There are five antibody isotypes (IgA, IgD, IgE, IgG, and IgM) from mice, same as humans. Each isotype has a different heavy chain. Isotypes are also called classes. Another name for antibodies is immunoglobulin, hence the suffix of 'Ig' to designate antibody classes and subclasses. Naive B cells produce IgM and IgD. During B cell maturation, through isotypic switching, a mature B cell will produce one of IgG, or IgA, or IgE isotypes and subclasses. Different isotypes have different half-lives in vivo, ranging from 12 hours to 8 days [1].

Figure 1. BALB/c mouse, a common mouse strain for mouse monoclonal antibody development. Courtesy of The Jackson Laboratory

Each heavy chain is formed by variable and constant regions. The constant regions have been designated as distinct genes, although they only form part of the immunoglobulin heavy chain. Table 1 lists mouse antibody classes, subclasses, gene names, and links to NCBI and IMGT gene databases.

For IgG2a and IgG2c isotypes: inbred mouse strains with the Igh1-b allele have IgG2c isotype instead of IgG2a [2]. The murine heavy chain locus has only one of these two isotype genes in addition to the other isotypes. Mouse IgG2a scaffold is sometimes used in recombinant antibody expression [3].

Figure 2. IgG, IgE, IgD, IgM, and IgA schematic representations.

Labome antibody database organizes antibody products from over 100 high-quality suppliers. Many of them provide monoclonal antibodies generated from mice.

Table 2 list a few examples of commercial antibodies, indicating antibodies are available with different isotypes.

Table 3 lists the different sets of isotype and subclass genes in some of the commonly used animal species in laboratories. IgG2a is absent in certain mouse strainslike C57BL, SJL, and NOD mice, which have IgG2c [4].

Antibody isotypes in vivo are very important in immune responses and effector mechanism. Mouse IgG isotypes are important to the biological functions of the immunoglobulin in vivo as well. Mouse IgG isotypes displayed marked differences in bactericidal (IgG3 >> IgG2b > IgG2a >> IgG1) and opsonophagocytic (IgG3 > IgG2b = IgG2a >> IgG1) activity when reacting with the P1.16 epitope on the outer membrane PorA protein of Neisseria meningitidis [5]. The establishment of mouse gut microbiome is likely enabled through mucosal IgA [6].

Human IgG subclasses differ in the size of their hinge regions and display different levels of spatial tolerance for the bivalent binding, with IgG3 having the longest amino acid hinge region and exhibiting the most flexible bivalent binding [7]. Human IgG1 to IgG4 subclasses are named after their serum prevalence [8].

Most antibody users seem to be not particular about the antibody classes and subclasses, this is especially true in western blot and immunoprecipitation. However in double labeling experiments and flow cytometry runs, such as in the case of secondary antibodies, the classes/subclasses should be considered (see Labome secondary antibody review for an in-depth discussion).

As a side note, a number of monoclonal antibody therapeutics such as Eculizumab and Pembrolizumab are human IgG4 (mouse does not have IgG4 isotype). Maun HR et al, for example, humanized mouse and rabbit anti-tryptase variants as IgG4 antibodies [9]. Compared to IgG1-3, IgG4 Fc does not activate complement cascade, and binds to Fc receptor with intermediate affinity. Its unique structural properties have been identified [10]. In addition, increased concentrations of human serum IgG4 are associated with specific diseases such as autoimmune pancreatitis [11]. Complement activation can also be minimized through specific mutations in IgG1 [12].

The isotype and subclass for a mouse antibody can be determined by specific anti-heavy chain constant region antibodies, usually through ELISA or flow cytometry, with appropriate isotype controls. These isotyping kits are available in different formats from many different suppliers. One of the most cited isotyping kits in the literature is AbDSerotec MMT1, see the isotyping kit.

An immunoglobulin molecule has two heavy chains and two light chains, linked together with intrachain and interchain disulfide bonds (with exceptions). In human IgA2, the heavy and light chains are not linked with disulfide bonds.

Heavy chains for IgA, IgD, and IgG have a constant region with three immunoglobulin (Ig) domains. For example, mouse IgA heavy chain constant region has 344 amino acid, and mouse IgG1 constant region has 324 amino acids. Other types of heavy chains may have a different number of immunoglobulin domains. For example, secreted and membrane-bound IgD heavy chain constant regions have two Ig domains (see Uniprot P01881 and P01882). Heavy chains for IgE and IgM have a constant region with four immunoglobulin domains. Mouse IgM heavy chain constant region has 475 amino acids. Heavy chains from all isotypes have a variable region with a single immunoglobulin domain.

