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Evaluation and Validation of the Detection of soluble Triggering Receptor Expressed on Myeloid Cells 1 by Enzyme-linked immunosorbent Assay.

Hasibeder A, Stein P, Brandwijk R, Schild H, Radsak MP - Sci Rep (2015)

Bottom Line: In addition, a soluble TREM-1 variant (sTREM-1) is detectable in sera or bronchoalveolar-lavage fluids from patients.Investigating potential confounding factors for sTREM-1 detection, serum heat-inactivation (HI) showed improved recovery compared to non-HI (NHI) serum, reproducible by addition of complement and re-heat-inactivation.Immunoassays for research use only are in general hampered by lack of standardization.

View Article: PubMed Central - PubMed

Affiliation: IIIrd Department of Medicine, Johannes-Gutenberg University Medical Center, Mainz, Germany.

ABSTRACT
Triggering receptor expressed on myeloid cells (TREM)-1 plays an important role in innate immune responses and is upregulated under infectious as well as non-infectious conditions. In addition, a soluble TREM-1 variant (sTREM-1) is detectable in sera or bronchoalveolar-lavage fluids from patients. Currently, various studies are difficult to compare, since the methods of detection by enzyme-linked immunosorbent assays (ELISA) vary among different research groups. In this study, we compared three different s-TREM-1 specific ELISAs and identified individual assay characteristics finding notable differences in sTREM-1 concentrations in part depending on the employed buffers. Investigating potential confounding factors for sTREM-1 detection, serum heat-inactivation (HI) showed improved recovery compared to non-HI (NHI) serum, reproducible by addition of complement and re-heat-inactivation. Hence we identified complement as a heat-sensitive confounder in some sTREM-1 ELISAs. We conclude that it is difficult to directly compare data of several studies, in particular if different ELISAs are engaged. Immunoassays for research use only are in general hampered by lack of standardization. Further standardization is needed until sTREM-1 ELISA is capable for better reproducibility of studies and clinical application.

No MeSH data available.


Related in: MedlinePlus

Effect of serum heat-inactivation on the detection of rh-sTREM-1 was assessed in different ELISAs.NHI and HI sera were diluted 1:4 and 2,000 pg/mL rh-sTREM-1 was added. Twofold serially dilution was performed and rh-sTREM-1 concentration was measured by the indicated ELISA. Mean and SD for three independent experiments each conducted in duplicates are shown. Values ranging beyond the designed standard curve were set to zero for graph creation and excluded of further calculations. Data were statistical tested for significant differences of rh-sTREM-1 of indicated concentrations in NHI and HI serum by two-tailed Wilcoxon matched-pairs test, followed by Bonferroni correction for multiple testing. *p < 0.05, **p < 0.01.
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f3: Effect of serum heat-inactivation on the detection of rh-sTREM-1 was assessed in different ELISAs.NHI and HI sera were diluted 1:4 and 2,000 pg/mL rh-sTREM-1 was added. Twofold serially dilution was performed and rh-sTREM-1 concentration was measured by the indicated ELISA. Mean and SD for three independent experiments each conducted in duplicates are shown. Values ranging beyond the designed standard curve were set to zero for graph creation and excluded of further calculations. Data were statistical tested for significant differences of rh-sTREM-1 of indicated concentrations in NHI and HI serum by two-tailed Wilcoxon matched-pairs test, followed by Bonferroni correction for multiple testing. *p < 0.05, **p < 0.01.

