Substances that alter the measurable concentration of the analyte or alter antibody binding can potentially result in immunoassay interference. Interfering, endogenous substances that are natural, polyreactive antibodies or autoantibodies (heterophiles), or human anti-animal antibodies together with other unsuspected binding proteins that are unique to the individual, can interfere with the reaction between analyte and reagent antibodies in immunoassay. Lipaemia, cross-reactivity, and exogenous interferences due to pre-analytical variation, matrix and equipment reaction also affect immunoassay. Interfering substances may lead to falsely elevated or falsely low analyte concentration in one or more assay systems depending on the site of the interference in the reaction and possibly result in discordant results for other analytes. The prevalence of interference is generally low in assays containing blocking agents that neutralise or inhibit the interference but is often higher in new, untested immunoassays. A wide range of analytes measured by immunoassay including hormones, tumour markers, drugs, cardiac troponin and microbial serology may be affected.
Interference in immunoassay may lead to the misinterpretation of a patient's results by the laboratory and the wrong course of treatment being given by the physician. Laboratories should put processes in place to detect, test and report suspected interferences. It is equally important that physicians communicate any clinical suspicion of discordance between the clinical and the laboratory data to the laboratory. The detection of interference may require the use of an alternate assay or additional measurements, before and after treatment with additional blocking reagent, or following dilution of the sample in non-immune serum. It is imperative that laboratories inform physicians of the follow-up procedure and report on the presence of any interference. The establishment of on-going laboratory-physician contact is essential to the continuing awareness of wrong patient results due to interference.
The analysis of protein biomarkers in urine is expected to lead to advances in a variety of clinical settings. Several characteristics of urine including a low-protein matrix, ease of testing and a demonstrated proteomic stability offer distinct advantages over current widely used biofluids, serum and plasma. Improvements in our understanding of the urine proteome and in methods used in its evaluation will facilitate the clinical development of urinary protein biomarkers. Multiplexed bead-based immunoassays were utilized to evaluate 211 proteins in urines from 103 healthy donors. An additional 25 healthy donors provided serial urine samples over the course of two days in order to assess temporal variation in selected biomarkers. Nearly one-third of the evaluated biomarkers were detected in urine at levels greater than 1ng/ml, representing a diverse panel of proteins with respect to structure, function and biological role. The presence of several biomarkers in urine was confirmed by western blot. Several methods of data normalization were employed to assess impact on biomarker variability. A complex pattern of correlations with urine creatinine, albumin and beta-2-microglobulin was observed indicating the presence of highly specific mechanisms of renal filtration. Further investigation of the urinary protein biomarkers identified in this preliminary study along with a consideration of the underlying proteomic trends suggested by these findings should lead to an improved capability to identify candidate biomarkers for clinical development.
The recent introduction of fluorescent bead-based technology, allowing the measurement of multiples analytes in a single 25–50 µl sample has revolutionized the study of cytokine responses. However, such multiplex approaches may compromise the ability of these assays to accurately measure actual cytokine levels. This study evaluates the performance of three commercially available multiplex cytokine fluorescent bead-based immunoassays (Bio-Rad's Cytokine 17-plex kit; LINCO Inc's 29-plex kit; and RnD System's Fluorokine-Multi Analyte Profiling (MAP) base kit A and B). The LINCO Inc kit was found to be the most sensitive assay for measuring concentrations of multiple recombinant cytokines in samples that had been spiked with serial dilutions of the standard provided by the manufacturer, followed respectively by the RnD Fluorokine-(MAP) and Bio-Rad 17-plex kits. A positive correlation was found in the levels of IFN-γ measured in antigen stimulated whole blood culture supernatants by the LINCO Inc 29-plex, RnD Fluorokine-(MAP) and RnD system IFN-γ Quantikine ELISA kits across a panel of controls and stimulated samples. Researchers should take the limitation of such multiplexed assays into account when planning experiments and the most appropriate use for these tests may currently be as screening tools for the selection of promising markers for analysis by more sensitive techniques.
