algorithm based on serum levels of PSA combined with other serum biomarkers may significantly improve prostate cancer diagnosis. Plasma glycan-binding IgG/IgM studies suggested that glycan patterns differ between normal and tumor cells. We hypothesize that in prostate cancer glycoproteins or glycolipids are secreted from tumor tissues into the blood and induce auto-immunoglobulin (Ig) production. A 24-glycan microarray and a 5-glycan subarray were developed using plasma samples obtained from 35 prostate cancer patients and 54 healthy subjects to identify glycan-binding auto-IgGs. Neu5Acα2-8Neu5Acα2-8Neu5Acα (G81)-binding auto-IgG was higher in prostate cancer samples and, when levels of G81-binding auto-IgG and growth differentiation factor-15 (GDF-15 or NAG-1) were combined with levels of PSA, the prediction rate of prostate cancer increased from 78.2% to 86.2% than with PSA levels alone. The G81 glycan-binding auto-IgG fraction was isolated from plasma samples using G81 glycan-affinity chromatography and identified by N-terminal sequencing of the 50 kDa heavy chain variable region of the IgG. G81 glycan-binding 25 kDa fibroblast growth factor-1 (FGF1) fragment was also identified by N-terminal sequencing. Our results demonstrated that a multiplex diagnostic combining G81 glycan-binding auto-IgG, GDF-15/NAG-1 and PSA (≥?2.1 ng PSA/ml for cancer) increased the specificity of prostate cancer diagnosis by 8%. The multiplex assessment could improve the early diagnosis of prostate cancer thereby allowing the prompt delivery of prostate cancer treatment.

     

Measuring distances in supported bilayers by fluorescence interference-contrast microscopy: polymer supports and SNARE proteins

Fluorescence interference-contrast (FLIC) microscopy is a powerful new technique to measure vertical distances from reflective surfaces. A pattern of varying intensity is created by constructive and destructive interference of the incoming and reflected light at the surface of an oxidized silicon chip. Different levels of this pattern are probed by manufacturing silicon chips with terraces of oxide layers of different heights. Fluorescence collected from membranes that are deposited on these terraces is then used to measure the distance of the fluorescent probes from the silicon oxide surface. Here, we applied the method to measure the distance between supported lipid bilayers and the surface of oxidized silicon chips. For plain fluid phosphatidylcholine bilayers, this distance was 1.7 +/- 1.0 nm. The cleft distance was increased to 3.9 +/- 0.9 nm in bilayers that were supported on a 3400-Da polyethylene glycol cushion. This distance is close to the Flory distance (4.8 nm) that would be expected for a grafted random coil of this polymer. In a second application, the distance of a membrane-bound protein from the membrane surface was measured. The integral membrane protein syntaxin1A/SNAP25 (t-SNARE) was reconstituted into tethered polymer-supported bilayers. A soluble form of the green fluorescent protein/vesicle-associated membrane protein (GFP-VAMP) was bound to the reconstituted t-SNAREs. The distance of the GFP from the membrane surface was 16.5 +/- 2.8 nm, indicating an upright orientation of the rod-shaped t-SNARE/v-SNARE complex from the membrane surface.     

An integrated chip for rapid, sensitive, and multiplexed detection of cardiac biomarkers from fingerprick blood

Cardiovascular diseases are the major cause of death among adults worldwide. Electrocardiogram (ECG) is a first test when a patient suffering from chest pain sees a doctor, however, it is lack of the required sensitivity. Standard assays to detect cardiac biomarkers, like enzyme-linked immunosorbent assay (ELISA) are sensitive, but suffer from important sample and reagent consumption in large-scale studies. Moreover they are performed in central laboratories of clinics and hospitals and take a long time, which is highly incompatible with the quick decisions needed to save a heart attack patient. Herein, we describe an integrated chip allowing rapid, sensitive, and simultaneous analysis of three cardiac biomarkers in fingerprick blood. The integrated chip is composed of a filtration chip for plasma separation from blood and a silicon nanowire (SiNW) array sensor chip for protein detection. These two chips are fabricated separately and bonded to form a single unit after alignment. The integrated chip is capable of reducing the dead volume of the sample by eliminating the tubing between the two chips. After the plasma is filtrated by the filtration chip, the SiNW sensor, spotted with three different antibodies, enabled us to detect three cardiac biomarkers, troponin T (cTnT), creatine kinase MM (CK-MM) and creatine kinase MB (CK-MB), simultaneously. The integrated chip is able to attain a low detection limit of 1 pg/ml for the three cardiac biomarkers from 2 μl blood in 45 min.     

