Frequently Asked Questions - Electron Microscopy

The test article supernatant, whether from a large or a small test article, is clarified by filtration or centrifugation, then concentrated by density gradient ultracentrifugation techniques.

The concentrated test article is then prepared by mixing a known concentration of latex spheres with the unknown number of virus particles from the test article. This mixture is mounted onto grids, negatively stained, and examined in the transmission electron microscope. Both latex spheres and virus particles are enumerated, and the concentration of virus particles in the test article is determined by using ratio formulas.

Test articles from cell banks, master seed stocks, or production seed stocks are examined using ultra-thin sectioning.  Test articles are prepared following standard written protocols, then examined by transmission electron microscope for the presence of virus or other microbial contaminants.  Examination is conducted at low and high magnifications appropriate for the test articles.

This assay allows for the determination and identification of viruses present within, or secreted from, the cells used to produce biopharmaceuticals.

The following is a general guideline for the type of immunolabeling procedure utilized, depending upon the location of the target protein.  Contact ABI to speak with a technical support representative about which immunolabeling procedure is most appropriate for your needs.

• Pre-Embedding Immunolabeling: Pre-Embedding Immunolabeling is used primarily for the detection of cell-surface associated antigens, as well as external envelope antigens of budding or extracellular virus particles. The sample must be unfixed and shipped by overnight delivery service to ABI at 4°C.
• Negative Stain Immunolabeling: Negative Stain Immunolabeling is performed on specimens found in concentrated suspensions, such as virus particles, cell fragments, macromolecules, and bacterial specimens. Both internal and surface-related target proteins on minute specimens can be immunolabeled using this procedure.
• Post-Embedding Immunolabeling: Post-embedding labeling is used exclusively for the immunolabeling of intracellular proteins found within cells and tissues.

The following is a general guideline for preparing samples according to the type of immunolabeling procedure utilized.  Contact ABI to speak with a technical support representative about preparing your samples.

• Pre-Embedding Immunolabeling: In pre-embedding immunolabeling, the cells are exposed to both the primary antibody and secondary antibody in situ, before normal fixation procedures. Because we work with the cells live or in situ, the cell line is sent to ABI fresh and without fixation in its normal cell culture media.
• Negative Stain Immunolabeling: Negative stain immunolabeling is performed on fresh specimens in suspension before chemical fixation is employed, allowing the antigenicity of the sample to be intensified. The specimens are sent either fresh or frozen by overnight carrier to ABI and are handled as a "live" sample.  Specimens to be examined by negative staining techniques may have to be concentrated before immunolabeling techniques.  Contact ABI to learn if your sample must be concentrated.
• Post-Embedding Immunolabeling: For post-embedding labeling procedures, immunolabeling is carried out on cells and tissues that have been fixed and embedded in a plastic resin. An embedded block containing the specimen is ultra-thin sectioned, allowing access to antigens as they become exposed on the surface of the sections.

When using this procedure, the cells and/or tissue must be fixed using a chemical fixative before shipment to ABI.  Contact ABI for more information about fixing your sample.

For immunolabeling experiments, regardless of what type of procedure is to be employed, it is the responsibility of the investigator to provide the primary antibody and information regarding the titer of the antibody.  ABI will provide the secondary antibody or conjugate used for immunolabeling the specimens.

Negative staining gives an investigator an ultrastructural view of particulate specimens which are found exclusively in suspension, including virus particles, lipoprotein particles, cell fragments, bacteria, and other macromolecules.  This procedure provides an intimate morphological view of the ultrastructure of specimens in suspension, including fine-surface details and interior components. Using certain procedures, it also permits the quantitation of particles per mL in suspension. No other method is known by which ultrastructural details of minute specimens can be visualized with high resolution.

Contact ABI and speak with a technical support representative to learn if Negative Stain electron microscopy is appropriate for your needs.

To provide the best results possible, each specimen to be examined must be at a minimum particle concentration of 2.0 × 10⁷ per mL.

If the sample is difficult to concentrate, ABI has a full-service BSL-3 laboratory where samples can be concentrated in a matter of a few hours to meet the specifications of negative staining techniques.

ABI offers negative staining quantitation services utilizing latex bead ratio methods to quantify virus in samples.  Samples can be from 1X cell culture fluids or from concentrated virus samples.

Negative staining is useful for quantitative and qualitative studies of viruses or other microbes. Specimen preparations are stabilized in electron-dense salts and examined by transmission electron microscopy. When used for virus quantification studies, Latex sphere ratio methods are used, with results expressed as viral particles per mL (based on the sample concentration as supplied).

The main advantage ultra-thin section microscopy has over any other type of service is that cells and tissues can be examined at an ultrastructural level. This technique provides details of the interior of the cells and tissues, including organelle structure, morphology of macromolecules, such as virus particles, and does so without causing distortion of the cells or tissues.

Contact ABI and speak with a technical support representative to learn if ultra-thin section electron microscopy is appropriate for your needs.

ABI will provide you with an appropriate aldehyde-based fixative, depending on the type of sample you are working with, along with a washing buffer and a protocol for sample fixation. ABI will send you this material free of charge.

Ultra-thin sectioning reveals fine details of cell and tissue ultrastructure as well as the structure of viruses or other present microbes.  Cells or tissues embedded in plastic are thin sectioned, mounted and stained, and examined by transmission electron microscopy.