A high affinity polyclonal antibody-based enzyme linked immunosorbent assay (ELISA) was developed for the quantification of zeranol in bovine urine. On the basis of urine matrix studies, the optimized dilution factors producing insignificant matrix interference were selected as 1:5 in pretreatment. In the improved ELISA, the linear response range was between 0.02 and 1 μg/ml , and the detection limit was 0.02 μg/ml for the assay. The overall recoveries and the coefficients of variation (CVs) were in the range of 82%~127% and 3.5%~8.8%, respectively. Thirty-six bovine urine samples spiked with zeranol (ranging from 0.2 to 10 μg/ml) were detected by the ELISA and liquid chromatography (LC) method, and good correlations were obtained between the two methods (R
2=0.9643). We conclude that this improved ELISA is suitable tool for a mass zeranol screening and can be an alternative for the conventional LC method for zeranol in bovine urine.
Zeranol; Enzyme linked immunosorbent assay (ELISA); Bovine urine
A new competitive electrochemiluminescence-based immunoassay for the type-2 brevetoxins in oyster extracts was developed. The assay was verified by spiking known amounts of PbTx-3 into 80% methanol extracts of Gulf Coast oysters. We also provide preliminary data demonstrating that 100% acetone extracts, aqueous homogenates, and the clinical matrixes urine and serum can also be analyzed without significant matrix interferences. The assay offers the advantages of speed (≃2h analysis time); simplicity (only 2 additions, one incubation period, and no wash steps before analysis); low limit of quantitation (conservatively, 50 pg/mL = 1 ng/g tissue equivalents); and a stable, nonradioactive label. Due to the variety of brevetoxin metabolites present and the lack of certified reference standards for liquid chromatography–mass spectrometry confirmation, a true validation of brevetoxins in shellfish extracts is not possible at this time. However, our assay correlated well with another brevetoxin immunoassay currently in use in the United States. We believe this assay could be useful as a regulatory screening tool and could support pharmacokinetic studies in animals and clinical evaluation of neurotoxic shellfish poisoning victims.
A high-pressure liquid chromatographic assay was developed for the quantitative analysis of a new cephalosporin, BMY-28142, in plasma and urine. The plasma method involved protein precipitation with acetonitrile and trichloroacetic acid followed by extraction of the acetonitrile into dichloromethane. After centrifugation, the organic phase was discarded, the aqueous solution was injected into a reverse-phase column, and peaks were detected at 280 nm. The urine method involved dilution of a urine sample with sodium acetate buffer (pH 4.25) and direct injection into the high-pressure liquid chromatography system. The assay validation data indicate that the assays for BMY-28142 in plasma and urine were specific, accurate, and reproducible. The analytical methods were applied to the determination of protein binding in human serum and to a pharmacokinetic study in rats. The results of the protein-binding study indicated that BMY-28142 was 16.3% bound to human serum proteins. In the pharmacokinetic study in rats, the maximum level in plasma of 38.7 micrograms/ml was achieved at 2.33 h after administration of a subcutaneous dose of 100 mg/kg. The levels in the plasma then declined with an elimination half-life of about 0.56 h. The mean values for the steady-state volume of distribution and total body clearance were 0.46 liters/kg and 11.9 ml/min per kg, respectively. The 0- to 24-h excretion of intact BMY-28142 in urine accounted for 88.6% of the dose.
A semiquantitative PCR assay for the detection of BK virus in urine was developed using primers for BK virus that specifically amplified BK but not JC virus. DNA was extracted from urine through treatment with proteinase K followed by DNA precipitation with sodium acetate. Semiquantitation was achieved by amplifying serial dilutions (1:1, 1:10, 1:100, and 1:1,000) of the urine specimens. Each assay included both positive (stock BK virus and previously positive patient urine) and negative (no template) controls. A urine sample was interpreted as positive if any of the serial dilutions showed amplification of the DNA fragment of the expected size. For some patient-derived samples, amplification of the expected-size fragment was achieved with a dilute template whereas no amplification was achieved with a concentrated template. This was attributed to interfering substances in the urine. PCR results were compared with urine cytology and shown to be more sensitive. Validation studies were performed at the University of Nebraska Medical Center, utilizing a separate qualitative PCR assay that detects both BK and JC virus and distinguishes between them by restriction enzyme digestion patterns. Of 46 urine samples analyzed using both methods, 22 were positive by both assays, 18 were negative by both assays, 5 were positive only by the Nebraska method, and 1 was positive only by our method. In comparison with the Nebraska PCR, our PCR assay had a sensitivity of 81% and specificity of 95%. For twenty-one (43%) of 49 immunocompromised patients, tests were postive when specimens were submitted because of clinical suspicion of BK virus infection.
