SpotMap

HCP Coverage

SpotMap is a complete solution for analysing HCP coverage with 2D gel electrophoresis and Western blot images.

  • Speed. Get a coverage % in less than 10 minutes.
  • Objectivity. Unmatched automation reduces the need for manual intervention.
  • Reproducible. Evidence of consistent coverage results between different users and labs.
  • Quantitation. Enables reliable, accurate quantitation of gel and blot images.
  • 21CFR ready. Help meet 21 CFR part 11 compliance for GMP/GLP labs from the captured image to final analysis.

SpotMap solves the common problems you face in 2D gel electrophoresis vs Western blot analysis for HCP coverage such as no reproducibility in results between users, hours of unnecessary spot matching and lack of confidence in the percentage coverage results.

It is consistently designed and updated in line with biopharma industry feedback making it the most complete solution to help you monitor your host cell protein (HCP) impurities and reach your coverage requirements.

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Trusted by over 30+ top biopharmaceutical companies who are already using SpotMap for:

HCP coverage analysis: Host cell protein (HCP) and anti-HCP antibody characterization via:

  • 2D gel and Western blotting
  • 2D Difference Gel Electrophoresis (2D DIGE)
  • 2D Differential In Blot Electrophoresis (2D DIBE)

Bioprocess improvement: Assess the purification of biologic production through comparison of multiple 2D gels.

Analytical development:Developing process-specific immunoassays for use in host cell protein (HCP) analysis of new biologics and biosimilars.

“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.”
Virginia Liu-Compton Research Associate @ Immunogen

Generate extensive data-driven insights

We believe it is important to give you the tools to view your data from a variety of perspectives with efficiency. SpotMap makes this possible by allowing you to pull new insights from your images using our efficient and easy-to-use interface, allowing you to generate your most complete report to date.

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Submit results with ease and confidence

Intuitive algorithms and an easy-to-use interface allow you to obtain an accurate idea of coverage in less than 10 minutes and a complete and reproducible coverage calculation and report in less than an hour. New users can get up and running with minimal training and maintain consistent coverage calculations.

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Highly automated coverage calculation

Combining the speed, flexibility and unmatched automation to get better, more objective results. No other software allows such advanced automation to achieve reproducible antibody-to-HCP coverage.

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What's New

SpotMap is always improving so you can get better, more objective results. We roll out new versions in response to the pharmaceutical industry and our biopharma customer requests. Below are just some of the new features in the latest version of SpotMap that industry leaders have asked for (we think you’ll agree).

Demo all new features

Advanced automation: We’ve addressed your need for more automatic functions and introduced new features and upgraded our algorithms to create unmatched automation for your 2D WB analysis to achieve reproducible results.

HCP antibody reactivity We’ve built new tools to verify your HCP antibody reactivity (without any additional setup). This tool helps researchers have a more complete understanding of the antigen quality.

Identify cross-reactivity Cross-reactivity interferes with accurate measurement of HCP coverage. Quickly visualise any suspected cross-reactivity in under 30 seconds.

21 CFR part 11 tool support Future proof your analysis results – by tracking, recording and authenticating your experiments. See Version Control integration with SpotMap for more information.

Try it for free for 14 days

Schedule your demo and you’ll also gain access to the very latest version to try for free

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Common Questions Answered

How do I compare visually different gels and Western blots?

SpotMap specializes in working with challenging images e.g. Western blots can be difficult to work with due to comparatively few spots present or the potential for spots to bloom or streak due to the detection method and positional variation often occurs on 2D gel images during the experimental procedure. SpotMap enables an efficient and reproducible alignment process by performing at the pixel level to provide direct and accurate comparison of images.

Can I download a trial of SpotMap to try with my own images?

Yes. You can download a trial version of the software after a quick online demonstration. The trial works with your own images.

How long does the trial last?

You can trial SpotMap for up to 14 days on your own images. You will have access to our analytical and support team should you have any additional questions, or require an additional run through of the software.

What data can I export?

SpotMap allows you to export a complete analysis report with particular attention given to reports needed in GMP/GLP and 21 CFR compliant laboratories. This includes:

  • Date of report, Author, Software Version
  • Original images, Properties, Image QC
  • Results of percent coverage
  • Number of spots common, additional and missing between images
  • Spot detection parameters and edits
  • Colour coded spot maps overlaid on the images identifying the location of Common, Missing and Additional spots
  • CSV data file containing all measurements of all spots for all images or a spot set
  • All analysis parameters used throughout
  • Manually added images
  • Complete audit trail documentation
  • Time-stamped audit trails, showing who and when projects are produced, opened and signed off

Speak to us about tracking and authentication in SpotMap for helping 21 CFR compliance or visit the Version Control page for more information.

