Webinar: Soluble Biomarkers for Alzheimer's Disease

Medix Biochemica recently hosted a webinar discussing soluble biomarkers for Alzheimer’s disease, featuring expert insights and updates on diagnostic tools and treatments. Our guest speaker and subject-matter expert, Professor Henrik Zetterberg from the University of Gothenburg, guided the audience through the latest advancements in Alzheimer's disease diagnostic development. Medix Biochemica’s R&D Manager, Dr. Emilia Galli, then discussed recent developments in antibodies targeting key analytes involved in Alzheimer’s disease pathology.

Here’s a look at what was discussed during the webinar.

Visit our post-webinar page to: Watch the full webinar, Download the slide deck and Download the Q&A's

The most exciting recent developments in fluid-based biomarkers for neurodegenerative disease diagnostics

Amyloid pathology

We all produce amyloid beta-protein 42 (Aβ42) and, when we’re young, it stays dissolved and can be found in biofluids such as cerebrospinal fluid (CSF) and blood. As we age, we either become amyloid non-depositors (keeping Aβ in our biofluids) or amyloid depositors, accumulating amyloid plaques in the brain, a hallmark of Alzheimer’s disease. 

Research shows that the onset of amyloid deposition in brain tissue occurs from around the age of 50. This indicates that the amyloid cascade in Alzheimer's disease starts from the age of 50, and it becomes possible to detect by amyloid positron emission tomography (PET) from the age of around 53 or 54. Compared to healthy controls, people with amyloid plaque pathology in the brain have approximately 50% lower levels of ​​the 42-amino acid long beta amyloid peptide in their biological fluids. 

Research shows that this peptide is quite sticky and is the core protein that builds up plaques in the brain. If Aβ42 starts to accumulate in brain tissue, its concentration in biological fluids decreases.  

Aβ42 on its own is a good biomarker, but it becomes much more effective in combination with the more water-soluble form, Aβ40. If we make a ratio of Aβ42 over Aβ40, we get a more effective test for detecting amyloid plaque pathology in the brain. Studies over the years have highlighted the difference between amyloid-negative and amyloid-positive people, allowing us to see the decrease in the blood Aβ42:40 ratio. 

A fully automated Cobas assay has been developed for the plasma Aβ42:40 ratio and, technically, the assay is highly effective at a group level. The challenge with plasma tests comes on an individual level, because it’s difficult to separate amyloid-negative and amyloid-positive people.  

This is why diagnosing amyloid pathology with a blood test is acceptable for group enrichment or screening, but not for individual diagnostics. 

Tau pathology 

Tau pathology is another key characteristic of Alzheimer's disease. In addition to Aβ plaques, Alzheimer’s disease patients also develop intraneural tangles composed of hyperphosphorylated and truncated tau protein. These patients show elevated levels of p-Tau in their lumbar CSF. Notably, this increase in p-Tau is specific to Alzheimer's disease, and is not observed in other neurodegenerative diseases that also feature tau tangles.  

There are several forms of p-Tau but the form that has gained the most attention recently is p-Tau217, due to its strong potential for staging Alzheimer’s disease: with the onset of amyloid pathology, p-Tau 217 levels rise early and then there is a stepwise increase that correlates with the disease stage. As a result, most laboratories are now focusing on developing assays for p-Tau217. Additionally, p-Tau181 and p-Tau205 are also valuable in biomarker-based staging of Alzheimer’s disease. 

Although it becomes even more effective when combined with a memory test, p-Tau217 can be used by itself to predict Alzheimer's disease and dementia in people with mild cognitive impairment. Notably, the p-Tau217 marker in plasma is comparable to levels measured in CSF, meaning that a simple plasma p-Tau217 test could potentially replace the need for invasive CSF testing. 

To summarize, diagnosing Alzheimer’s-type tau pathophysiology with a blood test is effective for group-level enrichment or screening as well as individual diagnostics. Plasma p-Tau assays are now being established as clinical chemistry tests in many labs around the world. 

Neurofilament light 

Neurofilament light (NfL) is a popular biomarker for detecting neurodegeneration. Levels of NfL are increased across multiple neurodegenerative diseases, with the highest levels seen in amyotrophic lateral sclerosis (ALS), frontotemporal dementia and Huntington’s disease. NfL levels normalize in response to successful treatment. 