A radioimmunoassay for the measurement of gonadotrophin releasing hormone (GnRH) in plasma and urine using readily available reagents was developed. The GnRH assay showed good precision, recovery, and parallelism over a wide range of GnRH concentrations with a sensitivity of 15 pg/ml. The assay was compared with a commercially available kit (Buhlmann Laboratories). Although the Buhlmann kit showed acceptable precision, recovery, sensitivity, and correlation with the developed GnRH assay for plasma samples, lack of parallelism of serially diluted plasma and urine samples was consistently observed, together with a poor correlation with the developed GnRH assay for urine, suggesting a matrix effect with the Buhlmann kit. The developed assay is suitable for measuring GnRH in samples obtained from patients receiving pulsatile infusions of GnRH. In contrast, the commercially available Buhlmann kit was unsuitable for measuring plasma GnRH as the kit had a top standard of only 160 pg/ml, well below the peak plasma concentration. It would not be possible to dilute samples for analysis because of the lack of parallelism of diluted samples compared with standards obtained with the Buhlmann assay.
The measurement of multiple protein biomarkers may refine risk stratification in clinical settings. This concept has stimulated development of multiplexed immunoassay platforms that provide multiple, parallel protein measurements on the same specimen.
We provide an overview of antibody-based multiplexed immunoassay platforms and discuss technical and operational challenges. Multiplexed immunoassays use traditional immunoassay principles in which high-affinity capture ligands are immobilized in parallel arrays in either planar format or on microspheres in suspension. Development of multiplexed immunoassays requires rigorous validation of assay configuration and analytical performance to minimize assay imprecision and inaccuracy. Challenges associated with multiplex configuration include selection and immobilization of capture ligands, calibration, interference between antibodies and proteins and assay diluents, and compatibility of assay limits of quantification. We discuss potential solutions to these challenges. Criteria for assessing analytical multiplex assay performance include the range of linearity, analytical specificity, recovery, and comparison to a quality reference method. Quality control materials are not well developed for multiplexed protein immunoassays, and algorithms for interpreting multiplex quality control data are needed.
Technical and operational challenges have hindered implementation of multiplexed assays in clinical settings. Formal procedures that guide multiplex assay configuration, analytical validation, and quality control are needed before broad application of multiplexed arrays can occur in the in vitro diagnostic market.
Aims: Urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) is a widely used biomarker of oxidative stress. However, variability between chromatographic and ELISA methods hampers interpretation of data, and this variability may increase should urine composition differ between individuals, leading to assay interference. Furthermore, optimal urine sampling conditions are not well defined. We performed inter-laboratory comparisons of 8-oxodG measurement between mass spectrometric-, electrochemical- and ELISA-based methods, using common within-technique calibrants to analyze 8-oxodG-spiked phosphate-buffered saline and urine samples. We also investigated human subject- and sample collection-related variables, as potential sources of variability. Results: Chromatographic assays showed high agreement across urines from different subjects, whereas ELISAs showed far more inter-laboratory variation and generally overestimated levels, compared to the chromatographic assays. Excretion rates in timed ‘spot’ samples showed strong correlations with 24 h excretion (the ‘gold’ standard) of urinary 8-oxodG (rp 0.67–0.90), although the associations were weaker for 8-oxodG adjusted for creatinine or specific gravity (SG). The within-individual excretion of 8-oxodG varied only moderately between days (CV 17% for 24 h excretion and 20% for first void, creatinine-corrected samples). Innovation: This is the first comprehensive study of both human and methodological factors influencing 8-oxodG measurement, providing key information for future studies with this important biomarker. Conclusion: ELISA variability is greater than chromatographic assay variability, and cannot determine absolute levels of 8-oxodG. Use of standardized calibrants greatly improves intra-technique agreement and, for the chromatographic assays, importantly allows integration of results for pooled analyses. If 24 h samples are not feasible, creatinine- or SG-adjusted first morning samples are recommended. Antioxid. Redox Signal. 18, 2377–2391.
