Frequently Asked Questions - Antibodies

All ABI antibodies are supplied frozen.

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.

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 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.

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 (ie., 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 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.