Tremendous progress in the development, characterization, and manufacturing of monoclonal antibodies (MAbs) has been made since 1976, the year when George J. F. Kohler and Cesar Milstein published their seminal paper (1) on the production of MAbs by producing hybrids between mouse splenocytes with their myeloma fusion partner. Kohler's and Milstein's outstanding contribution, for which they were awarded together with Niels K. Jerne the 1984 Nobel Prize in Physiology or Medicine, and—beyond all—their deliberate (and, alas, incomprehensible by today's standards) decision not to patent the hybridoma technology resulted in the rapid and widespread adoption of MAbs by both academia and industry.

As shown in Table 1, over the last 30 yr two new types of MAbs, recombinant and synthetic, have been developed and validated. Recombinant MAbs can be

From: Methods in Molecular Biology, vol. 378: Monoclonal Antibodies: Methods and Protocols Edited by: M. Albitar © Humana Press Inc., Totowa, NJ

Table 1

Progress in Antibody Development Since 1976



Antibody types

Polyclonal Monoclonal (animal)

Polyclonal Monoclonal (animal) Monoclonal (recombinant) Monoclonal (synthetic)

Diversity platform

Nonamino acid-based (e.g., aptamers)

Monoclonal antibodies: host species



Human Chicken


Expression systems

Mouse cells

Mouse and other mammalian cells



Plants (algae, higher plants)


Research Diagnostics






Therapeutic indications

Transplant rejection Inflammatory diseases Cardiovascular diseases Antiviral

produced in transgenic mice that carry human antibody gene loci inserted in their germ line (2), through bacteriophage display-based technologies yielding high quantities of high-affinity antibodies (3,4), and ribosome mRNA displays allowing for construction of high-member, high-affinity human immune repertoire antibody libraries (5). Synthetic antibodies (diabodies, triabodies, tetrabodies) are generated using chemical or molecular biological cross-linking to produce di-, tri-, and tetrameric multivalent conjugates exhibiting enhanced specificity and functional activity (6). Successful attempts have been made to replace amino acids by other biological molecules to create chemical diversity and produce nucleic acid-based molecules mimicking the most fundamental property—specific binding to target antigens—of natural MAbs (7). In the "classical" MAb family, the range of host species expanded from mouse-mouse hybridomas to mono- and heterohybridomas originating in mice, rats, hamsters, rabbits, humans, chickens, and plants. All these developments, accompanied by major breakthroughs in manufacturing processes (including highly efficient and animal-free expression bacterial systems) and regulatory environment made initially by Idec (Rituximab) and Genentech (Trastuzumab), led to drastic expansion of MAb-based diagnostics and therapeutics in several areas of modern medicine (8,9).

However, it has to be emphasized that mouse MAbs remain the primary "work horse" in the vast majority of research and diagnostics applications. According to broad antibody industry surveys, more than 95% of research MAbs are represented by mouse MAbs (10). The very process of generating mouse MAbs practically did not change since the early 1980s (11), and Kohler and Milstein's P3X63Ag8.653 myeloma fusion partner is still the gold standard in hybridoma development. The real challenge for a researcher entering the hybridoma field is not in reproducing routine cell fusion and protein purification protocols, but in avoiding common experimental mistakes resulting in the production of low- or nonspecific hybridomas with poor growth characteristics and low-affinity antibodies. For example, many research antibody Reagent companies continue manufacturing MAbs using mouse ascitic fluids. This approach, although seemingly attractive and inexpensive in the short term, results in the production (and subsequent use by these companies' customers in academia and industry) of MAbs irreversibly contaminated by (1) irrelevant and (2) immunologically active immunoglobulins originating from the host serum. Another example is provided by Reagent companies using affinity chromato-graphy protocols that include very low pH elution buffers, thereby leading to high yields of mostly inactive MAbs. Not only do these flaws in experimental design defeat the idea of monospecific and uniformly functional MAbs, but they cannot be quantified and accounted for in all downstream MAb applications. Therefore, this methodology chapter is built around the conventional and widely accepted hybridoma protocol, with a special emphasis on tissue culture and biochemical techniques aimed at producing truly monospecific and highly active mouse MAbs.

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