Antibody conjugation is usually achieved through chemical reaction, although specific enzymatic conjugation, such as sortase-based protein terminal conjugation [1, 2], or even through protein engineering [3], is gaining popularity. The preferred site for chemical conjugation on the antibody is the –NH₂ (amine) group of a lysine or the free –SH (sulfhydryl) group of cysteine [4]. While the conjugation chemistry treats all accessible residues equally, the number of molecules that can be conjugated is limited by the following:

• pH dependence of the reaction: Reaction conditions that allow only specific groups to react can be standardized to obtain an optimum Ab: reporter molecule ratio. E.g., at an alkaline pH, the reactivity of certain aliphatic amine groups is much higher than other groups. There is also a problem of overall antibody stability at alkaline pH. It is crucial to limit the reaction time under these conditions to avoid deterioration of the antibody. Conjugated cysteine residues are prone to fragmentation at alkaline pH [5]. This is why most commercial conjugation kits are designed to complete the conjugation reaction within 15- 30 minutes and usually target the primary amine group of lysine (see Table 5).

  Another option is to engineer antibodies to alter the pI from 8-9 to 5-6 [6]. This ensures that the antibody does not precipitate during the conjugation reaction.

• Size of the conjugate: Small molecules like haptens or drugs can be conjugated in a minimum ratio of antibody: conjugate::1:4 or even 1:10. Larger molecules like enzymes may be restricted to just two molecules per antibody. For oligonucleotides, the ratio is 1:1.
• Presence of the lysine and cysteine residues in the Fab which determine Ag binding and therefore should not be conjugated. On an average, IgG molecules have about 80 lysine residues of which 20 are present in solvent accessible sites. Cysteine residues are more uniformly distributed and are fewer in number. About 16 pairs of cysteine residues exist as 12 intra-chain and four inter-chain disulfide bonds. Inter-chain cysteine is usually targeted for conjugation due to its solvent accessibility [7].

To enhance the efficacy of conjugation the antibody may be altered by the introduction of unusual amino acids or chemical modification.

One method to reduce the number of undesirable conjugations is the replacement of cysteine with selenocysteine. This reduces the probability of conjugation occurring at inappropriate sites of the polypeptide chain(s) that disrupt the structure of the antibody (and therefore its affinity and/or specificity) [8].

In the case of antibody-drug conjugates, modification of glutamine residues using the enzyme transglutaminase, modification of aldehyde-tagged proteins at their C-terminal and introduction of unnatural amino acids such as p-acetylphenylalanine using cell-free systems have been employed to generate specific, stable and productive conjugates [9]. TH Pillow et al introduced LC-K149C, HC-L174C, and HC-Y373C cysteine residues into anti-HER2 7C2 and anti-CLL-1 antibodies to enable their conjugation with chimeric degraders [10].

One advantage of sulfhydryl groups is that they can be used to form a stable, cleavable disulfide (-S-S-) linkage. Most of the cysteines in antibodies are already present in the form of disulfides, and the available ones may not be at convenient positions. Hence sulfhydryl groups can be introduced at the site of primary amines – especially those of lysine residues- by modification with reagents such as Traut's reagent (2-iminothiolane), MBS, SPDP, SATA, and their derivatives. Some of the features of these reagents are given in Table 1.

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Figure 1. Modification of amines to sulfhydryls. A) Reaction with Traut’s reagent to generate a free sulfhydryl. B) Generation of a non-cleavable sulfhydryl using SMCC. C) Generation of a protected sulfhydryl using maleimide crosslinking.

Enzymes linked to antibodies form the basis of enzyme-linked immunosorbent assays (ELISAs) (see Table 2). These are among the most extensively used types of IA and are safer and easier than the RIA. They combine the specificity of antibody-antigen interaction with the sensitivity of enzyme assays. Moreover, since it can be performed in multiple well plates, several samples can be assayed simultaneously. Three main types of ELISAs have been developed to date, based on the substrate used. The earliest was the immunoassay using a colorimetric method for detection. However, this method typically detects 0.01 ng to 0.1 ng of antigen which is relatively low. The sensitivity of ELISAs can be improved using fluorogenic and chemiluminescent substrates.

Conjugation of enzymes to antibodies can be based on the linkage of the sugar or lysine groups in the enzyme. Sodium periodate oxidation of the sugars present on glycoproteins renders them susceptible to reaction with the terminal amine (–NH₂) of lysine residues in the antibody. This forms a stable covalent link between the glycoprotein and the antibody. This is the method of choice to conjugate enzymes like horseradish peroxidase (HRP) to antibodies [16].

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Figure 2. The sodium periodate method results in a Schiff’s base between the oxidised sugar of the enzyme and the –NH₂ from a lysine residue of the antibody.

Conjugation of urease to antibodies has been done by simple glutaraldehyde crosslinking. Glutaraldehyde, like dimethyl adipimate and the homologous dimethyl suberimidate, is homo-bifunctional crosslinking agents [17]. These molecules have the same active groups on both ends and a sufficiently long ‘linker’. These are generally used in protein-protein crosslinking, and therefore, antibody-enzyme conjugation. The reactive end groups target terminal amines (–NH₂) of lysine and hydroxylysine on both proteins.

