Highly Multiplexed Immunoassays for the Validation of Anti-GPCR Antibodies

Abstract

Antibodies targeting G protein-coupled receptors (GPCRs) are important tools to study the biology and pharmacology of this important superfamily of cell surface signaling proteins. However, generating anti-GPCR antibodies is challenging, partly due to the high sequence homology of related GPCRs. Immunoassays built on suspension bead arrays (SBAs) can be applied to validate anti-GPCR antibodies for flow-based assay applications by enabling a multi-parallel assessment of on-target and off-target binding. In this chapter, we describe how to immobilize an antibody library to generate SBAs. We further describe how a library of engineered GPCRs can be combined with the multiplexed SBA assay to validate the specificity of the bead-bound anti-GPCR antibodies. The SBA-based approach presented here offers a versatile and robust tool for multiplexed characterization of antibody binding selectivity and off-target interactions, as well as for mapping GPCR epitopes involved in antibody binding.

1. Introduction

Antibodies (Abs) are vital tools in human proteomics. They are used to study protein expression patterns in various body fluids or tissues, protein subcellular localization, splice variants, posttranslational modifications, and protein-protein interactions [1]. However, before any affinity reagent should be applied to a downstream analysis platform, it must be validated in an application-specific manner [2]. One relevant Ab-based multiplexing technology is called the suspension bead array (SBA) immunoassay. The SBA assay is used to study complex mixtures of analytes using color-coded microbeads that can be coupled to individual Abs and then pooled before sample incubation. Decoding of the beads is accomplished in a flow cytometer. The SBA assay enables highly multiplexed detection of up to 500 different analytes in solution and has been robustly applied to profile the proteome of human plasma samples [3]. The multiplexed SBA immunoassay approach allows for a parallel determination of the binding selectivity of Abs by identifying on-target and potential off-target interactions [4, 5].

Within an SBA, different unique bead populations incorporate different ratios of a red and an infrared dye, allowing for up to 500 bead populations of different color code signatures. Mixtures of these beads are used to create arrays in suspension, and their composition can be adjusted individually to address a scientific question in each assay. A flow cytometer analyzes the co-occurrence of the color code and any bead-bound reporter dye to display bead assigned interactions [6].

In the context of investigating the selectivity pattern of anti-G protein-coupled receptor (GPCR) Abs of interest, beads can be coupled with different Abs. Although many anti-GPCR Abs are commercially available, their specificity is generally not well characterized. Their utility in immunocapture assays is not generally reported, especially under conditions where functional receptor conformations are relevant. Developing specific anti-GPCR Abs presents a significant challenge for several reasons: (i) it can be challenging to purify high-quality, functional GPCRs to use as the immunogen, (ii) most of the secondary structure of a typical GPCR is hydrophobic and occluded in the plasma membrane or in a detergent-lipid micelle, (iii) the extracellular domain (ECD) of GPCRs can be poorly immunogenic, (iv) there is high homology among human and mouse GPCRs, and (v) there is high homology between closely related GPCRs such that anti-GPCR Abs tend to have high cross-reactivity [7].

A key challenge for applying the SBA assay to validate anti-GPCR Abs is the need for an extensive and high-quality library of solubilized GPCRs. Addressing this issue requires a plasmid library of GPCRs engineered in such a way to maximize expression, incorporate epitope tags to characterize all the GPCRs in the library in a standardized way, and minimally alter or disrupt the functional integrity of each receptor. Consequently, we have previously engineered a library of 215 Dual Epitope-Tagged (DuET) GPCRs representing all phylogenetic GPCR subfamilies, selecting tags that can be targeted by well-validated monoclonal Abs (mAbs) with high specificity and affinity [8]. Moreover, a miniaturized transfection pipeline performed on cells with a high transfection efficiency was developed, as this enables the generation of hundreds or thousands of solubilized cell-based samples that will comprise the analytes of an SBA assay. We identified and successfully employed a suspension-adapted cell line amenable to transfection to maximize the expression of the ectopically expressed membrane proteins (Fig. 1a).

Fig. 1.