Protein patterning by maskless photolithography on hydrophilic polymer-grafted surface

With the help of a microfabrication process and surface modification technology, a method of fabricating protein patterned chips was developed which can be utilized as a powerful tool for performing bioassays in a high-throughput manner. A digital micromirror array (MMA) system was used as a virtual photomask, so that a maskless photolithography process was able to be used to build patterned biomolecules on a chip by selective illumination onto the chip surface. We utilized the nitroveratryloxycarbonyl (NVOC) group as a photolabile protecting group for protein patterning. The NVOC-protected surface was selectively irradiated by a UV illuminator using an MMA. After removing the NVOC group, biotin was coupled to the NVOC-cleaved site, onto which a buffered streptavidin solution was eluted. At this point, we could obtain a streptavidin-patterned surface and observe the effect of the polymer-grafted surface in reducing nonspecific binding.     

Novel Prostate Cancer Biomarkers Derived from Autoantibody Signatures

BACKGROUND: Due to the low specificity of the prostate-specific antigen (PSA) assay and a high false positive rate, a large number of prostate cancer (PCA) biopsies are performed unnecessarily. Consequently, there is a need for new biomarkers that can identify PCA at any stage of progression while limiting the number of false positives. The use of autoantibody signature–developed biomarkers has proven to be an effective method to solve this problem. RESULTS: Using T7 phage–peptide detection, we identified a panel of eight biomarkers for PCA on a training set. The estimated receiver-operating characteristic (ROC) curve had an area under the ROC curve of 0.69 when applied to the validation set. Spearman correlations were high, within 0.7 to 0.9, indicating that the biomarkers have a degree of inter-relatedness. The identified biomarkers play a role in processes such as androgen response regulation and cellular structural integrity and are proteins that are thought to play a role in prostate tumorigenesis. CONCLUSIONS: Autoantibodies against PCA can be developed as biomarkers for detecting PCA. The scores from the algorithm developed here can be used to indicate a relative high or low risk of PCA, particularly for patients with intermediate (4.0 to 10 ng/ml) PSA levels. Since most commercially available assays test for PSA or have a PSA component, this novel approach has the potential to improve diagnosis of PCA using a biologic measure independent of PSA.     

Disposable polydimethylsiloxane/silicon hybrid chips for protein detection

This paper presents disposable protein analysis chips with single- or four-chamber-constructed from poly(dimethylsiloxane) (PDMS) and silicon. The chips are composed of a multilayer stack of PDMS layers that sandwich a silicon microchip. This inner silicon chip features an etched array of micro-cavities hosting polymeric beads. The sample is introduced into the fluid network through the top PDMS layer, where it is directed to the bead chamber. After reaction of the analyte with the probe beads, the signal generated on the beads is captured with a CCD camera, digitally processed, and analyzed. An established bead-based fluorescent assay for C-reactive protein (CRP) was used here to characterize these hybrid chips. The detection limit of the single-chamber protein chip was found to be 1 ng/ml. Additionally, using a back pressure compensation method, the signals from each chamber of the four-chamber chip were found to fall within 10% of each other.     

Superhydrophobic Properties of Nanostructured–Microstructured Porous Silicon for Improved Surface-Based Bioanalysis

Wettability is a fundamental property of a solid surface, which plays important roles in many industrial applications. The possibility to create well-controlled nonwetting states on silicon surfaces without photolithography-based processing can bring many advantages in the biotechnology and microfluidics areas. In this paper, superhydrophobic properties of macroporous–nanoporous structured silicon are reported. The superhydrophobic porous silicon layers are prepared by electrochemical etching of bulk crystalline silicon wafers. Altered anodization conditions provide surfaces with varying pore morphologies, yielding different wetting properties, ranging from highly wetting (nanoporous morphologies) to water-repellent surfaces (macroporous morphologies). Subsequent surface modification with a fluorocarbon coupling agent can further improve nonwetting properties and stabilize the surface for a long term. Contact angles as high as 176° were achieved on macroporous silicon and superhydrophobicity was maintained for several months without degradation. The porous surfaces have proven to be a very attractive substrate for protein microarrays. Fluorescence-based assay of immunoglobulin G in plasma is reported with a limit of detection of 1 pM, a spot size of 50 μm, and an array density of 15,625 spots per square centimeter. Macroporous surfaces have also been developed for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) applications, where the intrinsic hydrophobic surface properties confine the deposited sample to MALDI spots of less than 200 μm with well-defined MALDI crystals, providing a high-sensitivity readout. Furthermore, a superhydrophobic MALDI-TOF MS target anchor chip composed of nonporous anchor points surrounded by superhydrophobic porous areas for sample deposition and on anchor point confinement is reported. Such anchor chips allowed localized crystallization of large sample volumes (5 μL) improving the hydrophobic spot confinement strategy in terms of final MALDI crystal localization and readout sensitivity.     

