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Cell centrifugation

It is useful, and often necessary, to break apart the cell and separate the cellular organelles into individual fractions for further study. Once the organelles are separated it is easier to identify pathways of disease or basic biochemical functions within the cell. One easy, and relatively gentle (if performed properly) method to do this is via cell centrifugation. This process involves three main steps: homogenization of the cellular extract, differential sedimentation, and density gradient centrifugation. The homogenization step breaks apart the cell membranes and releases the organelles into one big cellular soup. Then, the first step of centrifugation begins with differential sedimentation. This results in a rough separation of organelles. The fractions are further separated into clean fractions by density gradient centrifugation, which uses a material gradient (often sucrose) to help separate the organelles by density during centrifugation. 

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Cell based assays

Cell based assays are used to quantify cellular function, measure how stimuli affect cells, or to localize an effect within the cell. The cells are live and intact, and require the use of fluorescent tags and chemiluminescent or colorimetric enzymes. The quantification is performed by flow cytometry or microscopy. This is very different from studies of protein or nucleic acid which require destruction of the cell and isolation of those components from cell lysate. A cell based assay is conducted entirely within live, intact cells. The goal is to understand a cellular process, localization of a molecule or drug to a cellular compartment, or to measure how cells react to a substance. Cell based assays are usually performed in tightly controlled cell lines to test for a wide range of behaviors:

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How does magnetic bead separation work?

Magnetic bead separation is a quick, efficient, clean process that scientists use to replace filtration, centrifugation and separation techniques. Magnetic beads and particles are used as carriers of antigens, antibodies, catalyzers, proteins and nucleic acids, enabling action on cells, bacteria, viruses and other biological entities.
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Magnetic DNA Purification: History and recent developments

 

Genomic sequencing and molecular analysis have become so standard to biological research that they are now all but required for work to be published in high profile journals. Outside the scientific realm, magnetic DNA purification is also fundamental to forensic analysis in the criminal justice system. Therefore, a method to rapidly extract and purify high-quality DNA and RNA from a variety of tissues is indispensable, and improvements to the technique are desired.

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Mistake #5 in CLIA IVD-kit manufacturing: Inappropriate safety precautions when working with magnetic fields

The first four mistakes we described in the last weeks are related to the production process of CLIA IVD-kits. However, even if you get a perfect reproducible, high performant process, it is a last mistake you should avoid. We have frequently see IVD-manufacturers to adopt solutions implying high safety risk for the operators and the equipment.

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Mistake #4 in CLIA IVD-kit manufacturing: Neglecting process scalability

When developing a CLIA IVD-kit, the initial focus is on the biomarker and how to coat the magnetic beads. Biomagnetic separation conditions usually get swept to one side.

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Mistake #3 in CLIA IVD-kit manufacturing: Defining the process based purely on the separation time

Not all mistakes made in CLIA-IVD kit manufacturing involve the magnetic rack itself. Besides the two mistakes we reviewed during the last weeks, the third mistake we have detected involves process validation. Biomagnetic separation processes are often validated solely by specifying a separation time.

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Mistake #1 in CLIA IVD-kit manufacturing: Blaming the magnetic beads

Product development is a time-consuming, expensive process for CLIA-IVD kit manufacturers. There are several steps involved: 

  • Selecting the biomarker
  • Choosing the right coupling 
  • Selecting the right magnetic bead 

You are well versed with the first two points but what is “the right bead”? Assuming you have the right biomarker and a perfect coupling, the ideal magnetic bead should have the following properties:

  • High recovery/fast separation, compatible with the timing of the analyzer step. It needs to be fast enough during large-scale production processes without high bead and coupled biomarker losses.
  • No aggregation problems. Beads should be easy to re-suspend. It makes no sense to separate quickly if several additional sonication steps are required, which are difficult processes to control/implement in large volumes.
  • Low kit-to-kit variability. Batch aliquots (typically less than a milliliter) of production batches (liters scale) must be consistent. If not, variability causes problems when interpreting the results in the analyzer.

 

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How can we specify a Biomagnetic Separation Process?

When a new CLIA-IVD kit is transferred from R&D to production, all the manufacturing protocols should be adapted to the new throughput and volume. Biomarker specifications, buffers and coating protocols would benefit from the cumulated experience in non-magnetic kits. Coupling an antibody to magnetic beads is quite similar to doing it in colloidal gold or latex particles. But the washing protocols using Biomagnetic Separation are something new. The use of classical (and dirty) centrifugation method makes not so much sense when we can use the magnetic properties of the beads. Similar reasoning applies to the use lateral flow filtration or other complex and time-consuming non-magnetic separation techniques.

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