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Separate But Equal


By : Quality Assurance Staff, AMTS, Inc.

In this installment we will take a close look at the characteristics of antibodies themselves and discuss the differences between them. Over many years of troubleshooting, validating, and assisting an array of different clinical and academic customers, we have discovered that throughout the IHC diagnostic learning process, there has been too much emphasis placed on composition rather than proper application. Many of the techs who practice IHC understand that an antibody contains a variable region and a constant region with multiple forms of immunoglobulins. However, those same techs often mistakenly associate a monoclonal antibody to a mouse and a polyclonal antibody to a rabbit. Misconceptions also exist with the general assumption that a monoclonal is more specific and a polyclonal is more sensitive. So what is the real difference between a monoclonal and polyclonal? Afterall, some monoclonals are manufactured to indirectly act like a polyclonal, and some polyclonals are being raised against synthetic peptides to mimic a monoclonal? The fact is that many of the differences that one might outline between a monoclonal antibody and a polyclonal antibody are simply misconceptions.

When a human gets sick, the body’s reaction to a foreign antigen is the activation and clonal extension of antigen-reactive B lymphocytes. Once matured into plasma cells, each clone of cells will secrete its own unique specific antibody. The body utilizes a series of clones to a specific region of the target protein in a monoclonal fashion all working in conjunction with one another to bind to many different sites of the same surface. This response is made in a polyclonal manner. How do we isolate one clone against the natural polyclonal response? This is where the key differences come to light.

The condensed form for the manufacturing procedure of a monoclonal is as follows: immunize the mouse with the desired antigen using an adjuvant to increase immune response. Once the spleen has enlarged, it is removed (splenectomy). The cells are then fused with the plasma membrane of a B-cell cancer (myeloma) which lost the ability to synthesize hypoxanthine-guanine-phosphoribosyltransferase (HGPRT-). The cell fusion is then transferred to a culture medium containing hypoxanthine plus aminopterin (a folic acid analog) and thymidine (HAT) making the cells fully dependent on HGPRT, thus losing their ability to synthesize an antibody molecule of their own. The logic here is that any unfused myeloma cells in HAT media will stop growing since they lack HGPRT and any unfused spleen cells will succumb to their limited life span leaving behind a hybridoma (a successful fusion.) The spleen cell component will then supply HGPRT and immortalize the myeloma.

Once a successful clone is created, it is assigned a number. Clone numbers, often seen in parentheses after the antibody name, do not represent what many would believe. Some might imagine that this implies the region of a gene, a specific amino acid sequence, or some industry secret. The truth is much less romantic. Successful fusions are a rare phenomenon, with the industry success rate for monoclonal production ranging between 40% and 60%. Ten 96 well plates with HAT solution are required in the process. Each plate has eight rows (A-H) and twelve columns (1-12.) So if plate number two position A-1 is a successful fusion then the clone designation is 2A1. The only number of any importance is that which follows a decimal point within the clone number, i.e. 2A1.2. The number is derived from the fact that the original cultures were started with multiple kinds of cells and more than one hybridoma may exist. One now has to isolate a single cell antibody-positive-culture and a subculture becomes required, namely a subclone designated 0.2. The positive antibody producing subclone, isolated from a single successful fusion is now a monoclonal and an isotype is established.

The real difference between monoclonal and polyclonal antibodies relates to the size of the animal to be immunized. Polyclonal antibodies are collected from the serum of the immunized animal and the immunoglobulin-containing fraction is purified. However, the antibodies cannot be isotyped because they are generated by a pool of activated B-cells and some are not directed against the protein of interest.

If this is all that is required to make a polyclonal, why not use the largest animal possible? The reason is that it is not always financially viable. One needs a cage for a rabbit and a farm for a goat. It is also needed to conduct multiple immunization intervals (called busters) on multiple animals. All of these productions bleeds need to be purified and tested, which add to the cost of manufacturing. In short, the smaller the animal, the lower the overhead.

