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Signal loss due to oligomerization in ELISA analysis of amyloid-beta can be recovered by a novel sample pre-treatment method.

Janssen L, Sobott F, De Deyn PP, Van Dam D - MethodsX (2015)

Bottom Line: However, ELISA was developed for monomeric proteins and may be ill-suited for detecting aggregates.Synthetic Aβ40 monomers, Aβ42 oligomers and biological samples from mice and humans were subjected to a chemical pre-treatment protocol with: trifluoroacetic acid (TFA), formic acid (FA) or hexafluoroisopropanol (HFIP) prior to ELISA analysis.In our study we have shown that: •Aβ oligomerization leads to epitope masking and steric hindrance and results in an underestimation of the total Aβ content with ELISA.•Chemically pre-treating samples to disaggregate oligomers can (partially) recover the signal loss.•This novel sample pre-treatment method could provide a more accurate ELISA measurement of the total Aβ concentration in samples with a high oligomer content.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neurochemistry and Behavior, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium.

ABSTRACT
According to the predominant theories, soluble amyloid-beta (Aβ) aggregates are the principal neurotoxic agents in Alzheimer's disease pathology, making them a popular target for the development of therapeutics and diagnostic markers. One of the most commonly used methods for determining the concentration of Aβ is ELISA. However, ELISA was developed for monomeric proteins and may be ill-suited for detecting aggregates. Therefore, we investigated the effect of aggregation on the ELISA measurement and developed a novel chemical pre-treatment method, designed to disaggregate Aβ peptides, to improve the ELISA measurement of the total Aβ concentration. Synthetic Aβ40 monomers, Aβ42 oligomers and biological samples from mice and humans were subjected to a chemical pre-treatment protocol with: trifluoroacetic acid (TFA), formic acid (FA) or hexafluoroisopropanol (HFIP) prior to ELISA analysis. In our study we have shown that: •Aβ oligomerization leads to epitope masking and steric hindrance and results in an underestimation of the total Aβ content with ELISA.•Chemically pre-treating samples to disaggregate oligomers can (partially) recover the signal loss.•This novel sample pre-treatment method could provide a more accurate ELISA measurement of the total Aβ concentration in samples with a high oligomer content.

No MeSH data available.


Related in: MedlinePlus

Results of ELISA measurements of Aβ40 monomer standard solution. Comparison of means (±SD) between the untreated (n = 7), PBS (n = 8), TFA (n = 8), HFIP (n = 8) and FA group (n = 7). Asterisks represent significant differences between the untreated group and treatment groups (post-hoc Bonferroni test; ***p < 0.001). The dashed line represents the standard’s theoretical concentration. Abbreviations: FA, formic acid; HFIP, hexafluoroisopropanol; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid.
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fig0010: Results of ELISA measurements of Aβ40 monomer standard solution. Comparison of means (±SD) between the untreated (n = 7), PBS (n = 8), TFA (n = 8), HFIP (n = 8) and FA group (n = 7). Asterisks represent significant differences between the untreated group and treatment groups (post-hoc Bonferroni test; ***p < 0.001). The dashed line represents the standard’s theoretical concentration. Abbreviations: FA, formic acid; HFIP, hexafluoroisopropanol; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid.

Mentions: Initially, we investigated whether the chemical treatments had any unwanted effects on the ELISA measurement of Aβ by subjecting our standard solution of synthetic Aβ 1-40 monomers (0.1 ng/ml) to the various treatments. ELISA results indicated significant, unwanted effects in some treatment groups (F4,37 = 42.813; p < 0.0001; Fig. 2). Post-hoc Bonferroni tests between the individual groups showed there was no significant difference between measurements of untreated standard and TFA-treated (p = 0.884) or FA-treated (p = 0.231) standard. On average, these three groups showed minimal deviation (<10%) from the theoretical standard concentration, demonstrating no adverse effect of these sample pre-treatments on sample recovery. The control group, treated with PBS, and the HFIP-treated group did appear to experience a significant reduction in signal due to the procedure, when compared to the untreated group (both p < 0.0001; Fig. 2). The fact that the PBS control group was affected and that there was no real difference between the PBS group and HFIP group (p = 1.000), seems to indicate that the observed effect is caused by the process of drying and reconstituting the samples, rather than by the chemical itself.