Urinary proteomic profiling has potential to identify candidate biomarkers of renal injury in infants provided an adequate urine sample can be obtained. Although diapers are used to obtain urine for clinical evaluation, their use for proteomic analysis has not been investigated. We therefore performed feasibility studies on the use of diaper-extracted urine for 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Pediatric waste urine (2–20 mL) was applied to gel-containing, non-gel and cotton-gauze diapers and then mechanically expressed. Urine volume and total protein were measured pre- and post-extraction. Proteins were separated via 2D-PAGE following application of urine (20–40 mL) to each matrix. 2D-PAGE was also performed on clinical specimens collected using each diaper type. Differences in the adsorption and retention of urine volume and protein were noted between matrices. Non-gel and cotton-gauze diapers provided the best protein/volume recovery and the lowest interference with the Bradford assay. 2D-PAGE was also successfully completed using urine samples from both cotton fiber matrices. Conversely, samples from low-gel diapers demonstrated poor protein separation and reproducibility. Diapers containing cotton-fiber matrices appear adequate for 2D-PAGE. Qualitative and quantitative analyses of resolved proteins using replicate, high resolution gels will be required, however, before diaper-extracted urine can be applied in proteomic profiling.
diaper; pediatric; proteomics; urine
Multiplex arrays are increasingly used for measuring protein biomarkers. Advantages of this approach include specimen conservation, limited sample handling, and decreased time and cost, but the challenges of optimizing assay format for each protein, selecting common dilution factors, and establishing robust quality control algorithms are substantial. Here, we use measurements of 15 protein biomarkers from a large study to illustrate processing, analytic, and quality control issues with multiplexed immunoassays.
We contracted with ThermoScientific for duplicate measurements of 15 proteins in 2322 participants from a community-based cohort, a plasma control, and recombinant protein controls using 2 custom planar microarrays with 6 (panel A) or 9 (panel B) capture antibodies printed in each well. We selected constituent analytes in each panel based on endogenous concentrations and assay availability. Protocols were standardized for sample processing, storage, and freeze-thaw exposures. We analyzed data for effects of deviations from processing protocols, precision, and bias.
Measurements were within reportable ranges for each of the assays; however, concentrations for 7 of the 15 proteins were not centered on the dose–response curves. An additional freeze-thaw cycle and erroneous sample dilution for a subset of samples produced significantly different results. Measurements with large differences between duplicates were seen to cluster by analyte, plate, and participant. Conventional univariate quality control algorithms rejected many plates. Plate-specific medians of cohort and plasma control data significantly covaried, an observation important for development of alternative quality control algorithms.
Multiplex measurements present difficult challenges that require further analytical and statistical developments.
Dried urine spots (DUS) have been reported to provide a simple screening tool for congenital cytomegalovirus (CMV) infection. We developed a standardized method for CMV quantification from DUS. Two applications of 20 μ L urine remained within the rim of the filter paper disc and were used to determine the analytical performance of Towne CMV spiked into urine and applied on the discs. The measurable range spanned 3.7 to ≥8.0 log10 copies/mL. The detection limit was 22 DNA copies/disc. Urine samples from congenitally-infected newborns and negative controls were either diluted 1:10 or applied on filter paper at the same volume. DNA copy number from DUS correlated well with copy number from 1:10 diluted urine, although there was a trend for lower levels from DUS (0.3 log10 difference). Our standardized method for CMV detection and quantification may facilitate CMV studies in resource-limited areas and allow for longitudinal monitoring of viral loads in treated infants.
The Bio-Plex® system utilizes xMAP technology to permit the multiplexing of up to 100 different analytes. Multiplex analysis gives researchers the ability to look at analytes simultaneously providing more information from less sample volume in less time than traditional immunoassay methods. Similar to ELISA, xMAP utilizes an antibody sandwich for detection but differs from ELISA in capture substrate and detection method. Rather than a flat surface, Bio-Plex®assays make use of differentially detectable bead sets as a substrate capturing analytes in solution and employs fluorescent methods for detection. These bead sets identify the analytes and detection antibodies are used to measure the quantity of analyte. The use of differentially detectable beads enables the simultaneous identification and quantification of many analytes in the same sample.
