Protocol for using an ELISA to detect total α-synuclein levels in Drosophila melanogaster lines expressing human α-synuclein point mutations

Summary

In Parkinson’s disease (PD), alpha-synuclein (α-syn) aggregation causes neuronal dysfunction and death, particularly of dopaminergic neurons, which is central to PD symptoms and progression. Here, we present a protocol for using a sandwich ELISA to quantify total α-syn levels in various Drosophila melanogaster genotypes expressing human α-syn point mutations associated with PD. We describe steps for aging flies, collecting fly heads, and preparing samples. We then detail procedures for ELISA setup and microplate reader analysis.

Graphical abstract

Highlights

• •Quantification of human α-synuclein in a Drosophila model with pathogenic mutations
• •Steps for Drosophila crosses to obtain the desired genotypes and sample processing
• •Guidelines for ELISA, data analysis, and interpretation

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

In Parkinson’s disease (PD), alpha-synuclein (α-syn) aggregation causes neuronal dysfunction and death, particularly of dopaminergic neurons, which is central to PD symptoms and progression. Here, we present a protocol for using a sandwich ELISA to quantify total α-syn levels in various Drosophila melanogaster genotypes expressing human α-syn point mutations associated with PD. We describe steps for aging flies, homogenization, collecting fly heads, and preparing samples. We then detail procedures for ELISA setup and microplate reader analysis.

Before you begin

α-synuclein is a neuronal protein that aggregates under pathological conditions to form inclusions called Lewy bodies.^1,2 These aggregates define a group of neurodegenerative disorders, including PD, collectively known as synucleinopathies. In PD, accumulation of α-synuclein contributes to motor and non-motor symptoms such as resting tremor, gait disturbances, cognitive decline, depression, and reduced quality of life.^3,4,5

Several pathogenic point mutations have been identified in human α-syn such as A30P, E46K, H50Q, G51D, A53E and A53T.^6,7,8,9 In this study, we compare flies expressing three pathogenic α-syn variants (E46K, G51D, and A53T) with flies expressing wild-type α-syn. Using the ELISA assay kit, we quantified total human α-syn levels across each genotype. This ELISA assay kit was previously used in an in vivo drug-screening experiment in Drosophila designed to identify inhibitors of α-syn aggregation (Sciortino and Banerjee, unpublished data) and reliably detected changes in α-syn levels between untreated controls and drug-treated groups.

Innovation

Existing methods for measuring α-syn levels, such as western blotting and immunohistochemistry, rely on relative signal intensity, lack precise quantification and require normalization to loading controls. Subtle yet biologically meaningful changes may be missed as signal intensity does not always scale linearly with protein concentration. In addition, differences in antibody quality, exposure times, staining procedures and imaging parameters across experiments or laboratories can introduce inconsistency thereby limiting reproducibility and statistical rigor.

This protocol introduces a sensitive ELISA-based assay that quantifies total human α-syn using a capture antibody and HRP-conjugated detection antibody. While the commercial ELISA kit used here is validated for mammalian brain homogenates and cell or tissue lysates prepared in RIPA lysis buffer, this protocol adapts the assay for Drosophila melanogaster head lysates prepared in 1% NP-40 buffer. Due to differences in detergent composition and extraction conditions between RIPA and NP-40 buffers, optimization of homogenization volume, dilution factors, and sample preparation steps were required. This modification extends the applicability of the assay to smaller volume samples in addition to analyzing invertebrate models.

The assay also enables detection in the picogram range, reduces sample requirements, and improves quantitative accuracy, sensitivity, and reproducibility compared to traditional approaches. Additionally, the plate-based format supports scalable, high-throughput analysis, providing a key methodological advantage for quantitative α-syn measurement.

Generating Drosophila stocks

Timing: A minimum of 2 weeks

• 1.
  Obtain Drosophila stocks from the Bloomington Drosophila Stock Center, Indiana University, Bloomington: https://bdsc.indiana.edu/index.html.

  Note: A fly stock containing an elav-GAL4 driver on the X chromosome (stock 458), in which the neuronal elav promoter drives broad GAL4 expression in post-mitotic neurons.

