A novel detection method of cleaved plasma high‐molecular‐weight kininogen reveals its correlation with Alzheimer's pathology and cognitive impairment

Figures

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  Fig. 1. Identification of monoclonal antibody specific for HKi/c (3E8), HKi (2B7), and HKc (4B12). (A) Western blot using commercially available anti-HK antibody (Abcam) under reducing conditions shows purified human HKi and HKc used for animal immunization and antibody screening. HKi is observed at 120 kDa and the HKc light chain fragment at 45 kDa. Representative Western blot is from three independent experiments. The direct ELISA with a fluorescent substrate using purified (B) 3E8 (HKi/c), (C) 2B7 (HKi), and (D) 4B12 (HKc) antibody (0.004–3 μg/mL). HKi and HKc (50 ng/well) were coated on the plate. The antibodies 3E8, 2B7, and 4B12 showed specificity for HKi/c, HKi, and HKc, respectively. Values are presented as mean ± SD of experiments performed in triplicate. Abbreviations: HKi, intact high-molecular-weight kininogen; HKc, cleaved high-molecular-weight kininogen; SD, standard deviation.

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  Fig. 2. HKi (2B7)- and HKc (4B12)-specific antibodies are able to discriminate between HKi and HKc in sandwich ELISAs and pull-down assay. (A) Sandwich ELISA with a fluorescent substrate coated with capture antibody 3E8. Plates were incubated with various ratios of HKi and HKc protein as indicated on the x-axis (0–50 ng/well). 2B7 and 4B12 can discriminate between HKi and HKc under the mixed condition of HKi/c in the sandwich ELISAs. Values are presented as mean ± SD from three independent experiments. (B) Pull-down assay with streptavidin beads. Biotinylated 3E8, 2B7, and 4B12 antibodies were incubated with or without HKi and HKc protein and then pulled down with streptavidin beads. Western blot analysis in representative samples was performed using commercial anti-HK antibody (Abcam), showing the selective binding of 3E8, 2B7, and 4B12 for HKi/c, HKi, and HKc, respectively. HKi and HKc proteins used in the assay (input, 125 ng each/lane) were shown in Fig. 1A (100 ng each/lane). Anti-Aβ antibody was used as a negative control. Replicates are biological replicates (n = 3). Abbreviations: HKi, intact high-molecular-weight kininogen; HKc, cleaved high-molecular-weight kininogen; Aβ,…

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  Fig. 3. Aβ42-induced contact system activation and HK cleavage in human plasma can be quantified by sandwich ELISAs. Plasma from healthy donors 1 and 2 was incubated with buffer (0.08% NH4OH/PBS) or Aβ42 (20 μM) for 60 minutes at 37°C. (A) Western blot using anti-HK antibody (Abcam) showed that Aβ42 induced HKi cleavage in both donors' plasma. Representative Western blot from three independent experiments. (B) HKi and (C) HKc sandwich ELISAs with 1:3000 sample dilution. In both donors, HKi levels were decreased in Aβ42-activated plasma (donor 1: 134.3 ± 5.9 vs. 1.1 ± 3.2, P = .0006; donor 2: 107.5 ± 13.7 vs. 2.9 ± 8.3, P = .0026) accompanied by the increase of HKc levels (donor 1: 9.7 ± 8.6 vs. 68.9 ± 4.0, P = .004; donor 2: 4.6 ± 7.3 vs. 98.6 ± 5.2, P = .0008) compared with those in vehicle-treated plasma. The change in HK levels detected in the ELISAs was consistent with the Western blot profiles. Values are presented as mean ± SD from three independent experiments. Comparisons between groups were performed using the two-tailed paired t-test. **P < .01; ***P < .001. Abbreviations: HKi, intact high-molecular-weight kininogen; HKc, cleaved high-molecular-weight kininogen; Aβ, β-amyloid;…

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  Fig. 4. Sandwich ELISAs revealed changes in HKi and HKc levels in AD patient plasma and a significant correlation to HKi and HKc levels obtained for these samples by Western blot. (A and B) The HKi and HKc ELISAs were performed with 1:3000 sample dilution in human plasma from 9 ND controls and 10 AD patients from University of Kentucky Sanders-Brown Center on Aging. HKc level was significantly increased (P = .0003), accompanied by the decrease of HKi level in AD patients (P = .0004), compared with that in ND controls. Data are presented as vertical scatter plots with medians, and experiments were performed in triplicate. Comparison between groups was performed using the two-tailed Mann-Whitney test. *** P ≤ .001. Each value in these individuals detected in the ELISAs is shown in Supplementary Fig. S1. (C and D) Correlation between ELISA values and Western blot profiles in HKi and HKc. The HKi and HKc ELISA values were normalized with the protein concentration in each plasma sample, and the Western blot profiles of HKi were normalized to transferrin [14]. There were significant correlations between ELISA values and Western blot profiles in HKi (r = 0.86, P < .0001) and HKc (r = −0.88, P <…

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  Fig. 5. Sandwich ELISAs demonstrate that HKi- and HKc-level changes in AD patient plasma correlate with dementia and neuritic plaque scores. The HKi (A, C, and E) and HKc (B, D, and F) ELISAs were performed with 1:3000 sample dilution and then normalized with the protein concentration in each plasma of 9 ND controls and 10 AD patients from University of Kentucky Sanders-Brown Center on Aging. For dementia scores, MMSE (A and B), for which 30 = no dementia and 0 = severe dementia, and CDR (C and D), for which 0 = no dementia and 3 = severe dementia, were used. (E and F) Postmortem CERAD neuritic plaque score of 0 corresponds with ND, (B) corresponds with probable AD, and (C) corresponds with definite AD. HKi and HKc level changes as detected by the ELISAs were significantly correlated with MMSE (HKi: r = 0.52, P = .022; HKc: r = −0.62, P = .005), CDR scores (HKi: r = −0.57, P = .011; HKc: r = 0.65, P = .003), and CERAD (HKi: r = −0.62, P = .005; HKc: r = 0.81, P < .0001). Correlations were analyzed using Pearson's correlation coefficient (r). Abbreviations: CERAD, Consortium to Establish a Registry for Alzheimer's Disease; MMSE, Mini Mental State Examination; CDR, Clinical Dementia…