Frequently Asked Questions
Monoclonal
13-210-100
Yes. We recommend Antigen Retrieval Citra Plus Solution from BioGenex. Follow the manufacturer’s instructions for the microwave antigen retrieval method for Formalin-fixed tissue sections.
Use HRP or Alk Phos Conjugated Anti-Rat IgG (H+L).
A 1:100 dilution is recommended (Range: 1:100-1:1000).
To use higher dilutions, titrate on KS + tissue sections for optimum use dilution.
Incubate the MAb for 60 minutes at room temperature (approximately 25°C).
Use Universal Blocking Buffer to inhibit endogenous tissue peroxidase or phosphatase before staining with primary antibody (KSHV/HHV-8 MAb to ORF73 (anti-LNA-1)).
Yes. Follow the immunostainer manufacturer’s instructions.
All ABI antibodies are supplied frozen.
Monoclonal antibodies are purified by protein-G 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.
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.
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.
A modified BCA 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.
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.
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 (i.e., part of the virion), try purified virus rather than an infected cell extract.
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 gel then transferred to a nitrocellulose membrane. The membrane is blocked with 5% non-fat dry milk in Phosphate Buffered Saline + 0.1% Tween-20 (PBST) and allowed to dry. The membrane is then cut into strips, and incubated with monoclonal antibody, at an appropriate dilution, in blocking buffer. The strip is washed in three changes of PBST for five - ten minutes each and incubated with diluted Goat anti-mouse IgG-AP in blocking buffer. The strip is washed as before and developed with BCIP/NBT as per manufacturer's instructions.
Elutriated monocyte suspensions are >90% pure when analyzed using several 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).
ABI customers with access to flow cytometry equipment have repeatedly confirmed ABI's monocyte purity estimates by this sensitive and reliable method.
ABI recommends seeding a minimum of 4 x 105 monocytes per cm2, to as many as 8 x 105 monocytes per cm2. Then, follow the culture recommendations provided with your shipment to establish mature macrophage monolayers.
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.
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.
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 are not recommended as a reliable method for ongoing research experiments (other than for research on the cryopreservation of macrophages).
"Purified Viral Lysate" is a gradient purified virus preparation that has been partially or completely lysed and solubilized by detergent treatment.
Viral lysates are inactivated. Inactivation is confirmed by cell culture infectivity assays indicated in the product’s Certificate of Analysis.
HIV-1IIIB 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.
Generally, ABI recommends the following virus preparations for the following applications:
- 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.
Although SDS-PAGE/Western blot is part of our QC regimen, ABI does not routinely provide SDS-PAGE and Western blot profiles on our viral products.
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.
Quantitated DNA controls are supplied at 1-2 × 104 copies/µL (with the exception of BK and JC polyomaviruses, which are supplied at 1-2 × 105 copies/µL). The copy number of Quantitated DNA controls is determined by real time PCR using the Roche LightCycler®.
The concentration of a concentrated solution of the purified DNA to be quantitated is determined by fluorometry using PicoGreen®. Fluorometry is preferred to absorbance as a measure of the concentration since interference by possible contaminants such as RNA, single-stranded DNA, oligonucleotides or other nucleic acid breakdown products is virtually eliminated using fluorescence with PicoGreen®.
From this concentration, the copy number of the concentrated DNA is calculated using the formula above. This copy number is used to prepare a standard curve from 101 to 107 copies/µL. A dilution of quantitated DNA is prepared at the approximate selling dilution. This dilution is run in triplicate against the standard curve in real time PCR with the Roche LightCycler® and the concentration calculated by the LightCycler® software.
ABI does not recommend storing dilutions of these controls under any conditions. All dilutions should be made immediately before use and used promptly. We have observed that dilutions used for standard curves typically “lose” copy number with time — sometimes as soon as an hour or so after dilution. This is especially common at dilutions less than 100-1000 copies per microliter.
The concentration of these DNAs is determined using fluorometry with the fluorescent dye PicoGreen®. These DNAs are intended primarily as positive controls in PCR reactions.
No. Since these controls contain both cellular and viral DNA in unknown and varying proportions, it is not possible to calculate or estimate number of viral copies/µL.