NfL blood measurement can be used to assess neurodegeneration at a group enrichment/screening level and in individual diagnostics, although CSF is likely to be more sensitive for revealing neurodegeneration than plasma. While a neurodegenerative disease cannot be excluded with an NfL plasma test, it can serve as a guiding tool to determine whether further examinations are needed. 

Glial activation 

Glial fibrillary acidic protein (GFAP) is a protein that is released from CNS astrocytes when they are activated, a process that is clearly linked to amyloid pathology. Amyloid activation of astrocytes is reflected in the plasma GFAP test. Since GFAP is unstable in the CSF matrix, a plasma GFAP test is more effective than a CSF test. Additionally, there are several candidate blood tests being explored for detecting microglial activation.  

Synaptic pathology 

There are numerous pre- and postsynaptic markers that can be measured in CSF, typically increasing in response to amyloid pathology.  

An exception is NPTX2 , which decreases in neurodegenerative diseases. NPTX2 shows potential as a synaptic function biomarker, and preliminary data suggests it could be detected in blood. 

Another promising synaptic marker is β-synuclein, which also shows encouraging data in blood-based measurements. 

Synuclein pathology 

To detect synuclein pathology, lumbar CSF from patients is spiked with recombinant alpha-synuclein and assessed using thioflavin T (THT) fluorescence. This method detects if misfolded alpha-synuclein forms beta-pleated sheets, indicating potential alpha-synuclein pathology.  

The approach could help diagnose synuclein-related neurodegenerative diseases, such as Parkinson's disease. The Swedish BioFINDER studies, involving over 1 900 individuals, explored the prevalence of Lewy body pathology, contributing to our understanding of synucleinopathies in large cohorts. 

TDP-43 pathology  

An aggregation-seed assay, similar to the RT-QuIC test for alpha-synuclein, has been developed to detect TDP-43 pathology. It has, however, been challenging to replicate consistently.  

Recently, a blood-based extracellular vesicle quantification of TDP-43 has shown promising results, suggesting the potential for a blood-based test for TDP-43 pathology. 

Simplified testing: DROP-AD 

Testing for Alzheimer's disease is being simplified through the use of dried blood spots, allowing for easy testing via a finger prick. This process is under development to make Alzheimer's disease testing more accessible. Because dried plasma cards are stable for up to six months at room temperature, they offer a convenient and effective method for detecting both Alzheimer's disease and neurodegeneration. 

Antibodies against Alzheimer's disease markers: Medix Biochemica’s recent launches and development pipeline  

Medix Biochemica has a long history, dating back to the 1980s, of developing high-quality, high-sensitivity monoclonal antibodies (mAbs) for the in vitro diagnostic (IVD) industry. In addition to the traditional hybridoma method, Medix Biochemica has adopted various novel methods – such as phage display and recombinant antibody expression – to increase success rates and speed up project timelines. 

Recognizing the importance of neurodegenerative disease diagnostics, we’ve put our efforts into developing mAbs which target relevant analytes, including:  

• Amyloid beta (Aβ)
• Phosphorylated tau (p-Tau)  
• Total tau (t-Tau)
• Central nervous system tau (CNS-Tau)
• Neurofilament light (NfL)  
• Glial fibrillary acidic protein (GFAP) 

We collaborate closely with scientific experts and our customers on mAb development projects to meet the specific needs of the IVD field. 

NfL mAb pairs 

For detecting neurodegeneration, we currently offer three mAbs specific to NfL. These mAbs can be paired in sandwich assays. By spiking recombinant NfL into human serum, we've demonstrated the sensitivity of these mAb pairs at clinically relevant concentrations for blood-based detection, even when using an unoptimized fluorescent immunoassay (FIA). 

About NfL mAbs: 

• High sensitivity, capable of detecting blood levels as low as 10 pg/ml with an unoptimized FIA  
• Kinetics measured by BLI (Octet) with free rNfL (LA666, Medix Biochemica) show strong binding
• Good correlation of CSF measurements to results with ‘gold standard’ mAb pair
• No cross-reactivity with NfM, NfH, S100B or NSE 

t-Tau mAb pairs 

Total tau is also a valuable biomarker for assessing neurodegeneration in Alzheimer's disease. Medix Biochemica currently offers three mAbs for t-Tau, which all work in pairs. These mAbs work very effectively with CSF samples from Alzheimer's patients.  