Schematic of workflow for validation of anti-GPCR antibodies by multiplexed suspension bead array assay. (A) Engineered GPCR constructs encoding GPCRs that have N- and C-terminal epitope tags were expressed in high-yield, suspension-adapted Expi293F mammalian cells. The cells expressing the GPCRs were then collected and the membranes were solubilized with dodecyl maltoside (DM) detergent, resulting in heterogeneous mixtures of solubilized membrane proteins including the GPCRs. (B) Abs were coupled to unique color-coded beads and pooled to generate SBAs (1). Aliquots of solubilized cell membrane samples were normalized to total protein concentration and incubated with the SBAs (2). Phycoerythrin (PE)-conjugated anti-1D4 mAb (PE-Ab) was used to detect the GPCRs captured by the Ab-coupled beads (3). The data were collected on a Luminex FlexMap 3D instrument (4). (Adapted from Dahl et al. [8]. Created in BioRender.com)

To address the need for validated anti-GPCR Abs, we focused on anti-GPCR Abs from the Human Protein Atlas (HPA). The HPA adopted a unique systematic pipeline approach to develop approximately 2400 polyclonal Abs (pAbs) for more than 600 GPCRs, using 50–150 amino acid-residue long peptide immunogens to generate pAbs in rabbits [9]. We tested the pAbs against the library of 215 solubilized, epitope-tagged GPCRs described above. We performed an SBA assay screen of 408 anti-GPCR Abs from the HPA and validated 248 of these Abs against 154 GPCR targets [8]. We found that ~61% of the Abs tested were selective for their intended target, ~11% bound off-target GPCRs, and ~28% did not bind any GPCR. The multiplexed nature of the SBA assay enabled us to uncover trends in Ab selectivity that can be exploited in the future for anti-GPCR Ab design.

In the following sections, we describe a procedure for the generation of solubilized cell membranes with overexpressed GPCRs (Fig. 1a) and bead-based arrays of Abs (Fig. 1b). We also describe how the arrays can be used in an assay to assess Ab selectivity for the intended protein target while identifying any off-target binders.

2. Materials

2.1. Preparation of Solubilized GPCRs

1. Tissue culture hood.

2. Tissue culture incubator with humidification and control of CO₂ levels.

3. Tissue culture flask shaker.

4. Expi293F cells (Thermo Fisher).

5. Expi293 Expression Medium (Thermo Fisher).

6. Expifectamine 293 Transfection Kit (Thermo Fisher).

7. 125 mL and 250 mL Erlenmeyer cell culture flasks and 12-well plates (all from Corning).

8. DuET Library GPCR constructs (available from Addgene) and empty vector construct.

9. 1.5 mL and 2 mL Eppendorf tubes.

10. Tabletop centrifuge.

11. Nutator mixing device.

12. Wash buffer: Phosphate-Buffered Saline (PBS) (10× PBS from Thermo Fisher) diluted in Milli-Q water.

13. Solubilization buffer: 50 mM HEPES, 1 mM EDTA, 150 mM NaCl, and 5 mM MgCl₂, pH 7.4, with 1% (w/v) n-dodecyl-β-D-maltoside (DM) and cOmplete Mini Protease Inhibitor.

14. Protein quantification assay (DC Assay kit, Bio-Rad).

15. Liquid nitrogen.

16. −80 °C freezer.

2.2. Coupling of Abs onto Beads

1. Beads: MagPlex^® magnetic microspheres (Luminex Corp).

2. Plates: 96-well half-area flat bottom polystyrene plates (Greiner Bio-One).

3. Plate shaker (Grantbio PHMP-4).

4. Plate magnet (LifeSep, 96F) (see Note 1).

5. Coupling buffer: 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.0; store at +4 °C for up to 1 month and at −20 °C for long term.

6. Activation buffer (1X): 100 mM monobasic sodium phosphate (Sigma), pH 6.2; store at +4 °C for up to 1 month and at −20 °C long term.

7. EDC solution: Prepare aliquots of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in tubes and store at −20 °C. Dissolve in activation buffer at 50 mg/mL concentration directly prior to usage (see Note 2).

8. S-NHS solution: Prepare aliquots of sulfo-N-hydroxysuccinimide (sulfo-NHS) in tubes and store at 4 °C. Dissolve in activation buffer at 50 mg/mL directly prior to usage (see Note 2).

9. Wash buffer: 0.05% (v/v) Tween 20 in 1× PBS, pH 7.4 (PBS-T).

10. Abs to be coupled: The Abs will be diluted to 0.02 µg/µL in 100 µL of the coupling buffer (2 µg of each Ab is used per coupling reaction, that is, per 5 × 10⁵ beads/ID (see Note 3)).

11. Detection Ab solutions for coupling efficiency evaluation: R-phycoerythrin (R-PE)-conjugated anti-species Abs diluted in PBS-T at a concentration of 0.5 µg/mL. For example, anti-mouse IgG- and anti-rabbit IgG-conjugated R-PE (Jackson ImmunoResearch, cat. 115-116-146 and 111-116-144, respectively) (see Note 4).

12. Storage buffer: Prepare 1X storage buffer from a 10× blocking reagent for ELISA (BRE, Roche Diagnostics, stored at −20 °C) by diluting in Milli-Q water and supplementing it with 0.1% (v/v) ProClin 300^™.

