Antibodies are immunoglobulin (Ig) glycoproteins produced by plasma cells (B cells) in response to foreign antigenic molecules (immunogens). The primary function of antibodies is to bind specifically to these foreign antigens to disable them and/or mark them for destruction by the immune system, thereby protecting the host from infection. There are several classes of antibodies. The first portion of this summary focuses on conventional full-size antibodies, the IgG and IgM class antibodies, which are heavily utilized in a multitude of research, diagnostic and therapeutic biomedical applications.

The basic unit of a conventional antibody is a four polypeptide unit consisting of two identical heavy chains and two identical light chains held together by disulfide bonds. The light chains are shorter, with lower molecular weights than the heavy chains. The general shape of an antibody is a Y, with a flexible hinge (interdomain) region at the center of the Y. The flexibility of the interdomain hinge region is important for the bivalent binding of an antibody [2], allowing the two binding pockets to interact with antigenic sites at variable distances. Each polypeptide chain has a constant region, which does not vary significantly among antibodies, and a variable region, which is specific to each particular antibody. The common notation for the light chain variable region is V_L and for the light chain constant region is C_L (Figure 1). The notation is similar for the heavy chain variable (VH) and constant regions (C_H) with C_H1, C_H2, and C_H3 denoting the different constant region domains of the heavy chain. Carbohydrates are normally attached to the C_H2 domains of the heavy chains. The fragment crystallizable (Fc) region contains only constant regions from the heavy chains (C_H), but the fragment antigen-binding region (Fab) includes both a constant domain and the variable domains of both the heavy and light chains (VH and C_L). The fragment variable region F_V region contains only the two variable domains (Figure 1). See mouse antibody for discussion on immunoglobulin isotypes, subclasses, and the number of immunoglobulin domains.

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Figure 1. The basic structure of a conventional full size antibody (left panel) and of common antibody fragments (right panel). From [1]

Each complete antibody has two antigen-binding pockets, located in the F_V regions, and can bind to two antigens (bivalent binding). However, if the two antigens are too close (≤3 nm), or too far apart (≥29nm), the antibody can only bind to one antigen (monovalent binding) [2]. There is a significant affinity change between monovalent and bivalent bindings with a 1,500-fold change in Kd values [2].

The structure of specific antibodies is available from the Structural Antibody Database (SAbDab), a curated database of publicly available antibody structures, as well as structural modelling tools, which is updated weekly from the Protein Data Bank (PDB) [3].

Conventional full-size antibodies have been utilized in research for protein detection via Western blot analyses [4], immunohistochemistry [5], and enzyme-linked immunosorbent assays (ELISA) [6] for decades. Full-size antibodies have also been developed for diagnostic applications such as pregnancy tests and detection of bacteria and viruses in blood, such as an ELISA that detects HIV. Additionally, conventional full-size antibodies are used commonly in disease therapeutics. For example, Infliximab is one antibody of many available that recognize tumor necrosis factor alpha (TNFα) and it is used in the treatment of Crohn's disease and rheumatoid arthritis [7, 8]. Trastuzumab, or Herceptin, is an antibody that binds to epidermal growth factor receptor 2 and is used in the treatment of metastatic breast cancer [9]. There are also several antibody-based therapies, including Muromonab [10], given to transplant recipients to prevent allograft rejection.

Though conventional full-size antibodies may both be used for therapeutic applications, there are advantages and disadvantages to using the complete antibody. An important advantage of conventional antibodies is the fact the Fc region engages the body's immune response, and can target bound antigens for destruction. This Fc region can also be a disadvantage in some clinical applications because the immune response that it typically elicits may be detrimental to the patient's health. Additionally, full-size antibodies cannot penetrate well into certain tissues due to their relatively large size [11]. In some cases, when using full-size antibodies for diagnostic applications the Fc domain can cause significant nonspecific binding, which may impair detection applications.

For many applications antibody fragments are preferable. Antibody fragments can be produced through chemical or genetic mechanisms. Chemical fragmentation utilizes reducing agents to break the disulfide bonds within the hinge region and digestion of the antibody with proteases including pepsin, papain, and ficin. Genetic construction of fragments offers the ability to create a multitude of fragment-containing molecules, each with unique binding and functional characteristics.

Chemical and protease digestion of full-size IgG or IgM antibodies yield antigen-binding fragments (Fab; Figure 1), and Fc fragments, comprised only of the heavy chain CH2, and CH3 domains. Biochemical methods of generating antibody fragments produce useful tools for diagnostic and therapeutic applications, but it is quite laborious and requires a large quantity of antibody starting material.