Each immunoglobulin domain is about 110 amino acid long. Each IgD and IgG heavy chain is about 450 amino acids, and each IgE and IgM heavy chain is about 550 amino acids. Each light chain (either kappa or lambda) has one constant immunoglobulin domain and one variable immunoglobulin domain, and is about 211 to 217 amino acids. The common IgG isotypes have a molecular weight of 150 KD. Under the reducing condition, each heavy chain is about 50 KD and each light chain is about 25 KD. IgM tends to exist in pentamer or hexamer form, where IgM molecules are covalently linked through disulfide bonds, which give rise to a molecular weight of 970 KD. Since immunoglobulins are glycosylated, the apparent molecular weights may differ, sometimes significantly, from the nominal molecular weights.

The mouse is the predominant animal host for monoclonal antibody production and also a host for polyclonal antibody generation. While typical mouse monoclonal antibody production involves immunizing mice (often BALB/c mice, for example [289, 290] ) with foreign antigens along with the commonly used Freund adjuvant [289] or the new, water-soluble QuickAntibody [291], mouse monoclonal hybridoma can also be obtained directly by fusion of unimmunized spleen cells from a specific mouse strain with auto-antibodies, as in the case of anti-SM Y12 antibodies [292]. Other strains such as C57BL/6 [289, 293], NMRI, C3H, and SJL, and other adjuvants like Chitosan, LQ, or incomplete Freund's adjuvant, and evan cells overexpressing an immunogen [293], can also be used [294]. Genetically engineered mouse strain can also be used as the host to develop specific antibodies, for example, an IgG monoclonal antibody with high binding specificity against GM3 ganglioside was developed through beta3Gn-T5 knockout mice [295].

As of June 5, 2022, Labome database lists 254742 mouse monoclonal antibodies against 12039 human genes, 7144 mouse genes, 6475 rat genes and a total of 11079 genes from other 156 species. Please note, due to rebranding (see a discussion about rebranding here), other databases may cite higher available numbers. However, Labome has the only database that strives to reduce the number of rebranded antibody products. For any antibody not available from suppliers or researchers, companies or core laboratories, for example, Antibody Production Services (APS Ltd) [293] and Innovagen [296], can custom-produce as fee-for-service.

Labome surveys the literature to develop Validated Antibody Database. Table 4 lists some of the most commonly cited mouse monoclonal antibodies among the articles Labome has curated. The most cited mouse antibody is likely to be the actin clone AC-15, which is commonly used as a western blot loading control.

The mouse is also a widely used research model, and many antibodies against mouse genes are available. As of June 5, 2022, Labome antibody database lists 106246 monoclonal antibodies against 8684 mouse genes, and 100849 polyclonal antibodies against 13590 mouse genes (see a summary of antibodies against mouse genes).

Antibodies against all these genes can be searched and compared at Labome website, for example, antibodies against mouse p53, mouse albumin, mouse cd31, mouse cd20, and mouse c-myc.

Most monoclonal antibodies are generated in mice. It is common to use anti-mouse secondary antibodies. Labome survey on secondary antibodies indicates that goat anti-mouse secondary antibodies are the most common choices. On the right panel of that webpage, you can see links to secondary antibodies against different mouse isotypes and light chains.

A point of concern is when a mouse antibody and an anti-mouse secondary antibody are used on mouse tissues. The secondary antibody tends to also recognize endogenous mouse immunoglobulins and generates a high background. This endogenous immunoglobulins should be blocked and saturated by a block reagent, such as an F(ab) fragment of an anti-mouse secondary antibody. Commercial kits, like "Mouse on Mouse” Blocking Reagent from Vector Laboratories (MKB-2213 [297] or BMK-2202 [298] ) or AffiniPure Fab fragment Donkey antimouse IgG from Biozol (JIM-715-007-003) [289], can accomplish this.

One of the important methods for generating humanized antibodies is through the immunization of transgenic mice with part of a human immunoglobulin genomic sequence. The entire human immunoglobulin variable-gene repertoire has been inserted into the mouse genome, enabling the generation of monoclonal antibodies with fully humanized variable regions [299], and in Regeneron VelocImmune mice [300, 301].