Mentions: Evaluating accuracy of indicated ELISAs serum interferences are assumed regarding recovery rates between 40 and 84%. To further investigate these interferences in defined sTREM-1 concentrations, we examined the effect of human serum from healthy donors and furthermore the effect of heat-inactivation262728 on detection of rh-sTREM-1 and dilution series in the different ELISAs. NHI and HI serum samples were employed at 1:4 dilutions and 2,000 pg/mL rh-sTREM-1, further 1:2 diluted in appropriate buffers. Figure 3A confirms the accuracy results of the Radsak ELISA, where only about half of the applied sTREM-1 standard amount can be detected after the addition of NHI serum. Aside in samples containing HI serum sTREM-1 standard was at a significantly higher amount detectable, indicating a positive effect of heat-inactivation of serum on sTREM-1 detection in a concentration dependent manner (2,000 pg/mL p = 0.005, 1,000 pg/mL p = 0.005, 500 pg/mL p = 0.005, 250 pg/mL p = 0.010, 125 pg/mL p = 0.094, 62.5 pg/mL p = 0.094, 31.25 pg/mL p = 0.115). Similar results were obtained when NHI and HI sera were engaged in R&D ELISA with pcb (2,000 pg/mL p = 0.005, 1,000 pg/mL p = 0.005, 500 pg/mL p = 0.005, 250 pg/mL p = 0.026, 125 pg/mL excluded, 62.5 pg/mL excluded, 31.25 pg/mL excluded) (Fig. 3B), with slight improvement of the performance when surrendering pcb (2,000 pg/mL p = 0.005, 1,000 pg/mL p = 0.005, 500 pg/mL p = 0.005, 250 pg/mL p = 0.005, 125 pg/mL excluded, 62.5 pg/mL excluded, 31.25 pg/mL excluded) (Fig. 3C). In contrast, performance of Hycult Biotech ELISA was completely independent of these heat-sensitive confounding factors (2,000 pg/mL p = 0.156, 1,000 pg/mL p = 0.063, 500 pg/mL p = 0.094, 250 pg/mL p = 0.156, 125 pg/mL excluded, 62.5 pg/mL excluded, 31.25 pg/mL excluded) (Fig. 3D). OD values ranging below the calculated ODmin had to be excluded of further calculations. Apart from that, the detectable differences between HI and NHI sera were only significant for several dilutions with correspondent high rh-sTREM-1 standard concentrations in the Radsak ELISA (Fig. 3A).


Evaluation and Validation of the Detection of soluble Triggering Receptor Expressed on Myeloid Cells 1 by Enzyme-linked immunosorbent Assay.

Hasibeder A, Stein P, Brandwijk R, Schild H, Radsak MP - Sci Rep (2015)

Effect of serum heat-inactivation on the detection of rh-sTREM-1 was assessed in different ELISAs.NHI and HI sera were diluted 1:4 and 2,000 pg/mL rh-sTREM-1 was added. Twofold serially dilution was performed and rh-sTREM-1 concentration was measured by the indicated ELISA. Mean and SD for three independent experiments each conducted in duplicates are shown. Values ranging beyond the designed standard curve were set to zero for graph creation and excluded of further calculations. Data were statistical tested for significant differences of rh-sTREM-1 of indicated concentrations in NHI and HI serum by two-tailed Wilcoxon matched-pairs test, followed by Bonferroni correction for multiple testing. *p < 0.05, **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4612298&req=5