Immunoassay; multiplex detection; ELISA; Bio-Plex®; xMAP; Luminex
The formation of reactive oxygen species (ROS) within cells causes damage to biomolecules, including membrane lipids, DNA, proteins and sugars. An important type of oxidative damage is DNA base hydroxylation which leads to the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 5-hydroxymethyluracil (5-HMUra). Measurement of these biomarkers in urine is challenging, due to the low levels of the analytes and the matrix complexity. In order to simultaneously quantify 8-oxodG and 5-HMUra in human urine, a new, reliable and powerful strategy was optimised and validated. It is based on a semi-automatic microextraction by packed sorbent (MEPS) technique, using a new digitally controlled syringe (eVol®), to enhance the extraction efficiency of the target metabolites, followed by a fast and sensitive ultrahigh pressure liquid chromatography (UHPLC). The optimal methodological conditions involve loading of 250 µL urine sample (1∶10 dilution) through a C8 sorbent in a MEPS syringe placed in the semi-automatic eVol® syringe followed by elution using 90 µL of 20% methanol in 0.01% formic acid solution. The obtained extract is directly analysed in the UHPLC system using a binary mobile phase composed of aqueous 0.1% formic acid and methanol in the isocratic elution mode (3.5 min total analysis time). The method was validated in terms of selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), extraction yield, accuracy, precision and matrix effect. Satisfactory results were obtained in terms of linearity (r2 > 0.991) within the established concentration range. The LOD varied from 0.00005 to 0.04 µg mL−1 and the LOQ from 0.00023 to 0.13 µg mL−1. The extraction yields were between 80.1 and 82.2 %, while inter-day precision (n = 3 days) varied between 4.9 and 7.7 % and intra-day precision between 1.0 and 8.3 %. This approach presents as main advantages the ability to easily collect and store urine samples for further processing and the high sensitivity, reproducibility, and robustness of eVol®MEPS combined with UHPLC analysis, thus retrieving a fast and reliable assessment of oxidatively damaged DNA.
Mass spectrometric assays have the potential to replace protein immunoassays in basic science, clinical research, and clinical care. Previous studies have demonstrated the utility of assays using multiple-reaction monitoring mass spectrometry (MRM-MS) for the quantification of proteins in biological samples and many examples of the accuracy of these approaches to quantify spiked analytes have been reported. However, a direct comparison of multiplexed assays using liquid chromatography-tandem mass spectrometry with established immunoassays to measure endogenous proteins has not been reported.
We purified the HDL from the plasma of 30 human subjects enrolled in a clinical nutrition research study and used label-free shotgun proteomics approaches to analyze each sample. We then developed two different 6-plex assays that used isotope dilution MRM-MS: one assay used stable isotope labeled peptides and the other used stable isotope labeled apolipoprotein A-I (apoA-I), the most abundant protein in HDL, as internal standards to control for matrix effects and mass spectrometer performance. The shotgun and MRM-MS assays were then compared with commercially available immunoassays for each of the six analytes.
Quantification by shotgun proteomics approaches correlated poorly with the six protein immunoassays. However, the MRM-MS approaches that used internal standard peptide or a single internal standard protein correlated well. In addition, MRM-MS approaches had good repeatability (<10% CV) and linearity.
Multiplexed MRM-MS assays correlate well with immunochemical measurements and have acceptable operating characteristics in complex samples. Our results support the proposal that MRM-MS could be used to replace immunoassays in a variety of settings.
