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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

Complement interferes in sTREM-1 ELISA.NHI and HI sera were 1:4 diluted and 4 U of reconstituted guinea pig complement (C’) was added to 240 μL-diluted sera. HI serum after addition of C’ was again heat-inactivated at 56 °C for 30 min to inactivate the complement. 2,000 pg/mL rh-sTREM-1 was added to each preparation and twofold serially dilution was performed. Shown are the results of extrapolated rh-sTREM-1 at 1,000 pg/mL tested by Radsak sTREM-1 ELISA. Mean and SD for three independent experiments each conducted in duplicates are shown. Statistical significance for indicated samples was calculated by two-tailed Wilcoxon matched-pairs test followed by Bonferroni correction for multiple testing. **p < 0.01.
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f4: Complement interferes in sTREM-1 ELISA.NHI and HI sera were 1:4 diluted and 4 U of reconstituted guinea pig complement (C’) was added to 240 μL-diluted sera. HI serum after addition of C’ was again heat-inactivated at 56 °C for 30 min to inactivate the complement. 2,000 pg/mL rh-sTREM-1 was added to each preparation and twofold serially dilution was performed. Shown are the results of extrapolated rh-sTREM-1 at 1,000 pg/mL tested by Radsak sTREM-1 ELISA. Mean and SD for three independent experiments each conducted in duplicates are shown. Statistical significance for indicated samples was calculated by two-tailed Wilcoxon matched-pairs test followed by Bonferroni correction for multiple testing. **p < 0.01.

Mentions: Complement components present in serum are known to react with several immunoglobulins29 and interference with detection in immunoassay can partly be eliminated by serum heat-inactivation26. Therefore rh-sTREM-1 concentration was measured in 1:4 diluted NHI and HI serum, as well as after the addition of guinea pig complement and re-heat-inactivation26 employing Radsak sTREM-1 ELISA. These experiments were again performed in 1:2 serially dilution of rh-sTREM-1. As shown above rh-sTREM-1 in NHI serum resulted in significantly increased recovery (p = 0.005) compared to HI serum (Fig. 4). Furthermore 4 U exogenous guinea pig complement was added to the 1:4 dilution of HI serum. Addition of complement resulted in significantly decreased rh-sTREM-1 concentration (p = 0.005) demonstrating that the positive effect of heat-inactivation on rh-sTREM-1 detection compared to NHI serum can be effaced through the addition of complement. To further test if re-heat-inactivation of HI serum with complement will restore measured rh-sTREM-1 amount we re-heat-inactivated HI serum with complement. Interestingly, rh-sTREM-1 after heat-inactivation of HI serum with complement was again significantly increased as compared to non heat-inactivated HI serum with complement (p = 0.005) (Fig. 4).


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)

Complement interferes in sTREM-1 ELISA.NHI and HI sera were 1:4 diluted and 4 U of reconstituted guinea pig complement (C’) was added to 240 μL-diluted sera. HI serum after addition of C’ was again heat-inactivated at 56 °C for 30 min to inactivate the complement. 2,000 pg/mL rh-sTREM-1 was added to each preparation and twofold serially dilution was performed. Shown are the results of extrapolated rh-sTREM-1 at 1,000 pg/mL tested by Radsak sTREM-1 ELISA. Mean and SD for three independent experiments each conducted in duplicates are shown. Statistical significance for indicated samples was calculated by two-tailed Wilcoxon matched-pairs test followed by Bonferroni correction for multiple testing. **p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Complement interferes in sTREM-1 ELISA.NHI and HI sera were 1:4 diluted and 4 U of reconstituted guinea pig complement (C’) was added to 240 μL-diluted sera. HI serum after addition of C’ was again heat-inactivated at 56 °C for 30 min to inactivate the complement. 2,000 pg/mL rh-sTREM-1 was added to each preparation and twofold serially dilution was performed. Shown are the results of extrapolated rh-sTREM-1 at 1,000 pg/mL tested by Radsak sTREM-1 ELISA. Mean and SD for three independent experiments each conducted in duplicates are shown. Statistical significance for indicated samples was calculated by two-tailed Wilcoxon matched-pairs test followed by Bonferroni correction for multiple testing. **p < 0.01.
Mentions: Complement components present in serum are known to react with several immunoglobulins29 and interference with detection in immunoassay can partly be eliminated by serum heat-inactivation26. Therefore rh-sTREM-1 concentration was measured in 1:4 diluted NHI and HI serum, as well as after the addition of guinea pig complement and re-heat-inactivation26 employing Radsak sTREM-1 ELISA. These experiments were again performed in 1:2 serially dilution of rh-sTREM-1. As shown above rh-sTREM-1 in NHI serum resulted in significantly increased recovery (p = 0.005) compared to HI serum (Fig. 4). Furthermore 4 U exogenous guinea pig complement was added to the 1:4 dilution of HI serum. Addition of complement resulted in significantly decreased rh-sTREM-1 concentration (p = 0.005) demonstrating that the positive effect of heat-inactivation on rh-sTREM-1 detection compared to NHI serum can be effaced through the addition of complement. To further test if re-heat-inactivation of HI serum with complement will restore measured rh-sTREM-1 amount we re-heat-inactivated HI serum with complement. Interestingly, rh-sTREM-1 after heat-inactivation of HI serum with complement was again significantly increased as compared to non heat-inactivated HI serum with complement (p = 0.005) (Fig. 4).

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