The antigen-binding fragments produced by biochemical digestion include Fab, (Fab')2, Fab', and F_V, all of which lack the Fc region. Monovalent F(ab) fragments have one antigen-binding site, whereas divalent (Fab')2 fragments have two antigen-binding regions that are linked by disulfide bonds. Two individual F(ab) fragments are produced when a full-size antibody is digested with papain enzyme. A F(ab')2 fragment, which retains a portion of the hinge region, is produced by pepsin digestion of IgG or IgM antibodies. Reduction of F(ab')2 fragments produces 2 monovalent Fab' fragments, which have a free sulfhydryl group that is useful for conjugation to other molecules. F_V fragments are the smallest fragment made from enzymatic cleavage of IgG and IgM class antibodies (Figure 1). F_V fragments have the antigen-binding site made of the V_H and V_L regions, but they lack the constant regions of Fab (C_H1 and C_L) regions (Figure 1, right panel). The V_H and V_L are held together in F_V fragments by non-covalent interactions. The fragments can be generated through commercially available kits, for example, F(ab')2 Fragmentation Kit from G-Biosciences [12].

Genetic engineering methods allow the production of single chain variable fragments (scFv), which are F_V type fragments that consist of the V_H and V_L domains linked by an engineered flexible linker peptide (Figure 1) [13]. Manipulation of the orientation of the V-domains and the linker length creates different forms of F_V molecules [14]. When the linker is at least 12 residues long, the scFv fragments are primarily monomeric (as shown in Figure 1) [14]. Linkers that are 3-11 residues long yield scFv molecules that are unable to fold into a functional F_V domain. These molecules associate with a second scFv molecule, which creates a bivalent diabody [15]. If the linker length is less than three residues, scFv molecules associate into triabodies or tetrabodies [14]. For example, Tao Y et al generated a VH-VL diabody with a short GGGGS linker and scFv with a long GTTAASGSSGGSSSGA linker [16]. Multivalent scFvs possess greater functional binding affinity to their target antigens as a result of having two more target antigen binding sites, which reduces the off-rate of the antibody fragment [17]. Minibodies are scFv-C_H3 fusion proteins that assemble into bivalent dimers [18]. Small scFv fragments with two different variable domains can be generated to produce bispecific bis-scFv fragments capable of binding two different epitopes concurrently [19, 20]. Genetic methods are also used to create bispecific Fab dimers (Fab₂) and trispecific Fab trimers (Fab₃) [20]. These antibody fragments are capable of simultaneously binding 2 or 3 different antigens, respectively. Researchers often use scFv fragments to stabilize protein complexes in protein structure studies [21].

In addition to conventional antibodies, camelid and shark (squalidae) species contain a subset of peculiar Heavy Chain Antibodies (hcAb) exclusively composed by heavy chain homodimers lacking light chains [22, 23]. The Fab portions of these antibodies, called VHH in camelids and VNAR in sharks, are the smallest antigen-binding regions naturally found [24]. Nanobodies are VHH-derived recombinant domains able to bind antigens, often cloned from VHH phage libraries such as those against betacoronarivus S proteins [25] or against SARS-CoV-2 spike protein RBD domain [26]. Their binding thermodynamics and structures have been studied ( [27] and reference therein). Nanobodies are very stable and can be easily produced in huge quantity by using common simple protein expression systems such as bacteria (functional conventional full-size antibodies are difficult to express properly in a bacterial system), thus representing a promising tool for research and therapeutic purposes, especially in the areas of super-resolution microscopy, mass spectrometry, and targeted protein degradation [28]. Nanobodies can also be delivered inside living cells through conjugated with peptides [29, 30], or in vivo [31], or expressed directly in vivo and recognize its targets in vivo, for example, VHH Z70 against tau nucleation [32]. Nanobodies against RFP or GFP, when conjugated with far-red Atto dyes, attained 118-fold magnification of fluorescent signals over GFP or RFP, and were used to generate whole-body mouse neuronal connectivity [33]. They have also been used to stabilize the active state of proteins in structural studies [34]. An expression system with multiple concatenated nanobodies against different influenza strains is being examined as a means to generate a universal flu vaccine [35]. Recombinant anti-IgG secondary nanobodies have great potential to replace widely used polyclonal secondary antibodies produced using animals [36].

