Surface Assay for Specific Detection of Soluble Amyloid Oligomers

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Surface assay for specific detection of soluble amyloid oligomers utilizing Pronucleon™ peptides instead of antibodies Evgenia G. Matveeva, Jonathan R Moll, Mariam M. Khan, Richard B Thompson, and Richard O Cliff ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00381 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017

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ACS Chemical Neuroscience

Surface Assay for Specific Detection of Soluble Amyloid Oligomers Utilizing Pronucleon™ Peptides instead of Antibodies Evgenia G. Matveeva1*, Jonathan R. Moll*, Mariam M. Khan*, Richard B. Thompson**, and Richard O. Cliff *

*Adlyfe, Inc., 9430 Key West Ave. Rockville, MD 20850, USA **University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, Baltimore, MD 21201, USA

Abstract Immunoassays such as ELISAs are widely used for diagnostics; however, antibodies as detection reagents may have insufficient selectivity and other shortcomings. We present a novel non-antibody based detection method based on binding target molecules to peptides (used as recognition molecules): a surface assay for A-beta oligomers employing a peptide comprising amino acid residues of the human βamyloid protein (Pronucleon™ peptide, PP) as capture agent. For convenience we term this “Pronucleon™ peptide linked immunosorbent assay”, or PLISA. Pronucleon™ peptides are amino acid sequence matched to target amyloids of interest, in particular soluble Aβ-1-42 amyloid protein oligomers, which are widely considered as an early biomarker for Alzheimer’s disease (AD) in body fluids. The Pronucleon™ peptide in PLISA is immobilized on the surface and substitutes for the capture antibody used in ELISA for binding the Aβ-1-42 oligomers present in the sample. We present data comparing

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Corresponding author. Current address: Cellphire, Inc., 9430 Key West Ave, Rockville, MD 20850, USA. E-mail address: [email protected] (Evgenia “Eva” Matveeva)

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synthetic oligomer PLISA assays in spiked buffer and body fluids (such as CSF, brain extracts, or whole blood), to ELISA, and demonstrate better selectivity of the PLISA to amyloid β–42 oligomers versus monomers and fibrils. The detection limit, calculated as the mean (blank) plus three standard deviations, was in the range of 0.35 - 1.5 pM2 (32-135 ng/L). Keywords: Pronucleon™ peptide, soluble amyloid beta oligomers, Alzheimer's disease biomarkers, surface assay, ELISA, PLISA

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Oligomers contained approximately 20 monomers (20-mers) in average - see Materials and Methods for more details.


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Introduction Enzyme-linked immunosorbent assays (ELISAs)3 are widely used tests in clinical practice and other fields. Unfortunately, antibodies have significant shortcomings as detection reagents. For instance, their production requires prior development of immunogens – molecules typically combining target molecule motifs and carrier molecules – and small variations in immunogen structure or antibody production conditions may result into large variations in antibody selectivity, especially in sandwich assays involving at least two types of antibodies. It is also challenging to immobilize antibodies in high yield without activity loss. Finally, antibodies as folded proteins are not stable to modest changes in temperature, pH, and other solution conditions, necessitating special care in their production, storage, and handling. For these reasons, other recognition molecules such as dendrimers, aptamers, peptide nucleic acids, and others (generically described as “receptors” or “ligands”) have been developed for assays of various kinds [1-9]. Peptides are used as one of the types of non-antibody alternative recognition molecules [6-9]. Alzheimer's disease (AD) is the most common neurodegenerative disorder, characterized by progressive memory and cognitive impairment. Alzheimer disease is the leading cause of dementia, affecting more than 30 million people worldwide: the estimated number of people with AD worldwide will be over 100 million by 2050, unless we are able to treat or prevent the disease [10-11]. Of Americans aged 65 and over, 1 in 8 has Alzheimer’s, and nearly half of people aged 85 and older have the disease [12]. It was shown that the amyloid β–peptide ending at residue 42 (Aβ-1-42) is deposited in AD brain first, and is the predominant form in senile plaques. Monomer Aβ is a product of normal metabolism and 3

Abbreviations used: AD, Alzheimer's disease; BSA, Bovine serum albumin; Bt, biotin; DMSO, dimethylsulphoxide; ELISA, enzyme-linked immunosorbent assay; F, fluorescein; FLAG, FLAG® peptide (DYKDDDDK); HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HRP, horseradish peroxidase; oligo, beta-amyloid oligomer; PBA, pyrene butyrate; PLISA, Pronucleon™ peptide linked immunosorbent assay; PP, Pronucleon™ peptide; NP-40, Tergitol; ThT, Thioflavin T; TMB, (3,3',5,5'-tetramethylbenzidine); SA, Streptavidin.


