SpotMap 2DHCP antibody coverage by 2D western
SpotMap 2D compares your 2D gel with the matching 2D western blot and reports the percentage antibody coverage, overall and for each quadrant of the blot.
SpotMap 2D is the original, industry-leading analysis software specifically designed from the ground up to automate and simplify the process of determining host cell protein (HCP) antibody coverage by 2D-PAGE and 2D Western **blot-**based techniques, including support for 2D-DIGE and 2D-DIBE. Ideal for high-throughput pharmaceutical and biotech environments, SpotMap 2D efficiently automates antibody coverage analysis, significantly reducing manual intervention, time taken for analysis and inter-operator variability.
In the regulator’s words: antibody coverage is the percentage of host cell protein spots on the 2D gel that the anti-HCP antibody reagent recognizes on the matching 2D western blot, assessed across the range of molecular weight and charge, and it is the orthogonal check used when you qualify the reagent behind your HCP ELISA (USP <1132>). SpotMap 2D reports that percentage, plus a separate score for each quadrant of the blot, so a weak corner of the map is visible rather than averaged away. For the method itself, see HCP coverage analysis using 2D western blot.
How to report SpotMap 2D in your methods: “Anti-HCP antibody coverage was determined by 2D western blot and calculated in SpotMap 2D (TotalLab Ltd, Newcastle upon Tyne, UK) as the percentage of 2D gel spots with a matching immunoreactive spot on the blot, reported overall and per quadrant.”
Developed through close collaboration with the pharmaceutical industry, SpotMap 2D delivers accurate, reproducible, and rapid HCP antibody coverage analysis you can trust. You can access some real-world case studies, posters, application notes, and technical evidence showing how SpotMap 2D is actively helping some of the world’s largest pharma companies here.
SpotMap 2D doesn’t try and force you into a specific workflow or to perform your analysis in a certain order or way, rather it simply provides all the tools you may require for host cell protein coverage analysis in an easy to use sandbox format. You know your experiment and your analysis better than any software ever could and we respect that.
We have had real difficulty matching reactions on blots to the corresponding areas on the gels. With our previous technology it genuinely wasn’t possible so adding the SpotMap software to our facility has helped greatly. […] Once we had the images put through SpotMap software it corresponded to areas that personally I would have never matched, which ended up being the correct areas.
SpotMap 2D is specifically designed from the ground up to analyze coverage – the percentage of immunoreactivity that an anti-HCP antibody has for the total population of HCPs present – rather than being repurposed 2D proteomics based software. It does this by comparing 2D gel or blot images of proteins detected with an anti-HCP antibody (i.e., a secondary image) against an image revealing all HCP antigen proteins (i.e., a primary image). SpotMap 2D is not limited to just comparisons between 2 images however, with the universal spotmap propagating to all images within a project. This allows users to compare multiple different blots and gels representing different antibodies, batches etc. simultaneously for quicker and easier candidate antibody selection. HCP coverage analysis can be useful in many applications:
The images used for coverage assessments can be generated from a host of different experimental workflows and SpotMap 2D supports all 2D-PAGE based practices including conventional 2D gel electrophoresis and Western Blotting, 2D differential in gel electrophoresis (2D-DIGE) and Immunoaffinity chromatography (IAC) followed by 2D differential in gel electrophoresis (2D-DIGE). Our SpotMap software has been in active development for more than a decade and in that time has been cited numerous times in proteomics and anti-HCP antibody generation best practice guides within the field (click to open):
 |
 |
We’ve been looking at different solutions for three months now and from what we just saw [demonstrated] this is already the best
When it comes to 2D Western blot and HCP coverage analysis, flexibility matters. Unlike competitor software, SpotMap 2D is truly vendor-neutral, supporting a wide range of image file types, including TIFF, GEL, MEL, IMG/INF, GSC, 1SC, JPEG, PNG and BMP files, ensuring you can analyze high-quality images from virtually any instrument. It seamlessly integrates with nearly any scanner, densitometry system, or gel documentation device on the market. Whether you’re working with a Cytiva Typhoon™, Sapphire FL Biomolecular Imager, Bio-Rad Gel Doc™, Fujifilm LAS™, or any other imaging system, SpotMap 2D adapts to your existing lab setup—not the other way around.
Officially Recommended by Merck for use with their Auto2D devices, SpotMap 2D gives scientists complete freedom in how they capture and analyze their HCP coverage data (click to read the full technical note):
Stop letting hardware compatibility dictate your software choices or workflows. With SpotMap 2D, you get complete freedom to use the imaging systems that work best for your lab—without compromise.