Resources

Additional SpotMap Questions

Can I check my image quality?

Yes. Images are quality checked and any issues/notes are available to view.

Can I account for gel/blot distortions?

Yes. Alignment makes it possible to accurately compare images by removing the positional variation introduced during the gel running, blotting and imaging process.

Can I identify spots of interest?

Yes. You can add text labels on the image. These can also point towards certain features on the gel/blot that are of interest.

Can I edit my images?

Yes. If your images require minor changes (flipping etc.) then you can edit them within the software without changing the original images.

Can I measure percentage coverage of 2D gels and Western blots?

Yes. Based upon the categorisation of spots, percentage coverage of one image vs another is calculated.

Can I compare spot measurements?

All spot measurements are available for export or may be viewed in the table within the software. Spot measurements include volume, peak height, area, background and position. If pI/MW information has been added to the image the estimated values are also available.

Can I calculate spot coverage?

Yes. A spot map is created of the fully resolved host cell protein. The presence of spots expressed on the blot is then used to calculate the relative coverage of the Western blot vs the 2D gel.

Can I export results?

Yes. A spot map is created of the fully resolved host cell protein. The presence of spots expressed on the blot is then used to calculate the relative coverage of the Western blot vs the 2D gel.

Gel documentation systems

Is it compatible with my imager/gel documentation system?

SpotMap is compatible with most imaging/gel documentation systems.

What image formats can I use?

It supports most industry-standard file formats, such as, JPG, PNG, GEL, MEL, TIFF or IMG/INF with both 8-bit and 16-bit. For analysing your gels we recommend storing them in a lossless format such as TIFF. For more recommendations on how to capture your gels view our image capture article. Read article

Analysis

What does the analysis mode do?

In the analysis mode, a master spot map identifying the location of all spots in the project is created and spots on each image are categorized as present or absent from this map. The present or absent settings are used to identify spots common, missing or additional to the images in a comparison and measure % coverage.

Why is a single spot map used for the analysis?

Alignment places corresponding spots in the same coordinate space on the images, this allows a single spot map to be used. Using a single spot map allows you to directly compare matching spots/features between images and easily identify spots absent from that given image.

What is spot coverage and how is this calculated?

Spot coverage is the percentage of spots in the comparison being made which are present on the secondary image. Percentage coverage is calculated against the chosen base image using the calculation:

Percentage coverage = (Common + Additional)/(Common + Missing + Additional) x 100

What is a base image?

The base image is the image against which the others are compared. The base image can be selected to be any other image in the analysis. The term secondary image refer to any image compared to the base image.

What does automatic spot detection do?

Using advanced imaging techniques an initial spot map is created of the selected image within the set parameters of smoothing and peak sensitivity. A rectangular or elliptical region of interest can be added to the analysis to target the spot detection.

Why are some spot edits required?

Sometimes the automatic spot detection results need to be refined. Spot outlines around the edge can be removed. Spot outlines in the middle can be added, deleted, merged or split. Additional spots can be added to the map. Our tests have shown that you can have different levels of editing (little or a lot) and still get similar coverage results. It is key to be consistent so that you get similar results across experiments. Using the spot assignment feature when editing spots will aid in categorising spots by pre setting the presence or absence of a spot on all other images.

How does the single spot map work in SpotMap?

Users should detect spots on the gel first as stained gels are often more defined than a western blot image. The spot outlines are then transferred to the blot (this should be done after alignment as it helps to see where the spots exist on the blot) then you can detect additional spots on the blot. This will give you one single spot map for determining matching and missing spots. You can watch this video for more information on how the spot map is generated.

Why select spots using filters

The filter option is usually used to objectively select spots you may wish to remove from the spot map or categorise as present or absent from the image. A filter clause can be created based upon a measurement of area, volume or peak height and set based on a specific value, a selected spot or chosen from a sliding scale. Selected spots can also be used to create a spot set.

What is a spot set?

A spot set can be created from a spot selection, manually chosen spots or predefined areas, i.e. quadrants. Results and spot measurements can be collated for each spot set.

Can I add molecular weight and pH markers?

Yes. Markers can be added to the map to identify the MW markers. A linear or non-linear pI strip can be added to the map.

How does identifying spots as present or absent allow results to be calculated?