  Note: A fly stock containing the UAS-hSNCA construct on the third chromosome (stock 8146), which expresses wild-type human α-syn (SNCA) under UAS control.

  Note: A fly stock containing the UAS-hSNCA.E46K construct on the second chromosome (stock 80043), which expresses human SNCA with familial Parkinson’s disease amino acid change, E46K, under UAS control.

  Note: A fly stock containing the UAS-hSNCA.A53T construct on the second chromosome (stock 95241) which expresses human SNCA with familial Parkinson’s disease amino acid change, A53T, under UAS control.

  Note: A fly stock containing the UAS-hSNCA.G51D construct on the second chromosome (stock 80044), which expresses human SNCA with familial Parkinson’s disease amino acid change, G51D, under UAS control.

  Note: The UAS-hSNCA.A53T (stock 95241) and UAS-hSNCA.G51D (stock 80044) stocks may contain flies carrying the CyO balancer chromosome, which produces a curly-wing phenotype. Balancer chromosomes are specially engineered chromosomes with multiple nested inversions that suppress recombination during meiosis and carry dominant visible markers such as curly wings. Most balancer chromosomes are lethal when homozygous.

  • a.
    Maintain stocks in vials with 10 ml of standard fly food.
• 2.
  Set individual crosses with the elav-Gal4 virgin females and males from the UAS fly lines described above.

  • a.
    Place 20–25 CO₂-anesthetized virgin females from the elav-Gal4 stock in standard food vials with 10 anesthetized males from the UAS stock.

    Note: Collecting female flies within 6–8 h of eclosion ensures that they have not been fertilized. For assistance in distinguishing males from females, we recommend Atlas of Drosophila Morphology: Wild-type and Classical Mutants.¹⁰

  • b.
    Transfer the parent crosses to fresh food vials daily for 5 days.

    • i.
      First, gently tap the flies on a mouse pad to collect the flies at the bottom of the vial.
    • ii.
      Quickly remove the flug and invert the original vial over a new food vial.
    • iii.
      Seal the new vial with a fresh flug.
  • c.
    Keep the original vials with eggs, larvae and pupa from the F1 generation.

    Note: Adult flies typically begin to eclose from their pupal cases approximately 12 days after mating. The parent crosses should be discarded within 10 days to prevent inter-generation contamination.

  • d.
    Begin collecting flies from the F1 progeny from the day of eclosion (day 0) in new food vials. Collect both males and female flies of the desired genotypes (Figure 1).

    Note: If any of the UAS parent lines carry the CyO balancer chromosome, collect only the non-curly-winged progeny. These flies will have the correct genotype, carrying both the UAS transgene and the GAL4 driver (Figure 1).

Figure 1.