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is not toxic; but as it forms multimeric and polymeric assemblies, it becomes toxic for neuronal cells in vitro and in vivo [13-15]. Soluble oligomers of amyloid β–peptides, in particular Aβ-1–42 oligomers, are considered to be a biomarker for AD in body fluids in serum and cerebrospinal fluid (CSF) [14-23]. High molecular weight Aβ-1–42 oligomers (ca. 80 kD vs 4 kD monomer molecular weight – approximately 20-mers) are elevated in CSF of Alzheimer's patients [22], but total CSF concentrations of all forms of Aβ-1–42 are decreased [24], hence it is very important to develop an assay capable of selectively detecting Aβ-1–42 oligomers and distinguishing them from the non-toxic monomers. It has proven difficult to develop antibodies selective for Aβ-1-42 oligomers, especially in the presence of monomers. There is wide consensus that any AD treatment would be most effective at early stages of the disease [25], making early AD detection particularly desirable as it is for many forms of cancer. We have previously reported the use of small fluorescently-labeled peptides (Pronucleon™ peptides, PP), to detect amyloid proteins in their beta-sheet conformation both in prion disease and in AD [26-29].These peptides undergo a sequence-specific conformational rearrangement in the presence of Abeta aggregates, resulting in a change in their fluorescence properties that can be monitored using standard laboratory instrumentation. We demonstrated specific binding of a variety of PP with slightly different structures (see Table 1) by various assays, including fluorescence anisotropy [28], fluorescence spectroscopy, immunoprecipitation, and ELISA (unpublished data). Here we present a novel surface assay based on PP used as capture agent, “Pronucleon Peptide linked immunosorbent assay” (PLISA). Pronucleon ligands (peptides) are amino acid sequence matched to target amyloids of interest. Ligand sequences are selected based on regions of the target protein known to undergo conformational changes associated with amyloid formation. Ligand sequence selection is the basis for our assay specificity. Pronucleon peptide is immobilized on the surface to substitute for an antibody to specifically capture the Aβ–42 oligomers from the sample; they are then quantified by standard means.


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Table 1.Pronucleon™ peptide structures. ADSequence XXX ADBiotin-KLVFFAEDVGSNKHAIIELMK(ε304 PBA)-NH2 ADPBA-KLVFF AEDVG SNK (biotin)HA 313 IIELM K-(PBA)

Comment (other abbreviation) P22 with biotin P22 with biotin attached by peptide linker #4 at side chain of K13

AD314

PBA-KLVFF AEDVG SNKHA IIELM K(PBA)-GGSGGS-biotin

P22 with biotin attached by peptide linker #3 flexible linker, biotin labeled at C-terminus

AD317

PBA-KLVFF AEDVG SNKHA IIELM K(PBA)-GLVPRGSG-biotin

AD321

PBA-KLVFF AEDVG SNKHA IIELM K(PBA)-PSGSPK(biotin)

P22 with biotin attached by peptide linker #2 thrombin-site linker, biotin labeled at Cterminus P22 with “kinked” linker, biotin labeled at C-terminus

AD324

PBA-KLVFF AEDVG SNKHA IIELM K(PBA)-EAAAK-biotin

AD352

PBA-KLVFF AKDVG SNKHA IIELM K(PBA)-GLVPRGSGK-biotin

AD353

PBA-KLVFF AEDVG SNKGA ISGLM K(PBA)-GLVPRGSGK-biotin

Bt-linker(9)- I32S

AD354

PBA-KLVFF AEDVG SNKHA IIELM K(PBA)rr-GLVPRGSGK-biotin

Bt-linker(9)- p22 + 2xArg

AD356

KLVFF AEDVG SNKHA IIELM QGLVPRGSGK-biotin

AD378

Fluorescein-AhxCLVFFAEDVGSNKGAIIGLMCrr-NH2

ADFluorescein-Ahx-CLVFF AEDVG SNKGA 379 IIGLM Crr-NH2 Bt = biotin; F = fluorescein; PBA = pyrene butyrate

P22 with biotin attached by peptide linker #1 helical linker, biotin labeled at C-terminus Bt-linker(9)- Italian Mutant

