Signal loss due to oligomerization in ELISA
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.
Oligomerization of amyloid-beta (Aβ) can lead to epitope masking and steric hindrance, which can result in an underestimation of the Aβ concentration in ELISA analysis (Fig. 1). We, therefore, decided to develop a chemical pre-treatment protocol to disaggregate Aβ oligomers in samples prior to analysis.
Three candidate chemicals with disaggregation properties, namely trifluoroacetic acid (TFA) , hexafluoroisopropanol (HFIP)  and formic acid (FA)  were selected from literature and applied to various samples to evaluate efficacy of the protocol and to identify the most promising option for future research. First, the pre-treatment protocol was tested with synthetic Aβ monomer solutions to assess any unwanted effects.
Next, we progressed to solutions of aggregated synthetic Aβ to determine the efficiency of the various treatments. Finally, we examined the compatibility of the protocol with biological samples, i.e., brain extract of the APP23 mouse model for AD and cerebrospinal fluid from human AD patients and control individuals.