A crossing scheme of Drosophila melanogaster lines to produce F1 progeny

Cross between the GAL4 driver line with the UAS transgenic line that results into the F1 progeny carrying both the GAL4 driver and the UAS construct.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Anti-Alpha-synuclein antibody	abcam	ab138501
Anti-GAPDH Monoclonal Antibody	Invitrogen	MA5-15738
LI-COR BIOTECH LLC IRDye 800 CW Donkey anti-Mouse IgG Secondary Antibody (0.5 mg)	Fisher Scientific	NC9744100
LI-COR BIOTECH LLC IRDye 800 CW Donkey anti-Rabbit IgG Secondary Antibody (0.5 mg)	Fisher Scientific	NC0250902
Chemicals, peptides, and recombinant proteins
NP-40	Fisher Scientific	9016-45-9
NaCl	Fisher Scientific	7647-14-5
HEPES	Millipore Sigma	7365-45-9
MgCl2	Millipore Sigma	7791-18-6
CaCl2	Fisher Scientific	10035-04-8
HPLC H2O	Fisher Scientific	7732-18-5
Roche cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail	Millipore Sigma	11836170001
Critical commercial assays
Sensolyte Anti- α-Synuclein (Human) ELISA kit	Anaspec	AS-55550-H
Experimental models: Organisms/strains
P{w[+mW.hs]=GawB}elav[C155]	Bloomington Stocks	458
w[∗]; P{w[+mC]=UAS-Hsap\SNCA.F}5 B	Bloomington Stocks	8146
w[∗]; P{w[+mC]=UAS-hSNCA.E46K}2	Bloomington Stocks	80043
w[∗]; P{w[+mC]=UAS-SNCA.A53T.U}2	Bloomington Stocks	95241
w[∗];P {w[+mC]=UAS-hSNCA.G51D}2	Bloomington Stocks	80044
w[1118]	Bloomington Stocks	5905
Software and algorithms
Prism v.10	GraphPad	graphpad.com
Microsoft Excel (version 16.89.1)	Microsoft Corporation	www.microsoft.com
SoftMax v. 7.1.2	SoftMax	Moleculardevices.com
Image Studio v. 6.1	LICORbio	www.licorbio.com/image-studio
Other
Narrow Plastic Fly Food Vials	Archon Scientific	D20101- CASE
Flugs for Narrow Plastic Vials	Genesee Scientific	49-102
Square-bottom Fly Food Bottles	Archon Scientific	D20601- CASE
Flugs for Stock Bottles	Genesee Scientific	49-100
CO2 Anesthetizing Pad	Genesee Scientific	59-114
Stemi 355 microscope	Zeiss	435066-9530-000
Flypad Frame	Genesee Scientific	59-118
Flystuff Blowgun	Genesee Scientific	54-104
FlyStuff Foot Pedal Complete System with Standard Flypad	Genesee Scientific	59-121C
Flystuff Drosophila Trays and Dividers	Genesee Scientific	32-122
Gas Regulator	Agilent	5183-4644
Carbon Dioxide Cylinder	Linde Gas and Equipment Company	CD50
Scalpel	Fine Science Tools	10003-12
Blade	Fine Science Tools	10011-00
Metal Probe	Fine Science Tools	10140-01
Micro Tissue Grinder (0.2 mL)	DWK Life Sciences	885470-0000
1.7 mL microtubes	Axygen	MCT-175-C
Eppendorf Research Plus 8-channel Multichannel Pipette 120-1200 μL	Pipette.com	ES-8-1200-R
Pipetman L Starter Kit	Gilson	F167370
Reagent Reservoir	Pipette.com	P8050
Insulated Centrifuge	Fisher Scientific	p-8471006
Ice Bucket Round 4 L Blue 1CS	Government Scientific Source	07210123
Kimwipes	Fisher Scientific	06-666
SpectraMax iD3	Molecular Devices	5054747 K
LI-COR ODYSSEY CLx	LI-COR	9140
TGX FastCast Acrylamide Kit, 12%	Bio Rad	1610173
Trans blot RTA mini 0.2 μm Nitrocellulose Transfer kit	Bio Rad	1704270

Materials and equipment

1% NP-40 extraction buffer composition

Reagent	Final concentration	Amount
NP-40	1%	100 μL
NaCl	100 mM	200 μL
HEPES (pH 7.5)	50 mM	500 μL
MgCl2	1 mM	10 μL
CaCl2	1 mM	10 μL
HPLC H2O	N/A	9.18 mL
Protease Inhibitor	N/A	1 tablet
Total	–	10 mL

Protease inhibitor should be stored at 2^oC to 8^oC. All other reagents can be stored at 22°C to 25°C for at least one year.

Step-by-step method details

Aging flies

Timing: 1–21 days

The purpose of this step is to collect and age the flies.

• 1.Collect an equal number of male and female flies resulting from the fly crosses described in step 2 d (Generating Drosophila Stock).
• 2.Maintain flies in food vials, transferring them twice a week into fresh food vials until the flies reach 21 days of age.

Preparation for homogenization

Timing: 10 min

This step ensures the homogenizers are ready for use before samples are introduced.

• 3.Use a separate pestle and glass homogenization tube for each sample in the ELISA assay.

Note: When selecting the pestle and tube, ensure the pestle makes direct contact with the tube to properly crush the fly heads.

• 4.Label each homogenizer with their respective genotype.
• 5.Pipet 40 microliters (μL) of NP-40 extraction buffer into each tube and place the tube and pestle on ice.