Bt-nonpyrenated p22 F-pep11a F-pep11b


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Results and Discussion The PLISA assay may be performed in a variety of formats (Figure 1) similar to ELISA and other immunoassays with the Pronucleon peptide (PP) essentially replacing one or more antibodies. In the examples described here, PP is used instead of capture antibody in a sandwich-format surface assay (Figure 1A and 1B). The PP is bound to the well surface by adding a tag to the PP (such as biotin or a small antigen such as fluorescein) and adding the peptide to wells with a previously immobilized tagcapturing agent (such as streptavidin in case of biotin, or an antibody against the small molecule tag). When a sample containing beta-amyloid oligomer (oligo) is added, PP captures the oligo, and the amount of bound oligo is quantified by subsequent treatment with another (or the same) PP labeled with reporter agent such as an enzyme a different tag (such as FLAG peptide) with subsequent addition of a secondary reporter anti-tag antibody (such as anti-FLAG antibody labeled with HRP) (Figure 1A; data not shown). Alternatively, PP could be used instead of only one antibody (sandwich-format assay), either capture (Figure 1B) or reporter (Figure 1C) antibody. A competitive format surface assay based on PP could also be developed (Figure 1D), with immobilizing oligo on the surface first, for example by using biotinylated oligo immobilized on a streptavidin (SA) coated surface. Next, PP is added and bound to the oligo on the surface; and finally, sample is added and oligo from the sample will compete with oligo immobilized on the surface, and remove some or all of the PP. The amount of the remaining PP will then be determined by either labeled anti-PP antibody, or a tag conjugated to PP itself.


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Figure 1. Various formats to use Pronucleon™ peptide (PP) as a ligand in a PLISA. A) PP is immobilized on a surface (for example via streptavidin-biotin binding) and captures target molecule (betaamyloid oligomer) from the sample; then another PP labeled with a tag is added, which binds to the target and then is quantified by adding anti-tag secondary antibodies labeled with enzyme (HRP) or other label (fluorophore). PP replaces both capture antibodies and reporter antibodies; B) PP is immobilized as in A and captures target; then anti-target antibody is added which is labeled itself (for example with HRP) or can be developed using secondary antibodies. PP replaces capture antibodies; C) antibody is immobilized on a surface and captures target molecule (beta-amyloid oligomer) from the sample; then PP labeled with a tag is added, which binds to the target and then is developed as in A. PP replaces reporter antibodies; D) target analyte is immobilized on the surface, then PP is added and binds to the immobilized target. Next, sample is added and target analyte from the sample competes with immobilized analyte for binding with PP and removes bound PP from the surface. A-C present sandwich assay formats and D presents competitive assay format.

Here we present results from PLISA using PP immobilized on the well surface by binding biotinylated PP to streptavidin attached to the surface, and anti-beta-amyloid antibodies (Ab 6E10) labeled with HRP used as reporter antibody. We compare this sandwich PLISA format with sandwich ELISA using the same HRP-labeled reporter antibody, and 6E10 capture antibody. Different PP's (structures in Table 1) exhibited a range of apparent affinities, with AD-317 and AD-354 among the best,


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with roughly a two-fold range in non-specific binding in the absence of oligo; also, the composition of the diluent and wash had a significant effect on non-specific binding. We tested various diluents such as fish gelatin, SuperBlock blocking buffer, BSA at various concentrations up to 5% for diluents, and various surfactants (such as NP-40, Tween 20, Triton X-100, at various concentration ranges) and salt (0 – 150 mM NaCl) additions for both diluents and wash solutions. Examples of the PLISA dose-response curves are presented in Figure 2 A (fish gelatin) and B (BSA). BSA and fish gelatin give better doseresponse compared to and SuperBlock (data not shown). Peptide AD-317 was tested with both fish gelatin and BSA, and detection limit (determined as the oligo concentration at the signal at the blank plus 3 standard deviations) was 50 pM for the fish gelatin (Figure 2A) and 100 pM for BSA (Figure 2B). However, larger error bars (one SD) and saturation at oligo concentrations above 2 nM were observed for fish gelatin dose-response curve compared to BSA (see AD-317 on Figures 2A and 2B).

Figure 2. PLISA dose-response signal (non-stopped TMB absorbance, Y axis) with various biotinylated PPs (streptavidin-biotin immobilization of PP was used to coat PP on the surface). All PPs were coated at 100 nM concentration. A) Washing solution was 20 mM HEPES pH 7.0 and 0.05 % NP-40. Oligo- PP diluent was 1 % fish gelatin in 0.05 % NP-40, 20 mM HEPES pH 7.0; B)


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washing solution was 20 mM HEPES pH 7.0, 15mM NaCl, 0.05 % NP-40. Oligo- PP diluent was 1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40.

