Rabbit antibody

The European rabbit (Oryctolagus cuniculus) is a widely used animal model in immunology. In current applications, rabbits are a major source of various monoclonal antibodies (mAb) and polyclonal antibodies (pAb). Polyclonal antibodies are a combination of different antibodies against a specific antigen. These different antibodies recognize different antigenic epitopes. In contrast, monoclonal antibodies recognize a specific antigen binding site and bind only to a single epitope.

According to the location of antigenic determinants, immunoglobulins can be classified as: isotype, allotype, and idiotype. Isotype: constant region determinants are called isotypic determinants or isotypes. The antigenic determinants of isotype antibodies are present in CH and CL. Based on the differences in CH, antibodies can be classified into classes and subclasses; based on the differences in CL, antibodies can be classified into types and subtypes. Allotype: Products of allelic forms of the same gene will have slightly different amino acid sequences in the constant regions, which are known as allotypic determinants. The sum of the individual allotypic determinants displayed by an antibody determines its allotype. Idiotype: the unique antigenic determinant structure in the variable region of antibodies produced by different B cells within the same individual. Each antibody's V region contains 5-6 individual-specific amino acid structures, known as idiotope. These idiotopes can induce the production of anti-idiotype antibodies as antigenic epitopes. If the idiotope is a structure near the Ab1 framework region, the induced antibodies are called Ab2α. Ab2α does not affect the binding of Ab1 to the antigen. If the idiotope is at the site where Ab1 binds to the antigenic epitope, the induced antibodies are also called Ab2β, which can mimic the antigen and competitively inhibit the binding of Ab1 to the antigen, acting as an "internal image" of the antigen. If the idiotope is at the site where Ab1 binds to the antigenic epitope, the induced antibodies can partially inhibit the binding of Ab1 and the antigen.

Polyclonal antibodies derived from rabbits have been widely used in various biomedical research, including immunohistochemistry, immunofluorescence, protein imprinting, and flow cytometry. In addition, some rabbit polyclonal antibodies have been used in clinical treatment. Monoclonal antibodies derived from rabbits have been approved for clinical in vitro diagnostic testing. The detected sources include tumor-associated antigens and Helicobacter pylori, among others. It is expected that in the near future, many rabbit monoclonal antibody-derived therapies will transition from preclinical research to clinical studies. Companies currently developing rabbit or rabbit-derived monoclonal antibodies include Abcam (RabMAb platform), Alcon, Apexigen, Cell Signaling Technology, Agilent Technologies, MAB Discovery, Lab Vision Corporation, Thermo Fisher Scientific (Invitrogen ABfinity recombinant rabbit antibodies), and Ventana Medical Systems.

Advantages of rabbit antibodies

As a widely used animal model in immunology, many standard procedures for rabbit-based immunological research and development have been established and fully validated. Many natural features of rabbit antibodies also give them unique advantages. First, taxonomically, rabbits belong to the order Lagomorpha, which is evolutionarily distant from the order Rodentia to which rats and mice belong. Rabbit antibodies can recognize antigenic epitopes that cannot be recognized by mouse antibodies, significantly increasing the number of available epitopes and facilitating the development of antibodies targeting both human and murine homologous proteins. Second, rabbits produce significant immune responses to small molecules and haptens, whereas rodents do not. Third, most of the mouse strains currently used are inbred, while rabbit inbred strains are lacking. The diversity in genetic backgrounds leads to a significant higher immune response diversity in rabbit. Immunohistochemistry results of mouse and rabbit monoclonal antibodies targeting the same human antigen show higher sensitivity of rabbit monoclonal antibodies. Fourth, the common monoclonal antibody production techniques require collection of B cells from the spleen, bone marrow, or blood. Since rabbits are significantly larger than mice, a higher number of B cells can be obtained from each rabbit. Fifth, the genetic mechanisms of B cell production and maturation in rabbits are significantly different from those in humans and mice. Sixth, rabbit IgG has only one subtype and easy for antibody sequencing, engineering, and humanization, which is crucial in antibody drug development.

The development of rabbit B cells and antibody repertoire

The rabbit's antibody response is driven by a series of interactions between immunogens, antigen-presenting cells (APCs), T cells, and B cells. After the initial immune response, naïve B cells are stimulated to differentiate into plasma cells that secrete antibodies. For most protein antigens, the first specific antibodies appear in the serum within five days after immunization, with the majority being IgM antibodies. Following the initial response, under the influence of cytokines secreted by T cells, B cells switch to produce IgG antibodies. Antibody concentration (titer) continues to rise, reaching a peak around two weeks after immunization, then slowly declining. With repeated exposure to the immunogen, the immune response and average affinity of the antibodies for the antigen sharply increase. In rabbits, antibody affinity maturation is achieved through clonal selection, somatic gene conversion (SGC), and somatic hypermutation (SHM).