Note: When placing the tube on ice, ensure that ice does not enter the tube.

Collecting fly heads

Timing: ∼45 min

The purpose of this step is to isolate fly heads of desired genotypes.

• 6.Take out the ELISA kit from the 4°C refrigerator and allow it to reach 23°C before use.
• 7.Anesthetize 3 male and 3 female flies of desired genotype per sample (n) on the CO₂ fly pad.
• 8.Use a scalpel to cut the fly heads from the respective genotypes.

Note: While cutting the fly heads, do not damage the fly eyes.

• 9.Transfer the fly heads into the chilled NP-40 extraction buffer using a probe.

Note: When placing the heads into their respective tubes, ensure they are immersed in the NP-40 extraction buffer.

• 10.Repeat steps 8–10 for collecting samples from each genotype.

Sample preparation

Timing: 1 h

The purpose of this step is to prepare cell lysates for downstream analyses.

• 11.Using a Kimwipe, remove any condensation on the pestle. Crush the fly heads to ensure effective cell and tissue disruption during homogenization.

Note: When crushing fly heads, make sure every head is homogenized to avoid inconsistencies between samples.

• 12.Turn on refrigerated centrifuge and set to 4°C
• 13.
  Let samples sit for 10 min on ice.

  • a.
    Transfer 40 μL of each sample into a clean Eppendorf tube.
  • b.
    Place samples in the centrifuge set at 4°C.
  • c.
    Centrifuge at 18,400 × g for 15 min.
• 14.After centrifugation, pipet 30 μL of sample, without disturbing the pellet, into new Eppendorf tubes labeled with the respective genotypes and keep on ice.

Note: If the pellet is disturbed, it is best to repeat the centrifugation step for 3–4 min.

ELISA day one

Timing: 1.5 h

The purpose of this step is to prepare the necessary components and incubate the samples and standards with the provided detection antibodies.

Note: Prepare additional volume of all buffers/solutions to account for extra wells and pipetting loss.

• 15.
  Pipet 25 μL of sample into an Eppendorf tube labeled with the respective point mutation.

  • a.
    Add 75 μL of NP-40 extraction buffer to prepare a master dilution stock for each sample.
• 16.Mix each master dilution stock by inverting, an spin down before use.
• 17.Combine 498.75 μL of component C (wash buffer provided in the kit) along with 1.25 μL of the master dilution stock of each sample in a labeled Eppendorf tube to prepare the sample solution for each point mutation (Table 1).
• 18.Invert and spin down each sample to ensure proper distribution.
• 19.
  Suspend Component B in 1 milliliter (mL) of Component C at a concentration of 100,000 picograms (pg) per mL (Table 1).

  • a.
    Invert tube gently several times, and let it sit at 23°C for 10–15 min.

Note: After adding 1 mL of Component C, use Component B to prepare the standards outlined in step 21 within 1 h, and then apply to ELISA plate outlined in step 23.

• 20.Using serial dilution, prepare 9 standards for the ELISA assay. Standard 1 (10,000 pg/mL), standard 2 (500 pg/mL), standard 3 (250 pg/mL), standard 4 (125 pg/mL), standard 5 (62.5 pg/mL), standard 6 (31.25 pg/mL), standard 7 (15.625 pg/mL) and standard 8 (7.8125 pg/mL).

Note: Only standards 2–8 should be used for the ELISA assay, as the protocol is optimized for the sample concentrations within this range. A successful standard curve should yield an R² value ≥ 0.95. If this criterion is not met, repeat the assay using freshly prepared, high-quality standards.

• 21.
  Arrange and label the strips of wells (Component A) of the ELISA plate (Table 1).

  • a.
    Reserve 14 wells for the standards, 3–5 wells per sample (n), and 3 wells for the blank readings (See Figure 2).

Note: Although only 300 μL is required to load three replicate wells (100 μL per well), 500 μL of each sample is prepared in step 17 to allow for additional technical replicates or to repeat loading, if needed.

