BACKGROUND
the advent of advanced molecular profiling techniques has enabled the derivation of molecular signatures that hold promise for more accurate, even individualized patient evaluation  <cit> .
a number of molecular signature assays are now being incorporated into clinical practice  <cit> , but the assays employed to monitor multiple targets per sample are, to date, rather complex and thus, expensive, and often require centralized processing and analysis.
we have previously employed a range of proteomic  <cit>  and genomic  <cit>  technologies to profile naturally voided urine samples with the aim of identifying disease-associated biomarkers that could be developed for the non-invasive detection of bladder cancer.
the objective of the current feasibility study was to test the ability of a customized multiplex assay system to accurately and simultaneously monitor three urinary protein biomarkers from one of our validated bladder cancer signatures  <cit> .
the levels of the three proteins, interleukin  <dig> , matrix metallopeptidase  <dig> , and vascular endothelial growth factor a , were quantitated in real-world urine samples and the diagnostic performance of the multiplex system was compared with analyses performed using three individual target commercial elisa kits.
the multiplex assay used q-plex™ technology , which involves the micro-spotting of individual groups of capture antibody in either a cartesian or polar coordinate system on the bottom of a  <dig> well plate with each spot being its own ‘micro elisa’ assay  <cit> .
micro-spotted systems have the advantage of higher assay sensitivities and faster reaction kinetics due to minimizing diffusion constraints for analyte/antibody binding  <cit> .
standard elisa incubation steps such as initial sample incubation, washing, secondary antibody incubation, washing, incubation are employed, but the labeling and reporting system used in the q-plex array™ is chemiluminescent.
chemiluminescent elisas have been shown to be more sensitive than colorimetric detection systems  <cit> .
in our study, the multiplex elisa assay was shown to be highly sensitive and to be consistently accurate across wells and plates.
most importantly, the assay achieved comparable diagnostic performance with respect to bladder cancer detection as the individual target commercial elisa assays  <cit> .
the system can be applied to a range of biological biomarker monitoring applications, and we are working to expand the multiplex platform to provide a high-throughput approach to quantitatively detect more comprehensive bladder cancer-associated diagnostic protein signatures in voided urine samples.
RESULTS
q-plex assay characterization
the physical components, a library of capture antibodies, and the secondary reagents of the q-plex™ system have undergone extensive optimization for consistent implementation  <cit> .
ranges for each analyte assay were evaluated by dilution of standards to determine upper ranges where high-end hook effect and apparent antibody saturation are avoided and lower ranges that are above detection limits.
lower limits of detection  were calculated based on 2× the standard deviation of the background of  <dig> negative wells.
lld for il <dig>  mmp <dig> and vegfa were  <dig>  pg/ml, 297 pg/ml and  <dig>  pg/ml, respectively, demonstrating sufficient sensitivity to detect proteins present in small amounts in voided urine samples.
the lld data for the individual elisa kits  were comparable in range for the most part.
any differences are presumably due to the antigen binding characteristics of the proprietary antibodies.
intra assay precision was measured with acceptance criteria of a coefficient of variation  of less than  <dig> .
median inter assay variability across all plates was also determined to be less than 15% cv for each analyte.
as the technology is an array, all components were checked for cross reactivity with other components in the antigen and antibody cocktails and confirmed to have less than  <dig> % cross reactivity.
lower limits of quantification  were determined to be the lowest point of the 10-point positive standard curve where the back-fit regression values were within 20% of the known value.
the minimal lloq, maximum and average lloq for il <dig>  mmp <dig>  and vegfa were  <dig>  pg/ml,  <dig>  pg/ml, and  <dig>  pg/ml; 35373 pg/ml, 52433 pg/ml, and 40782 pg/ml; and 19 pg/ml, 613 pg/ml, and 419 pg/ml, respectively.
only three of the  <dig> urine samples subsequently tested  fell outside the standard curve and required extrapolation.
urine sample analysis
after confirming the robustness of the multiplex assay for these specific biomarkers, it was used to test a voided urine sample set.
seventy-three clinical urine samples from md anderson cancer center orlando were made available for analysis.
demographics and disease characteristics of the entire cohort are summarized in table  <dig>  the ability of each of the test biomarkers within the multiplex array to predict the presence of bladder cancer was analyzed using nonparametric roc analyses, according to national cancer institute guidelines  <cit> .
urinary il <dig> was the most accurate single biomarker for bladder cancer detection with an auroc of  <dig>  , a sensitivity of 90%, specificity of 86%, ppv of 82% and npv of 92%.
