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Review
. 2013 Jun;59(6):903-16.
doi: 10.1373/clinchem.2013.202937. Epub 2013 Mar 21.

Clinical utility and analytical challenges in measurement of cerebrospinal fluid amyloid-β(1-42) and τ proteins as Alzheimer disease biomarkers

Ju-Hee Kang  1 , Magdalena Korecka, Jon B Toledo, John Q Trojanowski, Leslie M Shaw
Affiliations
Review

Clinical utility and analytical challenges in measurement of cerebrospinal fluid amyloid-β(1-42) and τ proteins as Alzheimer disease biomarkers

Ju-Hee Kang et al. Clin Chem. 2013 Jun.

Abstract

Background: Over the past 2 decades, clinical studies have provided evidence that cerebrospinal fluid (CSF) amyloid β(1-42) (Aβ(1-42)), total τ (t-τ), and τ phosphorylated at Thr181 (p-τ(181)) are reliable biochemical markers of Alzheimer disease (AD) neuropathology.

Content: In this review, we summarize the clinical performance and describe the major challenges for the analytical performance of the most widely used immunoassay platforms [based on ELISA or microbead-based multianalyte profiling (xMAP) technology] for the measurement of CSF AD biomarkers (Aβ(1-42), t-τ, and p-τ(181)). With foundational immunoassay data providing the diagnostic and prognostic values of CSF AD biomarkers, the newly revised criteria for the diagnosis of AD include CSF AD biomarkers for use in research settings. In addition, it has been suggested that the selection of AD patients at the predementia stage by use of CSF AD biomarkers can improve the statistical power of clinical trial design. Owing to the lack of a replenishable and commutable human CSF-based standardized reference material (SRM) and significant differences across different immunoassay platforms, the diagnostic-prognostic cutpoints of CSF AD biomarker concentrations are not universal at this time. These challenges can be effectively met in the future, however, through collaborative ongoing standardization efforts to minimize the sources of analytical variability and to develop reference methods and SRMs.

Summary: Measurements of CSF Aβ(1-42), t-τ, and p-τ(181) with analytically qualified immunoassays reliably reflect the neuropathologic hallmarks of AD in patients at the early predementia stage of the disease and even in presymptomatic patients. Thus these CSF biomarker tests are useful for early diagnosis of AD, prediction of disease progression, and efficient design of drug intervention clinical trials.

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Conflict of interest statement

Authors’ Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the author disclosure form. Disclosures and/or potential conflicts of interest:

Employment or Leadership: None declared.

Consultant or Advisory Role: L.M. Shaw, Innogenetics-Fujirebio through participation in the Alzheimer’s Disease Neuroimaging Initiative.

Stock Ownership: None declared.

Honoraria: L.M. Shaw, Siemens and NIA/NIH Alzheimer’s Disease Neuroimaging Initiative grant U01 AG0249.

Expert Testimony: None declared.

Patents: None declared.

Research Funding: None declared.