Signal loss due to oligomerization in ELISA analysis of amyloid-beta can be recovered by a novel sample pre-treatment method.

Janssen L, Sobott F, De Deyn PP, Van Dam D - MethodsX (2015)

Results of ELISA measurements of Aβ40 monomer standard solution. Comparison of means (±SD) between the untreated (n = 7), PBS (n = 8), TFA (n = 8), HFIP (n = 8) and FA group (n = 7). Asterisks represent significant differences between the untreated group and treatment groups (post-hoc Bonferroni test; ***p < 0.001). The dashed line represents the standard’s theoretical concentration. Abbreviations: FA, formic acid; HFIP, hexafluoroisopropanol; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4487349&req=5

fig0010: Results of ELISA measurements of Aβ40 monomer standard solution. Comparison of means (±SD) between the untreated (n = 7), PBS (n = 8), TFA (n = 8), HFIP (n = 8) and FA group (n = 7). Asterisks represent significant differences between the untreated group and treatment groups (post-hoc Bonferroni test; ***p < 0.001). The dashed line represents the standard’s theoretical concentration. Abbreviations: FA, formic acid; HFIP, hexafluoroisopropanol; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid.
Mentions: Initially, we investigated whether the chemical treatments had any unwanted effects on the ELISA measurement of Aβ by subjecting our standard solution of synthetic Aβ 1-40 monomers (0.1 ng/ml) to the various treatments. ELISA results indicated significant, unwanted effects in some treatment groups (F4,37 = 42.813; p < 0.0001; Fig. 2). Post-hoc Bonferroni tests between the individual groups showed there was no significant difference between measurements of untreated standard and TFA-treated (p = 0.884) or FA-treated (p = 0.231) standard. On average, these three groups showed minimal deviation (<10%) from the theoretical standard concentration, demonstrating no adverse effect of these sample pre-treatments on sample recovery. The control group, treated with PBS, and the HFIP-treated group did appear to experience a significant reduction in signal due to the procedure, when compared to the untreated group (both p < 0.0001; Fig. 2). The fact that the PBS control group was affected and that there was no real difference between the PBS group and HFIP group (p = 1.000), seems to indicate that the observed effect is caused by the process of drying and reconstituting the samples, rather than by the chemical itself.

Bottom Line: However, ELISA was developed for monomeric proteins and may be ill-suited for detecting aggregates.Synthetic Aβ40 monomers, Aβ42 oligomers and biological samples from mice and humans were subjected to a chemical pre-treatment protocol with: trifluoroacetic acid (TFA), formic acid (FA) or hexafluoroisopropanol (HFIP) prior to ELISA analysis.In our study we have shown that: •Aβ oligomerization leads to epitope masking and steric hindrance and results in an underestimation of the total Aβ content with ELISA.•Chemically pre-treating samples to disaggregate oligomers can (partially) recover the signal loss.•This novel sample pre-treatment method could provide a more accurate ELISA measurement of the total Aβ concentration in samples with a high oligomer content.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neurochemistry and Behavior, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium.

ABSTRACT
According to the predominant theories, soluble amyloid-beta (Aβ) aggregates are the principal neurotoxic agents in Alzheimer's disease pathology, making them a popular target for the development of therapeutics and diagnostic markers. One of the most commonly used methods for determining the concentration of Aβ is ELISA. However, ELISA was developed for monomeric proteins and may be ill-suited for detecting aggregates. Therefore, we investigated the effect of aggregation on the ELISA measurement and developed a novel chemical pre-treatment method, designed to disaggregate Aβ peptides, to improve the ELISA measurement of the total Aβ concentration. Synthetic Aβ40 monomers, Aβ42 oligomers and biological samples from mice and humans were subjected to a chemical pre-treatment protocol with: trifluoroacetic acid (TFA), formic acid (FA) or hexafluoroisopropanol (HFIP) prior to ELISA analysis. In our study we have shown that: •Aβ oligomerization leads to epitope masking and steric hindrance and results in an underestimation of the total Aβ content with ELISA.•Chemically pre-treating samples to disaggregate oligomers can (partially) recover the signal loss.•This novel sample pre-treatment method could provide a more accurate ELISA measurement of the total Aβ concentration in samples with a high oligomer content.

No MeSH data available.


Related in: MedlinePlus