Preclinical development of an immunoassay for the detection of TREM2: a new biomarker for Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau protein. The combination of biomarkers is crucial for AD diagnosis. The triggering receptor expressed on myeloid cells 2 (TREM2), a receptor expressed on microglia, is important in AD pathogenesis. Impairment of TREM2 function aggravates the toxic effects of amyloid plaques, and its activation has been shown to reduce Aβ burden and memory deficits. Increased levels of soluble TREM2 (sTREM2) in blood and cerebrospinal fluid is associated with AD. Therefore, TREM2 could serve as a non-invasive biomarker for AD. In this study, we developed a preclinical immunoassay to detect TREM2 for AD diagnosis. Highly sensitive and specific TREM2 antibodies were produced using the hybridoma technique. The three optimized immunoassays exhibited lower limit of quantitation (LLOQ) of 0.474, 0.807, and 0.415 ng/mL, respectively. These preclinical immunoassays showed high sensitivity and specificity. The sandwich enzyme-linked immunosorbent assay (ELISA) could potentially be used for AD diagnosis.

Keywords: Alzheimer’s disease; Biomarker; Detection; TREM2.

Figure 1

The antiserum titers were determined by indirect ELISA. Serum titers in mice #75, #86, and #66 were tested to measure immune response to TREM2. Serums of preimmune mice were used as negative control. (A) Titer of serum against TREM2 ECD Fc was tested by ELISA. (B) Titer of serum against TREM2 ECD His was tested by ELISA. The X-axis represented the serum dilution from 1/200 to 1/64,000, and the Y-axis represented the optical density at 450 nm. Error bars equaled ± one standard deviation (SD).

Figure 2

SDS-PAGE analysis of TREM2 antibodies. Lane M: protein marker. Lane 1–9: number corresponded to sample name, 3 ug, reduced. Number 1 corresponded to TREM2#46 mAb; Number 2 corresponded to TREM2#53 mAb; Number 3 corresponded to TREM2#57 mAb; Number 4 corresponded to TREM2#60 mAb; Number 5 corresponded to TREM2#63 mAb; Number 6 corresponded to TREM2#65 mAb; Number 7 corresponded to TREM2#69 mAb; Number 8 corresponded to TREM2#75 mAb; Number 9 corresponded to AL002.

Figure 3

The binding affinity of TREM2 mAbs to antigen TREM2. These mAbs were diluted from 10,000 to 0.001 ng/ml. Error bars equaled ± one standard deviation (SD).

Figure 4

Domains of human TREM2 protein.

Figure 5

Affinity and Epitope determination of TREM2 mAbs. (A) Absorbance was measured from double wells; error bars equaled ± one standard deviation (SD). (B) Epitope determination of TREM2 mAbs.

Figure 6

Representative results and a statistical report of TREM2 sandwich ELISA screening. (A-D) Capture antibodies were TREM2#24, #47, #54, #82. A total of 781 antibody pairs were screened, of which 76 antibody pairs showed OD 450 nm values greater than 3.0 ( more information in Supplementary Figure S3). (E) A statistical report detailing the characteristics of the selected antibody pairs was presented. These antibody pairs were methodically categorized by their capture antibodies, which were further subdivided into two distinct clusters based on epitope specificity. The isolated cluster consisted of antibody pairs that featured the capture antibody TREM2 #84, specifically targeting TREM2 (109–138) AA. All remaining antibody pairs recognized TREM2 (130–149) BSA.

Fig. 7

Establishment of TREM2 immunoassays by ELISA. Selected pairs were squared: capture Ab TREM2#24 paired detection Ab TREM2#75, capture Ab TREM2#47 paired detection Ab TREM2#11, capture Ab TREM2#83 paired detection Ab TREM2#21, capture Ab TREM2#85 paired detection Ab TREM2#75, and capture Ab TREM2#91 paired detection Ab TREM2#55. The curve represented the 4PL curve fitting. Error bars equaled ± one standard deviation (SD).

Fig. 8

Standard curves of three immunoassays. Selected pairs: capture Ab TREM2#91 paired detection Ab TREM2#55, capture Ab TREM2#83 paired detection Ab TREM2#21, and capture Ab TREM2#47 paired detection Ab TREM2#11. The binding sites of three pairs were different. Error bars equaled ± one standard deviation (SD).