Salt concentrations are important – we found that washing should be with at least 15 mM NaCl, and under 100 mM. Shorter incubation of HRP-conjugates (from 1-2 hours to 30 min) does not improve background but reduces the signal. Direct coating of the PP on the plate surface (by physical absorption) gave essentially no PLISA signal, perhaps due to unfavorable steric conditions of the PP interfering with binding to the large oligo. Figure 3A shows the dose-response PLISA of AD-317 PP coated by physical adsorption (at [PP] = 100 nM) compared with a non-coated plate: only a weak signal is seen at the highest [oligo] = 7.5 nM (patterned bars). Figure 3B shows the same PP coated on the plate by SA-biotin binding at various [PP] between 0 and 250 nM: even at low PP concentration of 10 nM one sees a good dose-response. PP is a hydrophobic molecule, and it should stick well to the plate surface; to check this factor we did a control experiment visualizing the PP itself (at zero oligo concentration) by using 4G8-HRP antibody conjugate instead 6E10-HRP conjugate: 4G8 antibodies (reactive to amino acid residues 17-24 of beta amyloid), recognize both PP and oligo, whereas 6E10 antibodies (reactive to amino acid residue 1-16 of beta amyloid), which do not bind to PP's (Fig. 3C and 3D). Figure 3C shows the 4G8-developed signals for uncoated wells as well as wells coated with PP (AD-317) by physical adsorption; Figure 3D shows corresponding signals for AD-317 coated via SA-biotin binding. These results indicate that AD-317 is actually present on the surface following immobilization by physical adsorption; however, it evidently is incapable of capturing the oligomer.


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Figure 3. PLISA dose-response signal (non-stopped TMB absorbance, Y axis) with capture PP (AD-317) coated directly on the plate by physical adsorption (A; [PP] in coating solution was 100 nM) or indirectly via biotin binding to streptavidin-coated plate (B, at various PP concentrations in coating solution). Washing solution was 20 mM HEPES pH 7.0 and 0.05 % NP-40. Oligo- PP diluent was 1 % BSA in 20 mM HEPES pH 7.0, 0.05 % NP-40. C and D show signal developed by 4G8-HRP antibodies at zero oligo concentration, for direct (C) and non-direct (D) peptide coating.

The effect of varying PP concentration in the coating solution on PLISA dose-response is presented in Figure 4: Figures 4A and 4B show dose-response, and Figure 4C shows PP amount on the surface (developed by 4G8 antibody) at various PP concentrations at coating. The significantly better doseresponse above about 20 nM PP is recapitulated in the 4G8 antibody quantification of the PP on the


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surface. Thus, to achieve good dose-response one need only apply PP at concentrations greater than 1020 nM.

Figure 4. PLISA dose-response signal (non-stopped TMB absorbance, Y ax) with capture PP (AD-317) coated via biotin-SA binding at various PP concentrations in coating solution (A, B). TMB was stopped at 34 min (Figure 4A, peptide range 500 - 0 nM) and at 56 min (Figure 4B, peptide range 150 – 0 nM. Washing solution was 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40; OligoPP diluent was 1 % fish gelatin in 0.05 % NP-40, 20mM HEPES pH 7.0. (C) shows signal developed by 4G8-HRP antibodies at zero oligo concentration at various PP concentrations for peptide range 500 – 0 nM.


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PLISA with a third method of PP immobilization on the surface was also tested; alternative immobilization involved first coating the well surface with anti-fluorescein antibodies, and then treating with a PP labeled with fluorescein, AD-379 (see Table 1). In this case fluorescein (F) was used not as usual (fluorophore), but as a small antigen for PP immobilization on the well surface. Both methods of PP immobilization, either via streptavidin-biotin binding, or via fluorescein - anti-fluorescein binding, give approximately similar dose-response curves with working range up to 50 nM oligo concentration (Figure 5), with better sensitivity for SA-biotin surface binding (Table 2). Also in Figure 5A, it is evident that the presence of cerebrospinal fluid has only modest effects.

Figure 5.PLISA dose-response examples with streptavidin- or anti-fluorescein-based immobilization: A) signal (non-stopped TMB) with capture PP coated by biotin-SA binding (at 100 nM AD-317 coating PP); washing solution was 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40; OligoPP diluent was 1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40 (grey circles). Black circles show the signal at 10 % CSF; B) signal (non-stopped TMB) with capture peptide coated by fluorescein - anti-fluorescein antibody binding (at 50 nM AD-379 coating PP) (grey circles). Washing solution was TBS-TT (25 mM Tris pH 7.6, 75 mM NaCl, 0.5 % Tween-20); Oligo- PP diluent was BBH-TT (1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, and 0.05 % NP-40). White circles show non-specific binding background (in absence of AD-379).