The viral copy number cannot be calculated exactly but may be estimated as follows. Using the formula and knowing that the molecular weight of the human genome is approximately 2 × 1012 g/mole, the number of copies of cellular DNA can be calculated to be approximately 15,000 copies/µL at a DNA concentration of 50 ng/µL. From the literature, there is reported to be 200-800+ copies/cell of HPV 16 per CaSki cell (600 copies/cell is the number commonly used). There are reported to be 10-50 copies of HPV 18 per HeLa cell (25-50 copies per cell is commonly used). Using 600 copies of HPV 16/CaSki cell, we can estimate approximately 9 x 106 copies/µL of HPV 16 in a 50 ng/µL DNA solution. Similarly, using 50 copies of HPV 18/HeLa cell, we can estimate approximately 750,000 copies/µL of HPV 18 in a 50 ng/mL DNA solution.
Viral transformed cell DNA controls are isolated from virus transformed cells by SDS/Proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. Therefore, these controls contain cellular genomic DNA with multiple copies of HPV 16 or 18 integrated in the cell genome. The concentration of these DNAs is determined using fluorometry with the dye PicoGreen®. These DNAs are intended primarily as positive controls in PCR reactions, although an estimation of the viral copy number can be obtained as described above.
Viral infected cell DNA controls are isolated from virus infected cells (not from purified virus) by by SDS/Proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. These controls contain both cellular and viral DNA in unknown and varying proportions. Therefore, it is not possible to determine the number of viral copies/µL. The concentration of these DNAs is determined using fluorometry with the dye PicoGreen®. These DNAs are intended primarily as positive controls in PCR reactions, not for quantitation.
Fluorometric determinations using PicoGreen® virtually eliminate any skewing of the absorbance readings by interfering contaminants such as RNA, single-stranded DNA, oligonulcotides or nucleotides.
ABI provides virus infected cell DNA controls, viral transformed cell DNA controls, and Quantitated DNA controls for viruses, bacteria, a parasite (Toxoplasma gondii) and a mollicute (Mycoplasma pneumoniae).
- Viral infected cell DNA controls are isolated from virus infected cells (not from purified virus); therefore, these controls contain both cellular and viral DNA in unknown proportions. They are supplied at a DNA concentration (viral plus cellular DNA) of 50 ng/µL.
- Viral transformed cell DNA controls are from cells transformed by human papillomavius (HPV) type 16 or type 18. They are supplied at a cellular DNA concentration of 50 ng/µL.
- Quantitated DNA controls (copy number controls) are prepared from purified viral DNAs which have concentrations determined by fluorometry. Quantitated DNA controls are supplied at 1-2 × 104 copies/µL (with the exception of BK and JC polyomavirus DNAs which are supplied at 1-2 × 105 copies/µL).
Yes, it is possible to calculate the approximate number of viral copies/µL for these DNA controls using the formula below:
Notice that the DNA concentration in the formula is in g/µL, not ng/µL.
- g/µL = ng/µL × 10-9
- 50 ng/µL = 50 × 10-9 g/µL
The cost per test depends on the volume used to saturate the solid phase already coated with antigen or antibody. A sufficient volume of Immunoassay Stabilizer must be present to completely cover unbound places on protein coated solid phase. If, in an ELISA assay, 100 µL of protein is used to coat each well, then approximately 150 µL per well of Immunoassay Stabilizer should be used. If other coating volumes are used, adjust the volume of Immunoassay Stabilizer accordingly.
No. The reagent has been developed to use as provided and should not be diluted further. Diluting it will weaken the stabilization effects and will raise the background.
In theory, yes. However, each protein must be checked individually for its characteristics in the presence of Immunoassay Stabilizer.
Yes. Use Immunoassay Stabilizer according to the protocols found on the Immunoassay Stabilizer product page. Allow the membrane to drip dry at room temperature. Use it while it is still wet, or completely dry it for future use.
Protein in aqueous solution depends upon interactions with water molecules to hold its shape. Removing the water forces the protein to change its conformation state to attain a low molecular energy state. This causes previously functional epitopes to be less accessible. Immunoassay Stabilizer essentially replaces the water as the protein is dried, allowing it to retain much of its fully hydrated structure and function.
The benefits of using Immunoassay Stabilizer will vary by application, but can be expected to improve the performance of an assay and can reduce the cost of making the assay.
