Following our quiz “how much are you rat” (you can click the link to test yourself before reading the rest of the article), we’d like to come back to the origins of rat-LOU species and give you the details of this unique animal. Time to discover the top facts about rat monoclonal antibodies.
Origin of rat species for antibody generation
In 1955, Maisin and Coll observed the appearance of lymphoid tumours in the ileocaecal region of a colony of rats maintained at the Institute of Cancerology of the University of Louvain, in Belgium. Curiously enough, Beckers, Maisin and their colleagues reported that some of these tumours were secreting immunoglobulins.
Fifteen years later, Bazin and Beckers started breeding these rats and called them after the name of the University. The rats from the University of Louvain became the first individuals of the rat-LOU species.
Two lines of this strain were then selected. The first one, characterized by a high rate of animals with tumours and named LOU/C. The second, named LOU/M with a low rate of tumours. The latter appears spontaneously at the age of 8 months and in the lymph nodes of the ileococcal region.
Rat Immune Globulin Allotypes
The molecules of a class or subclass of immunoglobulins do not always have the same antigenic determinants in all individuals of the same species. These determinants are called allotypes based upon Oudin's terminology. In this case, individuals of a species can be divided into two or more allotypic groups. In rats and mice, heavy chain allotypes have been described. In addition, in rats, two allotypes on the Kappa light chain have been demonstrated; one allotype of type 1a and one of type 1b.
LOU rats are examples of type 1a, while DA, OFA, Sprague, OKA rats are type 1b. The light chain allotype K, as we will see later, is the basis of the rat monoclonal antibody purification method and the hybrid LOU/C-OKA type 1b rat is used in this process.
But why did Pr. Bazin and his colleagues start working with rats?
Rat species offer several advantages for monoclonal antibody raising
- the first and main advantage is related to the rat antibody repertoire, which is really different from that of the mouse. Consequently, antibody production in rats is also different. For some antigens, only the rat responds or gives a better response than the mouse and the reverse is also true. Traditionally, we observed that rat species is a high responder against chemical compound and small entities like toxins and steroids, but also against difficult targets (epigenetic changes, neoepitopes…).
- the second advantage is related to the physico-chemical and biological properties of rat immunoglobulins. For example, antibodies of subclasses IgG1, IgG2a and IgG2b bind human and rabbit complement.
- An other advantage is related to the production of rat monoclonal antibodies in-vivo. Indeed, the volume of ascites obtained in a LOU/C rat is about 10 times greater than in mice and it is not uncommon to obtain between 100 and 150 mg of purified antibodies per animal, which reduces the cost price per mg produced.
- It’s possible to obtain antibodies of high purity in an efficient and economical way. This topic is detailed later in the article.
- Rat requires a reduced antigen amount for immunization, potentially saving costly raw material.
- Rat species is a perfect species to develop secondary antibodies and reveal primary mouse antibodies.
- Rat antibodies are found do not have the cross-reaction in immune detection of antigens out of a mouse background, which shown as a strong point in sandwich immunoassays.
- No special growth media is required: you can keep everything intact compared to mouse hybridoma cells without additional costs.
But how does SYnAbs manage to generate such monoclonal antibodies?
- Animals: LOU/C rats and LOU/M rats are immunized with antigens to obtain lymphoblasts from the spleen or lymph nodes. In fact, as a larger animal, rat permits to get access to lymph nodes organs, with a different immune response and changing immunization route.
- Immunizations: several immunization protocols are proposed. The quantities of antigens to be administered vary from 10 to 200 µg, depending on the nature of the antigen and the route of administration (subcutaneous, intraperitoneal, subcutaneous). SYnabs proprietary adjuvant is used to prepare the antigen to be administered. After a rest period, a final injection, without adjuvant, is usually given intravenously 3 days before fusion.
- Myeloma: SynAbs non-secreting myeloma IR983F is used as fusion partner cell. This lineage, which appeared in a LOU/C rat at the age of 72 weeks, has been adapted to in-vitro culture and made resistant to azaguanine and therefore HAT sensitive.
- Culture Media: commercially available media are used. The glutamine-free MEM medium with the addition of 1% HEPES 1M is used as a fusion medium. Fusion step has been optimized to generate a maximum of stable hybridoma clones. The ability of IR983F myeloma to fuse depends on the propagation conditions. It should be used when it is in the exponential phase of development. The medium for myeloma and hybridoma development is DMEM with added solutions of gentamycin (50 µg per ml), non-essential amino acids (1%) glutamine 200 mM (1%), sodium pyruvate (1%). The medium is mixed with solution of HAT (1%) after fusion and then HT thereafter (7 to 10 days after fusion).
- Cell culture & Screening: Hybridoma culture supernatant tests are usually performed 9-11 days after fusion. ELISA and FACS methods are used to detect hybridoma secreting specific monoclonal antibodies.
- Limit dilution: Positive hybridoma are developed and then cloned twice, by limit dilution or in agarose. The resulting clones are preserved in liquid nitrogen in the presence of 7.5% DMSO. Typing of monoclonal antibodies is performed, by ELISA, in order to determine the class or sub-class of the antibody and to ensure monoclonality of the hybridoma.
- Purification: Several methods for the purification of monoclonal antibodies are already proposed. This presentation describes only the original method of purification of rat monoclonal antibodies by affinity chromatography, but can be replaced by protein G for certain isotypes.
- We passed the harvest through an affinity chromatography column where an anti-allotype K1a monoclonal antibody (SYnAbs MARK-3 reference) is attached to the solid phase of Sepharose. This column retains only the monoclonal antibody, which is then eluted by an acidic buffer after washing the solid phase. Thus, a monoclonal antibody of purity comparable to that obtained by affinity chromatography on the antigen is obtained with several advantages. The first is the elimination of any non-specific reaction due to the presence of host rat immunoglobulins, the second is considerably economical since the column can be used several times regardless of the specificity of the antibody and the last one is the elimination of any potential bovine immunoglobulin present in serum.
In summary, the remarkable fusion capacity of IR983F myeloma, the immunological repertoire of the rat, which differs from that of the mouse, the stability of the generated hybridoma, and the remarkable quality of the purified monoclonal antibodies make rat model particularly interesting from a scientific, technical and economic point of view.
Want to know how to use rat potential for your specific project?