Recombinant Antibodies: A New Gold Standard
ABfinity™ Recombinant Antibodies Maximize Specificity, Sensitivity, and Consistency
Trade-Offs With Traditional Antibody Tools
The use of antibodies as research tools has been around for decades, and over time these antibodies have been refined to the point where they are specific, sensitive, and reliable. However, there is typically a trade-off between sensitivity and specificity when choosing antibodies—to get high sensitivity you often have to sacrifice specificity, and vice versa (Figure 1). Polyclonal antibodies can provide great sensitivity by virtue of being able to recognize multiple epitopes on a given antigen target, but this advantage also introduces potential artifacts such as increased nonspecific binding. Monoclonal antibodies, on the other hand, provide the highest specificity because they only recognize a single epitope, but this limited targeting results in fewer binding sites for the antibody on the antigen, which translates to reduced overall sensitivity.
Another concern with classic monoclonal and polyclonal antibodies is the variability inherent in their production. Polyclonal antibodies exhibit significant variation in bioactivity from lot to lot, introducing higher variability in your results. Monoclonal antibodies from hybridomas are also susceptible to cell line drift, resulting in loss of antibody production. The ABfinity™ recombinant antibodies provide uniformity and stability with every lot, enabling the highest performance available.
Figure 1. Comparison of the sensitivity and specificity of classic monoclonal and polyclonal antibodies and ABfinity™ recombinant monoclonal and oligoclonal antibodies.
What Are ABfinity™ Recombinant Antibodies?
ABfinity™ antibodies are recombinant antibodies produced by cloning the heavy and light chains of antibodies for specific targets and expressing these clones in mammalian cells. Once the DNA is cloned and banked, the protein sequence of the antibody will not change; the antibody bioactivity, specificity, and quality will be the same with every lot.
The process of making an ABfinity™ antibody starts with strategically designing the immunogen to provide the desired specificity. These immunogens are then used to immunize animals, whose plasma cells are screened for a positive immune response. The genes for the antibody heavy and light chains are then isolated, cloned, expressed, and screened for desired sensitivity and selectivity. DNA clones that produce positive functional test results are scaled up, purified, and passed through a final validation step that includes western blot analysis, indirect ELISA, and cellular staining (Figure 2) where applicable.
During the screening process, we identify many heavy and light chain variations that recognize the target of interest. The combination that provides the desired sensitivity and selectivity is selected as the ABfinity™ recombinant monoclonal antibody for that target. We also construct a “library” of the remaining antibody light and heavy chains that exhibited a positive response. The individual plasmids that make up this library are then scaled up, purified, and validated again to ensure the highest quality. The resulting population of antibodies derived from this validated library becomes the ABfinity™ recombinant oligoclonal antibody for the same target.
Figure 2. Immunocytochemical analysis with ABfinity™ recombinant oligoclonal antibodies showing nuclear localization of metadherin. U2OS cells were stained with anti–metadherin (C-term) ABfinity™ recombinant rabbit oligoclonal antibody (21HCLC) followed by Alexa Fluor® 488 goat anti-rabbit secondary antibody (green), as well as with Alexa Fluor® 594 Wheat Germ Agglutinin to label glycoconjugates on the cell membrane (red), and DAPI nuclear counterstain (blue).
ABfinity™ Monoclonals: Highly Consistent Performance
Monoclonal antibodies represent the peak of specificity for antibody tools; however, due to the nature of the production process required, lot-to-lot variation and cell-line drift are both potential issues. As a consequence, the performance of a traditional monoclonal antibody can change from lot to lot, requiring that you revalidate each lot before wasting valuable samples and time. Because ABfinity™ recombinant monoclonal antibodies (and ABfinity™ oligoclonals as well) are derived from cloned DNA sequences of the heavy and light antibody chains, they are not susceptible to cell-line drift or lot-to-lot variation, thus allowing for peak specificity and performance.
ABfinity™ Oligoclonals: The Best of Both Worlds
Polyclonal antibodies often show higher sensitivity than monoclonal antibodies because they recognize multiple antigenic sites on the target. However, lot-to-lot consistency is often a problem with standard polyclonal antibodies generated by immunizing an animal. Each immunization is likely to generate a different antibody profile, and therefore variation between lots can be very high. ABfinity™ recombinant oligoclonal antibodies comprise a selection of multiple different recombinant monoclonal antibodies, providing the best of both worlds—the sensitivity of a polyclonal antibody with the specificity of a monoclonal, all delivered with the consistency only found in a recombinant antibody.
The ABfinity™ oligoclonal antibody is functionally the same as a polyclonal antibody, recognizing multiple epitope sites on the target and therefore producing higher detection sensitivity for low-abundance targets when compared with monoclonal antibodies. The biggest advantage of the oligoclonal antibody, however, is that the identity of the light and heavy chains in the mixture is known, and this exact population can be produced in every lot (Figure 3), circumventing the biological variability typically associated with polyclonal antibody production.
Figure 3. Western blot analysis with ABfinity™ recombinant monoclonal antibodies showing lot-to-lot consistency. HeLa cell extracts separated on reducing gels were probed with four different lots of anti-SMAD2 ABfinity™ recombinant rabbit monoclonal antibody. The total protein concentrations in lanes A, B, and C were 2, 1, and 0.5 μg/ mL, respectively. The primary antibody was detected using the WesternBreeze® Chemiluminescent Kit (Anti-Rabbit).
Recombinant Technology Has Arrived
ABfinity™ recombinant antibodies set a high benchmark for specificity, sensitivity, and consistency. When maximal sensitivity is needed, ABfinity™ oligoclonal antibodies provide the sensitivity normally associated with polyclonal antibodies but with the specificity of monoclonal antibodies. Regardless of your choice, ABfinity™ recombinant monoclonal and oligoclonal antibodies provide a consistency that can only be achieved with recombinant technology. Learn more about ABfinity™ recombinant antibodies and their specificity.
Get a copy of this article as it appears in the print version of BioProbes 67.
Learn More About: