Frequently Asked Questions

Antibodies
How are the antibodies supplied?
All ABI antibodies are supplied frozen.
How pure are the antibodies, and how are they purified?
Monoclonal antibodies are purified by protein-A chromatography and are greater than 90% immunoglobulin by Coomassie Blue stained SDS-PAGE. No protein stabilizer is added. Polyclonal antibodies are generally supplied as undiluted/neat antiserum.
How should the antibodies be stored?
We recommend that all antibodies be aliquoted into use-size portions and stored frozen at -20° C or below. Avoid multiple freeze-thaw cycles as this will affect antibody activity. Do not dilute and freeze--make final dilution just prior to use.
How are the antibodies made?
All ABI monoclonal antibodies are made using standard hybridoma technology and are screened for function in immunofluorescence, ELISA, and Western blot prior to selection. Ascites is made in rats or Balb/c mice and is subsequently purified, tested in applicable assays, and made available for sale.
How is the protein concentration determined?
A modified Bradford protein assay is used with Bovine Gamma Globulin (BGG) as a protein standard. The use of BGG instead of BSA enables a more accurate determination of antibody concentration.
Will the antibodies work in IFA?
All ABI antibodies work in IFA. Each is tested using infected cells of the appropriate virus that have been acetone fixed, PBS washed, and air dried on a teflon-masked microscope slide. The antibody is diluted to 10 µg per milliliter (1:100) in 1X IFA Wash Buffer, applied at 15 µl per well, and incubated at 37°C in a humid chamber for 30 minutes. The slide is gently rinsed in a stream of 1X IFA Wash Buffer and washed twice for five minutes each in a stirred 1X IFA Wash Buffer bath. Goat anti-mouse Ig-FITC is applied at 1 to 2 drops per well and incubated for 30 minutes as before. The slide is rinsed and washed as before, dried, and 1 to 4 drops IFA Mounting Solution applied per well before applying a #1 thickness cover slip. The slide is then observed at 400X using a fluorescent microscope with a halogen lamp and fluorescein filter set.
Will the antibodies work in ELISA?
All ABI antibodies work in ELISA; however, it is critical to use an antigen preparation that has enough of the target protein at a sufficient purity to allow the antibody to "find" it. One of the major reasons for an antibody "not working" in ELISA is that quite often the antigen being used has so much non-relevant protein as compared to the target protein that the non-relevant protein occupies most of the available binding sites during coating. The target protein is not able to bind to the microplate plastic in sufficient quantity to allow detection by the antibody, and the results seem to indicate that the antibody did not work. Try a partial purification of the protein or, if the protein is structural (ie., part of the virion), try purified virus rather than an infected cell extract.
Will the antibodies work in Western blot?
Not all ABI antibodies work in Western blot. Please see the specification sheet for each antibody to determine its suitability for Western blot use. Epitopes that depend upon tertiary protein structure are made "unrecognizable" by the SDS that is used in the separation phase of the Western blot procedure. For those antibodies that do work in Western blot, the antigen is electrophoresed on a 4 - 15% polyacrylamide minigel at a density of approximately 16.7 µg of protein per centimeter of gel width and transferred to PVDF. The membrane is blocked with 1% calfskin gelatin/5% heat inactivated Normal Goat Serum in PBS/0.05% Tween 20 and allowed to air dry while suspended. The membrane is then cut into 3 mm strips, and one strip is incubated with one milliliter of 10 µg/ml (1:100) antibody in blocking buffer for two hours at 37°C. The strip is washed in three changes of PBST20 for five minutes each and incubated with 1 milliliter of diluted Goat anti-mouse IgG-AP (BioRad 170-6520) in 100 mM Tris 150 mM NaCl pH 8.0 supplemented with 5% heat inactivated Normal Goat Serum for one hour at 37°C. The strip is washed as before and developed with BCIP/NBT (BioRad 170-6432) per manufacturer's instructions. The dilution of 1:100 that is used in this assay is intended to check function only. It is not an "optimal" dilution that has been determined by ABI. The extent that an antibody can be diluted beyond 1:100 needs to be determined in the user's laboratory employing the user's exact methodology.
Cell Biology
Human Monocytes and Macrophages
Are human adult blood products tested for bloodborne pathogens?
All human adult blood cell products are tested for HIV-1, HIV-2, and HCV antibodies and for Hepatitis B surface antigen and HIV-1 core antigen by FDA approved tests.
Are source leukocytes from a single donor?
Yes. Source leukocytes are the residues of plateletpheresis of one human adult donor.
What is the relative quantity of T-cells, B-cells, and other specific white blood cell subpopulations in our mononuclear cell preparations?
Our mononuclear cell preparations are enriched for lymphocytes and monocytes. Monocytes represent 15-40% of the population. The remaining fraction consists of 15-40% B-cells, and 60-85% T-cells.
How pure are the elutriated monocyte suspensions?
Elutriated monocyte suspensions are >90% pure when analyzed by a variety of techniques. ABI documents purity during the elutriation process by multisizer analysis of purified monocytes, using statistical data to establish that >90% of the cell population has a mean cell size that is greater than small monocytes (approximately 8.5 microns). Final purity is confirmed by CD14 cell surface antigen distribution using immunofluorescence (IFA), with CD15 antigen tested by IFA as a control marker for granulocytes, which typically account for the majority of contaminating white blood cells in monocyte fractions. ABI customers with access to flow cytometry equipment have repeatedly confirmed ABI's monocyte purity estimates by this sensitive and reliable method.
How many cells do I plate per flask in order to establish mature macrophage cultures?
ABI recommends seeding a minimum of 4 x 105 monocytes per cm2, to as many as 8 x 105 monocytes per cm2. Then follow our culture recommendations that are provided with shipment to establish mature macrophage monolayers.
Can I trypsinize and subculture macrophages?
No. Trypsin or Trypsin-EDTA will not work to detach and disperse macrophages. Macrophages are terminally differentiated cells that are limited in their ability to be subcultured or expanded. The cells can be gently scraped and passaged, but many cells will not re-attach and cell division will not occur to yield confluent, mature monolayers. Therefore, it is best to plate the number of vessels (plates, flasks, etc.) needed and to plan NOT to subculture them.
How long will macrophages survive in culture?
Following our growth and handling recommendations, macrophage cultures can survive for 45-60 days before senescence. Cultures are most metabolically active up to about 30 days after seeding.
Can I cryopreserve and recover macrophages for future culturing?
Although successful cryopreservation and retrieval of functional macrophages have been documented in the literature, it is our experience that such manipulations give variable results and would not be recommended as a reliable method for ongoing research experiments (other than for research on the cryopreservation of macrophages).
Concentrated Viral Preparations
Which type of virus preparation is most suitable for: ELISA/Western blotting? Infectivity Assays and in vitro applications? Lymphocyte stimulation assays? Virus tropism/binding studies?
Generally, ABI recommends the following virus preparations for the applications listed:
  • ELISA/Western blotting: Purified virus or purified viral lysate.
  • Infectivity Assays and in vitro applications: Direct-pelleted or purified virus.
  • Lymphocyte stimulation assays: Custom prepared and inactivated purified virus.
  • Virus tropism/binding studies: Purified virus or custom prepared and inactivated purified virus.
What is meant by "Purified Viral Lysate"?
"Purified Viral Lysate" indicates a gradient purified virus preparation that has been partially or completely lysed and solubilized by detergent treatment, coupled with additional post-lysis treatment steps to increase purity and/or enhance inactivation efficiency.
Are viral lysates inactivated? How is that confirmed?
Viral lysates are inactivated, and inactivation is confirmed by cell culture infectivity assays.
For HIV-1, what is the relative amount of p24, gp120, and gp160 in a milligram of purified virus?
For HIV-1 IIIB, "single-banded" gradient purified virus will yield the following relative distribution of virus specific antigens: p24: 8-15% of total protein gp120: 0.10 - 1.0% of total protein gp160: 0.10 - 1.0% of total protein
Can you send me an SDS-PAGE and Western blot profile of your virus product?
Although PAGE/Western blot is part of our QC regimen, as a routine, ABI does not provide SDS-PAGE and Western blot profiles on our viral antigens.
For HIV-1, how can I equate the amount of p24 or RT activity to infectious units?

As a rule, it is not advisable to equate p24 or reverse transcriptase activity in terms of infectious equivalents for HIV, unless repeated infectivity assays of the same stock are performed and correlated with p24 or RT levels. Although the relative p24 or reverse transcriptase activity is positively correlated with virus infectivity in most cases, a true measure of virus infectivity (i.e., TCID50, plaque assay, LD50) as the basis for R & D is the most appropriate way to ensure reproducibility of results.

Immunoassay Stabilizer
How much does it cost to use?
The cost per test is dependent on the volume per well that is used to coat the solid support with antigen or antibody. The important point here is that a sufficient volume of Immunoassay Stabilizer must be present to completely immerse all adsorbed protein. If 100 µl is used to coat each well, approximately 125 µl per well of Immunoassay Stabilizer should be used. If other coating volumes are used, adjust the volume of Immunoassay Stabilizer accordingly.
Can I dilute Immunoassay Stabilizer?
No. The reagent has been developed for 1X use. Diluting it will dilute the stabilization effects as well as raise the assay background signal.
Will it work for all proteins?
In theory, yes. However, each protein must be checked individually for its characteristics in the presence of Immunoassay Stabilizer.
Can I use it for membrane assays?
Yes. Use Immunoassay Stabilizer as described after adsorbing your antigen or antibody. Allow the membrane to drip dry then transfer it to a desiccator.
How does immunoassay stabilizer work?
Proteins in aqueous solution depend upon interaction with water molecules to hold their shape. Removing the water forces the protein to change its conformation in order to attain a low molecular energy state. This causes previously functional epitopes to be rendered immunologically unrecognizable. Immunoassay Stabilizer essentially replaces the water as the protein is dried allowing the protein to retain much of its fully hydrated structure and function.
How much will it help my assay?
The benefit of using Immunoassay Stabilizer will vary by application but can be expected to improve the assay function and stability or considerably reduce the cost of making the assay.
Do I need to block non-specific sites after coating?
No. The Immunoassay stabilizer contains a blocking protein.
How important is the drying step?
The drying of the protein is crucial to retention of immunological activity over time. Residual water allows molecular movement and disrupts the stasis of desiccated storage. It is recommended that the protein be air dried to remove excess moisture and dried in a desiccated cabinet at room temperature overnight. In addition, packaging the dried, immobilized protein in a vapor barrier pouch with a desiccant is necessary for optimum stability.
Can I package the dried immunoassay plates in plastic zipper bags?

No. Plastic zipper bags allow air and gas interchange. The protein will reacquire water and lose stability. Heat sealable Mylar pouches are recommended.

Can immunoassay stabilizer be custom made?

Yes, Advanced Biotechnologies can custom manufacture immunoassay stabilizer with high quality ingredients of your choice to fit your assay requirements. The minimum volume is 100 liters.

Infectious Disease Antigens
How are the antigens supplied?
All ABI antigens are supplied frozen on dry ice. Vial sizes of 1.0 milliliter and 25 milliliter are offered to enable manufacturers to move directly from evaluation/qualification to production without incurring additional freeze-thaw cycles.
How pure are the antigens, and how are they purified?
ABI offers a great variety of antigens from viruses to pathogenic bacteria in different purities to address individual assay/cost requirements. We recommend that you contact us with your assay specifications so that we may suggest the appropriate form and purity. Purification methodology varies according to the individual product requirements as do the buffers in which the antigens are frozen.
How should the antigens be stored?
We recommend that all antigens be stored frozen at -70°C or below. Avoid multiple freeze-thaw cycles as this will affect antigen activity. Do not dilute and freeze--make final dilution just prior to use.
How are the antigens made?
ABI infectious disease antigens are made in infected cells using cell culture. Recombinant antigens are made in various systems, generally yeast or baculovirus. All are screened for function in ELISA and Western blot prior to release.
How is the protein concentration determined?
A modified BCA (Pierce Chemical) protein assay is used where Bovine Serum Albumin (BSA) is the protein standard.
Will the proteins work in ELISA?
All ABI infectious disease antigens work in ELISA; however, it is critical to use an appropriate primary antibody and conjugate dilution. Double-block titrations should be performed to determine the optimum use dilutions in your system.
Will the antigens work in Western blot?
All ABI infectious disease antigens work in Western blot according to the considerations mentioned for ELISA. For reference use, ABI uses the following procedure for Western blot (to be developed with human antiserum): The antigen is electrophoresed on a 4 - 15% polyacrylamide minigel at a density of approximately 15 µ of protein per centimeter of gel width and transferred to PVDF. The membrane is blocked with 1% calfskin gelatin/5% heat inactivated Normal Goat Serum in PBS/0.05% Tween 20 and allowed to air dry while suspended. The membrane is then cut into 3 mm strips, and one strip is incubated with one milliliter of 1:100 primary antibody in blocking buffer for one hour at 37°C. The strip is washed in three changes of PBST20 for five minutes each and incubated with 1 milliliter of diluted Goat anti-human IgG-AP (BioRad 170-6521) in 100 mM Tris 150 mM NaCl pH 8.0 supplemented with 5% heat inactivated Normal Goat Serum for one hour at 37°C. The strip is washed as before and developed with BCIP/NBT (BioRad 170-6432) per manufacturer's instructions.
Services
Electron Microscopy
Bioprocess Monitoring
How are small and/or large test articles prepared for virus quantitation services?

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 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.

How are test article cells, master, or production seed stocks evaluated using electron microscopy techniques?

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.

How are final results of these services reported?

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.

Immunolabeling
How do I know which type of immunolabeling procedure should be employed to properly label my target antigen of interest?
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.
How do I prepare my samples for immunolabeling procedures?

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.

Who provides the primary and secondary antibodies for immunolabeling services? How much volume of antibody should I provide?

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.

Due to some of the complexities that are involved with successfully performing immunolabeling techniques, we ask that before sending in your samples for immunolabeling services, you call and discuss your project with us directly over the telephone.
Negative Stain
How do I determine if negative stain electron microscopy is the right type of service to answer the questions I have in regard to my samples in suspension?

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.

Can biohazardous specimens be examined using this technique?

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.

Do my specimens in suspension need to be concentrated, and what kind of sample volume is required?

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.

Does Advanced Biotechnologies offer quantitation services for virus-related samples?
ABI offers negative staining quantitation services utilizing a technique developed over the past fifteen years.
What is Negative Stain Electron Microscopy?

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

Ultra-Thin
How do I determine if ultra-thin section electron microscopy is the right type of service to answer the questions I have regarding my cell culture or tissue samples?

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.

How do I prepare samples for ultra-thin section analysis?
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.
What do I receive in the way of data after my samples are completed?

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.

How long is the turn-around time for sample results?
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.
What is Thin-Section Electron Microscopy?

Thin-Section 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.

Test Kits
How are the kits supplied?
All ABI research use kits are supplied on cold packs. All necessary reagents are included as well as complete instructions on how to perform the assay and interpret the results.
What is the shelf life?
All kit reagents have been stabilized for a minimum of 18 months at 2 - 8°C storage from time of manufacture.
How should the kits be stored?
We recommend that all kits be stored at 2 - 8°C. Do not freeze.
What assay formats are available?
Immunofluorescence and ELISA kits are currently available in IgG format.
Are quantity discounts and automatic regular shipments offered?
There are a number of different options available. Please contact us for more information.
What equipment will I need to perform the assay?
Equipment needs are discussed in the assay instruction booklet. If you need to know equipment requirements prior to ordering, please order a package insert for the kit that you need.
How is the assay validated for "correct" results?
Both ELISA and IFA assays are optimized against uninfected control extract to show reactivity below the assay cutoff. In addition, Western blot and/or cell culture is used to confirm results. Normal sample sets where N >100 are used to establish specificity.
Viral Proteins
How are the proteins supplied?
All ABI proteins are supplied frozen on dry ice.
How pure are the proteins, and how are they purified?
All of the proteins, whether natural or recombinant, are purified to greater than 95% as judged by Coomassie Blue stained SDS-PAGE. Purification methodology varies according to the individual product requirements, as do the buffers in which the proteins are frozen.
How should the proteins be stored?
We recommend that all proteins be aliquoted into use-size portions and stored frozen at -70°C or below. Avoid multiple freeze-thaw cycles as this will affect protein activity. Do not dilute and freeze--make final dilution just prior to use.
How are the proteins made?
Natural proteins are made in infected cells using cell culture. Recombinant proteins are made in various systems, generally yeast or baculovirus. All are screened for function in ELISA and Western blot prior to selection.
Will the proteins work in ELISA?
All ABI proteins work in ELISA; however, it is critical to use an appropriate primary antibody and conjugate dilution. Double-block titrations should be performed to determine the optimum use dilutions in your system.
Will the proteins work in Western blot?
All ABI proteins work in Western blot according to the considerations mentioned for ELISA. For reference use, ABI uses the following procedure for Western blot (to be developed with monoclonal antibodies): The antigen is electrophoresed on a 4 - 15% polyacrylamide minigel at a density of approximately 4 µg of protein per centimeter of gel width and transferred to PVDF. The membrane is blocked with 1% calfskin gelatin/5% heat inactivated Normal Goat Serum in PBS/0.05% Tween 20 and allowed to air dry while suspended. The membrane is then cut into 3 mm strips, and one strip is incubated with one milliliter of 10 µg/ml (1:100) antibody in blocking buffer for two hours at 37°C. The strip is washed in three changes of PBST20 for five minutes each and incubated with 1 milliliter of diluted Goat anti-mouse IgG-AP (BioRad 170-6520) in 100 mM Tris 150 mM NaCl pH 8.0 supplemented with 5% heat inactivated Normal Goat Serum for one hour at 37°C. The strip is washed as before and developed with BCIP/NBT (BioRad 170-6432) per manufacturer's instructions.
I'm getting good Western blot reactivity, but at the wrong molecular weight. What is wrong?
It is rare for recombinant proteins to be the same molecular weight as their natural counterpart. Sequences are either deleted or added as a function of the restriction enzyme sites in the nucleic acid or the expression vector used to produce the proteins. Additionally, there are sometimes oligomers and partial cleavage products. Neither affects the function, but sometimes they will cause confusion. To alleviate doubt, use a monoclonal antibody to demonstrate the protein in a Western blot or ELISA. Please see the specification sheet for each protein for its correct molecular weight.