The present or absent categorisations from the spot map allow comparisons of images to be made. When any two images (the base and the secondary) are compared the spot presence/absence settings are used to determine which spots are Common, Missing or Additional and are used to calculate percentage coverage.

Base image Secondary image
Common YES YES
Missing YES NO
Additional NO YES
Absent NO NO

Results and Data Access

What does the results screen show?

The results screen presents an overview of the percentage coverage and numbers of spots common and additional to the base image. There is a small heat map which identifies where the spots common, additional and missing are in relation to the whole image, selecting “View Comparison” will open the base image and selected image in the analysis window in the colour scheme of Common, Additional and Missing spots. Results for spot sets can also be viewed in the results screen.

Can I export data?

Yes. You can use our automatic report generator to export a clear, detailed and complete report straight out of the software. Individual images and tables can be exported separately for reports and presentations, giving you full control of your presentation report and results.

Can I annotate the image?

Yes. You can add text labels to the spot map in the analysis screen, these can point towards certain features of the map which are of interest.

Questions about spot detection

How does the single spot map work in SpotMap?

This video explains how the single spot map works for present and absent assignment.


Watch video

How can I ensure the quantitative accuracy of my HCP coverage analysis?

In this video we show you how automatic spot outlines in SpotMap obtain an objective, accurate and quantitative analysis and how you can use 3D mode to validate the software you currently use for HCP coverage.


Watch video

Images

What is Image QC?

When adding images to the project they are quality checked and any issues/notes are highlighted for you immediately before any further analysis is attempted. Image QC flags any potential issues with:

  • Bit-depth
  • Image file size
  • Saturation
  • Manual editing of the images

Poor image quality can make analysis of your gels and blots difficult and may reduce the objectivity of your analysis.

What are the benefits of using high quality images?

Quantification of spot intensities may seem less important when only looking at spot numbers and coverage results. However, good quality images also improve:

  • Spot Detection
  • Filtering options when selecting small/low spots to remove
  • Definition of spots on the blot (no saturation)
  • Automatic alignment algorithm

Can I edit my images in the software?

Yes.Quick edits are available below each image to invert the colour intensities and flip or crop the images. More edits can be performed in the image editor window, accessed by right clicking on the image and selecting “Edit” from the menu. It is recommended that image edits are performed within the software as this will not affect the raw data of the images.

What problems does image editing solve?

Images which are different in size, shape and orientation are hard to compare directly. Poor image quality can make analysis of your 2D gels and Western blots difficult and reduce the objectivity of your results.

What edit options are available?

  • Flip images horizontally and vertically
  • Rotate images 90 degrees clockwise and anti-clockwise
  • Invert image
  • Translate image
  • Crop image
  • Rotate image a variable angle
  • Resize images to same size

Alignment

What is Alignment?

Alignment takes two images and overlays them in the same coordinate space so that a spot on the image to be aligned will be in the same location as the matching spot on the target image.

What problem does it solve?

Alignment makes it possible to accurately compare images by removing any positional variation introduced during the gel running, blotting and imaging processes.

How do I know Alignment is correct and complete?

Our 1 minute online tutorials are designed for to help you understand and learn the different visual cues in SpotMap and to understand the different software tools you can use to ensure you are confident using Alignment.


Watch video

Why is alignment better than spot matching techniques?

Using separate spot detection on each image and then matching each spot between the images is very inefficient. Alignment makes it possible to overlay spots from one image to another, placing them in the correct location.

How is alignment different to warping?

Warping and alignment both aim to remove the positional variation introduced during the gel running, blotting and imaging process. However warping is only accurate to the spot level whereas alignment is accurate to near pixel level.

Do I need to align DIGE images?

Alignment is not necessary for DIGE images. However, for low MW proteins there may be a shift due to the different dye, in this case alignment can be used.

Can I “fix” alignment after spot detection?

Yes. The alignment window can be opened at any point in the project to align or correct the alignment of an image. Once the “Apply” button has been clicked the alignment will update. The images can be initially aligned prior to or following spot detection.

How does Alignment work?

Automatically generated or manually added vectors between features on each image are used to create a map between the images. If the images have similar spot patterns then this can be fully automatic using our fast alignment algorithm.

What is the difference between automatic alignment vectors and manual vectors?

Automatic alignment attempts to look at the spot patterns between the images and match up similar spots/features and puts in automatically generated vectors. Manual vectors are added by users to features that they think are matched. These can be added before automatic alignment, which helps the algorithm, after automatic alignment to fix areas or simply on their own if automatic alignment fails or is difficult.

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