Our t-Tau products: 

• Anti-h Tau R13301 SPTN-5  
• Anti-h Tau R13302 SPTN-5
• Anti-h Tau R13303 SPTN-5  

About t-Tau mAbs: 

• Good correlation of CSF measurements to CE-marked assay
  
• Kinetics measured by BLI (Octet) with sensor-bound antigen show strong binding to t-Tau 

In addition to the tau expressed in the brain, the peripheral body produces a larger variant of tau and these levels are not affected by Alzheimer’s disease pathology. Our t-Tau mAbs most likely detect this peripheral tau as well, as confirmed by testing with peptides spanning the different sequences of these variants. This is why we don’t recommend using these antibodies for blood-based t-Tau test development.  

We do, however, have an ongoing project to develop CNS-specific tau mAbs. The project aims to target the epitope that differs between brain-derived tau and peripheral tau. 

p-Tau mAbs 

For the determination of tau pathology in clinical samples, we offer mAbs specific for the best-studied phosphorylation sites related to Alzheimer's disease. We offer three phosphorylation-specific mAbs that work paired with our t-Tau mAbs for the detection of p-Tau. 

Our p-Tau products: 

• p-Tau181: Anti-h p-Tau181 R13321 SPTN-5
• p-Tau217: Anti-h p-Tau217 R13323 SPTN-5
• p-Tau231: Anti-h p-Tau231 R13322 SPTN-5  

About p-Tau mAbs: 

• For anti-h p-Tau181 R13321, detection of native protein in CSF has been verified with correlation to known concentration measured with CE-marked assay
• Kinetics measured by BLI (Octet) with sensor-bound antigen show strong binding 

GFAP mAbs 

For analyzing neuroinflammation, we offer four mAbs targeting GFAP. These mAbs work in pairs and their epitope falls within the area considered the most relevant to Alzheimer's disease diagnostics.  

• Sensitivity reaches appr. 100 pg/ml with unoptimized FIA
• Kinetics measured by BLI (Octet) with free GFAP show strong binding
• No or minor cross-reactivity with homologs vimentin, desmin or pheripherin 

Our GFAP products: 

• Anti-h GFAP R13501 SPTN-5
• Anti-h GFAP R13502 SPTN-5
• Anti-h GFAP R13503 SPTN-5
• Anti-h GFAP R13504 SPTN-5

Aβ mAbs 

Aβ is one of the earliest biomarkers used in clinics to assess Alzheimer's disease pathology. We have focused on developing antibody pairs for detecting the C-terminus of Aβ(1-42) and Aβ(1-40), as well as mAbs for N-terminus. We have succeeded in finding candidates for all of three epitopes and are now in the recombinant cell line development phase. We expect to launch at the beginning of 2025. 

We already have preliminary data on the performance of these mAbs with CSF samples for the Aβ(1-40) pair. Detection of native protein in CSF has been verified with correlation to known concentration measured with CE-marked assay.  

​Detection of native Aβ(1-42) in CSF has been more challenging to prove, due to the analyte’s sticky nature and lower levels. 

Conclusions 

• Blood-based biomarkers: Tests like p-Tau, NfL, GFAP and synuclein markers offer potential for diagnosing Alzheimer’s and other neurodegenerative diseases.
• TDP-43 detection: New blood-based extracellular vesicle quantification shows promise for detecting TDP-43 pathology, although challenges remain in replication.
• Simplified testing: Dried blood spots (DROP-AD) via finger-prick are being developed to make testing for Alzheimer’s disease more accessible.
• Early detection: These advancements could enable earlier diagnosis, improving the chances for timely treatment and better outcomes.
• Widespread screening: With simplified methods and accessible testing, there is potential for broader screening and more personalized care for people with neurodegenerative diseases. 

The webinar concluded with an informative Q&A session. Medix Biochemica would like to extend our thanks to our presenters and to all who attended and participated. 

Visit our post-webinar page to: Watch the full webinar, Download the slide deck and Download the Q&A's

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Contact us for more information about our biomarkers for Alzheimer’s disease and other neurodegenerative diseases 

References: 

1. Soluble biomarkers for Alzheimer’s disease webinar August 2024. YouTube. Accessed December 5, 2024. https://www.youtube.com/watch?v=aW8EOGzUf88.