Jun 14, 2026
Strip Immunoblot Assay
- Rebekah Honce1
- 1University of Vermont
Protocol Citation: Rebekah Honce 2026. Strip Immunoblot Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx451kl8j/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: September 21, 2025
Last Modified: June 14, 2026
Protocol Integer ID: 227832
Keywords: strip immunoblot assay understanding the antibody response, antibody response to viral infection, strip immunoblot assay understanding, strip immunblot, antibody, specific antibody, throughput the strip immunblot, mouse serum, viral infection, antibody response, lcmv, quantitative determination of seropositivity, virus, outbreak, viral, assay, outbreak setting
Funders Acknowledgements:
NIH NIAID
Grant ID: R01AI171408
NIH NHLBI
Grant ID: T32HL076122
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Abstract
Understanding the antibody response to viral infection is essential for surveillance, diagnostics, and epidemiological research. However, current methods to detect virus-specific antibodies are often resource-intensive and impractical for deployment in outbreak settings or in field-based studies. This protocol presents an economical and high-throughput the strip immunblot assay (SIA) for detecting anti-lymphocytic choriomeningitis virus (LCMV) antibodies in mouse serum. The expected results include quantitative determination of seropositivity.
Materials
2-mercaptoethanol Sigma-Aldrich #M3148
Laemmli sample buffer Use 4X stock for a thicker sample viscosity to help with loading
2D gel Invitrogen #NP0346BOX
Transfer stacks Invitrogen #IB23002
Western incubation dish BioRad #1703902
Blocking buffer Thermo #37530
Washing buffer 0.5% Tween-20 in TBS
AP-conjugated Secondary antibody (IgG or IgM)
Developing reagent Invitrogen #WP20001, keep blot in dark after developing
Safety warnings
Preparation of the antigen lysate should be done under appropriate viral tissue culture methods including needed PPE. After inactivation with lysis buffer, lysate can be used on the benchtop. All sera samples should be heat inactivated at 57°C for 20 minutes prior to use on the benchtop.
Ethics statement
For development of this protocol, all animal procedures (X2-022) were approved by the University of
Vermont Animal Care and Use Committee (IACUC) and complied with the Guide for the Care and Use of Laboratory Animals. All experiments were conducted in a biosafety level 3 laboratory. Investigators were required to wear appropriate respiratory equipment (Versaflo PAPR, 3M). Mice were housed in HEPA-filtered, negative pressure, vented isolation containers and handled only within class 2A biosafety cabinets.
Before start
Assumes you have already generated antigen lysate for immunoblotting.
Antigen preparation
To the antigen source (clarified cellular lysates, purified virions, or recombinant purified protein), add 1:1 4X Laemmli sample buffer with 10% BME for final concentration of 2X sample buffer and 5% BME.
Heat at 100°C for 10 minutes.
Quickspin to collect sample droplets from top of snapcap tube.
Load 250 µL of prepared lysate to the large well of a 2D gel. Load 5 µL of SeeBlue2 ladder (or pre-stained ladder of choice) to the small well.
Perform electrophoreses at 100 V for approximately 1 hour.
Transfer gel to membrane using the iBlot system.
Using a ruler, draw a line with a pen approximately 0.5 cm from the top edge of the blot.
Using the ruler and a razor blade, cut strips. Strips should be approximately 2 mm in width, starting from the far edge of the blot. This works best using a pane of glass as a cutting surface. Discard the first 5 mm of the far edge.
Store strips in blocking buffer at 4°C until use.
Strip immunoblotting
Allocate the pre-blocked strips to a western incubation dish.
Cover with 500 uL blocking buffer.
Add desired volume of sera or blood sample to well. Recommend to use a volume of 2 uL to 10 uL.
Incubate overnight with rocking at 4°C.
Following day, carefully decant sample.
Rinse 3X with TBS-T, followed by 3 5-minute rinses with TBS-T.
Add 500 uL of 2° antibody to each lane and incubate at room temperature in the dark with rocking for at least 1 hour.
Rinse 3X with TBS-T, followed by 3 5-minute rinses with TBS-T.
Remove remaining washing buffer with a pipette.
Add 500 uL developing agent to blot and incubate in the dark for 30 minutes or until color develops.
Remove strips from incubation dish and mount on paper to scan. Store in dark.
Protocol references
Hjelle B, Jenison S, Torrez-Martinez N, Herring B, Quan S, Polito A, Pichuantes S, Yamada T, Morris C, Elgh F, Lee HW, Artsob H, Dinello R. Rapid and specific detection of Sin Nombre virus antibodies in patients with hantavirus pulmonary syndrome by a strip immunoblot assay suitable for field diagnosis. J Clin Microbiol. 1997 Mar;35(3):600-8. doi: 10.1128/jcm.35.3.600-608.1997. PMID: 9041397; PMCID: PMC229635.
Acknowledgements
Assay developed with Jillian German, Ella Botten, Chloe Schiff, Phil Eisenhauer, Inessa Manuelyan, and Jason Botten