No. The Immunoassay Stabilizer contains a blocking protein.
Drying the protein is crucial to maintaining immunological activity over time. The optimum stability can be only achieved when well-dried protein is appropriately stored in airtight pouches along with desiccants/indicator.
No. Plastic zipper bags allow air and gas interchange, causing the protein to lose stability and activity. Heat-sealable, airtight pouches are recommended instead.
Yes. ABI can manufacture custom immunoassay stabilizer solutions with high quality ingredients of your choice to fit your assay requirements. A 100 L minimum applies to orders. Contact ABI for more information about ordering custom Immunoassay Stabilizer formulas.
All ABI antigens are supplied frozen on dry ice. Vial sizes of 1 mL and 25 mL are offered to enable manufacturers to move directly from evaluation/qualification to production without incurring additional freeze-thaw cycles.
ABI offers a variety of antigens, from viruses to pathogenic bacteria, in different purities to address individual assay/cost requirements. Purification methodology varies according to the individual product requirements, as do the buffers in which the antigens are frozen.
We recommend that you contact us with your assay specifications so that we may suggest the appropriate form and purity.
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.
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.
A modified BCA protein assay is used with Bovine Serum Albumin (BSA) as the protein standard.
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.
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 monoclonal antibodies): The antigen is electrophoresed on a 4 - 15% polyacrylamide gel then transferred to a nitrocellulose membrane. The membrane is blocked with 5% non-fat dry milk in Phosphate Buffered Saline + 0.1% Tween-20 (PBST) and allowed to dry. The membrane is then cut into strips, and incubated with monoclonal antibody, at an appropriate dilution, in blocking buffer. The strip is washed in three changes of PBST for 5 - 10 minutes each and incubated with diluted Goat anti-mouse IgG-AP in blocking buffer. The strip is washed as before and developed with BCIP/NBT as per manufacturer's instructions.
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.
Although the turnaround time does somewhat depend on the number of samples submitted, it is usually less than 2-4 weeks from the date ABI receives the samples, signed quote, purchase order, and any other applicable materials. Contact ABI for more information about turnaround times pertaining to your order.
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 must be packaged and shipped according to current shipping regulations.
ABI does not handle or process the following specimens:
- Biosafety Level-4 specimens
- Microbes and toxins on the Select Agents and Toxins Registry
- Any other high-risk airborne agents
If you are unsure whether your specimen falls into one of these categories, please contact ABI to speak with a member of our technical support staff.
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.
Final results of electron microscopy services generally include 4 - 6 low and high magnification electron micrographs, which are photographed and labeled according to the final results obtained.
Results also include a final report consisting of detailed observations and summaries of the methods used.
All ABI research use kits are supplied on cold packs. Kits include the necessary reagents and complete instructions for performing the assay and interpreting the results.
Store all kits at 2 - 8°C. Do not freeze.
IgG IFA and ELISA kits are currently available in 96-well (12 x 8-well strip) format.
Many options are available. Contact ABI for more information.
Equipment needs are discussed in the assay instruction booklet. If you need to know equipment requirements prior to ordering, download the instruction booklet from the appropriate kit’s product page, or contact ABI to order a booklet.
The assay is considered valid if positive and negative controls and specimen diluent results are within the specification range. For ELISA kits, please see the “Quality Control” section of the product booklet. For IFA kits, please see the “Interpretation of Results” and “Additional Information on Interpretation” sections of the product booklet.
All ABI proteins are supplied frozen on dry ice.
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.
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.
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 reactivity in ELISA and Western blot prior to selection.
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.
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 gel then transferred to a nitrocellulose membrane. The membrane is blocked with 5% non-fat dry milk in Phosphate Buffered Saline + 0.1% Tween-20 (PBST) and allowed to dry. The membrane is then cut into strips, and incubated with monoclonal antibody, at an appropriate dilution, in blocking buffer. The strip is washed in 3 changes of PBST for 5-10 minutes each and incubated with diluted Goat anti-mouse IgG-AP in blocking buffer. The strip is washed as before and developed with BCIP/NBT as per manufacturer's instructions.
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.
A modified BCA protein assay is used with Bovine Serum Albumin (BSA) as the protein standard.