urinary mmp <dig> data generated an auroc of  <dig>  , sensitivity of 45%, specificity of 76%, ppv of 58% and npv of 65%, and vegfa as an individual analyte was noted to have an auroc of  <dig>  , sensitivity of 17%, specificity of 95%, ppv of 71% and npv of 61%.
through combinatorial analysis of all  <dig> biomarkers using optimal cutoff values defined by youden index calculations, the auroc  for the diagnostic panel using the multiplex array was  <dig>  .
the combination assay achieved an overall sensitivity of  <dig> , specificity of  <dig> , ppv of  <dig>  and npv of  <dig>  for bladder cancer classification.
spearman correlation coefficient was not lower than  <dig>  for any biomarker, thus comparison of the results of the q-plex™ assay to those of the commercial elisa kits directed towards il <dig>  mmp <dig> and vegfa were encouraging .
the ability of each of the test biomarkers of the individual commercial elisa assays to predict the presence of bladder cancer was analyzed using nonparametric roc analyses.
as with the multiplex assay, urinary il <dig> was the most accurate single biomarker when monitored by individual elisa with an auroc of  <dig>  , a sensitivity of 87%, specificity of 86%, ppv of 82% and npv of 90%.
urinary mmp <dig> data generated an auroc of  <dig>  , sensitivity of 45%, specificity of 76%, ppv of 58% and npv of 65%, while vegfa was noted to have an auroc of  <dig>  , sensitivity of 16%, specificity of 95%, ppv of 71% and npv of 61%.
through combination of data from all  <dig> biomarkers, the auroc for the diagnostic panel from the elisa assays was  <dig>  , with an overall sensitivity of  <dig> , specificity of  <dig> , ppv of  <dig>  and npv of  <dig>  for bladder cancer classification.
thus, the comparison of multiplex results with standard elisa of these three diagnostic biomarkers showed similar results and trends.
*, p <  <dig>  comparing cancer cohort to benign cohort for both q-plex™ array and commercial elisa assay.
the quansys multiplex elisa is now being optimized to measure the remaining proteins of our highly accurate bladder cancer-associated diagnostic panel comprised of  <dig> biomarkers  <cit> .
we are currently producing specific antibodies  for the  <dig> analytes in our diagnostic panel, which can potentially be incorporated into a quansys bioscience custom array system for an integrated bladder cancer detection assay.
the ability to analyze multiple proteins per well, in addition to dozens of samples per microplate, results in marked improvements in efficiency and considerable cost savings compared to single analyte elisa.
our study has several limitations.
notably, the study cohort of  <dig> subjects was relatively small, and only three targets from our validated 10-biomarker panel were tested in this study.
however this is a proof-of-principle study in which we investigated the potential for multiplexing analyte detection in real-world urine samples.
lastly, as part of a biomarker discovery and validation program, archived urines were retrieved from tissue banks for analysis.
prolonged storage can result in protein degradation and mute the performance of diagnostic protein tests on such samples.
thus, validation of the performance of the multiplexed detection of a bladder cancer biomarker panel in freshly voided urines will be required.
there is growing demand to integrate multiplex signatures to obtain favorable assay properties, such as reduced sample volume, decreased processing time, low cost analysis and low reagent consumption.
several multiplex protein services are available .
several studies have reported that the multiplex elisa procedures appear suitable and reliable for tissue lysate and serum  <cit> .
while there are inherent, usually subtle differences between the various multiplex technologies, the overall technique is rapid, cost effective, and reliable.
the reliability may be due in part to specific capture and detection antibody pairs that are optimized for the specific protein in question without cross-reactivity.
the q-plex system combines powerful technology that allows the simultaneous determination of the expression levels of many proteins in biologic samples .
the continued identification of biomarker proteins, which are associated with diagnosis or prognosis, will result in this technology being more widely adopted.
CONCLUSIONS
based on these encouraging preliminary data, we believe that the q-plex technology is a viable new platform that can be exploited to be a simple, yet accurate tool to quantitate a panel of diagnostic proteins in biologic specimens, in this case the detection of diagnostic bladder cancer biomarkers in voided urine samples.
importantly, this novel platform has acceptable inter assay variability, as well as very sensitive levels of detection.
the platform can be readily implemented in a clinical laboratory and may be used as the basis for further work by incorporation of our entire bladder cancer-associated diagnostic signature.