SpotMap played an integral role in our comparison of proteomes of different cell lines during the process of choosing a proper antigen for antibody development. The ability to overlay gels and assign numbers to each protein spot was especially useful in determining similarities and differences between gels without duplicating identified spots. One feature that I found to be especially useful was the 3D view which allowed for a better visual of spot intensity when determining spots. The table of identified spot for each image also made it easy to identify which spots are unique or common to the different images. With this software, my team was able to make a more informed choice on the best candidate to use for our purposes.
Present your 2D images, your way, using SpotMap 2D’s data access tool. Our multichannel color overlay image allows you to blend multiple images together to easily visualize similarities and differences between your 2D gels and Western blots. High DPI export to basically any other piece of software means you can share our easy to interpret display mode with colleagues or customers easily.
And of course, you can fully customize your report with the images you choose – raw grayscale, annotated, multichannel overlay, spot outline on/off, your call.
SpotMap fully complies with:
When integrated with our AuditSafe compliance module, users are able to control system access through unique passwords or Windows Active directory integration, record user activity, define users define project sign-off procedures to ensure compliance with various QC/QA requirements.
I have found the software very easy to use and has been exactly what I needed to compare western blots and pick the spots from my gel, which have now been sent off for mass spec.
Complex sample preparation and running procedures cause distortions such as faint spots, overlapping spots, saturated spots and streaks. You can overcome these challenges during your analysis easily within SpotMap by enhancing these visual distortions using our range of advanced visualization tools, allowing a more informed and confident decision where necessary. Our tools include interactive 3D views, user configurable secondary image overlay (multichannel overlay) or full color overlay mode. See your host cell protein expression data like never before. Our multichannel color overlay lets you blend multiple images with your preferred color scheme to easily visualize similarities and differences between 2D gels and blots.
Both spot analysis software generated a final coverage that was between 44 – 59%. However there was much more variation in coverage using BioRad's PDQuest than using SpotMap. Workflow between the two analysis systems varied by about 2 hours – analysis took approximately 1 hour using the SpotMap software and approximately 3.5 hours using the PDQuest software
In 2D Western blot-based Host Cell Protein (HCP) coverage studies, understanding how your ELISA antibody performs across diverse protein subsets is critical-especially for low-molecular-weight, low-immunogenicity HCPs that often evade detection. That’s where SpotMap’s automated quadrant analysis can put you ahead. Unlike competitor tools, which require manual definition of quadrant parameters, SpotMap automatically and accurately splits your blot into coverage quadrants, enabling faster, more consistent evaluations with zero guesswork or user variability.
While competitors offer the ability to perform quadrant-based analysis, their workflows require manual configuration and are more prone to user bias and inconsistency. SpotMap 2D gives scientists a smarter, faster path to accurate antibody validation.
Whether you’re validating a new anti-HCP ELISA or optimizing existing assays, SpotMap 2D delivers unmatched clarity and reproducibility.
We use intuitive, automated algorithms and an easy-to-use interface to enable users to obtain an accurate coverage percentage in around 10 minutes. This enables users to achieve extremely reproducible antibody-to-host cell protein coverage, even between different users and labs. It provides reliable, accurate quantitation of gel and blot images so that you can achieve objective results. Users are also able to verify host cell protein antibody reactivity, giving you a more complete understanding of the antigen quality. To see how 2D Western blot coverage compares with 2D-DIGE, antibody affinity extraction and ELISA-MS, read HCP antibody coverage methods compared. If you’re looking for software to support HCP coverage analysis, make bioprocess improvements or achieve analytical development then request a free demo of SpotMap. You’ll receive guidance on how to achieve reproducible results, tips for aligning challenging gels and Western blots and knowledge on the software tools to use on more challenging images, such as faint spots. 
SpotMap helps support our internal and external client HCP projects, generating coverage data to inform both antibody and ELISA kit development. We love how intuitive the software is to use and how much easier and quicker it is to generate data compared to other software packages on the market. The decreased analysis time really aids our efficiency when we have dozens of blots to analyze for client projects and cuts down the time burden analysis can entail. Manual spot-editing features, such as the ability to quickly separate merged spots in a highly customizable manner with a single click, contribute to this decreased analysis time and enable us to increase our workflow.
In some cases your anti-HCP antibody can have non-specific binding with your drug substance or other product within your sample, leading to an artificially inflated coverage score as these spots are included within the reported coverage % but are not actually detected host cell proteins.