Newer potential biomarkers in prostate cancer

Prostate-specific antigen (PSA) screening has led to a significant rise in the number of men diagnosed with prostate cancer and an associated increase in biopsies performed. Despite its limitations, including a positive predictive value of only 25%-40%, PSA remains the only generally accepted biomarker for prostate cancer. There is a need for better tools to not only identify men with prostate cancer, but also to recognize those with potentially lethal disease who will benefit from intervention. A great deal of work has been done worldwide to improve our knowledge of the genetics behind prostate cancer and the specificity of PSA by developing assays for different PSA isoforms. Common genetic alterations in prostate cancer patients have been identified, including CpG hypermethylation of GSPT1 and TMPRSS2:ERG gene fusion. Serum and urine detection of RNA biomarkers (eg, PCA3) and prostate cancer tissue protein antibodies (eg, EPCA) are being evaluated for detection and prognostic tools. This article reviews some of the promising developments in biomarkers.     

Immunomodulative properties of conjugates composed of detoxified lipopolysaccharide and capsular polysaccharide of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O135 bound to BSA-protein carrier

O135 serotype Vibrio cholerae isolated from Slovak river was used as a source of surface polysaccharide antigens. Following detoxification procedure, fractions of polysaccharides were separated by size exclusion chromatography. Two resultant fractions were the capsular polysaccharide (M _w ∼ 197,000 Da) and the lipopolysaccharide fragment (M _w ∼ 13,300 Da). These materials were used for preparation of four novel glycoconjugates. Two of them containing detoxified lipopolysaccharide as antigen were prepared by original chemical method using the new biocompatible polymer as carrier of antigen. Additionally, other two conjugates were prepared by direct linking of capsular and detoxified lipopolysaccharide antigens to the protein carrier using adipic acid dihydrazide spacer. The immunogenicities (induced IgM, IgG, IgA antibodies) of all conjugates were determined by enzyme-linked immunosorbent assay. Polymer containing conjugates elicited higher levels of specific anti-lipopolysaccharide IgM and IgG antibodies in comparison with other conjugates without polymer carrier. Enhanced IgM vibriocidal activity of mice antisera was also evident here.     

Vaccination of prostatectomized prostate cancer patients in biochemical relapse,with autologous dendritic cells pulsed with recombinant human PSA

This study was conducted in prostate cancer patients in biochemical relapse after radical prostatectomy, to assess the feasibility, safety, and immunogenicity of therapeutic vaccination with autologous dendritic cells (DCs) pulsed with human recombinant prostate-specific antigen (PSA) (Dendritophage-rPSA). Twenty-four patients with histologically proven prostate carcinoma and an isolated postoperative rise of serum PSA (>1 ng/ml to 10 ng/ml) after radical prostatectomy were included. The patients received nine administrations of PSA-loaded DCs by combined intravenous, subcutaneous, and intradermal routes over 21 weeks. Postbaseline blood tests were performed at months 1, 3, 6, 9, and 12 (PSA levels), at months 6 and 12 (circulating prostate cancer cells), at month 6 (anti-PSA IgG and IgM antibodies), and at up to eight time points before, during, and after immunization (PSA-specific T cells). Circulating prostate cancer cells detected in six patients at baseline were undetectable at 6 months and remained undetectable at 12 months. Eleven patients had a postbaseline transient PSA decrease on one to three occasions, predominantly occurring at month 1 (7 patients) or month 3 (2 patients). Maximum PSA decrease ranged from 6% to 39%. PSA decrease on at least one occasion was more frequent in patients with low Gleason score (p=0.016) at prostatectomy and with positive skin tests at study baseline (p=0.04). PSA-specific T cells were detected ex vivo by ELISpot for IFN- in 7 patients before vaccination and in 11 patients after vaccination. Of the latter 11 patients, 5 had detectable T cells both before and during the vaccination period, 4 only during the vaccination period, while 2 patients could for technical reasons not be assessed prevaccination. No induction of anti-PSA IgG or IgM antibodies was detected. There were no serious adverse events or otherwise severe toxicities observed during the trial. Immunization with Dendritophage-rPSA was feasible and safe in this cohort of patients. An immune response specific for PSA could be detected in some patients. A notable effect was the disappearance of circulating prostate cells in all patients who were RT-PCR positive before vaccination.Scientific correspondence should be addressed to B. Barrou; editorial correspondence to M.L. Ericson.     