 the advent of advanced molecular profiling techniques has enabled the derivation of molecular signatures that hold promise for more accurate, even individualized patient evaluation. a number of molecular signature assays are now being incorporated into clinical practice, but the assays employed to monitor multiple targets per sample are, to date, rather complex and thus, expensive, and often require centralized processing and analysis. we have previously employed a range of proteomic and genomic technologies to profile naturally voided urine samples with the aim of identifying disease-associated biomarkers that could be developed for the non-invasive detection of bladder cancer. the objective of the current feasibility study was to test the ability of a customized multiplex assay system to accurately and simultaneously monitor three urinary protein biomarkers from one of our validated bladder cancer signatures. the levels of the three proteins, interleukin, matrix metallopeptidase, and vascular endothelial growth factor a, were quantitated in real-world urine samples and the diagnostic performance of the multiplex system was compared with analyses performed using three individual target commercial elisa kits. the multiplex assay used q-plex™ technology, which involves the micro-spotting of individual groups of capture antibody in either a cartesian or polar coordinate system on the bottom of a well plate with each spot being its own ‘micro elisa’ assay. micro-spotted systems have the advantage of higher assay sensitivities and faster reaction kinetics due to minimizing diffusion constraints for analyte/antibody binding. standard elisa incubation steps such as initial sample incubation, washing, secondary antibody incubation, washing, incubation are employed, but the labeling and reporting system used in the q-plex array™ is chemiluminescent. chemiluminescent elisas have been shown to be more sensitive than colorimetric detection systems. in our study, the multiplex elisa assay was shown to be highly sensitive and to be consistently accurate across wells and plates. most importantly, the assay achieved comparable diagnostic performance with respect to bladder cancer detection as the individual target commercial elisa assays. the system can be applied to a range of biological biomarker monitoring applications, and we are working to expand the multiplex platform to provide a high-throughput approach to quantitatively detect more comprehensive bladder cancer-associated diagnostic protein signatures in voided urine samples.  q-plex assay characterization the physical components, a library of capture antibodies, and the secondary reagents of the q-plex™ system have undergone extensive optimization for consistent implementation. ranges for each analyte assay were evaluated by dilution of standards to determine upper ranges where high-end hook effect and apparent antibody saturation are avoided and lower ranges that are above detection limits. lower limits of detection were calculated based on 2× the standard deviation of the background of negative wells. lld for il mmp and vegfa were  pg/ml, 297 pg/ml and  pg/ml, respectively, demonstrating sufficient sensitivity to detect proteins present in small amounts in voided urine samples. the lld data for the individual elisa kits were comparable in range for the most part. any differences are presumably due to the antigen binding characteristics of the proprietary antibodies. intra assay precision was measured with acceptance criteria of a coefficient of variation of less than. median inter assay variability across all plates was also determined to be less than 15% cv for each analyte. as the technology is an array, all components were checked for cross reactivity with other components in the antigen and antibody cocktails and confirmed to have less than % cross reactivity. lower limits of quantification were determined to be the lowest point of the 10-point positive standard curve where the back-fit regression values were within 20% of the known value. the minimal lloq, maximum and average lloq for il mmp and vegfa were  pg/ml,  pg/ml, and  pg/ml; 35373 pg/ml, 52433 pg/ml, and 40782 pg/ml; and 19 pg/ml, 613 pg/ml, and 419 pg/ml, respectively. only three of the urine samples subsequently tested fell outside the standard curve and required extrapolation. urine sample analysis after confirming the robustness of the multiplex assay for these specific biomarkers, it was used to test a voided urine sample set. seventy-three clinical urine samples from md anderson cancer center orlando were made available for analysis. demographics and disease characteristics of the entire cohort are summarized in table  the ability of each of the test biomarkers within the multiplex array to predict the presence of bladder cancer was analyzed using nonparametric roc analyses, according to national cancer institute guidelines. urinary il was the most accurate single biomarker for bladder cancer detection with an auroc of, a sensitivity of 90%, specificity of 86%, ppv of 82% and npv of 92%. urinary mmp data generated an auroc of, sensitivity of 45%, specificity of 76%, ppv of 58% and npv of 65%, and vegfa as an individual analyte was noted to have an auroc of, sensitivity of 17%, specificity of 95%, ppv of 71% and npv of 61%. through combinatorial analysis of all biomarkers using optimal cutoff values defined by youden index calculations, the auroc for the diagnostic panel using the multiplex array was. the combination assay achieved an overall sensitivity of, specificity of, ppv of and npv of for bladder cancer classification. spearman correlation coefficient was not lower than for any biomarker, thus comparison of the results of the q-plex™ assay to those of the commercial elisa kits directed towards il mmp and vegfa were encouraging. the ability of each of the test biomarkers of the individual commercial elisa assays to predict the presence of bladder cancer was analyzed using nonparametric roc analyses. as with the multiplex assay, urinary il was the most accurate single