Frequently Asked Questions - Infectious Disease Antigens

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.

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.

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 (Pierce Chemical) protein assay is used where Bovine Serum Albumin (BSA) is 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 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.