Mass spectrometry; multiple reaction monitoring; endogenous; proteins; high density lipoprotein; targeted proteomics
We report validation of the first high-pressure liquid chromatography isotope-dilution mass spectrometry method to measure sulforaphane (SFN) and its glutathione-derived conjugates in human urine. As epidemiological evidence continues to mount that the consumption of a diet rich in cruciferous vegetables may reduce the risk of certain cancers, the development of analytical methodologies to accurately measure isothiocyanates (ITCs) and their subsequent metabolic products becomes paramount. SFN, the principal ITC produced by broccoli, is an effective chemopreventive agent with multiple modes of action. SFN and SFN conjugates have often been measured collectively utilizing a cyclocondensation assay with 1,2-benzenedithiol. More recently, some of the major SFN conjugates have been determined using mass spectrometry. Here, triple-quadrupole mass spectrometry has been coupled with the use of stable isotope-labeled internal standards of D8-SFN and all four D8-SFN mercapturic acid pathway conjugates to provide an accurate, precise, sensitive, and specific method for analysis of these compounds. Using urine samples collected during an earlier intervention with broccoli sprouts, the concentrations of SFN, SFN-cysteine, and the mercapturic acid SFN-N-acetylcysteine were sufficiently high such that as little as 50 nL of urine was required for analysis. Although each study participant received an equivalent dose of broccoli sprout preparation, the interindividual conversion of the precursor glucosinolate to SFN varied over 100-fold. These 98 urines provided an ideal sample set for examining the robustness of the assay. The mean urinary concentrations ± standard deviations in overnight voids following ingestion of the first dose were 4.7 ± 5.1, 0.03 ± 0.05, 0.06 ± 0.06, 18 ± 15, and 42 ± 23 nmol/mg creatinine for SFN, SFN-glutathione, SFN-cysteine-glycine, SFN-cysteine, and SFN-N-acetylcysteine, respectively. This method determines SFN and all four SFN glutathione-derived metabolites with minimal sample preparation and will be extremely useful in understanding the role of SFN-rich foods in preventing cancer and other chronic diseases.
Protein biomarker candidates from discovery proteomics must be quantitatively verified in patient samples before they can progress to clinical validation. Here we demonstrate that peptide immunoaffinity enrichment coupled with stable isotope dilution mass spectrometry (SISCAPA-MRM) can be used to configure assays with performance suitable for candidate biomarker verification. As proof of principle, we configured SISCAPA assays for troponin I (cTnI), an established biomarker of cardiac injury, and interleukin 33 (IL-33), an emerging immunological and cardiovascular marker for which robust immunoassays are currently not available.
We configured individual and multiplexed assays in which peptides were enriched from digested human plasma using antipeptide antibodies. Assay performance was established using response curves for peptides and proteins spiked into normal plasma. We quantified proteins using labeled peptides as internal standards, and we measured levels of cTnI in patients who underwent a planned myocardial infarction for hypertrophic obstructive cardiomyopathy.
Measurement of cTnI and IL-33 proteins from trypsin-digested plasma was linear from 1.5 to 5000 μg/L, with imprecision <13% for both proteins, processed individually or multiplexed. Results correlated well (R=0.89) with a commercial immunoassay.
We used an established biomarker of cardiac injury and an emerging biomarker to demonstrate how SISCAPA can detect and quantify changes in concentration of proteins present at 1–10 μg/L in plasma. Our results demonstrate that these assays can be multiplexed and retain the necessary precision, reproducibility, and sensitivity to be applied to new and uncharacterized candidate biomarkers for verification of low-abundance proteins in blood.
Previously, we used a proteomics approach for the discovery of new diagnostic markers of acute appendicitis (AA) and identified LRG that was elevated in the urine of children with AA and enriched in diseased appendices. Here, we sought to evaluate the diagnostic utility of enzyme-linked immunoassay (ELISA) of urine LRG in a blinded, prospective, cohort study of children being evaluated for acute abdominal pain.
Urine LRG concentration was measured using a commercially available LRG ELISA, and selected ion monitoring (SIM) mass spectrometry (MS). Urine LRG test performance was evaluated blindly against the pathologic diagnosis and histologic grade of appendicitis.
Urine LRG was measured in 49 patients. Mean urine LRG concentration measured using commercial LRG ELISA was significantly elevated in patients with AA, but exhibited an interference effect. Direct measurements using SIM MS demonstrated that LRG was elevated more than 100-fold in patients with AA as compared to those without, with the receiver operating characteristic area under the curve of 0.98 (95% CI = 0.96-1.0). Among patients with AA, elevations of urine LRG measured using ELISA and SIM MS correlated with the histologic severity of appendicitis.
Urine LRG ELISA allows for discrimination between patients with and without AA, but exhibits limited accuracy due to immunoassay interference. Direct measurements of urine LRG using SIM MS demonstrate superior diagnostic performance. Development of a clinical-grade urine LRG assay is needed to advance the diagnostic accuracy of clinical evaluations of appendicitis.
