For reasons of price and performance, polyclonal antibodies are often the reagents of choice in laboratory applications. Affinity Biologicals offers an extensive line of antibodies to haemostasis related proteins. Although ABI does offer some murine monoclonal antibodies, most antibodies are polyclonal antibodies raised in sheep and goats. Because polyclonal antibodies contain antibodies to multiple recognition sites (epitopes) on an antigen the apparent affinity and avidity of antigen-antibody interactions is very high. This results in greater sensitivity and also means that the antigen is more likely to be recognized after denaturation or proteolytic processing. These are important considerations for applications such as immunoblotting from denaturing gels such as SDS-PAGE and in immunohistochemistry of fixed cells and tissues. Polyclonal antibodies are excellent for use in immunoassays where high sensitivity is required and are frequently the preferred reagent for immunoprecipitation techniques and activity neutralization assays. At ABI we have had good success using our polyclonal antibodies immobilized to resins to specifically immuno-deplete target proteins from citrated plasma and to recover the target protein from these resins with excellent recovery of activity.

Most Affinity Biologicals antibodies are available in several different formats, each differing in the level of refinement.

• Whole IgG is the base level product and refers to IgG antibodies purified from antiserum (usually sheep or goat) using multiple precipitation and dialysis steps. The purity of the IgG is typically 90% and is provided in a solution of HEPES buffered saline containing 50% glycerol (v/v). The titre is essentially the same as the starting antiserum and each vial typically contains the amount of IgG recovered from one millilitre of antiserum. This level of product is generally intended for use in applications such as immunoprecipitation, immunoelectrophoresis, immuno-depletion and activity neutralization assays.
• Affinity-purified IgG are antibodies further purified using immobilized antigen. Affinity-purification is an enrichment process in which whole IgG is passed over a resin to which antigen has been covalently attached. Non-reactive antibodies are washed away and specific antibodies are eluted under conditions that disrupt the antigen-antibody interaction. The result is a very high-purity product with a substantially higher titre than whole IgG. This level of product is intended for applications such as immunoblotting, immunostaining of cells and several types of immunoassays where the higher signal-to-noise ratio achieved with this enriched product is required.
• Enzyme labeled IgG is either whole IgG or affinity-purified IgG that has been conjugated with Horseradish Peroxidase as a reporter enzyme. These products are intended for use as labeled primary antibodies in applications such as immunoassay and immunoblotting.

Polyclonal antibodies typically recognize numerous binding sites on protein antigens, which in concert produce very high affinity binding constants. In many cases, antigens are recognized in denaturing as well as non-denaturing conditions. In general, applications in which polyclonal antibodies work well include:

•   ELISA (direct, double antibody or competitive)
•   Gel Precipitation (immunoelectrophoresis, immunodiffusion)
•   Neutralization of antigen activity
•   Depletion of antigen by immune-affinity chromatography
•   Immunoblotting (Western blotting) from SDS-PAGE
•   Immunostaining (ImmunoHistoChemistry)
•   Flow cytometry

It should be noted that while many of our antibodies may be suitable in the above applications, only direct ELISA and Gel Precipitation testing is done on every lot of antibody as part of product quality control and approval.  ABI will guarantee performance in direct ELISA and Gel Precipitation applications only, except where otherwise indicated.

The optimal concentration of antibody for any particular application will vary depending on the antibody format as well as the detection method used. ABI strongly suggests that each antibody be titrated by the end user to determine the optimal working concentration for each particular application. However, based on our experience we can make some general recommendations regarding antibody concentration ranges that can be used as a starting point in optimizing assay conditions.

The following chart contains general recommendations only and may not apply to all our products.

All antibody products are dispensed with a 5% excess volume.

ABI antibodies are supplied in a solution of HBS-glycerol (10mM HEPES, 0.15M NaCl, pH 7.4, 50% glycerol (v/v)) to increase the long-term stability of the IgG. The glycerol acts to stabilize the IgG by reducing hydration and also helps to suppress microbial growth. We recommend storage between -10°C and -20°C. At these temperatures the solution will not freeze. The solution of HBS-glycerol allows multiple withdrawals from the vial without the need for numerous freeze-thaw cycles that reduce antibody potency. We recommend that the vial be warmed to ambient temperature and gently mixed before pipetting to reduce the viscosity. The antibodies are supplied in microcentrifuge tubes and residual reagent may be gathered from the walls and cap with a brief centrifugation. Glycerol is hygroscopic and therefore care should be taken to ensure that caps are re-fastened securely with the O-ring intact before putting the vial back in the freezer.

Any of our antibodies can usually be dialyzed into any buffer according to customer specifications. A special handling fee of 20% of the purchase price applies to orders of 10 vials or less.