Some definitions first for a general audience
: Usually the serum product of an immunized animal. Heterogeneous because they comprise
- Various antibodies specific for a variety of antigens, some specific for different epitopes of the same antigen,
- Different antibody classes and sub-classes, i.e., , and
- Wide range of antibody concentrations ( ) and affinities for their respective antigens.
(mAbs): Products of a hybridoma clone ( ), derived from a single antibody-secreting B cell fused to a myeloma cell line. Homogeneous. In essence, product of a cellular factory spewing out that one mAb. Thus, the secreted antibody is mono-specific, with a single affinity to the single epitope it binds on the antigen, and is of a single antibody class ( ).
Epitope: Site that an antibody binds on an antigen.
Affinity: Strength of antibody binding to antigen.
Avidity: Sum of affinities of multiple antigen-binding sites on an antibody.
Polyclonal antibodies: Pros & Cons
Unlike mAbs, polyclonal antibodies
- Can be produced faster and more cheaply.
- Can bind their target antigens under a variety of salt and pH concentrations so are more stable.
- Comprise antibodies specific for different epitopes of a given antigen. Outcome is higher antibody affinity, i.e., antigen-binding sensitivity.
- Easier to couple to a variety of labels such as enzymes, , etc.
Such attributes make polyclonal antibodies a better option in assays
- For binding proteins sensitive to conformational changes or denaturation, or which are polymorphic.
- In (IP) and (ChIP) assays.
- For detecting low concentrations of a given protein.
- As capture antibody in sandwich (Enzyme-Linked Immunosorbent Assay), one of the most widely used assays to detect and quantify antigens. For capture and not for detection. ELISA assays using them for detection are more susceptible to dramatic changes when diluting sera since a polyclonal serum contains varying antigen specificities and affinities at different concentrations (titers). OTOH, diluting mAbs doesn’t affect their affinity/avidity so easier to interpret that a change in reaction results from change in antigen concentration. While mAbs have a single specificity, heterogeneity of polyclonal antibodies means varying affinities even against the same epitope which makes assessing their specificity much more complicated. Unlike polyclonal antibodies, using mAbs for detection in ELISAs allows calibration, and therefore, standardization.
However, major drawbacks of polyclonal antibodies are
- Batch-to-batch variability since each batch is typically product of one or few immunized animals. OTOH, being products of immortalized cells, practically limitless amounts of mAbs can be produced.
- Binding multiple epitopes on a given antigen increases scope for cross-reactivity. This necessitates affinity purification ( ) before using them in assays.
- Consequence of polyclonal antibodies comprising different antigenic specificities and affinities is higher background signal in various assays, i.e., lower specificity.
Some Therapeutic Applications of Polyclonal antibodies
Polyclonal antibodies are widely used forto prevent acute rejection in transplant recipients. In transplants, one of the major logistical problems is severe, chronic shortage of living donors. As a result, criteria have steadily expanded to include organs previously precluded from consideration. These include greater tissue mismatches, and organ and tissue donations after cardiac death. Problem with using expanded criteria organs is higher probability of rejection. This necessitates using more powerful immunosuppressive therapy, often lifelong.
Some polyclonal antibody preparations have become mainstays among immunosuppressive regimens used to prevent early, acute rejection in solid organ transplants. Two main types of polyclonal antibody preparations are widely used, one sourced from rabbits, the other from horses (). They are ( , , , 4, , )
- Thymoglobulin®, a rabbit anthymocyte globulin (rATG).
- ATG-Fresenius, a rabbit anthymocyte globulin.
- ATGAM® or eATG, equine antithymocyte globulin.
- Lymphoglobulin®, horse ATG.
Thymoglobulin®, a rabbit anthymocyte globulin (rATG)
- First licensed in April 1984 in Europe and in 1999 in USA (6).
- Manufactured by Genzyme/Sanofi.
- Is polyclonal IgG anti-human thymocyte globulin.
- Rabbits are immunized with human (developing T cells).
- Is widely used in solid organ transplants.
- Was given to ~ half of all new kidney transplant recipients in the US between 2000 and 2009 (7).
- Initially used in kidney transplants, today it’s used in a variety of solid organ transplants such as liver, heart, lung, pancreas, intestinal as well as hematopoietic stem cell transplantation and aplastic anemia ( , ).
- Manufactured by Neovii Biotech (formerly Fresenius Biotech) (4).
- Rabbits are immunized with a human line.
ATGAM® or eATG, equine antithymocyte globulin (5)
- Horses are immunized with human T cells and their antibodies harvested from their serum.
- Developed by in the 1950s.
- started using it in the 1960s.
- Registered for use in kidney transplantation in the US since 1981.
- First commercially available ATG in Europe and USA.
- Manufactured by Pfizer (previously Pharmacia Upjohn).
Lymphoglobulin®, Horse ATG
- Manufactured by Genzyme/Sanofi.
To avoid early, acute rejection, high-risk transplant recipients, usually defined as glucocorticoid-resistant, are typically given these antibodies starting shortly before the transplant and continuing immediately afterward for a few weeks.
While exact mechanism by which these complex reagents immunosuppress is unknown, ADCC () likely causes rapid and widespead lysis of T cells though elimination of other cells such as NK cells is also possible. This Rx also entails serious risks which include , , , higher infection risk, and many others ( , , 4, , 8, ).
1. Gaber, A. Osama, et al. “Rabbit antithymocyte globulin (thymoglobulin): 25 years and new frontiers in solid organ transplantation and haematology.” Drugs 70.6 (2010): 691-732.
2. Penninga, Luit. Immunosuppressive Polyclonal and Monoclonal T-cell Antibody Induction Therapy for Solid Organ Transplant Recipients: Systematic Reviews with Meta-analyses and Trial Sequential Analyses of Randomised Clinical Trials: Ph. D. Thesis. Afsnit 7812, Blegdamsvej 9, 2100 Ø, 2014.
3. Chen, Huifang, Qian, Shiguang. “Current Immunosuppressive Therapy in Organ Transplantation.” CURRENT IMMUNOSUPPRESSIVE THERAPY IN ORGAN TRANSPLANTATION: 51.
4. Nishihori, Taiga, et al. “Antithymocyte globulin in allogeneic hematopoietic cell transplantation: benefits and limitations.” Immunotherapy 8.4 (2016): 435-447.
7. Cai, J., and P. I. Terasaki. “The current trend of induction and maintenance treatment in patient of different PRA levels: a report on OPTN/UNOS Kidney Transplant Registry data.” Clinical transplants (2009): 45-52.
8. Bamoulid, Jamal, et al. “Anti-thymocyte globulins in kidney transplantation: focus on current indications and long-term immunological side effects.” Nephrology Dialysis Transplantation (2016): gfw368.)
9. Malvezzi, Paolo, Thomas Jouve, and Lionel Rostaing. “Induction by anti-thymocyte globulins in kidney transplantation: a review of the literature and current usage.” Journal of nephropathology 4.4 (2015): 110.