2.3. Pooling of Ab-Coupled Beads

1. Plate shaker (Grantbio PHMP-4).

2. Plate magnet (LifeSep).

3. Low-binding 5 mL microcentrifuge tubes.

4. Magnetic tube holder (Dynal, MGC-S).

5. Sonication bath (Branson Ultrasonic Corp.).

6. Wash buffer: 0.05% (v/v) Tween 20 in 1x PBS, pH 7.4 (PBS-T).

7. ProClin 300^™ (Sigma-Aldrich).

8. Storage buffer (same as in Subheading 2.2).

2.4. Coupling Efficiency Test and Assay Procedure

1. Assay plates: 96-well or 384-well half-area flat bottom polystyrene plates.

2. Assay buffer: PBS containing 0.5% polyvinyl alcohol (PVA) (w/v), 0.8% polyvinylpyrrolidone (PVP) (w/v), 0.1% casein (w/v), and 10% purified rabbit IgG (cat. P120–101, Bethyl).

3. Solubilization assay buffer: 50 mM HEPES, 1 mM EDTA, 150 mM NaCl, and 5 mM MgCl₂, pH 7.4, with 0.1% (w/v) DM.

4. Wash buffer: 0.05% (v/v) Tween 20 in 1× PBS, pH 7.4 (PBS-T).

5. Solubilized GPCRs: solubilized GPCRs diluted to a final concentration of 2 µg/µL in assay solubilization buffer.

6. Detection buffer: 1× blocking reagent for ELISA (BRE, Roche Diagnostics) containing 0.1% DM, 0.1% Tween 20, and 10% rabbit IgG.

7. Detection Ab solution: R-PE-conjugated anti-tag Ab, or R-PE-conjugated anti-species Ab for coupling test. Conjugated Abs can be purchased (i.e., Jackson ImmunoResearch) or the Ab (i.e., anti-1D4 mAb) can be conjugated to R-PE with a kit. The detection Ab is diluted in the detection buffer directly before use (see Notes 4 and 5). We use R-phycoerythrin (R-PE)-conjugated Abs directed against the tag of the protein fragments or R-PE-conjugated Abs directed against the species of the Abs coupled to the beads for the coupling test diluted in PBS-T at a concentration of 0.5 µg/mL.

8. Luminex instrument: FlexMap 3D allows for a multiplexing of up to 500 IDs and assay in a 384-well plate.

3. Methods

3.1. Generation of Solubilized, Heterologously Expressed GPCRs

1. Culture and transiently transfect Expi293F cells according to manufacturer’s instructions in Erlenmeyer cell culture flasks. We recommend conducting transfections in 12-well plates in which each well contains 1.25 mL of cells at a concentration of 3 × 10⁶ cells/mL. Each well is transfected with 4 µL of Free-Style MAX Reagent and 0.25 µg of GPCR plasmid DNA, keeping constant the total transfected plasmid DNA at 1.5 µg/well by adding empty vector pcDNA3.1(+).

2. Harvest cells 72 h after transfection by washing cells twice with ice-cold PBS and centrifuging at 100 RCF for 10 min for each wash.

3. Resuspend the cell pellets in solubilization buffer and incubate them for 2 h at +4 °C with nutation. We recommend resuspending the pellets in 250–550 µL of solubilization buffer.

4. Clarify solubilized lysates by centrifugation at 22,000 RCF for 20 min at +4 °C.

5. Transfer solubilized lysates to a microcentrifuge tube and quantify total protein content by Protein DC assay according to the manufacturer’s specifications.

6. Flash-freeze solubilized lysates in liquid nitrogen, ensuring proper safety protocols are adhered to, before storage at −80 °C.

3.2. Coupling of Abs on Beads

In the following, a method for the coupling of Abs onto magnetic beads is described. The coupling is intended to include at least 24 bead IDs in parallel and is therefore performed in microtiter plates. Magnetic plates or tube holders attract and temporarily retain the beads during specific steps. Depending on the future usage of the beads, they can be stored in microtiter plates or pooled and transferred to tubes.

1. Distribute the different bead IDs in desired portions (e.g., 40 µL = 5 × 10⁵ beads/ID) into the wells of a half-area bottom plate, and wash the beads with 80 µL of activation buffer (see Notes 1 and 6).

2. Add 50 µL of activation buffer to each well.

3. Prepare fresh solutions of NHS and EDC in activation buffer. Calculate the use of 0.5 mg of each substance per bead ID for each coupling, and prepare a mixture by combining 10 µL NHS solution, 10 µL EDC solution, and 30 µL of activation buffer per bead ID (i.e., per well) such that there is enough for all wells (see Notes 2 and 7).

4. Distribute 50 µL of the prepared EDC-NHS mixture to each well.

5. Incubate the microtiter plate for 20 min at room temperature in the dark, under permanent and gentle mixing on a shaking table (650 rpm), and wash thereafter three times with 100 µL of coupling buffer/well.