There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.
For decades, immunoassays have provided the framework for protein biomarker studies in clinical medicine and in therapeutic monitoring for drug development. At the same time, investigators have uncovered many issues that make immunoassays unreliable in many human serum and plasma samples. LC-MS/MS after tryptic digestion of proteins is potentially an attractive solution, but the sensitivity of the method is not sufficient to measure many important low-abundance proteins directly. The use of antipeptide antibodies to immunoenrich peptides of interest can improve the sensitivity of the approach, greatly simplify the matrix enabling shortened chromatographic runs, and facilitate the multiplexed quantification of analytes, which could reduce the costs of quantitative protein measurements in complex specimens. We provide an overview of the method and the steps needed to develop an assay. In addition, we review the efforts to make this method generally more applicable.
The clinical sensitivity of nucleic acid amplification tests may be determined by analytical sensitivity and inhibitors in patient samples. We established endpoints for detection of propagated Chlamydia trachomatis L2 434, diluted according to swab and urine protocols for APTIMA Combo 2 (AC2), ProbeTec ET (PT), and Amplicor (AMP) assays. AC2 was 1,000-fold more sensitive than PT and 10-fold more sensitive than AMP on mock swab specimens. For urine, AC2 analytical sensitivity was 100-fold greater than those of the other assays. Spiking an aliquot of each clinical-trial sample from 298 women demonstrated inhibition rates in first-void urine (FVU), cervical swabs (CS), and vaginal swabs (VS) of 12.1%, 12.8%, and 10.4% for AMP; 27.2%, 2%, and 2%, for PT; and 0.3%, 1.7%, and 1.3% for AC2. Inhibition of our C. trachomatis spike and the PT or AMP amplification controls from the manufacturers showed less than 50% correlation. Using an infected-patient reference standard (a specimen positive in at least two tests or a single test positive in two of three samples) in AC2, the VS identified 68/69 (98.6%) infected women compared to CS (89.9%) or FVU (81.2%). Significantly fewer women were identified by PT (65.2%, 63.8%, and 66.7%) or AMP (65.2%, 59.4%, and 56.5%) with the three specimens. By individual specimen type, AC2 confirmed virtually all PT- and AMP-positive specimens, but rates of AC2 confirmation by AMP or PT ranged from 62.9 to 80.3%. The AC2 test identified significantly more women infected with C. trachomatis (P = 0.001). Vaginal swabs appear to be the specimen of choice for screening.
The use of targeted proteomics to identify urinary biomarkers of kidney disease in urine can avoid the interference of serum proteins. It may provide better sample throughput, higher sensitivity, and specificity. Knowing which urinary proteins to target is essential. By analyzing the urine from perfused isolated rat kidneys, 990 kidney origin proteins with human analogs were identified in urine. Of these proteins, 128 were not found in normal human urine and may become biomarkers with zero background. A total of 297 proteins were not found in normal human plasma. These proteins will not be influenced by other normal organs and will be kidney specific. The levels of 33 proteins increased during perfusion with an oxygen-deficient solution compared to those perfused with oxygen. The 75 proteins in the perfusion-driven urine have a significantly increased abundance ranking compared to their ranking in normal human urine. When compared with existing candidate biomarkers, over ninety percent of the kidney origin proteins in urine identified in this study have not been examined as candidate biomarkers of kidney diseases.
Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.
We have detected differences in metabolite levels between doped athletes, clean athletes, and volunteers (non athletes). This outcome is obtained by comparing results of measurements from two analytical platforms: UHPLC-QTOF/MS and FT-ICR/MS. Twenty-seven urine samples tested positive for glucocorticoids or beta-2-agonists and twenty samples coming from volunteers and clean athletes were analyzed with the two different mass spectrometry approaches using both positive and negative electrospray ionization modes. Urine is a highly complex matrix containing thousands of metabolites having different chemical properties and a high dynamic range. We used multivariate analysis techniques to unravel this huge data set. Thus, the several groups we created were studied by Principal Components Analysis (PCA) and Partial Least Square regression (PLS-DA and OPLS) in the search of discriminating m/z values. The selected variables were annotated and placed on pathway by using MassTRIX.