Another factor in costs is the time it takes to bring an antibody to market. A monoclonal may take 62 weeks to bring to market while a polyclonal is substantially less. However, a hybridoma cell line from a successful monoclonal fusion is banked in liquid nitrogen and is immortal whereas the rabbit or goat required for a polyclonal antibody will eventually die. At that point, the whole project would need to be restarted from scratch, introducing an inherent variability to the new lot.

So which is the better antibody, monoclonal or polyclonal? Five to ten years ago, the answer would have been that it depends on the antibody, but monoclonal would often be the best choice. Today, the industry has evolved to maximize the strength of each. For example, many great monoclonal targets are being used to mimic a polyclonal. Such would be the case in Cluster Differentiating markers, because it would be difficult or impossible for a single clone to identify multiple targets from multiple cells such as activated lymphocytes, immature lymphocytes, B-cells, T-cells, and subsets of cells. Thus, multiple monoclonal antibodies are mixed in a cocktail (panoramic antibody) and function together as a polyclonal. A great example of this would be CD45 (see Figure 1). One monoclonal is raised against a cytoplasmic domain region while another clone is raised against the paracytoplasmic region of the same protein, such as the case in many cytokeratin markers.

While monoclonal antibodies sound like the best choice, especially given the ability to mimic polyclonal antibodies, there are many instances in which the domain target lies intracytoplasmic (viral inclusions and multifocal regions) where a monoclonal would not be able to bind. Furthermore, poorly immunogenic and highly conserved proteins do not lend themselves to illicit a monoclonal generation. Adding to a polyclonal’s strength, they are raised against small synthetic peptide antigens which are highly specific regions of a target protein. These peptides are conjugated to a carboxylated polyesterene bead as a carrier. Because synthetic peptides are available in highly purified forms, the vast majority of active B-cells are against a single region. This makes the polyclonal antibodies behave more like a monoclonal in specificity with the added sensitivity of a true polyclonal (multi foci specificity of the same target). The Sandwich-ELISA test is a prime example, where a polyclonal is used to capture another antibody, combining together to create the antigen. Afterwards a monoclonal antibody is used to detect that antibody.

These are the real reasons why a mouse is used in the production of monoclonal antibodies and why there are limitations to the size of the animal in the production of polyclonal antibodies. The constraints are unfortunately due to overhead costs and not because of biological reasons. From our perspective, the real potential of both monoclonal and polyclonal formats is yet to be fulfilled. Someday these wonderful molecules will cure diseases and open doorways to medical diagnostics in ways we have yet to imagine.

While monoclonal antibodies sound like the best choice, especially given the ability to mimic polyclonal antibodies, there are many instances in which the domain target lies intracytoplasmic (viral inclusions and multifocal regions) where a monoclonal would not be able to bind. Furthermore, poorly immunogenic and highly conserved proteins do not lend themselves to illicit a monoclonal generation. Adding to a polyclonal’s strength, they are raised against small synthetic peptide antigens which are highly specific regions of a target protein. These peptides are conjugated to a carboxylated polyesterene bead as a carrier. Because synthetic peptides are available in highly purified forms, the vast majority of active B-cells are against a single region. This makes the polyclonal antibodies behave more like a monoclonal in specificity with the added sensitivity of a true polyclonal (multi foci specificity of the same target). The Sandwich-ELISA test is a prime example, where a polyclonal is used to capture another antibody, combining together to create the antigen. Afterwards a monoclonal antibody is used to detect that antibody.

These are the real reasons why a mouse is used in the production of monoclonal antibodies and why there are limitations to the size of the animal in the production of polyclonal antibodies. The constraints are unfortunately due to overhead costs and not because of biological reasons. From our perspective, the real potential of both monoclonal and polyclonal formats is yet to be fulfilled. Someday these wonderful molecules will cure diseases and open doorways to medical diagnostics in ways we have yet to imagine.

Figure 1.

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