Considerations in Antibody Selection
As protein reagents, antibodies offer considerable flexibility in detecting specific targets.
However, this also means multiple factors can influence finding the ideal match, depending on the application and experimental conditions.
The following are common considerations users must make when comparing and selecting different types of antibodies.
Monoclonal vs Polyclonal Antibodies
Antibody clonality offers two common categories of antibodies used in research, each with unique advantages.
Monoclonal antibodies, which arise from a single clonal expansion of hybridoma cells, are homogenous, of a single isotype, and are selective to only one epitope of a target immunogen.
They are generally easier to mass produce and offer greater batch-to-batch consistency.
In contrast, polyclonal antibodies are heterogeneous antibody populations that can bind multiple epitopes of a given antigen.
This feature of polyclonals maximizes the potential to return a good signal, which can be useful in the early studies of a target lacking an optimized protocol.
A downside to polyclonals is the variation across batches, and validation of every lot may be more challenging for manufacturers or end users.
1
Read this guide to learn more about
choosing between monoclonal and polyclonal antibodies.
Recombinant Antibodies
Developed more recently,
recombinant antibodies
are a type of engineered antibody produced in vitro by stable cell lines.
Recombinant antibodies have many advantages such as being renewable, animal origin-free, lot-to-lot consistency, and adaptable.
They can also be engineered to achieve higher affinity binding than B-cell-generated antibodies.
Unlike hybridomas, they do not suffer from genetic instability over time.
Recombinant antibodies have been found to exhibit better performance over monoclonals and polyclonals in applications such as Western blotting, immunoprecipitation, and immunofluorescence.
1,2
Read this article for more
advantages and applications of recombinant antibodies.
Antibody Validation
Prospective antibody buyer should check for validation for their chosen application and species.
Validation provides proof that an antibody recognizes its specific target and works for a given application.
Thus, validation should include example data or figures from the application of interest.
When reviewing validation data, consider the five pillars for antibody validation, which have been proposed to ensure research reproducibility.
The pillars outline the use of genetic strategies, orthogonal strategies, independent antibodies, tagged proteins, and immunocapture followed by MS analysis.
3
On the supplier’s antibody datasheet, users should also examine the supplied protocol.
Antibody performance depends on several experimental variables and even a highly validated antibody can perform poorly if the wrong protocol is utilized.
Other useful information on the datasheet include a listing of known cross-reactivities and any links to references that cite the use of the antibody.
For more information, read our
guide on antibody validation.
Users should also be familiar with aspects of validation that are unique to certain applications. Some examples are highlighted below:
Flow cytometry:
Antibody performance can vary among live cells, dead cells, or cells that have been fixed or permeabilized.
An intact plasma membrane may prevent antibodies from accessing intracellular targets.
As flow cytometry is also dependent on cellular populations, validation using appropriate cell types is also important.
4
Western blot:
All Western blot validation data should include size markers and positive/negative controls.
In addition, to determine antibody reactivity, lysates from cells overexpressing the target protein, such as cell-free systems, stable or transiently transfected cell lines, are often used.
However, detecting native proteins in cells or tissues can provide greater value in assessing antibody performance.
5
For more information, read our
antibody guide for Western blotting.
ELISA:
For ELISA methods that use a pair of antibodies, such as for capture and detection, it is best for the pair to be validated together to ensure accurate results.
6
It is also good practice to examine the reference materials provided by ELISA kit suppliers, such as protocols, which can dictate the optimal experimental conditions to ensure reproducibility.
Immunofluorescence:
In immunofluorescence applications, including IHC, antibody performance is dependent on the nature and state of the cell or tissue samples.
It is important to observe target detection, specificity, and localization using appropriate cell and tissue types and whose target expression levels are known.
The experimental conditions of the validation, such as the nature of tissue fixation, should also be considered.
Antibody Companies
With so many companies available, it is important to know what to look for from an antibody supplier.
As mentioned above, users should carefully examine the validation data provided by the antibody supplier, as well as other information provided in the product datasheet.
Suppliers doing due diligence will provide a wealth of information to the benefit of the prospective buyer.
These can include quality control tests, protocols, usage recommendations, and citations of the product in the literature.
Read some tips on
how to begin choosing an antibody.
More antibody tools at your disposal
In addition to antibodies, additional products can be used to support various applications in immunodetection.
Antibody labeling kits
enable the direct conjugation of labels to antibodies, which are useful for those looking for more customization.
Labeling kits can be used to conjugate magnetic beads, detection enzymes, specific fluorophores, and other specialized labels.
For researchers seeking to collect specific antibodies,
antibody purification kits
can be used to isolate polyclonal or monoclonal antibodies from various sources, such as serum, ascites, culture media, or hybridoma cell line supernatants.
When certain needs lie outside your team's area of expertise, various
services in antibody production and development
are available.
These services, which include production of monoclonal, polyclonal, and recombinant antibodies, purification, sequencing, and labeling, are often customizeable to the unique requirements of the project.
References
1. Biddle M, Stylianou P, Rekas M, et al. Improving the integrity and reproducibility of research that uses antibodies: a technical, data sharing, behavioral and policy challenge. MAbs. 2024;16(1):2323706. doi:10.1080/19420862.2024.2323706
2. Ayoubi R, Ryan J, Biddle MS, et al. Scaling of an antibody validation procedure enables quantification of antibody performance in major research applications. Elife. 2023;12:RP91645. Published 2023 Nov 23. doi:10.7554/eLife.91645
3. Uhlen M, Bandrowski A, Carr S, et al. A proposal for validation of antibodies. Nat Methods. 2016;13(10):823-827. doi:10.1038/nmeth.3995
4. Easthope E. Antibody Validation for Flow Cytometry. Biocompare. 2023 Nov 23 [cited 2024 May]. Available from: https://www.biocompare.com/Editorial-Articles/579837-Antibody-Validation-for-Flow-Cytometry/
5. Validating Antibodies for Western Blotting. Biocompare. [cited 2024 May]. Available from: https://www.biocompare.com/Reproducibility/336768-Validating-Antibodies-for-Western-Blotting/
6. Easthope E. Improving ELISA Performance. Biocompare. 2020 Feb 27 [cited 2024 May]. Available from: https://www.biocompare.com/Editorial-Articles/560326-Improving-ELISA-Performance/