Detection of Amylin-β-amyloid Hetero-Oligomers by Enzyme-Linked Immunosorbent Assay

Materials and reagents

Biological materials

1. Mouse anti-human-Aβ_(1-16) antibody (BioLegend, catalog number: 803002)

2. Rabbit anti-amylin P2 antibody (not commercially available, see [23])

3. Mouse anti-human-total-Aβ (BioLegend, catalog number: 800720)

4. Aβ40 synthetic peptide (Anaspec, catalog number: AS-24235)

Reagents

1. ELISA assay diluent (5×) (BioLegend, catalog number: 421203)

2. Dimethyl sulfoxide (DMSO) (Sigma, catalog number: D8418)

3. Na₂CO₃ (Sigma, catalog number: S7795)

4. NaHCO₃ (Sigma, catalog number: S5761)

5. 10× PBS (Corning, catalog number: 46-013-CM)

6. 10% Tween-20 (Bio-Rad, catalog number: 1610781)

7. Aβ₄₀ synthetic peptide (Anaspec, catalog number: AS-24235)

8. 3,3',5,5' tetramethylbenzidine (TMB) substrate (Thermo Scientific, catalog number: 34028)

9. Stop solution (Thermo Scientific, catalog number: N600)

Solutions

1. Bicarbonate buffer (see Recipes)

2. PBST washing buffer (see Recipes)

Recipes

1. Bicarbonate buffer (250 mL)

Note: Adjust the solution’s pH to 9.6. Use distilled water as diluent.

2. PBST washing buffer (500 mL)

Note: Dilute 50 mL of 10× PBS to 1× using 450 mL of distilled water as diluent before preparing PBST washing buffer.

Laboratory supplies

1. 96-well clear flat-bottom polystyrene high bind microplate (Corning, catalog number: 9018)

2. Sealing film, polyester (VWR, catalog number: 89134-430)

3. Absorbent paper towels (Pacific Blue Basic, catalog number: 23504)

4. epT.I.P.S. standard 20–300 μL pipette tips (Eppendorf, catalog number: 022492047)

5. 1.5 mL standard line microcentrifuge tubes (VWR, catalog number:10025-726)

6. 50 mL reagent reservoirs (Corning, catalog number: 4870)

7. 8-channel 30–300 μL multichannel pipette (USA Scientific, catalog number: 7108-3300)

Equipment

1. xMark microplate absorbance spectrophotometer (Bio-Rad, catalog number: 1681150)

Software and datasets

1. Microsoft Excel (2019)

2. Microplate Manager Software 6 (MPM) (Bio-Rad, v6.3)

Procedure

This protocol has been validated using frozen and freshly prepared samples. The homogenization buffers and protocol used previously are described in [23]. Preparation of samples in other buffers or using other homogenization methods may be possible so long as the conditions do not disrupt amylin-Aβ binding interactions.

Unless otherwise noted, bring all reagents to room temperature prior to use. Determine ELISA plate layout/template prior to use; we recommend using the first two columns (16 wells) for the standard curve (8 standards in duplicate). Dilute 5× assay diluent to 1× final concentration using 1× PBS prior to use. A multichannel pipette is used for the addition of all solutions, except the standards/controls, which are added individually. A graphical summary of the protocol is shown in Figure 1.

Figure 1. Graphical summary of ELISA protocol. General overview of the present ELISA protocol, highlighting key steps. Note that after the addition of the stop solution, the plate must be read in a spectrophotometer, which is not depicted in this summary figure. Data analysis steps are not shown.

A. Coating with capture antibodies

1. Dilute the mouse anti-human-Aβ_(1-16) antibody to a final dilution of 1:400 using bicarbonate buffer and coat the wells of the 96-well plate to be used for the standards with 100 μL.

2. Dilute the rabbit anti-amylin P2 antibody to a final dilution of 1:400 using bicarbonate buffer and coat all sample wells of the plate with 100 μL.

3. Seal the plate with sealing film and incubate overnight at 4 °C without shaking.

B. Loading standards and samples

1. Prepare the standards.

a. Resuspend the Aβ₄₀ peptide in a sufficient amount of DMSO to prepare the 1 ng/mL stock solution.

b. Serial dilute the stock in 1× assay diluent to create the following concentrations (in ng/mL): 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1562. Use assay diluent buffer as a blank (“0”) standard.

2. Remove the plate sealer and decant antibody solutions by gently shaking the plate (upside down) over a sink. Remove any residual solution by gently tapping the plate on an absorbent paper towel.

a. When tapping the plate, tap hard enough that any residual solution is removed from the wells and the wells are dry.

b. Repeat this method of washing/tapping for all following solution decanting steps.

3. Wash the plate with 300 μL of PBST washing buffer per well. Remove any remaining wash buffer by tapping the plate on an absorbent paper towel, as above. An automatic plate washer may also be used if available.

4. Add 300 μL of 1× assay diluent to each well and incubate for 1 h at room temperature.

5. Decant blocking solution and remove any residual solution as described above.

6. Wash the plate with 300 μL of PBST washing buffer per well 1–2 times. Decant and tap after each wash, as described above.

7. Add 100 μL of the Aβ₄₀ peptide standard to the appropriate wells (coated with the anti-human-Aβ_(1-16) antibody only). Include 100 μL of 1× assay diluent as the “0” standard/blank, as described above.

8. Dilute samples to 1:2 final dilution using 1× PBS for a final volume of 200 μL (100 μL per well, in duplicate). Plate 100 μL of diluted sample to the appropriate wells (coated with the anti-amylin P2 antibody).