6. Continue by adding 100 µL of the Ab solution to the activated beads without interruption. Then, incubate for 2 h at room temperature in the dark, under permanent and gentle mixing on a shaking table (650 rpm).

7. Wash the Ab-coupled beads three times with 100 µL of wash buffer/well.

8. Add 50 µL of storage buffer (1×) to each well prior to the bead storage at +4 °C in the dark overnight.

3.3. Bead Mixture Preparation

Theoretically, suspending the starting amount of 5 × 10⁵ beads/ID in 100 µL of storage buffer after the coupling procedure yields a bead concentration of 500 beads/ID per µL. We recommend preparing bead array mixtures where the final bead concentration to be distributed into an assay plate is 100 beads/ID per µL. Thus, a bead array mixture should be prepared so that each bead ID is diluted 1:5 into a final volume enough for an extra 10% in terms of the number of assay wells (see Note 8). The Ab-coupled beads can be stored in the microtiter plate without pooling at +4 °C in the dark for at least 6 months (assuming no evaporation or contamination). The following protocol describes the workflow for pooling the beads to generate an SBA, which can be stored in the dark at +4 °C for up to 6 months under ideal conditions (see above).

1. Centrifugate the microtiter plate(s) containing the Ab-coupled beads at 205 RCF for 1 min.

2. Transfer the entire volume from each well (50 µL) into a LoBind tube on a magnet. Remove excess storage buffer manually as needed.

3. Add 50 µL of storage buffer to each well and repeat the transfer process to maximize bead counts per ID. This wash step can be repeated once or twice. Continue to remove excess storage buffer manually as needed.

4. After the pooling is complete, adjust the final volume of the bead array mixture with 1× storage buffer. Use a magnetic tube holder if any extra storage buffer needs to be removed.

5. Vortex and sonicate the bead array mixture for 30–60 s and store it at +4 °C in the dark until usage.

3.4. Coupling Efficiency Test

The presence of Abs immobilized on beads can be confirmed after the coupling if they have been produced in a species for which R-PE-conjugated affinity reagents are available (see Subheading 2.4, item 7). High output signals (measured as arbitrary units of median fluorescence intensity (MFI)), typically of at least 1000 MFI, or at least sixfold higher than background, indicate a successful coupling.

1. Distribute 5 µL of bead mixture to each well of an assay plate (see Note 9). It is recommended to test the bead mixture in duplicate or triplicate for each species-specific R-PE detection Ab.

2. Prepare the detection Ab solutions and add 45 µL of these solutions to the wells with bead mixture (see Note 10).

3. Incubate for 30 min at room temperature under permanent and gentle mixing on a shaking table (650 rpm).

4. Wash the assay plate three times with 100 µL of wash buffer/well.

5. Add a final 100 µL of the wash buffer to each well before the plates are analyzed with the Luminex instrumentation.

6. Set the software protocol to count all bead IDs used in the coupling over a 60-s time-out interval in a volume of 100 µL (see Note 11).

3.5. Assay Procedure

The protocol is for a 384-well plate or a 96-well half-volume assay plate.

1. Distribute 5 µL of bead mixture to each well of an assay plate (see Notes 8, 9, and 10).

2. Distribute 32.5 µL of assay buffer to each well of an assay plate.

3. Thaw the solubilized GPCR samples on ice. Prepare 2 µg/µL dilutions of each sample in solubilization assay buffer such that the total volume prepared is at least 10% more than necessary.

4. To each well in the assay plate (already containing the bead mixture and assay buffer), dispense 12.5 µL of diluted solubilized GPCR sample.

5. Gently vortex the assay plate and centrifugate for 1 min at 205 RCF.

6. Incubate overnight at +4 °C in the dark.

7. Wash 6× with 60 µL wash buffer/well.

8. Add 50 µL of the detection Ab solution and vortex the assay plate.

9. Incubate for 1 h at +4 °C in the dark.

10. Wash 3× with 60 µL wash buffer/well, and add a final 60 µL of the wash buffer to each well before the plates are analyzed with the Luminex FlexMap 3D instrument.

11. Select the utilized bead IDs in the Luminex instrument software and count at least 100 beads per ID. We suggest using the MFI for further data processing.

12. Analytes with a bead count above or equal to 35 counts are deemed reliable. If the MFI recorded for the Ab targeting the overexpressed GPCR in the sample surpasses a defined threshold above the background signal (representing samples lacking the intended GPCR), with no signals surpassing the threshold from any other GPCR-containing sample, the Ab is identified as selective for its intended GPCR target. Data normalization, centering, and/or transformation can be applied as necessary.