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In order to test the selectivity of PLISA and ELISA, we compared PLISA and ELISA by performing both assays in the same day, by the same person using otherwise identical reagents, plates, and instruments. Both assays were sandwich format assays, and the only difference was the capture agent: for PLISA it was PP (AD-317) coated onto the well surface via SA-biotin binding, and for ELISA it was anti-β-amyloid antibody 6E10. The respective dose-response curves are presented in Figure 6. Working range is approximately up to 50 nM oligo for ELISA (Figure 6), and up to about 100 nM for PLISA (data not shown). ELISA is less sensitive (compared to PLISA) at higher oligo concentrations, and more sensitive at lower oligo concentrations. Detection limits, determined as the oligo concentration from dose-response curve, corresponding to the average blank signal plus 3 SD, are presented in Table 2 and reach 0.35 – 0.75 pM oligo for PLISA, equivalent to 30 ng/mL of oligo. Table 2 demonstrated that detection limits are similar for PLISA and ELISA when same reagents, in particular detection antibodies and labels, and same plates ate used. The best PLISA detection limit is about 15 times higher than ELISA sensitivity of 2.2 ng/mL at blank + 3SD reported in [30].

Figure 6. PLISA versus ELISA dose-response examples: signal (non-stopped TMB absorbance) with capture PP coated by biotin-SA binding (at 100 nM AD-317 coating PP); washing solution was


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20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40; Oligo-PP diluent was 1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40 (grey circles). Black circles show the signal at 10% CSF.

Table 2. Detection limits of PLISA and ELISA. Peptide immobilization AD-317 (100 mM) by biotin-streptavidin binding

Detection limit (PLISA) 10 pM (non-stopped TMB measured at 71 min); 1 pM (stopped TMB)

Detection limit (ELISA)

7.5 pM (stopped TMB)

Experiment conditions (oligomer diluent) 1% BSA in 20mM HEPES pH 7.0, 15 mM NaCl, 0.05% NP-40

AD-317 (100 mM) by biotin-streptavidin binding

75 pM (non-stopped TMB measured at 50 min); 15 pM (stopped 45 pM (stopped TMB) TMB)

10% CSF in 1% BSA in 20mM HEPES pH 7.0, 15mM NaCl, 0.05% NP-40

AD-378 (50 nM) by fluorescein-antifluorescein antibodies binding

10 pM (non-stopped TMB measured at 63 min); 10 pM (stopped TMB)

1% BSA, 1% sucrose in 50mM HEPES pH 7.4, 150 mM NaCl, 0.5% Tween-20

AD-379 (50 nM) by fluorescein-antifluorescein antibodies binding AD-379 (50 nM) by fluorescein-antifluorescein antibodies binding AD-379 (50 nM) by fluorescein-antifluorescein antibodies binding AD-379 (25 nM) by fluorescein-antifluorescein antibodies binding

0.7 pM (stopped TMB measured at 60 min); 1 pM (stopped TMB)

1% BSA, 1% sucrose in 50mM HEPES pH 7.4, 150 mM NaCl, 0.5% Tween-20 1% BSA, 1% sucrose in 50mM HEPES pH 7.4, 150 mM NaCl, 0.5% Tween-20 1% BSA, 1% sucrose in 50mM HEPES pH 7.4, 150 mM NaCl, 0.5% Tween-20 1% BSA, 1% sucrose in 50mM HEPES pH 7.4, 150 mM NaCl, 0.5% Tween-20

0.35 pM (non-stopped TMB measured at 51 min) 1.5 pM (stopped TMB at 45 min)

3 pM (stopped TMB at 45 min)

The PP used in the PLISA gives it significantly better specificity than the ELISA with the canonical 6E10 antibody for recognizing oligomer versus monomer or fibril forms of Abeta 1-42.


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Selectivity data for PLISA versus ELISA for the Aβ-1-42 oligomer, compared to Aβ-1-42 monomer and Aβ-1-42 fibril forms, are presented in Table 3 and demonstrate much less cross reactivity for PLISA for both fibril and monomeric form. PLISA and ELISA (same conditions and reagents except of the peptide replacing capture antibodies) was run using oligomer only, or monomer only (matching to oligomer set of concentrations), or fiber only (matching to oligomer set of concentrations), and then the ratio of the signals was calculated, for example PLISA at 7.5 nM oligomer to PLISA at 150 nM monomer, or PLISA for fiber at weight/volume concentration matching 150 nM monomer to PLISA at 150 nM monomer. Table 3. Specificity of PLISA and ELISA. monomer/oligomer signal in %

