A guide to selecting high-performing antibodies for Alpha-1-antitrypsin (UniProt ID: P01009) for use in western blot, immunoprecipitation and flow cytometry

Limitations

Inherent limitations are associated with the antibody characterization platform used in this study. Firstly, the YCharOS project focuses on renewable (recombinant and monoclonal) antibodies and does not test all commercially available A1AT antibodies. YCharOS partners provide approximately 80% of all renewable antibodies, but some top-cited polyclonal antibodies may not be available through these partners. We encourage readers to consult vendor documentation to identify the specific antigen each antibody is raised against, where such information is available.

Secondly, the YCharOS effort employs a non-biased approach that is agnostic to the protein for which antibodies have been characterized. The aim is to provide objective data on antibody performance without preconceived notions about how antibodies should perform or the molecular weight that should be observed in western blot. As the authors are not experts in A1AT, only a brief overview of the protein’s function and its relevance in disease is provided. A1AT experts are invited to analyse and interpret observed banding patterns in western blots. Thirdly, YCharOS experiments are not performed in replicates primarily due to the use of multiple antibodies targeting various epitopes. Once a specific antibody is identified, it validates the protein expression of the intended target in the selected cell line, confirms the lack of protein expression in the KO cell line and supports conclusions regarding the specificity of the other antibodies. All experiments are performed using master mixes, and meticulous attention is paid to sample preparation and experimental execution. In instances where antibodies yield no signal, a repeat experiment is conducted following titration. Additionally, our independent data is performed subsequently to the antibody manufacturers internal validation process, therefore making our characterization process a repeat.

Lastly, as comprehensive and standardized procedures are respected, any conclusions remain confined to the experimental conditions and cell line used for this study. The use of a single cell type for evaluating antibody performance poses as a limitation, as factors such as target protein abundance significantly impact results. Additionally, the use of cancer cell lines containing gene mutations poses a potential challenge, as these mutations may be within the epitope coding sequence or other regions of the gene responsible for the intended target. Such alterations can impact the binding affinity of antibodies. This represents an inherent limitation of any approach that employs cancer cell lines.

Antibodies

All A1AT antibodies are listed in Table 2, together with their corresponding Research Resource Identifiers (RRID), to ensure antibodies are cited properly.¹⁷ Secondary antibodies used in this study are provided in Table 3. To ensure consistency with manufacturer recommendations and account for proprietary formulations (where antibody concentrations are not disclosed), antibody usage is reported as dilution ratios rather than absolute concentrations.

Cell culture

All cell lines used in this study are listed in Table 1, alongside their corresponding RRIDs, to ensure proper citation.¹⁸ Cells were cultured in Minimum Essential Medium (MEM) Eagle (Sigma-Aldrich #M0325-500ML) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific #A5256801), 1% sodium pyruvate (Thermo Fisher Scientific #11360070), and 1% antibiotic/antimycotic solution (Capricorn Scientific #AAS-B). All cell lines used in this study were routinely tested for mycoplasma contamination and were confirmed to be mycoplasma-free.

CRISPR/Cas9 genome editing

The Hep G2 SERPINA1 KO clone was generated using low-passage cells. Prior to ribonucleoprotein transfection, 20,000 cells were plated in each well of a 48-well plate and incubated overnight to allow attachment. in vitro guide RNA was generated with the HighYield T7 sgRNA synthesis kit (Jena Bioscience #RNT-105), followed by incubation with DNase I-XT (New England Biolabs #M0570) and quick CIP (New England Biolabs #M0525) to remove DNA and 5′ phosphorylation respectively. RNA was purified using the Monarch spin RNA cleanup kit (New England Biolabs #T2040) and 125ng of guide RNA (target sequence: CACTCACGATGAAATCCTGG) was combined with 625ng of spCas9. Ribonucleoprotein transfection was then carried out using the Lipofectamine CRISPRMAX Cas9 transfection reagent (Thermo Fisher Scientific #CMAX000008) according to the manufacturer’s protocol. The culture medium was replaced the following day, and single-cell isolation was performed on day three. Single cells were plated into 96-well plates and clonally expanded.

Antibody screening by western blot

Hep G2 WT and SERPINA1 KO cells were washed three times in Hanks’ balanced salt solution (HBSS) (Capricorn Scientific #HBSS-2A) and serum deprived for 48-hours in MEM media without phenol red (Thermo Fisher Scientific #51200046) supplemented with 1% sodium pyruvate, 1% antibiotic/antimycotic solution and 1% L-glutamine (Thermo Fisher Scientific #A2916801). Culture medium was then collected and centrifuged for 500 × g, for 10 minutes at 4°C to eliminate cells and larger contaminants, then for 4500 × g, for 10 minutes at 4°C to eliminate smaller contaminants. Conditioned medium was then concentrated by centrifugation at 4000 × g for 30 minutes at 4°C using Amicon Ultra 15mL centrifugal filters with a 3 kDa molecular weight cut off (Sigma Aldrich #UFC900396). Culture media was then supplemented with 1× protease inhibitor cocktail (Cell Signaling Technology #7012).

For lysate preparation, Hep G2 WT and SERPINA1 KO cells were washed three times in phosphate buffered saline (PBS) (Thermo Fisher Scientific #70011044) and lysed in RIPA buffer containing 1× of protease inhibitor cocktail, sodium orthovanadate and phenylmethylsulfonyl fluoride (Santa Cruz Biotechnology #sc-24948). Lysates were sonicated (40% amplitude for 5 seconds) three times and incubated for 30 minutes on ice prior to centrifugation at 20,000 × g for 1 hour at 4°C.