The development of the rabbit's B-cell repertoire differs significantly from other mammals. The most accepted model divides the developmental process into three steps. First, the generation of a neonatal B-cell repertoire through B lymphopoiesis occurs in the embryonic liver and omentum, and switch to the bone marrow after birth. The development of the neonatal B-cell repertoire begins between the second and third weeks of gestation. Second, the primary 'pre-immune' B-cell repertoire is produced within the first two months after birth in the gut-associated lymphoid tissue (GALT). Finally, the secondary 'immune' B-cell repertoire is generated upon activation after B cells bind to antigens. In the bone marrow of adult rabbits, B lymphopoiesis is very limited, indicating that B cell generation is mainly confined to the early developmental process. In the adoptive transfer model of rabbit-to-rabbit transfer, rabbit B cells can be transferred to the host's stem cell niches. Therefore, rabbit B cells have a long lifespan and the potential for self-renewal, maintaining the stability of the rabbit's antibody repertoire throughout their lives.

Mice and human bone marrow B lymphocyte generation occurs continuously throughout life, and antibody diversity is mainly generated through the recombination of VH, DH, and JH gene segments. In contrast, species such as rabbits, sheep, and chickens only have short-term B lymphocyte generation during early development. The antibody repertoire in these species is mainly diversified in GALT.

According to the primary B cell development model proposed by Ehlich et al., rabbits' B cells first undergo heavy chain rearrangement, followed by light chain rearrangement. This model suggests that VH-D-JH recombination of the heavy chain occurs first, followed by the expression of the pre-B-cell receptor (pre-BCR). Upon stimulation of the pre-B-cell receptor, VL–JL light-chain recombination is started. The number of proB and preB cells in rabbits peaks at 2-3 weeks after birth, then steadily decreases, significantly reducing by 16 weeks of age, at which point almost all B-lineage cells in the bone marrow are B cells. The increase in bone marrow B cells may be due to peripheral B cells returning to the bone marrow rather than continuous B lymphocyte generation. ProB and preB cells are almost nonexistent in adult individuals, indicating that B lymphocyte generation in rabbits has essentially ceased by adulthood. The majority of B cells in adult rabbits are generated through B lymphocyte generation occurring between birth and 16 weeks of age.

The presence of functional pre-BCR in proB and preB cells, as well as the orderly rearrangement of H and L chain genes, indicates that rabbit B cell development primarily occurs through a classical or ordered pathway. H and L chain genes do not rearrange simultaneously. There are up to 200 VH genes in the VH gene library used for VH-D-JH heavy chain rearrangement, but most of these genes are not expressed or are expressed at low levels. There are 10-20 D genes and 4-5 JH genes used for VH-D-JH heavy chain rearrangement. Some genes in these gene clusters are preferentially used in the recombination process. For example, VH1 is used in approximately 80-90% of heavy chain rearrangements. The VH1 gene encodes three allelic variants of the VH region, VHa1, VHa2, and VHa3. The remaining 10-20% of heavy chain rearrangements use VHx, VHy, or VHz genes. Additionally, there is a preference for the use of D2a, D2b, D1, and JH4. To partially compensate for the decreased diversity caused by the preferential use of VH, D, and JH genes, non-templated nucleotide addition (N-nucleotides) occurs at the VH-D and D-JH junctions during heavy chain rearrangement in rabbits. The third complementarity-determining region of the heavy chain (HCDR3) in rabbit antibodies contains two VH-D and D-JH junctions, making it the most variable of all six CDRs. The average length of rabbit HCDR3 is 15±4 amino acid residues, while the average length of mouse HCDR3 is 11±2 amino acid residues and that of humans is 15±4 amino acid residues. Therefore, the similarity between rabbit HCDR3 and human HCDR3 is higher than that between rabbit and mouse.

The diversity of heavy chains in the B-cell repertoire of neonatal rabbits can be compensated by different light chains. Rabbit light chains are divided into two types: λ light chains and κ light chains. There are two isotypes of κ light chains, K1 and K2. The K1 type of κ light chain contains four allotypes, b4, b5, b6, and b9. The constant region sequences of these rabbit κ light chain allotypes have higher amino acid sequence diversity than the allotypes of human antibodies. K1 is the most common isotype, accounting for 70-90% of the light chains. The remaining light chains are K2 and λ type light chains. A characteristic of the K1 type light chain is the intrachain disulfide bond formed by cysteine at position 80 in the variable region and cysteine at position 171 in the constant region. This feature is absent in the light chains of human and mouse antibodies, and may contribute to the stability of rabbit antibodies. Light chain rearrangement in rabbit antibodies is more diverse than heavy chain rearrangement. The junctions of the recombinant VJ genes in rabbits contain particularly long N nucleotide sequences. Therefore, the average length of LCDR3 in rabbits is 12±2 amino acid residues, while the average length of LCDR3 in humans is 9±1 amino acid residues.