Figures

Fig. 1
Fig. 1. Pathogenic process of core proteins (Aβ and τ proteins) associated with the pathogenesis of AD
(A), Full-length APP, a transmembranous protein, can be cleaved by α-,β-, or γ-secretases (sec.) (indicated by small arrows in the left upper panel), which recognize specific sites of Aβ peptide, into nonamyloidogenic or amyloidogenic Aβ peptide fragments, soluble APPs, and intracellular peptide fragments (α- and β-sec.–generated carboxy-terminal fragments or α- and β-CTFs and the γ-secretase–generated APP intracellular domain). β-sec. cleaves at position 1 in the Aβ sequence, whereas α-sec. and γ-sec. have multiple probable cleavage sites around amino acids at position 13–16 or 17–20 and 33–42 of Aβ, respectively. α-sec. cleaves full-length APP (pathway 1) to generate soluble APPα and CTFα. APP is cleaved by β-sec. followed by α-sec. to generate nonamyloidogenic small fragments of Aβ sized from 13 to 16 (Aβ1–13–Aβ1–16), the soluble form of APPβ (sAPPβ), and CTFα (pathway 2). If full-length APP is cleaved by β-sec. and γ-sec. [an enzyme complex consisting of presenilin, nicastrin, PEN2 (presenilin enhancer 2), and APH1 (anterior pharynx defective 1)], amyloidogenic Aβ1–42 and other types of carboxy-terminal truncated Aβ fragments (Aβ1–17, 1–18, 1–19, 1–20, Aβ1–33, 1–34, 1–37, 1–38, 1–39, 1–40), sAPPβ, and APP intracellular domain are generated (pathway 3). The sticky Aβ1–42 is prone to aggregate into various types of toxic oligomer with a buried carboxy-terminus and is a pathologic substrate of amyloid plaque formation with other truncated Aβ peptides, including Aβ1–40. (B), When τ proteins, which normally bind to and stabilize microtubules, are hyperphosphorylated by several serine/threonine kinases [e.g., Cdk5 (cyclin-dependent kinase 5), GSK3 (glycogen synthase kinase 3), and MARK (microtubule-affinity-regulating kinase)], the hyperphosphorylated τ proteins no longer bind microtubules and self-aggregate to form PHFs. Depletion of τ from microtubules results in their instability, thereby leading to impaired axonal transport and synaptic dysfunction. The progressive accumulation of PHFs leads to their aggregation into intracellular NFTs and neuropil threads.
Fig. 2
Fig. 2. Amino acid sequences of epitopes for capture and detector mAbs used in Aβ1–42, t-τ, or p-τ181 measurement by ELISA and xMAP immunoassay platforms
Blue-colored antibody and small globe-tailed red-colored antibody indicate capture antibody and biotinylated detector antibody, respectively. Upper panel: a primary capturing 21F12 mAb in the Innotest kit-ELISA system or a 4D7A3 mAb in the INNO-BIA AlzBio3 kit-xMAP system binds specifically to the C-terminus of Aβx–42 peptides. The biotinylated 3D6 mAb is used in both immunoassay systems as a detector antibody binding to the N-terminus of Aβ1–42. The combination of 21F12–3D6 and 4D7A3–3D6 mAbs specifically quantifies the Aβ1–42 in both platforms without cross-reactivity with Aβ1–40 or truncated Aβ peptides. Red-colored sequences are epitopes for capture and detector antibodies. Middle panel: for CSF t-τ quantification, the AT120 capture mAb binding to the proline-rich domain in the center of all isoforms of human τ (3R/0N, 3R/1N, 4R/0N, 4R/1N, 3R/2N, and 4R/2N; the isoform illustrated in this Fig. is 4R/2N) independent of their phosphorylation is used both in the Innotest-ELISA and the INNO-BIA AlzBio3 kit. Following the binding of biotinylated detection mAbs (HT7 and BT2 for ELISA, HT7 for xMAP), the antigen–antibody complex is detected by a peroxidase-labeled streptavidin (ELISA) or a streptavidin-phycoerythrin fluorochrome (xMAP). The epitope sequence for each mAb is presented as a small black box. Lower panel: to detect p-τ181, the HT7, which binds to all isoforms, is used as a capture (ELISA) or a detector mAb (xMAP), and the AT270, which is specific to τ phosphorylated at the Thr181 position (underlined), is used as a detector (ELISA) or a capture mAb (xMAP). The epitope sequence for each mAb is presented as a numbered small black box.
Fig. 3
Fig. 3. Distribution of sensitivity and specificity reported in clinical studies for discrimination of AD from controls or other non-AD types of dementia and for predicting the progression of MCI to AD
(A), Sensitivity and specificity for the diagnosis of AD. Controls include healthy elderly study participants free of dementia or controls with nonneurodegenerative disorders free of neurodegenerative disease. (B), Sensitivity and specificity for differential diagnosis of AD versus other non-AD type dementias, including FTLD, DLB, VaD, and/or PDD. (C), Sensitivity for patients with MCI whose disease progresses to AD (pMCI) and specificity for MCI patients with stable cognitive function (sMCI). Three Comb: Aβ1–42 + t-τ + p-τ181. Dotted lines indicate 85% of sensitivity and specificity. We identified studies through a systematic search in PubMed with search terms of “cerebrospinal fluid,” “biomarker,” “Alzheimer disease,” “sensitivity,” and “specificity” and from the references of retrieved studies (see the Supplemental References for these selected references in the Data Supplement that accompanies the online version of this report at http://www.clinchem.org/content/vol59/issue6). It should be noted that the disease severity of AD and the follow-up periods of MCI patients across the studies documenting sensitivity and specificity of CSF AD biomarkers are variable.
Fig. 4
Fig. 4. Flow chart for qualification and standardization of CSF AD biomarker measurement and multidisciplinary efforts for future clinical applications
Boxes shaded white, gray, or pink indicate processes have been accomplished to a large degree, are underway and ongoing, or need to be accomplished, respectively. Blue boxes indicate principal entities or partners who have responsibility or have a major role in each indicated activity. Several approaches for the modeling of combined biomarkers are emerging, although additional studies supporting the clinical utility of combined biomarkers are required. The Coalition Against Major Diseases (CAMD) (www.c-path.org/camd.cfm) is making a major collaborative effort in support of the qualification by the US Food and Drug Administration (FDA) of CSF AD biomarkers for use as an enrichment tool in clinical trials. WW-ADNI, World-Wide Alzheimer’s Disease Neuroimaging Initiative (www.adni-info.org); Alz Assn, Alzheimer’s Association (www.Alz.org); PPMI, Parkinson’s Progression Markers Initiative (www.ppmi-info.org).
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