Rat is often considered as a pest but yet few people really know him. Rat is one of the predominant animal hosts for monoclonal antibody production, second to its rodent brother, mouse, and followed by rabbit.
The term 'rat' encompasses multiple species, including the famous species Norway rat (Rattus norvegicus), and black rat (Rattus rattus). Following our quiz “how much are you rat” (you can click the link to test yourself before reading the rest of the article and being spoiled), 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 city. The rats from the University of Louvain La Neuve became the first individuals of the rat-LOU species. Each time a clone name starts with “LO”, you can be 100% sure it’s a rat monoclonal antibody coming from SYnAbs company.
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.
Subclasses of rat immunoglobulines
Same as human and mouse, rat has five antibody isotypes (IgA, IgD, IgE, IgG, and IgM). But in mice the IgG class is divided into five sub-classes (IgG1, IgG2A, IgG2B, IgG2C and IgG3) whereas in rat there are only four (IgG1, IgG2A, IgG2B, IgG2C).
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 in the first place?
Why start using rat monoclonal antibodies?
Because rat species is a perfect species to develop secondary antibodies and reveal primary mouse antibodies.
Each time, you work with mouse species for preclinical experiment or R&D tests, a secondary antibody of a different species will get you a better recognition of your primary antibody. In fact, mouse antibodies are seldom used to produce antibodies against mouse antigens due to their tolerance of syngeneic antigens. Rats can so provide a large number of spleen B cells that are available for fusion with myeloma cells, which are extremely suitable for generating mAbs against mouse antigens.
What are the main advantages of rat monoclonals?
- The first 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, neo-epitopes, transmembrane proteins…).
- The second advantage is related to the physicochemical 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.
- Rat species requires a reduced antigen amount for immunization, potentially saving costly raw material.
- 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.
- As a larger animal, rat will permit you to run different immunization routes (tail vein, footpad,...) and also get access to different organs including lymph nodes and bone marrow organs, with a different immune response.
Can I generate a hybridoma of rat with mouse myeloma?
In general, hybridoma is a really difficult method to develop monoclonal rat antibody due to the lack of stable fusion partner. To our knowledge, all ratXmouse hybridoma clones grow slowly and will be gradually lost during the clonal selection step due to their genetic instability.
At SYnAbs, our scientists has developed IR983 the rat myeloma cell line coming from rat-LOU species. For each fusion, we’re ensuring a ratXrat homo-hybridoma clone with high productivity and stability features.
The way to generate rat hybridoma is described as the following:
- Animals: LOU/C rats and LOU/M rats are immunized with antigens to obtain lymphoblasts from the spleen, lymph nodes and bone marrow.
- 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 3 days before fusion. We use to vary the antigen types, playing with our proprietary technologies: SYnDNA for plasmid immunization, SYnPEP for conformational peptide strategy and SYnCELL for syngeneic cell immunization.
- 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 up to 4000 stable clones per fusion. 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.
Can I purify rat monoclonal antibody with classical protein A?
It’s not possible to purify rat monoclonal antibody with classical protein A capture step. The flip side of the coin is that SYnabs has been able to develop a rat antibody that specifically recognize residual protein A. This tool is used by our CDMO partners who want to discriminate free protein A and protein A linked to the purified antibody.
SYnAbs passed the cell culture 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 four main advantages:
- The first advantage 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,
- the last one is the elimination of any potential bovine immunoglobulin present in serum.
As an alternative, you can use protein G or protein L but if you perform your culture in serum conditions there’s a notable risk you bind bovine antibodies present into the serum. The best solution is to perform immunoaffinity capture step, using MARK reference for instance, a secondary antibody of mouse targeting all rat isotypes.
Conclusion on rat monoclonal antibodies
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.