If you know your therapeutic drug substance exhibits some cross-reactivity with your anti-HCP antibody, SpotMap 2D allows you to detect those spots and treat them separately to the “true” HCP positive spots using its innovative Product Spots function.
Simply mask the area on the blot image where your drug substance spots appear (based off either experience from previous runs or from the pI and MW ladder information present in the image) and SpotMap 2D will allow you to create a new group of spots from those product spots. You can now toggle their inclusion using the standard spot sets controls to see how their inclusion would have inflated your overall anti-HCP coverage % score. SpotMap 2D allows you to simultaneously increase accuracy in analysis whilst accounting for antibody cross-reactivity.
The best way to ensure you’re purchasing the latest version of SpotMap with the highest level of technical support is directly from us here at TotalLab by using the contact us form. We can accept payment in GBP (£), USD ($) or EUR (€). We do appreciate that some of our customers prefer sourcing products locally in a local currency or prefer to deal in their native language which may not be English. To facilitate this, TotalLab work with a global network of authorized distributors which you can find here.
SpotMap 2D is software for host cell protein (HCP) antibody coverage analysis using 2D gel electrophoresis and 2D western blots. It calculates the coverage percentage of anti-HCP antibodies, a key step in validating HCP ELISA reagents for biologics.
SpotMap 2D compares spot patterns between a total protein 2D gel and the corresponding 2D western blot, automatically matching spots and reporting the percentage of host cell proteins recognized by the antibody reagent. Alignment of the blot to the gel is automatic; see 2D gel image alignment explained for how it works and how to check it.
Yes. A free trial of SpotMap 2D is available from TotalLab so you can evaluate it on your own 2D gel and western blot coverage data.
SpotMap has been used in published host cell protein coverage work across the biopharmaceutical industry. Browse the SpotMap reference papers.
Immunoaffinity chromatography enhances detection and purification of host cell proteins, delivering reliable results for biopharmaceutical quality control.
Read More >
How HCP impurities are measured and controlled in biologics: ELISA, 2D Western blot coverage and LC-MS compared, the risks each one manages, and the software that supports HCP quantification and antibody coverage.
Read More >
Understanding your HCP antibody coverage is essential to validating your ELISA for host cell protein detection. This article explains how coverage impacts assay reliability, regulatory compliance, and effective antibody selection in biologics and biosimilar development.
Read More >
The scanner settings that decide whether a gel or blot image can be quantified: resolution and dpi, 16-bit vs 8-bit depth, dynamic range and saturation, file formats, and a pre-scan checklist for coverage work.
Read More >
Where positional variation between gels and blots comes from, what pixel-level alignment does to remove it, why aligning before spot detection beats matching afterward, and how to check alignment quality before you trust a coverage score.
Read More >
Discover how 2D Western blotting enhances Host Cell Protein (HCP) coverage analysis in biopharmaceutical research. Learn the key steps, from protein separation and transfer to PVDF membranes to antibody staining and data analysis. See how TotalLab's SpotMap software improves HCP detection and quantification, ensuring regulatory compliance and accurate results.
Read More >
Good image capture is critical to guarantee optimal performance of automated image analysis packages and generate reliable scientific data. This technical document provides a brief guide to the range of different image acquisition devices currently in use for gel applications, defines some the important technical factors required to generate digital images of a quality suitable for automated image analysis and recommendations for producing good 2D gel images.
Read More >
Discover what host cell proteins (HCPs) are, why they matter in biologic drug development, and how they’re analyzed for safety. Learn about common HCP analysis methods, challenges, and strategies to manage risks, with insights on tools from Totallab.
Read More >
How 2D gel electrophoresis works, step by step: isoelectric focusing, SDS-PAGE, staining and image analysis, with protocol values, a 1D vs 2D comparison, applications, limitations and troubleshooting.
Read More >
2D Western blot, 2D-DIGE and DIBE, antibody affinity extraction and ELISA-MS compared as methods for measuring anti-HCP antibody coverage, with the USP 1132 context and guidance on when each method fits.
Read More >
Here we present the characterization of a process specific anti-Pichia pastoris HCP antibody using 2 different analysis platforms: Biorad’s PDQuest and TotalLab’s SpotMap.
Read More >
SpotMap 2D software automates 2D-PAGE host cell protein coverage analysis. Explore case studies, posters, and testimonials from pharmaceutical researchers demonstrating accuracy, reproducibility, and trusted real-world performance to support your lab’s QC and research workflows.
Read More >
There is no regulatory threshold. Where the 70 percent figure came from, why 2D gel and LC-MS coverage numbers are not comparable, and what to report with any coverage claim.
Read More >