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Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA.

Frampton JP, White JB, Simon AB, Tsuei M, Paczesny S, Takayama S - Sci Rep (2014)

Bottom Line: This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients.The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced.Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Michigan.

ABSTRACT
Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

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Related in: MedlinePlus

Bland-Altman analysis for (a) HGF, (b) elafin, (c) ST2 and (d) TNFR1.The dashed horizontal lines represent the 2SD confidence intervals and the solid horizontal line represents the mean difference between assay formats. We observed discrepancies between the two assay formats, particularly at the higher biomarker concentrations.
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f6: Bland-Altman analysis for (a) HGF, (b) elafin, (c) ST2 and (d) TNFR1.The dashed horizontal lines represent the 2SD confidence intervals and the solid horizontal line represents the mean difference between assay formats. We observed discrepancies between the two assay formats, particularly at the higher biomarker concentrations.

Mentions: Bland-Altman analysis was used to evaluate the agreement between ATPS-ELISA and standard single ELISA (Figure 6). We observed proportional and magnitude biases between the ATPS-ELISA and single ELISA formats, indicating that there were discrepancies between the two methods. These discrepancies were generally tolerable at lower biomarker concentrations, for example, below 2,500 pg/mL for HGF, 36,000 pg/mL for elafin, 4,700 pg/mL for ST2 and 1,800 pg/mL forTNFR1 (i.e., concentrations that approximate the clinically important thresholds for GVHD diagnosis). Although a larger patient cohort would be required to determine clinical cutoffs for our assay, the approximate clinical cutoffs can be obtained from previous biomarker studies for HGF33, elafin9, ST216 and TNFR114. It is important to note that although the Bland-Altman analyses indicated discrepancies between the two assay formats, this does not necessarily indicate that one format is superior to the other. To provide a comparison of the performance of the two methods, we conducted receiver operating characteristic (ROC) analysis (Figure S3). In our hands, the ATPS format slightly outperformed the single ELISA format, as assessed by the areas under the curves, indicating that the ATPS method might provide superior sensitivity and specificity. Larger experiments involving greater numbers of biomarkers and additional samples, along with experimenter-to-experimenter comparisons, will be required to conclusively prove this assertion. However, these experiments are beyond the scope of the present study, which focuses on the development of a promising new technology for detection antibody patterning.


Aqueous two-phase system patterning of detection antibody solutions for cross-reaction-free multiplex ELISA.

Frampton JP, White JB, Simon AB, Tsuei M, Paczesny S, Takayama S - Sci Rep (2014)

Bland-Altman analysis for (a) HGF, (b) elafin, (c) ST2 and (d) TNFR1.The dashed horizontal lines represent the 2SD confidence intervals and the solid horizontal line represents the mean difference between assay formats. We observed discrepancies between the two assay formats, particularly at the higher biomarker concentrations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4007081&req=5

f6: Bland-Altman analysis for (a) HGF, (b) elafin, (c) ST2 and (d) TNFR1.The dashed horizontal lines represent the 2SD confidence intervals and the solid horizontal line represents the mean difference between assay formats. We observed discrepancies between the two assay formats, particularly at the higher biomarker concentrations.
Mentions: Bland-Altman analysis was used to evaluate the agreement between ATPS-ELISA and standard single ELISA (Figure 6). We observed proportional and magnitude biases between the ATPS-ELISA and single ELISA formats, indicating that there were discrepancies between the two methods. These discrepancies were generally tolerable at lower biomarker concentrations, for example, below 2,500 pg/mL for HGF, 36,000 pg/mL for elafin, 4,700 pg/mL for ST2 and 1,800 pg/mL forTNFR1 (i.e., concentrations that approximate the clinically important thresholds for GVHD diagnosis). Although a larger patient cohort would be required to determine clinical cutoffs for our assay, the approximate clinical cutoffs can be obtained from previous biomarker studies for HGF33, elafin9, ST216 and TNFR114. It is important to note that although the Bland-Altman analyses indicated discrepancies between the two assay formats, this does not necessarily indicate that one format is superior to the other. To provide a comparison of the performance of the two methods, we conducted receiver operating characteristic (ROC) analysis (Figure S3). In our hands, the ATPS format slightly outperformed the single ELISA format, as assessed by the areas under the curves, indicating that the ATPS method might provide superior sensitivity and specificity. Larger experiments involving greater numbers of biomarkers and additional samples, along with experimenter-to-experimenter comparisons, will be required to conclusively prove this assertion. However, these experiments are beyond the scope of the present study, which focuses on the development of a promising new technology for detection antibody patterning.

Bottom Line: This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients.The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced.Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Michigan.

ABSTRACT
Accurate disease diagnosis, patient stratification and biomarker validation require the analysis of multiple biomarkers. This paper describes cross-reactivity-free multiplexing of enzyme-linked immunosorbent assays (ELISAs) using aqueous two-phase systems (ATPSs) to confine detection antibodies at specific locations in fully aqueous environments. Antibody cross-reactions are eliminated because the detection antibody solutions are co-localized only to corresponding surface-immobilized capture antibody spots. This multiplexing technique is validated using plasma samples from allogeneic bone marrow recipients. Patients with acute graft versus host disease (GVHD), a common and serious condition associated with allogeneic bone marrow transplantation, display higher mean concentrations for four multiplexed biomarkers (HGF, elafin, ST2 and TNFR1) relative to healthy donors and transplant patients without GVHD. The antibody co-localization capability of this technology is particularly useful when using inherently cross-reactive reagents such as polyclonal antibodies, although monoclonal antibody cross-reactivity can also be reduced. Because ATPS-ELISA adapts readily available antibody reagents, plate materials and detection instruments, it should be easily transferable into other research and clinical settings.

Show MeSH
Related in: MedlinePlus