biomarker when monitored by individual elisa with an auroc of, a sensitivity of 87%, specificity of 86%, ppv of 82% and npv of 90%. urinary mmp data generated an auroc of, sensitivity of 45%, specificity of 76%, ppv of 58% and npv of 65%, while vegfa was noted to have an auroc of, sensitivity of 16%, specificity of 95%, ppv of 71% and npv of 61%. through combination of data from all biomarkers, the auroc for the diagnostic panel from the elisa assays was, with an overall sensitivity of, specificity of, ppv of and npv of for bladder cancer classification. thus, the comparison of multiplex results with standard elisa of these three diagnostic biomarkers showed similar results and trends. *, p <  comparing cancer cohort to benign cohort for both q-plex™ array and commercial elisa assay. the quansys multiplex elisa is now being optimized to measure the remaining proteins of our highly accurate bladder cancer-associated diagnostic panel comprised of biomarkers. we are currently producing specific antibodies for the analytes in our diagnostic panel, which can potentially be incorporated into a quansys bioscience custom array system for an integrated bladder cancer detection assay. the ability to analyze multiple proteins per well, in addition to dozens of samples per microplate, results in marked improvements in efficiency and considerable cost savings compared to single analyte elisa. our study has several limitations. notably, the study cohort of subjects was relatively small, and only three targets from our validated 10-biomarker panel were tested in this study. however this is a proof-of-principle study in which we investigated the potential for multiplexing analyte detection in real-world urine samples. lastly, as part of a biomarker discovery and validation program, archived urines were retrieved from tissue banks for analysis. prolonged storage can result in protein degradation and mute the performance of diagnostic protein tests on such samples. thus, validation of the performance of the multiplexed detection of a bladder cancer biomarker panel in freshly voided urines will be required. there is growing demand to integrate multiplex signatures to obtain favorable assay properties, such as reduced sample volume, decreased processing time, low cost analysis and low reagent consumption. several multiplex protein services are available. several studies have reported that the multiplex elisa procedures appear suitable and reliable for tissue lysate and serum. while there are inherent, usually subtle differences between the various multiplex technologies, the overall technique is rapid, cost effective, and reliable. the reliability may be due in part to specific capture and detection antibody pairs that are optimized for the specific protein in question without cross-reactivity. the q-plex system combines powerful technology that allows the simultaneous determination of the expression levels of many proteins in biologic samples. the continued identification of biomarker proteins, which are associated with diagnosis or prognosis, will result in this technology being more widely adopted.  based on these encouraging preliminary data, we believe that the q-plex technology is a viable new platform that can be exploited to be a simple, yet accurate tool to quantitate a panel of diagnostic proteins in biologic specimens, in this case the detection of diagnostic bladder cancer biomarkers in voided urine samples. importantly, this novel platform has acceptable inter assay variability, as well as very sensitive levels of detection. the platform can be readily implemented in a clinical laboratory and may be used as the basis for further work by incorporation of our entire bladder cancer-associated diagnostic signature. 
the following analytes, interleukin, matrix metallopeptidase, and vascular endothelial growth factor a were monitored using q-plex, a customized multiplex elisa system from quansys biosciences, and individual target commercial elisa kits. based on these encouraging preliminary data, we believe that the q-plex technology is a viable platform that can be exploited as an efficient, highly accurate tool to quantitate multiplex panels of diagnostic proteins in biologic specimens. here, we tested the performance of a new, multiplex protein array platform to quantitate three bladder cancer-associated proteins in urine samples. the q-plex assay achieved an overall diagnostic sensitivity of and specificity of, and the individual target elisa assays achieved an overall sensitivity of and specificity of. the ability to accurately measure multiple proteins simultaneously in a single assay has the potential to markedly improve the efficiency of a myriad of clinical assays.
 the ability to accurately measure multiple proteins simultaneously in a single assay has the potential to markedly improve the efficiency of a myriad of clinical assays. here, we tested the performance of a new, multiplex protein array platform to quantitate three bladder cancer-associated proteins in urine samples. the following analytes, interleukin, matrix metallopeptidase, and vascular endothelial growth factor a were monitored using q-plex, a customized multiplex elisa system from quansys biosciences, and individual target commercial elisa kits. the performance of the two approaches was compared by evaluating the diagnostic accuracy of the biomarker assays in samples from a cohort of subjects of known bladder cancer status.  the combination biomarker panel analyses revealed an auroc value of for the q-plex assay, and for the combination of the single-target elisa assays. the q-plex assay achieved an overall diagnostic sensitivity of and specificity of, and the individual target elisa assays achieved an overall sensitivity of and specificity of.  based on these encouraging preliminary data, we believe that the q-plex technology is a viable platform that can be exploited as an efficient, highly accurate tool to quantitate multiplex panels of diagnostic proteins in biologic specimens. biomarkersbladder cancerq-plexproteinurine 