Production of an Anti-Prostate-Specific Antigen Single-Chain Antibody Fragment from Pichia pastoris

Prostate-specific antigen (PSA) is a widely used marker for screening and monitoring prostate cancer. Because PSA levels are normally quite low, an antibody-based assay must be used to detect PSA. However, not all PSA-specific antibodies bind equally well to PSA or to its different isoforms. Therefore, a better understanding of how PSA interacts with PSA-specific antibodies is of considerable clinical interest. B80.3 is a widely used murine monoclonal anti-PSA antibody (IgG), which has very high affinity for both free and α-anti-chymotrypsin complexed PSA. More importantly, its gene sequence is known—making it one of only two anti-PSA antibodies that has been fully cloned and sequenced. To better elucidate the interaction between PSA and B80.3, a single-chain antibody fragment, derived from the variable domain of B80.3 (scFvB80), was cloned into a pPIC9 vector and expressed in Pichia pastoris. The secreted protein was purified using a three-step protocol beginning with a 50% ammonium sulfate precipitation step, followed by a T-gel thio-affinity step and concluding with a simple anion-exchange (DE52) filtration step. NMR studies indicate the protein is correctly folded while competitive enzyme-linked immunosorbant assays show that the purified scFvB80 has approximately 20% of the activity of the full-length B80.3 antibody. The protocol described here provides a quick and convenient route to prepare large quantities of very pure anti-PSA antibody fragments (15–20 mg/L culture medium) for detailed structural and biophysical characterization.     

Enhanced detection sensitivity of pegylated CdSe/ZnS quantum dots-based prostate cancer biomarkers by surface plasmon-coupled emission

We demonstrate the fabrication and detection of quantum dots (QDs)-based prostate specific antigens (PSAs) cancer protein biochips by using enhanced surface plasmon-coupled emission measurements (SPCE). The PSAs are immobilized on a SiO(2)-protected thin gold substrate and pegylated QDs which conjugated with antibodies of PSA are used as fluorescent probes. Due to the excellent brightness of the QDs and the high directionality of emission, as well as the high light collection efficiency of SPCE, the limit of detection (LOD) is down to 10 fg/mL (equal to 0.3 fM) for the PSA chips by using QDs-based cancer protein. We expect that this QDs-based SPCE measurement system with the low LOD supplies a great potential for detecting various cancer biomarkers that are present in only low concentrations within the human body.     

Dynamic thiol/disulphide homeostasis before and after radical prostatectomy in patients with prostate cancer

Abstract

Thiol groups are important anti-oxidants and essential molecules protecting organism against the harmful effects of reactive oxygen species (ROS). The aim of our study is to evaluate thiol–disulphide homeostasis with a novel recent automated method in patients with localized prostate cancer (PC) before and six months after radical prostatectomy (RP). 18 patients with PC and 17 healthy control subjects were enrolled into the study. Blood samples were collected from the controls subjects and patients before and six months after RP. Thiol–disulphide homeostasis was determined using a recently developed novel method. Prostate-specific antigen (PSA), albumin, total protein, total thiol, native thiol, disulphide and total antioxidant status (TAS) were measured and compared between the groups. Native thiol, total thiol and TAS levels were significantly higher in the control group than the patients before RP (p?<?.001). There was a non-significant increase in the native thiol, total thiol and TAS levels in the patients six months after RP in comparison to the levels before RP (p values .3, .3 and .09, respectively). We found a significant negative correlation between PSA and thiol levels. Our study demonstrated that the decreased thiol and TAS levels weakened anti-oxidant defence mechanism in the patients with PC as indicated. Increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the aetiopathogenesis of prostate cancer.     

The RFA regulatory sequence-binding protein in the promoter of prostate-specific antigen gene

To assure what sequence associated with the androgen regulation, a 15 bp region at the upstream of the ARE of prostate-specific antigen (PSA) promoter, termed RFA, was found indispensable for androgen receptor (AR)-mediated transactivation of PSA promoter. In transfection and CAT assays, some nucleotides substitution in RFA could significantly decrease the androgen inducibility for PSA promoter. The in vitro DNA binding assay demonstrated that RFA bound specifically with some non-receptor protein factors in prostate cell nucleus, but the mutant type of RFA lost this ability, so RFA might be a novel accessory cis-element. The RFA-binding proteins were isolated and purified by affinity chromatography using RFA probes. SDS-PAGE and preliminary protein identification showed these proteins possessed sequence high homology with multifunctional protein heterogeneous nuclear ribonucleoprotein A1, A2 (hnRNP A1, A2). RFA-binding proteins possibly cooperate with AR-mediated transactivation for PSA promoter as coactivator. The study results will facilitate further understanding the mechanism and tissue specificity of PSA promoter.     