A limited repertoire of neonatal B cells undergoes further diversification between the 4th and 8th week after birth, forming a primary pre-immune B-cell repertoire. This postnatal B cell diversification is extremely rare and has only been found in rabbits and pigs so far. The diversification of B cells in this process is mainly accomplished by two mechanisms, SGC and SHM. SGC is mainly used for antibody gene diversification in chickens and rabbits, primarily replacing large nucleotide sequences with previously unused VH gene segments. High-throughput sequencing results show an average replacement nucleotide sequence length of 59±36 nucleotides. Therefore, most of the unexpressed VH genes also play an important role in the diversity of the heavy chain. The frequency of heavy chain SGC in rabbits is 23%, much higher than in humans (2.5%) and mice (0.1%). Most non-templated nucleotide additions in VH-D-JH recombination belong to out-of-frame junctions. The nature of SGC is DNA homologous recombination, with the majority being in-frame replacements and extensions. At this stage of development, SHM is also a common phenomenon. The diversity of the rabbit κ-type light chain in the primary pre-immune B-cell repertoire is also increased through SGC and SHM. SGC of rabbit κ-type light chains occurs more frequently (32%) than heavy chains, and the average length of replacement is longer (86±48 nucleotides). The generation of the primary pre-immune B-cell repertoire is highly dependent on GALT. GALT absorbs pathogens from the gastrointestinal tract through specialized gastrointestinal epithelial cells called M cells and presents antigens to B cells, leading to B cell stimulation, differentiation, and proliferation. Surgical removal of GALT tissues in rabbits, such as the Peyer's patches, the sacculus rotundus, and the appendix, will result in severe immunodeficiency. Rabbits raised in a sterile environment also exhibit developmental abnormalities in GALT.

The secondary immune B-cell repertoire is generated following antigen-dependent B cell stimulation. Similarly, through SGC and SHM to increase the diversity of heavy and light chains, the B-cell repertoire targeting specific antigens is further expanded. Mice have four IgG isotypes: IgG1, IgG2a, IgG2b, and IgG3. Humans have four IgG isotypes: IgG1, IgG2, IgG3, and IgG4. Rabbits, on the other hand, have only one IgG isotype. Rabbits have at least 10 functional IgA isotypes, which is the most diverse IgA system known. In contrast, mice have only one IgA isotype, and humans have only two. The unique characteristics of B cell development and antibody repertoire make rabbits a valuable source for producing high-affinity and specific antibodies.

Rabbit IgG

There are five different classes of immunoglobulins in mice and humans. The type of immunoglobulin is determined by its heavy chain, with IgG being Cγ, IgM being Cμ, IgA being Cα, IgE being Cε, and IgD being Cδ. However, rabbits have only four classes, with no IgD found. The most abundant class of immunoglobulin in rabbit serum is IgG, with a serum concentration of 5-20 mg/mL, followed by IgA with a serum concentration of 3-4 mg/mL. The size of rabbit IgG molecules is approximately 150 kDa, composed of two identical light chains and two identical heavy chains. The two light chains and two heavy chains assemble into a Y-shaped molecule, displaying two antigen-binding sites. The antigen-binding sites are located in the variable regions (VH and VL) of the heavy and light chains. These variable regions have similar three-dimensional structures, but in each variable region, the hypervariable region forms three loops that protrude from one end of the molecule, forming the antigen-binding site. The hypervariable region is also known as the complementarity-determining region (CDR). Therefore, each binding site is composed of six CDRs, three from the light chain and three from the heavy chain. The variable region of the antibody can be further subdivided into CDR (CDR1-CDR3) and FR (framework region, FR1-FR4), so the structure of the variable region can be simply represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable region of the antibody is used to recognize and bind to antigens, while the constant region is used to initiate downstream effects, such as antibody-dependent cell-mediated cytotoxicity (ADCC). The light chain consists of the variable domain (VL) at the N-terminus and the constant domain (CL) following it. The variable region contains three complementarity-determining regions (CDRs). The heavy chain is composed of the N-terminal variable domain and the three constant domains (CH1, CH2, and CH3) following it. CH1 and CH2 are connected by a flexible hinge region. The amino acid sequence of the flexible hinge region is APSTCSKPTCP or APSTCSKPMCP. Three disulfide bonds can be formed in the flexible hinge region, one between the heavy chains and the other two between the light chains and the heavy chains. Rabbit IgG's light chain is divided into κ light chain and λ light chain, with κ light chain further divided into K1 and K2. The most common κ light chain, K1, contains an additional disulfide bond that links the variable region and the constant region. Compared to mouse and human IgG, rabbit IgG often has fewer amino acids in the N-terminus and D-E loop and has additional disulfide bonds in the variable region of the heavy chain.