Feb 24, 2025
High-throughput 96-well plate-based porcine antibody isolation protocol
Peer-reviewed method
- John Byrne1,
- Christina Bourne1,
- Sitka Eguiluz1,
- Stephanie Langel2,
- Elisa Crisci1
- 1Department of Population Health and Pathobiology, North Carolina College of Veterinary Medicine, Raleigh, NC;
- 2Center for Global Health and Diseases, Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH
- PLOS ONE Lab ProtocolsTech. support email: plosone@plos.org
Protocol Citation: John Byrne, Christina Bourne, Sitka Eguiluz, Stephanie Langel, Elisa Crisci 2025. High-throughput 96-well plate-based porcine antibody isolation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvmee6ng3p/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 05, 2024
Last Modified: February 24, 2025
Protocol Integer ID: 101284
Keywords: Antibody, Pig, Swine, Porcine, Resin, Chromatography, IgA, IgG, Isolation, High Throughput
Funders Acknowledgements:
Bill and Melinda Gates Foundation
Grant ID: INV-22595D
Abstract
This protocol describes a modified column chromatography technique to isolate both IgG and IgA from pregnant/lactating swine to use in a variety of immunoassays. This high throughput method allows for both a rapid and consistent isotype yield from individual animals and different tissue samples.
Materials
Glycine, 0.2M buffer soln., pH 2.5 Cat# J61855.AP (Thermo Scientific)
Tris Hydrochloride, 1M Solution (pH 8.0/Mol. Biol.) Cat# BP1758-500 (Fisher Scientific)
Ethanol, Absolute (200 Proof), Molecular Biology Grade Cat# BP2818500 (Fisher Scientific)
Tris-EDTA, 1x Solution, pH 8.0 ± 0.1 Cat# 77-86-1 (Fisher Scientific)
Sodium chloride, ACS, 99.0% min Cat# 7647-14-5 (Fisher Scientific)
Pierce Protein G Agarose Cat# 20399 (Thermo Scientific)
Peptide M / Agarose Cat# gel-pdm-5 (InvivoGen)
CaptureSelect IgA-XL Affinity Matrix Cat# 2943972010 (Thermo Scientific)
Ultra-pure water - In House
Corning Mediatech Cell Culture Phosphate Buffered Saline (10X) Cat# MT-46013CM (Fisher Scientific)
Universal 200ul Pipette tips Cat# 76322-144 (VWR)
BrandTech Scientific 781722, 96-Well Plates, immunoGrade, Clear Transparent, Flat Bottom Cat# 91-415F (Genesee Scientific)
Multiscreen 96 well Plate, hydrophobic PVDF membrane Cat# MSIPS4W10 (Millipore Sigma)
Thermo Scientific NanoDrop 2000c Spectrophotometer
Sorvall Legend XTR (Refrigerated), 120V (Thermo Scientific)
Sorvall Legend Micro 21R Microcentrifuge (Thermo Scientific)
Before start
Prepare 1x PBS
Prepare 0.1M Glycine
Prepare 70% ETOH
Prepare Ab Binding Buffer (10mM TRIS, 1mM EDTA, 300mM NaCl)
Sample Preparation
Sample Preparation
1m
1m
Quickly vortex all samples to ensure no clotting from serum and no fat blockages from milk
Centrifuge all samples at Room temperature for 2000 x g, Room temperature, 00:01:00
1m
Add 100 µL sample (serum/milk/colostrum) to 100 µL Ab Binding Buffer
- DO NOT ADD ANY PRECIPITATE TO THE MIX
Resin Plate Preparation
Resin Plate Preparation
3m
3m
Remove the Multiscreen 96 well ELISpot Plate from packaging
Place the Multiscreen 96 well ELISpot Plate on top of a 96-Well immunoGrade ELISA Plate ensuring the ejection ports of the ELISpot plate are in line with the ELISA plate wells
Add 50 µL Protein G, CaptureSelect IgA, or Peptide M to each well
- ONLY USE ONE RESIN PER WELL
Wash all wells by adding 50 µL 70% ETOH to each well
Centrifuge the plate stack at 4000 x g, Room temperature, 00:03:00
3m
Discard flow through from the bottom plate
Wash all wells by adding 200 µL 1x 7.4pH PBS to each well
Centrifuge the plate stack at 4000 x g, Room temperature, 00:03:00
3m
Discard flow through from the bottom plate
Restack the plates
Antibody Isolation
Antibody Isolation
41m
41m
Add 100 µL Sample (serum/milk/colostrum) / Ab Binding Buffer mix per well to each plate
Place the plate on a plate shaker and shake 600 rpm, Room temperature , 00:30:00
30m
Remove the plate stack from the shaker and centrifuge the plate at 4000 x g, Room temperature, 00:03:00
3m
Discard the flow through from the bottom plate
Restack the plates
Wash all wells by adding 200 µL Ab Binding Buffer to each well
Centrifuge the plate stack at 4000 x g, Room temperature, 00:03:00
3m
Discard the flow through from the bottom plate
Antibody Isolation
Antibody Isolation
41m
41m
Repeat Steps 19-21 3 times
Antibody Isolation
Antibody Isolation
41m
41m
Place a new 96-Well immunoGrade ELISA Plate under the Multiscreen 96 well ELISpot Plate to ensure that collected antibody are pure
Add 100 µL 0.1M Glycine and incubate 00:02:00 , Room temperature
2m
Elute the antibodies by centrifuging the plate stack at 4000 x g, Room temperature, 00:03:00
3m
Repeat step 24 and 25
Add 20 µL 1M TRIS to each well to neutralize the solution
Antibody Concentration and Storage
Antibody Concentration and Storage
11m
11m
Label one Amicon ultra 0.5 50k Centrifuge filter for each sample
Remove the 220 µL glycine-TRIS solution from the well and place into the corresponding Amicon ultra 0.5 50k Centrifuge filter
Centrifuge the filter tubes at 12000 x g, Room temperature, 00:05:00
5m
Wash each sample with 400 µL 1x 7.4pH PBS
Centrifuge the filter tubes at 12000 x g, Room temperature, 00:05:00
5m
Label one Amicon ultra 0.5 50k Centrifuge collection tube to store each sample
Invert the Amicon ultra 0.5 50k Centrifuge filter into the new labeled storage tube
Centrifuge the filter tubes at 1000 x g, Room temperature, 00:01:00
- this should yield ~ 100ul of isolated antibody
1m
Remove the filter from each tube and store isolated antibodies at 4 °C until later use
Quantify Antibody concentration
Quantify Antibody concentration
Use NanoDrop, select the "Protein A280" program
Pipette 1 µL isolated antibody on the NanoDrop pedestal
Analyze the sample
Record published mg/mL