SpotMap

Examples and Evidence

We work closely with our customers, who include Regeneron, GSK Belgium and ImmunoGen, to ensure they get the best from SpotMap.

For you to evaluate the software we will work with you, with a non-disclosure agreement in place, and provide a bespoke analysis report. This will demonstrate how SpotMap can determine HCP coverage with your 2D gel and Western blot data set.

Below are two examples of the analysis reports we can provide to show you the benefits of in-house HCP-antibody characterisation.

 Example Analysis Report: SpotMap analysis of a challenging 2D gel vs Western blot.

 

The images used in this example were challenging to analyse due to low colour depth, presence of non-spot features and streaking on the blot image: issues which are commonly faced in analysis of 2D gels vs Western blots.

The simple workflow of SpotMap provided results quickly and objectively. Within an hour relative coverage of the 2D gel by the Western blot was calculated as 18% and 702 spots were detected.

 Inter-user variation of percentage coverage is within a range of 2% using an optimised analysis process

 

The reproducibility of coverage results obtained by SpotMap was assessed across three data sets of varying difficulty, by three users of varying expertise.

Application Note: Anti-HCP antibody characterisation using SpotMap for analysis of 2D SDS-PAGE and Western blots.

 

SpotMap was used to characterise two pooled samples of antiserum by comparison of Western blots and a 2D gel. Percentage coverage of each antiserum was calculated and compared; pool 1 had broader reactivity at 44% coverage vs 39% coverage by pool 2 antiserum. Coverage was also compared between low and high molecular weight regions and the specific spots against which each antiserum sample had activity were compared.

 

SpotMap software used to minimise inter-user and inter-lab variation of anti-HCP antibody percentage coverage results

 

Users with a range of experience at two locations analysed two data sets of a 2D gel vs Western blot and the percentage coverage results were compared.

The coefficient of variation for each analysis was within 10%. Key areas to be considered for future analysis and creation of an SOP are also addressed.

Originally presented at WCBP 2016.

Technical Note: Removing positional variation introduced during the experimental procedure to accurately compare 2D gel and Western blot.

Alignment is the key feature of SpotMap which removes the positional variation introduced during the experimental procedure, allowing direct comparison of 2D gels with Western blots. Alignment of 2D gels and Western blots can be challenging when few spots are present on the blot. This report presents the results of a data set analysed following direct alignment of the Western blot to the 2D gel and indirect alignment of the 2D gel and Western blot to an intermediate image. Percentage coverage results varied by 1% between each alignment method.

 

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