Protein concentration was quantified using the Pierce BCA protein assay (Thermo Fisher Scientific #23225) and 30 μg of protein was used for both protein lysates and concentrated cell culture medium. Samples were combined with Laemmli sample buffer (Bio-Rad #1610747) containing 2-mercaptoethanol (final concentration 355 mM) (Sigma Aldrich #M7522) before being heated at 65°C for 10 minutes. Samples were then loaded in precast 4-20% WedgeWell Tris-Glycine Plus midi gels (Thermo Fisher Scientific #WTG42020BOX) alongside Prime-Step prestained broad range protein ladder (BioLegend #773302). SDS-PAGE was then performed in SureLock Tandem Midi Gel tanks (Thermo Fisher Scientific #STM1001) and run at 200V for 1 hour with Tris/Glycine/SDS buffer (Bio-Rad #1610772). Proteins were then transferred to 0.2μm supported nitrocellulose membranes (Cytiva #10600015) using a Criterion blotter with plate electrodes (Bio-Rad #17004070) run at 85V for 45 minutes. Proteins on the blot were then visualised with Ponceau S staining (Thermo Fisher Scientific #161470250) which was scanned to show alongside individual western blots. Blots were blocked with 5% milk for 1 hour in Tris-buffered saline containing 1% Tween 20 (TBST) (Thermo Fisher Scientific #J77500.K2). Primary antibodies were then incubated overnight at 4°C in 5% milk TBST with gentle shaking. Following three ten-minute washes with TBST, horseradish peroxidase (HRP) conjugated secondary antibodies were incubated at a dilution of 1/10000 (0.1μg/mL) in TBST with 5% milk for 1 hour at room temperature followed by three ten-minute washes with TBST. Membranes were then incubated with either Pierce ECL (Thermo Fisher Scientific #32106) for 1 minute or Clarity Western ECL substrate (Bio-Rad #1705061) for 5 minutes prior to detection with the ImageQuant LAS 4000.

Antibody screening by immunoprecipitation

Antibody-bead conjugates were prepared by adding 2 μg of antibody to 1 mL of Pierce IP Lysis Buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) (Thermo Fisher Scientific #87788) in a microcentrifuge tube, together with 30 μL of protein A (for rabbit antibodies) or protein G (for mouse antibodies) (Thermo Fisher Scientific #10002D and #10004D respectively). Tubes were rocked for 1 hour at 4°C followed by two washes to remove unbound antibody. Culture media from Hep G2 WT were collected as described in the western section above. 0.5 mL aliquots at 1mg/mL of culture medium were incubated with an antibody-bead conjugate for 18 hours at 4°C. The unbound fractions were collected, and beads were subsequently washed three times with 1.0 mL of IP buffer and processed for SDS-PAGE and western blot on precast midi 4-20% Tris-Glycine polyacrylamide gels.

Antibody screening by flow cytometry

Hep G2 WT and SERPINA1 KO cells were detached, and five million cells were labelled with CellTracker green or violet fluorescent dyes, respectively (Thermo Fisher Scientific, #C7025 and #C10094). WT and KO cells were then centrifuged at 300 × g, for 10 minutes and resuspended in PBS containing 1% bovine serum albumin (BSA) (Sigma-Aldrich, A9647). Cell populations were then combined at a 1:1 ratio, centrifuged and fixed on ice for 20 minutes using 800 μL of 4% PFA in PBS (Thermo Fisher Scientific #J19943.K2). Following fixation, 1.2 mL of 1% BSA in PBS was added to the tube, vortexed and centrifuged at 600 × g for 15 minutes at 4°C. Cells were then permeabilised in 400 μL PBS with 0.1% Saponin (Sigma-Aldrich #558255) for 10 minutes at room temperature, centrifuged at 600 × g for 15 minutes at 4°C and then blocked with 5% goat serum (Sigma-Aldrich #G6767), 1% BSA, 0.1% saponin in PBS for 30 minutes on ice. After the blocking step 400,000 cells were aliquoted into individually labelled tubes, centrifuged at 600 × g for 15 minutes at 4°C and incubated in 150 μL of 1% BSA, 0.1% Saponin PBS with primary A1AT antibodies for 30 minutes on ice. 500 μL of 1% BSA, 0.1% saponin PBS was then added to each tube, vortexed and centrifuged at 600 × g for 15 minutes at 4°C. Cells were then incubated with their corresponding Multi-rAb CoraLite^® Plus 647 secondary antibodies (0.83 μg/ml) (Proteintech #RGAR005 and #RGAM005) in 150 μL of 1% BSA, 0.1% saponin PBS for 30 minutes on ice. 500 μL of 1% BSA, 0.1% saponin PBS was then added to each tube, vortexed and centrifuged 600 × g for 15 minutes at 4°C.

Tubes were then resuspended in 1 ml of 1% BSA in PBS and data was acquired using the Attune NxT flow cytometer. Data was analysed using FlowJo with the following gates. The cell population was first gated on FSC-A vs SSC-A, within that gate single cells were selected by FSC-A vs FSC-H and then KO and WT cells were isolated by BL1-A vs VL1-A using a quadrat gate. Quantification of antibody staining was then observed in the RL1-A channel and histograms merged to demonstrate the staining intensity between the two populations compared to the two secondary only controls. The figure was then assembled using Adobe Illustrator 2024.