fiber / oligomer signal in %

[oligomer],

[monomer],

nM

nM

PLISA

ELISA

PLISA

ELISA

7.5

150

19

80

12

37

2.5

50

6

70

12

40

1.5

30

17

112

N/A

N/A

0.835

16.7

11

60

22

41

0.3

6

14

111

N/A

N/A

0.06

1.2

N/A

113

N/A

N/A

The presence of CSF does not significantly alter the PLISA dose-response curve (Figures 5A, 6) – we can see slight reduction of the signal at 10% CSF. PLISA also works in many brain extracts – we tested human brain TBS, PBS, TBS + Triton X-100, PBS + Triton X-100, and formic acid extracts (all at 10%, spiked with our synthetic oligo). Only formic acid extract showed no signal; all other brain extracts


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showed dose-response, but displaying higher background and reduced signal compared to spiked buffer (data not shown).

Materials and Methods Materials Bovine serum albumin (BSA), Tween-20, Tergitol (NP-40), 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), dimethylsulphoxide (DMSO), and other buffer components such as sucrose and NaCl were purchased from Sigma-Aldrich. PolySorb ELISA 96-well plates were purchased from NUNC. Streptavidin (SA) and One-step Ultra-TMB-ELISA substrate was purchased from Pierce. Pronucleon™ peptides, PP (Adlyfe, AD-XXX, various structures – see Table 1) were prepared by solid-phase 9-fluorenylmethyloxycarbonyl (Fmoc) synthesis by SciLight (Beijing, China). Stock solutions of peptides, at 200 µM in HFIP stored at -80 C were thawed and diluted 10-fold in water and then to desired concentration in appropriate buffer before use. Aβ1-42 monomer peptides were obtained from AnaSpec, Inc. (Fremont, CA, USA). Monomer peptides were dissolved in DMSO at a concentration of 2.5 mg/ml and sonicated for 10 min at room temperature in a Branson 1510 water bath sonicator. Concentration was determined using Pierce 660 nm protein assay (Thermo-Scientific), and the peptide stored in DMSO at a concentration of ~500 µM. Samples were divided into aliquots and stored at -80 C. Thioflavin T (ThT) staining and HPLC was used to confirm that the sample was monomeric. To determine the molecular size of Aβ1−42 monomers or oligomers, samples were chromatographed on two gel filtration HPLC columns in tandem (GE/Pharmacia G75 Superdex 75 10/300 GL and Superose 6 10/300) in PBS mobile phase on an Agilent 1100 HPLC instrument. The elution time of the SDS oligomer was compared to molecular weight standards to determine its average native molecular weight.


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Amyloid Beta Fibrils: Aβ1-42in the fibril form was synthesized by Adlyfe, Inc. [27], and a stock solution (in 10% DMSO in 40 mM sodium phosphate pH 7.4 100 mM NaCl, PBS) at 55 µM monomer equivalent concentration (stored at + 4 C) was diluted in appropriate buffer before use. SDS-derived soluble oligomers (oligo) were produced at Adlyfe, Inc. [28]; for use, lyophilized samples were re-constituted in a small volume of water. The average oligo molecular weight was determined to be ~80 kDa, equivalent to a 20-mer [28] (see HPLC details above). Monoclonal antibodies to human sequence Aβ designated 6E10 (reactive to amino acid residue 116 of beta amyloid), and 4G8 (reactive to amino acid residues 17-24 of beta amyloid), as well as horseradish peroxidase (HRP)-labeled 6E10, and HRP-labeled 4G8 were purchased from Covance (NJ, USA).

Peptide-capture based PLISA surface assay: streptavidin-biotin immobilization Immunoassay plates (96-well) were coated with streptavidin (100 µL/well, 5 µg/ml streptavidin in 50 mM Tris buffer, pH 7.4) overnight at room temperature in a humid chamber, and blocked with blocking buffer (250 µL/well, 0.05 % Tween-20, 1 % BSA, 0.15 M NaCl in 50 mM Tris, pH 7.4) for at least 2 hours at room temperature (or +4 C overnight). Next, plates were washed three times with TBST buffer (300 µL/well, 20 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween-20) and once with distilled water, and incubated with biotinylated peptide (such as AD-317: Table 1) in diluent buffer (100 µL/well, 1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05% NP-40) for 2 hours at room temperature. Then plates were washed again three times with NP-salt wash solution (20 mM HEPES pH 7.0, 10 % PBS, 0.05 % NP-40 (300 µL/well)) and once with distilled water, and incubated with sample (target Aβ oligomer or interfering substance in diluent buffer) for 1-3 hours or overnight at room temperature. Next, plates were washed again with NP-salt wash solution and water (as described above), and the oligomers