9. Add 100 μL of 1× assay diluent as a blank for the samples (for wells originally coated with the P2 antibody).

10. Seal the plate and incubate overnight at 4 °C.

C. Developing the assay and data collection

1. Prepare the HRP-conjugated anti-human-total-Aβ detection antibody by diluting to a final dilution of 1:400 in 1× assay diluent.

2. Remove the plate sealer, decant the standards and samples, and remove any remaining solution, as above.

3. Wash the plate with 300 μL of PBST washing buffer per well 3 times. Decant and tap after each wash, as above.

4. Add 100 μL of prepared detection antibody per well and incubate at room temperature for 1 h.

a. It is recommended to remove the TMB from 4 °C at this time, to allow it time to acclimate to room temperature before use.

5. Decant the antibody and remove the residual solution as above.

6. Wash the plate with 300 μL of PBST washing buffer 3–4 times with decanting and tapping.

7. Add 100 μL of TMB substrate (at room temperature) per well. Incubate for approximately 30 min until standard wells show gradation and sample wells show a signal. Check the plate after 20 min of incubation and then every few minutes to prevent oversaturation of signal intensity.

8. Add 50 μL of stop solution per well in the exact same order in which the substrate was added.

9. Read the plate at 450 nm in a spectrophotometer.

Data analysis

Following data acquisition using MPM6 software, data are exported to Excel for further analysis. In each experiment, samples should be run in duplicate or triplicate for technical replicates. Average the values for all duplicate/triplicate wells. Subtract the average absorbance value of the standard blank wells (the wells with only 1× assay diluent coated with anti-human-Aβ_(1-16) antibody in step A1) from all standard wells. Subtract the average absorbance value of the sample blank (the wells with only 1× assay diluent coated with anti-amylin P2 antibody in step A1) from all sample wells to remove background and non-specific signal. Create a standard curve using the absorbance values vs. standard concentrations and generate the linear regression equation. The concentration of samples can then be interpolated from the standard curve regression line. A detailed description of the data analysis method appears in our previous report [23] in the “Experimental procedures” section. An overview of the data analysis method is shown in Figure 2.

Figure 2. Data analysis overview. After exporting the values to Excel, values for duplicate wells are averaged together. For all standard values, the average of the “0” standard is subtracted. For all samples, the “1× assay diluent” average is subtracted. Then, the standard values are used to create a standard curve and generate a linear regression equation, which can be used to calculate sample values.

Validation of protocol

This protocol has been used and validated in the following research article:

• Kotiya et al. [23]. Rapid, scalable assay of amylin-β amyloid co-aggregation in brain tissue and blood. Journal of Biological Chemistry (Figure 1, panels E–F; Figure 2 panels A–E; Figure 3, panels B–E; Figure 4 panels A–C; Figure 6 panels A–E).

General notes and troubleshooting

General notes

1. For convenience, we have prepared an example plate layout (Table S1) showing standards and samples plated in duplicate. We have also included a blank plate layout, which can be filled out with sample identifiers (Table S2).

2. The inherent instability and amyloidogenicity of both amylin and β-amyloid can cause variability between measurements. We strongly recommend that all samples that are to be compared against one another be run on the same assay plate. If necessary, samples can be run as singlets in two separate plates, and the values averaged together.

3. Following the acquisition of concentrations of samples (in ng/mL), we recommend normalizing each value to the respective sample’s total protein concentration to account for individual differences and variances that occur as a result of sample preparation. We recommend reporting final values as “ng amylin/mg total protein” (or similar).

4. Though not required, we recommend wrapping the plate with aluminum foil to prevent light interference and smudging/fingerprinting on the plate’s clear bottom, which would interfere with absorbance readings.

5. Placing the plate on a plate shaker during any incubation step is not recommended; conduct all incubation and washing steps without shaking.

6. When adding solutions, we recommend always adding solutions (wash buffer, TMB, stop solution, etc.) from top to bottom, ensuring duplicate/triplicates receive each solution simultaneously.

7. We recommend initially diluting samples two-fold with 1× PBS, as described in the protocol above. Samples may need to be run with higher (if the signal is too strong) or lower (if the signal is too weak) dilution factors according to amylin-Aβ concentration in the prepared sample.

8. Although Aβ₄₀ does not form amyloid by itself rapidly [24], we recommend aliquoting Aβ₄₀ stock to prevent frequent freeze/thaw cycles, which can impact aggregation formation.

Troubleshooting

Problem 1: Oversaturation of standards or samples.

Possible cause: Insufficient number of washing and/or decanting steps.

Solution: Modify the number of washes during sections B and C and ensure proper decanting to remove excess/residual solution from each well before adding the next solution.

Problem 2: No (or little) gradation between standards.

Possible cause: Multiple freeze/thaw cycles of Aβ standards, incorrect preparation, or dilution.

Solution: Minimize the number of freeze/thaw cycles of the Aβ standards by aliquoting stock peptide. Only thaw the peptide standard when prepared to run the assay. Pipette the standards carefully and ensure no bubbles are present when loading the standards in the wells. If necessary, run repeat experiments with the standard curve plated in triplicate.

Supplementary information

The following supporting information can be downloaded here:

1. Table S1. Example plate layout

2. Table S2. Blank plate layout

Acknowledgments

F.D. acknowledges the following funding sources: National Institutes of Health R01 NS116058, R01 AG057290, R01 AG053999.

This protocol was adapted and modified from Kotiya et al. [23].

Competing interests

The authors declare no competing interests.

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