f3: Effect of serum heat-inactivation on the detection of rh-sTREM-1 was assessed in different ELISAs.NHI and HI sera were diluted 1:4 and 2,000 pg/mL rh-sTREM-1 was added. Twofold serially dilution was performed and rh-sTREM-1 concentration was measured by the indicated ELISA. Mean and SD for three independent experiments each conducted in duplicates are shown. Values ranging beyond the designed standard curve were set to zero for graph creation and excluded of further calculations. Data were statistical tested for significant differences of rh-sTREM-1 of indicated concentrations in NHI and HI serum by two-tailed Wilcoxon matched-pairs test, followed by Bonferroni correction for multiple testing. *p < 0.05, **p < 0.01.
Mentions: Evaluating accuracy of indicated ELISAs serum interferences are assumed regarding recovery rates between 40 and 84%. To further investigate these interferences in defined sTREM-1 concentrations, we examined the effect of human serum from healthy donors and furthermore the effect of heat-inactivation262728 on detection of rh-sTREM-1 and dilution series in the different ELISAs. NHI and HI serum samples were employed at 1:4 dilutions and 2,000 pg/mL rh-sTREM-1, further 1:2 diluted in appropriate buffers. Figure 3A confirms the accuracy results of the Radsak ELISA, where only about half of the applied sTREM-1 standard amount can be detected after the addition of NHI serum. Aside in samples containing HI serum sTREM-1 standard was at a significantly higher amount detectable, indicating a positive effect of heat-inactivation of serum on sTREM-1 detection in a concentration dependent manner (2,000 pg/mL p = 0.005, 1,000 pg/mL p = 0.005, 500 pg/mL p = 0.005, 250 pg/mL p = 0.010, 125 pg/mL p = 0.094, 62.5 pg/mL p = 0.094, 31.25 pg/mL p = 0.115). Similar results were obtained when NHI and HI sera were engaged in R&D ELISA with pcb (2,000 pg/mL p = 0.005, 1,000 pg/mL p = 0.005, 500 pg/mL p = 0.005, 250 pg/mL p = 0.026, 125 pg/mL excluded, 62.5 pg/mL excluded, 31.25 pg/mL excluded) (Fig. 3B), with slight improvement of the performance when surrendering pcb (2,000 pg/mL p = 0.005, 1,000 pg/mL p = 0.005, 500 pg/mL p = 0.005, 250 pg/mL p = 0.005, 125 pg/mL excluded, 62.5 pg/mL excluded, 31.25 pg/mL excluded) (Fig. 3C). In contrast, performance of Hycult Biotech ELISA was completely independent of these heat-sensitive confounding factors (2,000 pg/mL p = 0.156, 1,000 pg/mL p = 0.063, 500 pg/mL p = 0.094, 250 pg/mL p = 0.156, 125 pg/mL excluded, 62.5 pg/mL excluded, 31.25 pg/mL excluded) (Fig. 3D). OD values ranging below the calculated ODmin had to be excluded of further calculations. Apart from that, the detectable differences between HI and NHI sera were only significant for several dilutions with correspondent high rh-sTREM-1 standard concentrations in the Radsak ELISA (Fig. 3A).

Bottom Line: In addition, a soluble TREM-1 variant (sTREM-1) is detectable in sera or bronchoalveolar-lavage fluids from patients.Investigating potential confounding factors for sTREM-1 detection, serum heat-inactivation (HI) showed improved recovery compared to non-HI (NHI) serum, reproducible by addition of complement and re-heat-inactivation.Immunoassays for research use only are in general hampered by lack of standardization.

View Article: PubMed Central - PubMed

Affiliation: IIIrd Department of Medicine, Johannes-Gutenberg University Medical Center, Mainz, Germany.

ABSTRACT
Triggering receptor expressed on myeloid cells (TREM)-1 plays an important role in innate immune responses and is upregulated under infectious as well as non-infectious conditions. In addition, a soluble TREM-1 variant (sTREM-1) is detectable in sera or bronchoalveolar-lavage fluids from patients. Currently, various studies are difficult to compare, since the methods of detection by enzyme-linked immunosorbent assays (ELISA) vary among different research groups. In this study, we compared three different s-TREM-1 specific ELISAs and identified individual assay characteristics finding notable differences in sTREM-1 concentrations in part depending on the employed buffers. Investigating potential confounding factors for sTREM-1 detection, serum heat-inactivation (HI) showed improved recovery compared to non-HI (NHI) serum, reproducible by addition of complement and re-heat-inactivation. Hence we identified complement as a heat-sensitive confounder in some sTREM-1 ELISAs. We conclude that it is difficult to directly compare data of several studies, in particular if different ELISAs are engaged. Immunoassays for research use only are in general hampered by lack of standardization. Further standardization is needed until sTREM-1 ELISA is capable for better reproducibility of studies and clinical application.

No MeSH data available.


Related in: MedlinePlus