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quantified by treatment with labeled antibody (6E10-HRP at 500 ng/mL in diluent buffer, 100 µL/well, incubation for 1 hour at room temperature) with subsequent wash (with NP-salt wash solution and water as described above) and addition of the HRP substrate tetramethylbenzidine (TMB) (100 µL/well). The plate was incubated at room temperature in the dark on a shaker for 15 minutes, and then measured absorbance at 650 nm several times 30, 40-45, and 60 minutes after addition of TMB. If needed the reaction was stopped either when the maximal absorbance signal reached 0.65, or 60 minutes after TMB addition, with 2N sulfuric acid, 50 µL/well) and reading absorbance at 450 nm.

Peptide-capture based PLISA surface assay: anti-fluorescein antibody immobilization Immunoassay plates (96-well) were coated with anti-fluorescein antibodies (100 µL/well, 5 µg/ml in 100 mM NaHCO3 buffer, pH 9) overnight at room temperature in humid chamber, and blocked with blocking buffer (250 µL/well, 0.05 % Tween-20, 1 % BSA, 0.15 M NaCl in 50 mM Tris, pH 7.4) for at least 2 hours at room temperature (or +4 C overnight). Next, plates were washed three times with washing solution (250 µL/well, PBS + 0.05% NP-40), and incubated with peptide conjugated with fluorescein (such as AD-378) in diluent buffer (100 µL/well, 1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40) for 2 hours at room temperature. The plates were washed again three times with washing solution and incubated with sample (target Aβ oligomer or interfering substance in diluent buffer) overnight at room temperature. Next, plates were washed again with washing solution (3 times) and developed with labeled antibody (6E10-HRP at 500 ng/mL in diluent buffer, 100 µL/well, incubation for 1 hour at room temperature) with subsequent wash (3x with washing solution and 1x distilled water) and addition of the TMB substrate (100 µL/well, stopping the reaction with 1-2 N sulfuric acid, 50-100 µL/well).


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Absorption measurements Absorption measurements were recorded using a Tecan Safire2 multi-detection microplate reader, both for the non-stopped and stopped TMB reactions.

Data analysis Each point on the dose-response graphs shown the average of 3 replicates. Error bars show plus and minus one SD. Detection limits for PLISA and ELISA were determined as the oligo concentration at the signal at the blank plus 3 standard deviations.

Conclusions We developed a novel assay format utilizing a non-antibody based detection method: a surface assay based on the highly specific structure-selective binding of Pronucleon™ peptide (PP) (used as a substitute for an antibody), “Pronucleon™ peptide linked immunosorbent assay” (PLISA). PLISA is a structure-selective assay and discriminates well for the Aβ soluble oligomer (early stage Alzheimer’s disease biomarker) state against the Aβ monomeric and fibril forms. PLISA works in body fluids (CSF, brain extracts). The detection limit, calculated as the mean (blank) plus three standard deviations, was approximately 0.35 - 1.5 pM. Abeta 42 molecular weight is 4514, hence the detection limit of 0.35 pM equals to 0.35*20 = 7 pN (in monomer units) and 7*4514= 32000 pg/L, or 32 ng/L. Similarly, 1.5 pM equals to 135 ng/L oligomer. Cross-reactivity of PLISA signal for monomer (monomer/oligomer %) for PLISA varied between 6-19 %, compared to 60-113 % for ELISA (using same reagents and conditions), hence PLISA offers better selectivity for a specific test for oligomer.


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Author Contributions: EGM is the primary author and performed most of the PLISA and ELISA experiments; JRM provided the synthetic chemistry for both the peptide analysis and oligomer preparation; MMK performed the PLISA and ELISA experiments; RBT and ROC provided technical oversight and review in addition to manuscript preparation.

Funding Sources: This research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors. All work under this publication was paid for with private funding obtained by Adlyfe, Inc.

Conflict of Interest: The authors declare the following competing financial interest(s): These authors (EGM, JRM, MMK and ROC) declare competing financial interests. The author RBT declares no competing financial interest.


