- Aren’t monoclonal antibodies always better than polyclonal antibodies?
- What is meant by “whole IgG” and “affinity-purified” antibody formats?
- What is the best concentration to use these antibodies for ELISA and immunoblotting applications?
- What is provided in the VWF-EIA or other paired antibody products for ELISA? Are these actual kits?
- I am interested in antibodies that will react or cross-react with hemostasis related proteins in other species. Will some of the antibodies raised to human antigens cross-react with the rabbit or rat counterparts?
- Will these polyclonal antibodies recognize the unactivated, activated and complexed forms of my protein? What about recombinant constructs of protein fragments?
- Why are ABI antibodies supplied in a solution of buffered glycerol. What are the practical implications of working with such a viscous solution?
- Are the ABI antibodies available in solvents other than HBS-glycerol?
Aren’t monoclonal antibodies always better than polyclonal antibodies?
Not true. Properly prepared and characterized polyclonal antibodies are excellent reagents and when affinity purified they can easily compete with monoclonal antibodies in most applications. 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. The result is greater sensitivity, which also means that the antigen is more likely to be recognized even after denaturation or proteolytic processing. This is an important consideration in applications such as immunoblotting from denaturing gels such as SDS-PAGE and in immunohistochemistry on fixed tissue sections. 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 the purification of target proteins from the resins in high purity with excellent recovery of activity. For reasons of price and performance polyclonal antibodies are often the reagents of choice.
What is meant by “whole IgG” and “affinity-purified” antibody formats?
Most all of our polyclonal antibodies are available in several formats, which refer to the level of purification or refinement. The first level format is referred to as “whole IgG” and is an IgG fraction prepared directly from antiserum. The titre is essentially the same as the starting antiserum but in the purified whole IgG format it is a more stable product. Affinity-purified IgG is an enrichment process in which whole IgG is passed over immobilized antigen. Non reactive antibodies are washed away and specific antibodies are then eluted. This is a highly purified product with a substantially higher titre than whole IgG.
What is the best concentration to use these antibodies for ELISA and immunoblotting applications?
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.
|Format||Immunoblotting (ECL detection)||ELISA (capture)||ELISA (detecting)|
|Whole IgG (IG)||5-25 ug/ml||20-50 ug/ml||NA|
|Affinity-Purified (AP-IgG)||0.5-5 ug/ml||10 ug/ml||NA|
|IgG Peroxidase (HRP)||5 ug/ml||NA||10 ug/ml|
|AP-IgG Peroxidase (AP-HRP)||1 ug/ml||NA||2 ug/ml|
What is provided in the VWF-EIA or other paired antibody products for ELISA? Are these actual kits?
The paired antibody EIA products are not diagnostic kits or Analyte Specific Reagents. They consist of paired capture and detecting antibodies that have been titrated and optimized for use in “in-house” or “home-brew” sandwich style ELISA assays in a research setting. The product as provided contains sufficient capture and detecting antibodies for five full 96-well microplates and contains a detailed protocol sheet containing directions for use, recipes for solutions and sources for additional materials required. These products are intended for the experienced user and provide an economical alternative to more expensive pre-manufactured kits.
I am interested in antibodies that will react or cross-react with hemostasis related proteins in other species. Will some of the antibodies raised to human antigens cross-react with the rabbit or rat counterparts?
In some cases yes. In general polyclonal antibodies have wider species cross-reactivity than murine monoclonal antibodies. The species cross-reactivity of an antibody depends on factors such as the evolutionary distance of the test species from the host animal and the species from which the antigen was purified as well as the application in which the antibodies are being used. Some proteins are more highly conserved among mammalian species and antibodies raised to such proteins tend to demonstrate a greater degree of species cross-reactivity. For example, the goat anti-human von Willebrand Factor (vWF) has been shown to cross react with vWF from rabbit and rat plasma while the sheep anti-human F.VIII seems to react with human F.VIII only. Wherever possible we have tried to purify the antigen from the target species and to make antibodies to these protein antigens. ABI is committed to providing the hemostasis researcher with a complete line of immunoassay tools for human and animal research. Information from historical lots on typical species cross-reactivity for the different antibodies in our product line is available in a table in the Technical Information section. This is an ongoing process so if you can’t find what you’re looking for please contact us.
Will these polyclonal antibodies recognize the unactivated, activated and complexed forms of my protein? What about recombinant constructs of protein fragments?
In general, polyclonal antibodies are less sensitive to conformational and minor structural changes within the antigen protein than are monoclonal antibodies. Due primarily to the very high affinities and the multiple recognition sites with which polyclonal antibodies can bind the antigen proteins, even complexed forms (such as enzyme-inhibitor complexes) and fragments (such as proteolytic cleavage products or recombinant products) are still recognized by polyclonal antibodies.
Why are ABI antibodies supplied in a solution of buffered glycerol. What are the practical implications of working with such a viscous solution?
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 -10Ã‚Â°C and -20Ã‚Â°oC. 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. Do not store in frost-free freezers.
Are the ABI antibodies available in solvents other than HBS-glycerol?
Yes, any of the antibodies can usually be dialyzed into any buffer according to customer specifications. A special handling fee may apply to orders of 10 vials or less.