Welcome to neotem Bioanalytics

Exploring the Invisible: TEM expertise for powerful sample analysis

Negative staining: Clarity in shadows


VLP 1 neotem_1
Bakterium_50000x
Parapoxvirus


For the detection of adventitious agents in bulk harvest samples and the qualitative and quantitative analysis of suspended particles in liquid sample matrices, we utilise the method of negative staining TEM (nsTEM).

nsTEM:
The test item is embedded in a heavy metal salt solution which acts as a contrasting agent deflecting electrons due to its high electron density. As a result the embedded particles appear bright against a dark background when imaged using transmission electron microscopy.

nsTEM is also ideally suited to gain visual feedback on your sample quality in the various steps of upstream and downstream processing (e.g. detect background particles, cell debris, sample impurities, aggregates).

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Our state-of-the-art method of QnsTEM for the detection of adventitious agents in unprocessed bulk harvest samples

Retrovirus-like particles are released directly into the cell culture media and should be detected. Furthermore, additional adventitious agents which could have been introduced to the process should also be detected. Unprocessed cell-free culture supernatant is thus investigated using negative staining TEM. 

After separating the cells from the test item via centrifugation, possible virus or virus-like particles can be concentrated in the supernatant by ultracentrifugation. A defined volume of this test item is then stained with heavy metal salts. Biological structures are embedded in the staining agent and not directly stained (negative staining). These structures then appear bright against a dark background when visualising with TEM.

Quantitative Negative Staining TEM (QnsTEM) is an improved approach designed
to enhance the sensitivity, accuracy and robustness of virus detection and quantification in
biological suspensions. QnsTEM eliminates the need to mix beads into the sample by utilizing
reference grids for calibration, thereby reducing variability and improving reproducibility. The
method provides a state of the art and more reliable alternative to existing TEM
based techniques for adventitious agent detection in bulk harvest samples and for the
qualitative investigation of particles. The QnsTEM method enables accurate and precise
quantification of virus particles by transmission electron microscopy using a validated
calibration approach, which has been validated using well characterized reference materials with titers determined by qPCR.

To ensure that the test is executed in the defined parameters of the validation, system suitability criteria (SSC) were defined. Only after all SSCs have been successfully passed, the test item grid is then screened for adventitious agents.
 
Results and the corresponding Final Reports (CoA) can be issued within 5 working days.