• 22.Add 100 μL of each standard or sample to the designated wells. Load standards in duplicate and samples in triplicate (See Figure 2).
• 23.
  Spin down component G to ensure all contents can be retrieved from the bottom of the tube (Table 1).

  • a.
    Dilute Component G 200-fold with component C.
  • b.
    Add 50 μL of the resulting preparation to each well.
• 24.Cover the ELISA plate with the provided adhesive film and incubate at 4°C for 16 h.

Note: This allows α-syn in the samples to bind to the plate-coated capture antibodies and to simultaneously associate with the HRP-conjugated detection antibody.

Pause point: 16 h

Table 1.

Components of Anaspec ELISA kit

Component A	Anti-α-synuclein 8-well strips
Component B	α-synuclein Standard, human (1 μg)
Component C	1X Sample Dilution Buffer
Component D	10X Wash Buffer
Component E	TMB Color Substrate Solution
Component F	Stop Solution
Component G	Detection Antibody
Component H	Adhesive plate covers

Figure 2.

Representative ELISA plate layout

The figure shows the ELISA plate with the placement of standards (yellow), blank controls (grey), and experimental samples loaded in triplicate for each genotype across the 96-well plate. Figure created using BioRender.com.

ELISA day two

Timing: 1 .75 h

The purpose of this step is to perform washes and add the substrate solution before terminating the reaction.

• 25.Remove the ELISA assay kit components and allow them to reach 23°C
• 26.Prepare the required amount (350 μL per well for six washes) of 1x wash buffer by diluting Component D (10X wash buffer) 1:10 with deionized (DI) water (Table 1).
• 27.Remove and discard adhesive film on the ELISA plate.
• 28.
  Remove all liquid from the ELISA plate by gently blotting it on paper towels.

  • a.
    Wash each well six times with 350 μL of wash buffer.

Note: Allow each wash to sit for 15 s before removing it.

• 29.
  Add 100 μL of Component E (Table 1) into each well.

  • a.
    Incubate at 23°C for 18 min until a blue gradient is observed.

CRITICAL: Cover the ELISA plate during incubation, as Component E is light sensitive.

• 30.Add 50 μL of Component F (stop solution) into each well. A color change from blue to yellow should be observed (Table 1).

CRITICAL: The plate must be read within 20 min of adding Component F to all the wells.

Expected outcomes

The expected outcome of this protocol is the quantification of total human α-syn concentrations (pg/mL) in Drosophila samples using ELISA assay (Figure 3; also refer to Table S1). Flies expressing human α-syn WT serve as a positive control, confirming that the assay reliably detects the α-syn protein. The w¹¹¹⁸ genotype serves as a negative control, as these flies and Drosophila in general, do not express α-syn endogenously. Among the α-syn variants, flies expressing the E46K and A53T mutations are expected to exhibit higher α-syn concentrations compared to α-syn WT and the G51D mutation. In contrast, the G51D mutation is expected to show comparatively lower α-syn levels, suggesting reduced accumulation or stability of this variant. The results from the ELISA assay are further validated by western blotting analysis (Figure S1; Table S2). Together, these findings demonstrate that the ELISA assay can detect differences in α-syn levels in Drosophila carrying various disease-associated mutations in synucleinopathies.

Figure 3.

Quantification of human α-syn concentrations in specified genotypes of Drosophila by ELISA

Total α-syn levels (pg/mL) were measured from the brains of Drosophila expressing human α-syn WT and human α-syn point mutations, G51D, A53T, and E46K pan-neuronally using the elav-Gal4 driver. The w¹¹¹⁸ genotype served as a negative control. Data are presented as mean ± SEM from n = 3 biological replicates per genotype. Statistical comparisons were performed using one-way ANOVA followed by Dunnett’s multiple comparisons test. ns = not significant, α-syn^WT versus α-syn^G51D; ∗∗p=0.0027, α-syn^WT versus α-syn^A53T; ∗∗∗p=0.0004, α-syn^WT versus α-syn^E46K; ∗p=0.0459, α-syn^WT versus w¹¹¹⁸.