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Figure 1. Various formats to use Pronucleon™ peptide (PP) as a ligand in a PLISA. A) PP is immobilized on a surface (for example via streptavidin-biotin binding) and captures target molecule (beta-amyloid oligomer) from the sample; then another PP labeled with a tag is added, which binds to the target and then is quantified by adding anti-tag secondary antibodies labeled with enzyme (HRP) or other label (fluorophore). PP replaces both capture antibodies and reporter antibodies; B) PP is immobilized as in A and captures target; then anti-target antibody is added which is labeled itself (for example with HRP) or can be developed using secondary antibodies. PP replaces capture antibodies; C) antibody is immobilized on a surface and captures target molecule (beta-amyloid oligomer) from the sample; then PP labeled with a tag is added, which binds to the target and then is developed as in A. PP replaces reporter antibodies; D) target analyte is immobilized on the surface, then PP is added and binds to the immobilized target. Next, sample is added and target analyte from the sample competes with immobilized analyte for binding with PP and removes bound PP from the surface. A-C present sandwich assay formats and D presents competitive assay format. 233x142mm (150 x 150 DPI)



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Figure 2. PLISA dose-response signal (non-stopped TMB absorbance, Y axis) with various biotinylated PPs (streptavidin-biotin immobilization of PP was used to coat PP on the surface). All PPs were coated at 100 nM concentration. A) Washing solution was 20 mM HEPES pH 7.0 and 0.05 % NP-40. Oligo- PP diluent was 1 % fish gelatin in 0.05 % NP-40, 20 mM HEPES pH 7.0; B) washing solution was 20 mM HEPES pH 7.0, 15mM NaCl, 0.05 % NP-40. Oligo- PP diluent was 1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40. 112x111mm (150 x 150 DPI)


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Figure 2. PLISA dose-response signal (non-stopped TMB absorbance, Y axis) with various biotinylated PPs (streptavidin-biotin immobilization of PP was used to coat PP on the surface). All PPs were coated at 100 nM concentration. A) Washing solution was 20 mM HEPES pH 7.0 and 0.05 % NP-40. Oligo- PP diluent was 1 % fish gelatin in 0.05 % NP-40, 20 mM HEPES pH 7.0; B) washing solution was 20 mM HEPES pH 7.0, 15mM NaCl, 0.05 % NP-40. Oligo- PP diluent was 1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40. 112x111mm (150 x 150 DPI)



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PLISA dose-response signal (non-stopped TMB absorbance, Y axis) with capture PP (AD-317) coated directly on the plate by physical adsorption (A; [PP] in coating solution was 100 nM) or indirectly via biotin binding to streptavidin-coated plate (B, at various PP concentrations in coating solution). Washing solution was 20 mM HEPES pH 7.0 and 0.05 % NP-40. Oligo- PP diluent was 1 % BSA in 20 mM HEPES pH 7.0, 0.05 % NP40. C and D show signal developed by 4G8-HRP antibodies at zero oligo concentration, for direct (C) and non-direct (D) peptide coating. 245x182mm (150 x 150 DPI)


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Figure 4. PLISA dose-response signal (non-stopped TMB absorbance, Y ax) with capture PP (AD-317) coated via biotin-SA binding at various PP concentrations in coating solution (A, B). TMB was stopped at 34 min (Figure 4A, peptide range 500 - 0 nM) and at 56 min (Figure 4B, peptide range 150 – 0 nM. Washing solution was 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40; Oligo- PP diluent was 1 % fish gelatin in 0.05 % NP-40, 20mM HEPES pH 7.0. (C) shows signal developed by 4G8-HRP antibodies at zero oligo concentration at various PP concentrations for peptide range 500 – 0 nM. 213x175mm (150 x 150 DPI)



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Figure 5.PLISA dose-response examples with streptavidin- or anti-fluorescein-based immobilization: A) signal (non-stopped TMB) with capture PP coated by biotin-SA binding (at 100 nM AD-317 coating PP); washing solution was 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40; Oligo-PP diluent was 1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40 (grey circles). Black circles show the signal at 10 % CSF; B) signal (non-stopped TMB) with capture peptide coated by fluorescein - anti-fluorescein antibody binding (at 50 nM AD-379 coating PP) (grey circles). Washing solution was TBS-TT (25 mM Tris pH 7.6, 75 mM NaCl, 0.5 % Tween-20); Oligo- PP diluent was BBH-TT (1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, and 0.05 % NP-40). White circles show non-specific binding background (in absence of AD-379). 211x112mm (150 x 150 DPI)


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Figure 6. PLISA versus ELISA dose-response examples: signal (non-stopped TMB absorbance) with capture PP coated by biotin-SA binding (at 100 nM AD-317 coating PP); washing solution was 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40; Oligo-PP diluent was 1 % BSA in 20 mM HEPES pH 7.0, 15 mM NaCl, 0.05 % NP-40 (grey circles). Black circles show the signal at 10% CSF. 204x94mm (150 x 150 DPI)



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Surface Assay for Specific Detection of Soluble Amyloid Oligomers

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