一种葡聚糖芯片的再生方法、表征及其应用*

表面等离子体共振（surface plasmon resonance, SPR）生物传感器,作为一种适时快捷,无需标记的生物分子相互作用研究工具,已广泛应用于生物化学分析与研究。羧甲基化葡聚糖修饰的CM5传感芯片是Biacore 系列仪器应用最为普遍的核心部件,目前CM5芯片主要从法玛西亚公司购买,价格昂贵,且一旦共价交联的受体分子失活,就不能重复利用。阐述了一种简便、低成本、用于SPR生物传感器的葡聚糖修饰金膜芯片的再生方法及其表征和应用。用此方法再生的芯片能被循环伏安法和原子力显微镜很好地表征,并成功地用于抗前列腺特异性抗原(prostate-specific antigen,PSA)固定和PSA检测, 同时测定了PSA与其抗体之间的动力学和亲和常数。     

Proteomic analysis of conditioned media from the PC3, LNCaP, and 22Rv1 prostate cancer cell lines: discovery and validation of candidate prostate cancer biomarkers

Early detection of prostate cancer is problematic due to the lack of a marker that has high diagnostic sensitivity and specificity. The prostate specific antigen (PSA) test, in combination with digital rectal examination, is the gold standard for prostate cancer diagnosis. However, this modality suffers from low specificity. Therefore, specific markers for clinically relevant prostate cancer are needed. Our objective was to proteomically characterize the conditioned media from three human prostate cancer cell lines of differing origin [PC3 (bone metastasis), LNCaP (lymph node metastasis), and 22Rv1 (localized to prostate)] to identify secreted proteins that could serve as novel prostate cancer biomarkers. Each cell line was cultured in triplicate, followed by a bottom-up analysis of the peptides by two-dimensional chromatography and tandem mass spectrometry. Approximately, 12% (329) of the proteins identified were classified as extracellular and 18% (504) as membrane-bound among which were known prostate cancer biomarkers such as PSA and KLK2. To select the most promising candidates for further investigation, tissue specificity, biological function, disease association based on literature searches, and comparison of protein overlap with the proteome of seminal plasma and serum were examined. On the basis of this, four novel candidates, follistatin, chemokine (C-X-C motif) ligand 16, pentraxin 3 and spondin 2, were validated in the serum of patients with and without prostate cancer. The proteins presented in this study represent a comprehensive sampling of the secreted and shed proteins expressed by prostate cancer cells, which may be useful as diagnostic, prognostic or predictive serological markers for prostate cancer.     

Development of a Chip/Chip/SRM platform using digital chip isoelectric focusing and LC-Chip mass spectrometry for enrichment and quantitation of low abundance protein biomarkers in human plasma

Protein biomarkers are critical for diagnosis, prognosis, and treatment of disease. The transition from protein biomarker discovery to verification can be a rate limiting step in clinical development of new diagnostics. Liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM MS) is becoming an important tool for biomarker verification studies in highly complex biological samples. Analyte enrichment or sample fractionation is often necessary to reduce sample complexity and improve sensitivity of SRM for quantitation of clinically relevant biomarker candidates present at the low ng/mL range in blood. In this paper, we describe an alternative method for sample preparation for LC-SRM MS, which does not rely on availability of antibodies. This new platform is based on selective enrichment of proteotypic peptides from complex biological peptide mixtures via isoelectric focusing (IEF) on a digital ProteomeChip (dPC) for SRM quantitation using a triple quadrupole (QQQ) instrument with an LC-Chip (Chip/Chip/SRM). To demonstrate the value of this approach, the optimization of the Chip/Chip/SRM platform was performed using prostate specific antigen (PSA) added to female plasma as a model system. The combination of immunodepletion of albumin and IgG with peptide fractionation on the dPC, followed by SRM analysis, resulted in a limit of quantitation of PSA added to female plasma at the level of ～1-2.5 ng/mL with a CV of ～13%. The optimized platform was applied to measure levels of PSA in plasma of a small cohort of male patients with prostate cancer (PCa) and healthy matched controls with concentrations ranging from 1.5 to 25 ng/mL. A good correlation (r(2) = 0.9459) was observed between standard clinical ELISA tests and the SRM-based assay. Our data demonstrate that the combination of IEF on the dPC and SRM (Chip/Chip/SRM) can be successfully applied for verification of low abundance protein biomarkers in complex samples.