Nanobodies have the unique ability to cross the blood-brain barrier [37, 38] ; however, nanobodies tend to be processed and cleared very quickly from the body [39]. Nanobodies can be used for methods like immunoprecipitation, for example, RFP-Trap MA from Chromotek [40], or coupled to fluorescent proteins to track intracellular targets in live cells in real-time [41].

About 10% of bovine immunoglobulins contain an unusually long third heavy-chain complementarity-determining region (CDR 3H) with a large number of cysteine residues [42-44]. These cysteines pair to form disulfide bonds which leads to a stalk-and-knob like structure in the antigen binding domain [44]. This exceptionally long CDR3H domain with the long-stalk contributes to the diversity of bovine antibody specificity.

Intrabodies, or intracellular antibodies, refer to antibodies or their fragments (usually of scFv design) expressed directly inside cells or in animals in vivo through an expression vector. For example, Dong JX et al developed several nanobodies against neuronal proteins for intracellular expression as intrabodies [45]. One important consideration/caveat is the reducing intracellular environment which diminishes the affinity of an antibody or antibody fragment whose binding with the antigen is dependent on intradomain disulfide bonds. Another consideration is that intrabodies tend to aggregate. Kabayama H et al designed intrabodies with a net engative charge even at the lowest cytoplasmic pH 6.6 to generate ultra-stable cytoplasmic antibodies [46]. Intrabodies are used as alternatives to pharmacological inhibitors to target specific endocytic participants. For instances, expression of a single-chain variable fragment (scFv) derived from the 3B12A monoclonal antibody against the TDP-43 nuclear export signal in HEK293A cells or after in utero electroporation of its expression vector promoted the proteolysis of TDP-43 aggregates in cultured cells and embryonic mouse brain [47]. Virus-mediated delivery into the nervous system of an scFv antibody against the RNA recognition motif 1 of TDP-43 reduced microgliosis in a mouse model of acute neuroinflammation and mitigated cognitive impairment, motor defects, TDP-43 proteinopathy, and neuroinflammation in transgenic mice expressing TDP-43 mutations linked to amyotrophic lateral sclerosis [48]. The potential of intrabodies as a therapeutical modality remains to emerge.

An antibody fragment can also be linked with a cell-import tag, such as an IPTD tag [49], to facilitate its entry into cells.

Antibody fragments offer certain advantages over a full-size antibody for some applications. This topic was reviewed by Nelson [50]. One advantage of fragments over full-size antibodies is that antibody fragments are smaller than conventional antibodies and generally lack glycosylation, allowing their production in prokaryotic expression systems, which provide time and cost savings. Additionally, fragments are small enough to infiltrate into some tissues that full-size antibodies are unable to penetrate, which aids in many therapeutic and immunohistochemical procedures [11]. Furthermore, the lack of Fc domain is a substantial advantage for primary antibodies used in immunohistochemistry and other detection applications because they have greatly reduced non-specific binding to the Fc receptor. One scFv that is commonly used in diagnostics is the NC10 antibody against influenza neuraminidase. The MOC-31 antibody against epithelial cell adhesion molecule Ep-CAM is an scFv commonly used in as a cancer therapy. Diabodies, triabodies and tetrabodies have potential uses in applications such as radioimmunotherapy and diagnostic in vivo imaging [51]. However, fragments that lack the Fc domain are degraded in the body much more rapidly than conventional antibodies [52], and are unable to elicit Fc-mediated cytotoxic processes unless they are conjugated to an effector moiety [53], which requires further optimization for antibody fragment-based therapeutics.

Although various antibody fragments offer certain advantages, they are not commonly utilized in experiments. In the more than 60,000 articles manually curated by Labome only a few articles cited applications of antibody Fab fragments. The Roche anti-digoxigenin Fab antibody fragments ( 11093274910) and anti-fluorescein Fab antibody fragments ( 11426338910) are generated in sheep and produced through digestion with papain. Anti-digoxigenin Fab fragments were used for in situ hybridizations since the RNA probes are labeled with digoxigenin [54-56]. They were also used to perform protein folding analysis to study the process of single calmodulin molecule folding through single-molecule force spectroscopy [57]. F(ab) fragments of anti-mouse secondary antibodies are often used to block endogenous mouse Ig during immunostaining, for example, AffiniPure Fab fragment Donkey antimouse IgG from Biozol (JIM-715-007-003) [58].