Quantification and statistical analysis

• 1.Set the plate reader to measure absorbance at 450 nm, and include a 10s orbital shake before the reading.
• 2.
  Export the data into an Excel spreadsheet.

  • a.
    Calculate the average of the standard replicates.
  • b.
    Subtract the average of the three blank readings from each averaged standard.
• 3.Plot the averaged standard readings (y-axis) against their corresponding standard concentrations (x-axis) to generate the standard curve used to calculate α-syn concentrations in the samples (Figure 4).
• 4.
  Average the sample readings for each n (Table S1).

  • a.
    Subtract the average blank value to correct for background signal.
• 5.Calculate the sample concentration (pg/mL) using the slope and intercept from the standard curve as follows:

$$\left[\alpha -\text{syn}\right]=\frac{\text{average}\text{sample}\text{OD}-\text{intercept}}{\text{slope}}\mathrm{x}1600$$

Note: The total dilution factor of 1600 accounts for the 1:4 NP-40 dilution (step 15a), and the subsequent 1:400 dilution (step 17).

Figure 4.

ELISA standard curve used to determine α-syn concentrations in the Drosophila samples

Known concentrations of α-syn standards (pg/mL) were plotted against absorbance values measured at 450 nm. A linear regression analysis was used to generate an equation for sample concentration calculation (R² = 0.97).

Limitations

This ELISA assay protocol is specialized for detecting total human α-syn levels and cannot be used to detect aggregated forms of α-syn that may exist in pathological conditions such as oligomeric and fibrillar forms. In addition, this protocol is optimized for Drosophila melanogaster and has not been tested with other animal models. The microplate provided in the kit is coated with an antibody raised against the full-length α-syn protein. In our complementary western blot analyses, however, the detection antibody recognizes an epitope corresponding to amino acids 118–123 (VDPDNE). If a sample contains modifications, truncations, mutations, or binding interactions within or near this epitope region, the antibody binding may be compromised.

Troubleshooting

Problem 1

Low signal detection for standards and samples (related to steps 17–19, 23a and 24).

Potential solution

Verify that the 16 h incubation at 4°C is performed correctly, as incorrect incubation time or temperature reduces binding between α-syn, the capture antibody, and the HRP-conjugated detection antibody. Ensure that the detection antibody was diluted correctly according to step 23a (ELISA assay day one). If the signal intensity remains low, increase the amount of starting material to raise the total protein concentration.

Problem 2

High background signal affecting data sets (related to step 28a).

Potential solution

Ensure that all wells are washed 6 times with 1x wash buffer and blotted on paper towels after each wash.

Problem 3

The standard curve R² value is too low (related to step 19, 20 and 22).

Potential solution

Properly vortex and spin down each standard before and after every serial dilution, as incomplete mixing can result in a non-linear standard curve. Ensure Component B is fully dissolved and equilibrated to 23°C for 10–15 min, as incomplete dissolution can lead to inaccuracies during serial dilution.

Resource availability

Lead contact

Further questions and information for reagents and resources should be directed to and will be fulfilled by the lead contact, Swati Banerjee (banerjees@uthscsa.edu).

Technical contact

Technical issues can be directed to the technical contact, Marco Sciortino (sciortino@uthscsa.edu).

Materials availability

This study did not generate new reagents.

Data and code availability

This study did not analyze/generate datasets/code.

Acknowledgments

We thank the Bloomington Drosophila Stock Center, Indiana University, for the Drosophila stocks used in this study. This work was supported by funds from the National Institutes of Health/National Institute of Neurological Disorders and Stroke grant R01NS134867, the Perry and Ruby Stevens Parkinson’s Disease Center of Excellence, and the Long School of Medicine, UT San Antonio Health Science Center.

Author contributions

M.S. developed the protocol, conducted the experiments, analyzed the data, and wrote the original draft of the manuscript. R.V. conducted experiments to validate the findings. H.T. helped with optimizing the protocol and analyzing the data. S.B. conceptualized and supervised the project, provided resources, acquired funding, reviewed and edited the manuscript, and communicated with the journal. All authors contributed to reviewing and editing the manuscript.

Declaration of interests

The authors declare no competing interests.