Editing Hybridoma production (section)

=== Introduction ===

The hybridoma technology presented in this protocol was first described by Köhler and Milstein<sup>1</sup> in 1975 and, except for some technical improvements, the main procedure has not changed dramatically during the last 40 years<sup>2</sup>. The aim of this protocol is to explain a more appropriate immunization strategy, a standard method for the generation of monoclonal antibodies, and an example for a validation method (ELISA).

Antibodies are incredible tools and contribute to a wide range of technological approaches, such as flow cytometry, magnetic cell sorting, or immunofluorescence, as well as to diagnostic and therapeutic options for disease monitoring and treatment<sup>3</sup>. The commercial availability of monoclonal antibodies for desired targets is demonstrated by the presence of over 24 different web databases with nearly countless quantities of antibodies or antibody-related products<sup>4</sup>. In 2015, antibody molecules were part of an international discussion<sup>5-7 </sup>due to serious problems in proper validation and characterization of commercially available antibodies.

It can be difficult and expensive to find specific antibodies for a targeted antigen, and often, they do not have the affinity or specificity needed. Although generating an antibody is still time-consuming and requires skilled personnel to develop and validate the antibody, producing an antibody individually might better than buying one.

Due to the fact that antibody production is time-consuming and requires experience, alternative methods for the production of binding molecules were developed to overcome these problems. The most commonly used alternative method is the recombinant production of single-chain antibodies via phage display. The genes for the variable binding region are extracted from cells and combined with the coating protein of a phage. The single chain is then expressed on the surface of a bacteriophage and screened in several panning steps<sup>8</sup>. The production of single-chain antibodies is a bit faster, but it also requires a skilled experimentalist. The disadvantages of some recombinant single-chain antibodies are poor stability and a lack of suitability for ''in vitro'' diagnostics. In most diagnostic tests, an Fc-receptor for detection is necessary, which needs to be added to a recombinant single-chain antibody afterwards. Again, this is time-consuming and even more complex than the hybridoma technique. In ''in vitro'' diagnostics, full-length monoclonal mouse and rabbit antibodies have been demonstrated to be the best choice.

One of the major steps in generating monoclonal antibodies must be done before the work in the lab starts: the design of the immunoconjugate. Questions that need to be addressed are: What is the physical composition of the target in the final application, and which matrices are present? Which concentration will the target have in the application? What is the final application, and what are the requirements that the antibody must fulfill?

Always take into account that if a linear peptide fragment is used, it also has to be linear in the final epitope in the target of choice; otherwise, the antibody will not bind. Of course, independent of the screening method, antibodies could be selected to recognize different antigenic formats in different applications, but this must be validated very precisely. These are the reasons why antibody development and validation are such ambitious processes.

The choice of the antigenic format for immunization is fundamental for antibody development and determines the success or failure of this process. Once the mice express a relevant antibody titer, the spleen cells are isolated and fused with myeloma cells. The most common myeloma cell lines for murine monoclonal antibody development are X63-Ag 8.653<sup>9</sup> and Sp2/0-Ag 14<sup>10</sup> from a Balb/c mouse strain. The cells descend from a malignant B cell lymphoma and were selected because they do not secrete any of their own heavy or light chains. The cells can be adapted to a ratio between 1:10 and 10:1 (splenocytes versus myeloma cells). In this protocol, the cells were adapted to a ratio of 3:1 and fused by polyethylene glycol (PEG) and electrofusion, according to Stoicheva and Hui<sup>11</sup>.

The fusion of B cells and myeloma cells is a random process. Therefore, hybrids of two B lymphocytes or two myeloma cells could be generated, but those hybrids would not be able to survive for a long time in culture. The cells undergo a hypoxanthine, aminopterin, and thymidine (HAT) selection, by which only fused hybridoma cells can survive due to the possibility of using the ''de novo'' pathway of pyrimidine synthesis. For the generation of monoclonal antibodies, it is necessary to obtain a cell line originating from one mother cell. The monoclonality is ensured by limiting the dilution techniques and the microscopic analysis of cell growth. The hybridoma culture supernatants are screened for specific antibody production, mostly by ELISA or flow cytometry, and the best binders are selected. After mass culture and purification, the antibody molecule can finally be characterized and validated for the desired application.