Frequently Asked Questions - Electron Microscopy

For virus concentration, detection, and quantitation of large and small-volume test articles, the test article supernatant is concentrated by either ultracentrifugation and direct pelleting or by sucrose density gradient purification and ultracentrifugation.

The concentrated test article to be quantitated is prepared by mixing a known concentration of latex spheres and 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 and production seed stocks are prepared for ultra-thin sectioning, following standard written protocols, and are then examined for the presence of virus or other microbial contaminants in the transmission electron microscope at high magnifications.

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

The final results include six-to-eight high-magnification electron micrographs, which are photographed and labeled according to the final results obtained.

The final report includes complete documentation of all assays, techniques, procedures, references, and calculations utilized in performing these services. Also included in this report is a precise, interpretive summary of the final results obtained.

The following is a general guideline for the type of immuno-labeling procedure utilized, depending upon the location of the target protein:

• 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.
• 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.

For 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 would be sent to ABI fresh in their normal cell culture media.

Negative stain immunolabeling is performed on fresh specimens in suspension before chemical fixation is employed, thus allowing for the antigenicity of the sample to be intensified. The specimens would be sent to ABI either fresh or frozen and would be handled as a "live" sample.

Specimens to be examined by negative staining techniques may have to be concentrated before immunolabeling techniques.

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, thus allowing access to antigens as they become exposed on the surface of the sections.

Following this procedure, the cells and/or tissue must be fixed using a chemical fixative before shipment to ABI. Extensive testing of the fixatives and chemicals used during processing is performed by both ABI and the investigator to maximize the probability of successful immunolabeling.

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, as well as information regarding the recommended concentrations and/or dilutions to be used for the experiment.

ABI will provide the secondary antibody or conjugate used for immunolabeling the specimens.

We strongly recommend that the investigator provide at least 200 microliters of the antibody to be utilized for immunolabeling procedures, although the total volume needed does depend on both the type of service to be performed and the strength of the primary antibody being used.

The process of negative staining allows 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 gives 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 milliliter in suspension. No other method is known by which ultrastructural details of minute specimens can be visualized with high resolution.

The Electron Microscopy Laboratory at Advanced Biotechnologies Inc was designed for work with biohazardous specimens. Fresh or frozen Biosafety Level-3 specimens can be shipped to the laboratory where they are prepared following written protocols.

Biohazardous specimens need to be packaged and shipped according to current shipping regulations.

ABI does not handle or process Biosafety Level-4 specimens or any other high-risk airborne agents.

In order to provide the best results possible, it is very important that each particular specimen to be examined is at a minimum concentration of 2.0 × 10⁷ per milliliter.

If concentration of the sample is difficult, 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 a technique developed over the past fifteen years.

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.

There is no other method available to clearly and safely visualize ultrastructural details of biological samples.

ABI will provide you with an appropriate aldehyde-based fixative, depending on the particular type of sample you are working with, along with a washing buffer and an in-depth protocol detailing all the manipulations to be done during sample fixation. ABI will send you this material free of charge.

You will receive at least eight electron micrographs per sample, printed on 8" by 10" paper, giving you a precise, overall view of your samples. Electron micrographs are taken at a range of magnifications, normally ranging from 2,500x to 100,000x, depending on the type of sample being examined.

You will also receive an in-depth report, detailing the materials and methods utilized in preparing your samples, as well as an interpretive summary of the results of your samples.

Although the turn-around time does somewhat depend on the number of samples submitted, it usually takes less than 10 working days from the date ABI receives the samples.