B Shen et al performed whole mount staining of mouse half bones with Alexa Fluor 647-AffiniPure F(ab')2 fragment donkey anti-chicken IgY at 1:250, Alexa Fluor 488-AffiniPure F(ab')2 fragment donkey anti-rabbit IgG at 1:250, Alexa Fluor 488-AffiniPure F(ab')2 fragment donkey anti-rabbit IgG (at 1:250 (all from Jackson ImmunoResearch) [59]. Invitrogen Alexa Fluor 546-conjugated goat anti-rabbit IgG F(ab')2 fragment was used to perform immunohistochemistry to investigate the role of sFLT-1 in the maintenance of the avascular photoreceptor layer in mouse models [60] and Invitrogen Alexa Fluor 488 F(ab')2 fragment of rabbit anti-goat IgG (H+L) was used in immunohistochemistry to investigate the mechanism of tip link regeneration in auditory hair cells [61]. F(ab')2 secondary antibodies from Jackson ImmunoResearch are commonly used [62].

Fc receptors (FcRs) are molecules expressed primarily on/in innate immune cells, that recognize and bind the Fc domain of antibodies and thereby initiate a cell-based immune response. The diverse functions of FcRs reflect the wide range of protective or modulating roles of antibodies, including mediating the neutralization and clearance of targeted substrates, as well as the programming of adaptive immunity [63]. The biological functions of FcRs are regulated by immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs), that act as the receptor’s interface with activating and inhibitory signaling pathways, respectively. Thus, signaling by ITAMs can elicit cell activation, phagocytosis and endocytosis, whereas signaling by ITIMs has an inhibitory effect on cell activation [64]. FcRs have been described for all classes of immunoglobulins and some of them are discussed below.

This family includes FcγRI, FcγRII, FcγRIII and their isoforms. They are responsible for antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ACDP) [63].

Another receptor that binds IgG is neonatal Fc receptor (FcRn), which is involved in the transfer of passive humoral immunity from a mother to her fetus. FcRn also protects IgG from degradation in vivo, explaining their long half-life in the serum [65]. This phenomenon has led to the development of better therapeutic antibodies by introducing alterations in the Fc region to promote the Fc-FcRn interaction.

They include the high-affinity FcεRI, which is capable of binding monomeric IgE, and the low-affinity C-type lectin FcεRII, which interacts preferentially with complex IgE. FcεRI mediates the immediate hypersensitivity response of many allergic reactions by stimulating degranulation and the release of a range of inflammatory mediators on mast cells and basophils [66]. FcεRII exists both in a membrane-bound form that delivers a downregulating signal for IgE synthesis [66], as well as soluble fragments that generate an opposing upregulation on IgE synthesis [67]. Its role in transcytosis of IgE-allergen complexes in human airway and the intestinal epithelium is actively being investigated as a potential target for allergic airway inflammation as a result of food allergies [68, 69].

The sole member of the IgA receptor group, FcαRI, is expressed only in cells of the myeloid lineage. It plays a role in both pro- and anti-inflammatory responses depending on the state of the IgA bound. While binding of secretory IgA (SIgA) present at mucosal sites has anti-inflammatory effects including prevention of pathogen invasion, binding of serum IgA leads to inflammatory responses. FcαRI also regulates neutrophil viability depending on the inflammatory microenvironment [70].

TRIM21 can be distinguished from other FcRs as it shows a broad antibody specificity. It can bind IgG, IgM and IgA [71-73]. It is also expressed by cells of most histogenic lineages [74]. TRIM21 participates in antibody-mediated interference of viral replication by targeting cytosolic virus-antibody complexes for proteasomal degradation.

Binding of Fc domains to FcRs can have undesired effects in monoclonal antibody-based analytical methods such as immunohistochemistry (IHC), fluorescence activated cell sorting (FACS) and chromatin immunoprecipitation (ChIP). Non-specific binding to FcRs may introduce background noise that can lead to detection of false positives. Solutions to deal with this problem include the use of (i) isotype controls for gating, (ii) serum to broadly compete for the receptors involved in non‐specific binding, or (iii) purified IgG to block Fc receptors specifically [75]. Innovex Fc Receptor blocker #NB309 can be used to block paraffin or frozen sections during IHC or IC experiments [76] or BD Biosciences #553142 [76, 77], Miltenyi Biotec Fc receptor block [78, 79] for flow cytometry. For example, Chopra S et al blocked FcγR binding with 5 μg/ml TruStain fcX (anti-mouse CD16/32, clone 93) from BioLegend during flow cytometry and cell sorting [80].

Dr. Macarena Fritz Kelly from São José dos Campos, SP, Brazil, contributed the section about Fc receptors in September, 2018.