The advantage of using these crosslinking agents is the ease of carrying out the reaction. The proteins to be cross-linked are incubated with the reagent in a suitable amine-free buffer, and the high molecular weight proteins are separated by column chromatography. The obvious disadvantage is the non-specific crosslinking that leads to the formation of large aggregates of proteins that does not serve the purpose.

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Figure 3. Homobifunctional crosslinking agents for enzyme-antibody conjugation.

The urease conjugate has been used in research [18] and some diagnostic tests for hepatitis [19] and syphilis [20]. The detection method for urease activity involves a colorimetric transition from yellow to purple with the evolution of ammonia in the presence of bromocresol purple. This is useful to determine empirical values. However, quantification of results can be done with β-galactosidase conjugated antibodies which also give a low background for animal cells [21]. Cell-ELISA (a precursor to labeling cells for flow cytometry), now prefers the use of β-galactosidase conjugated antibodies to urease-conjugated antibodies.

These conjugates are used in ELISAs, Western blots/dot blots, immunohistochemistry (IHC) and recently, targeting solid tumors [22].

Fluorescent dyes have the property of absorbing one wavelength of electromagnetic radiation and then emitting the same at a longer wavelength. This Stoke’s shift is crucial for the detection of small quantities of the target compound/metabolite/ protein.

Fluorescently labeled antibodies give specificity to the binding and can be used for localization. In fluorescence labeling, a succinimidyl-ester functional group attached to a fluorophore core targets primary amines on the antibody to form a stable covalent linkage [23]. The degree of labeling of an antibody depends on the number of free primary amine groups and whether the fluorophore sterically affects the specificity, avidity or affinity of binding. Since there is a limit to the number of fluorophores that can be attached to an antibody, there is no standard that can be associated with this family of conjugated antibodies. In short, the problems with fluorochrome-labeled antibodies is too few or many fluorophores, non-specific staining and loss of antibody-antigen specificity or affinity [24]. This is why fluorophore-labeled antibodies are used more for immunohistochemistry and FACS rather than for quantitative immunoassays. Table 3 gives the natural fluorescent dyes which have been conjugated to antibodies.

Most other commercial dyes including Alexa dyes, Texas Red, are derived from these dyes. Table 4 gives a list of fluorochromes and their emission wavelength.

Commercial kits offer fluorescent dyes that have an isothiocyanate modification. These dyes can be coupled to amine groups in a single step using an ‘activation’ reagent which is generally succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC). At high pH (usually pH 9.0), a highly concentrated solution of SMCC in dimethylformamide is incubated with the isothiocyanate modified fluorophore. This modifies the antibody and gets it ready to conjugate to the fluorescent tag. The tag is added, incubated for an hour at room temperature. Then a quenching reagent does away with the unused fluorescent probe. It is advisable to purify the labeled antibody by HPLC to remove the unconjugated probe. For example, Rigau M et al conjugated an anti-BTN2A1 mAb to Alexa Fluor-647 by amine coupling from Thermo Fisher, and conjugated an anti-BTN3A antibodyto R-phycoerythrin with a sulfo-SMCC heterobifunctional crosslinker [25]. Dong JX et al conjugated an anti-Homer1 nanobody to Alexa 647 using succinimidyl-Alexa 647 from Thermo Fisher (A20186) [26]. L Cantuti-Castelvetri et al labeled anti-NRP1 monoclonal antibodies with DyLight™ 488 NHS ester from Thermo Fisher Scientific [27].

A significant disadvantage of fluorochrome-labeled antibodies is the high background scattering (noise) and non-specific fluorescence from biological molecules that may be present in the sample. These can interfere with the measurements and limit the use of such probes. Under such conditions, lanthanide conjugated antibodies serve as ideal fluorophores with their long Stokes’ shifts and the ability to remain excited for milliseconds [28]. Han G et al published three protocols for antibody conjugation with lanthanides and other rare-earth elements [29]. For mass cytometry, Fluidigm Corporation provides kits for conjugating antibodies. Rosshart SP et al used Maxpar® antibody conjugation kits from Fluidigm Corporation to study microbiota and immune responses in laboratory mice born to wild mice [30] ; so did Guo CJ et al for their conjugation of antibodies for mass cytometry [31].

The lanthanides (sometimes called the rare earth elements) are a group of 14 ‘f-block’ elements of the Periodic table from atomic number 57(lanthanum) to 71 (lutetium). Of these, europium (Eu), dysprosium (Dy) and gadolinium (Gd) have been mainly used in biomedical imaging [32]. All these elements show 4f-5d, 4f-4f transitions, with absorption in the UV range and emission in the visible range.

Lanthanides can form chelates that generate detectable chemiluminescence. Polyaminocarboxylates (PACs), for example the linear ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) and their cyclic counterparts 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,8,11- tetraazacyclotetradecane-1,4,8-triacetic acid (DO3A) are inexpensive metal ion chelators, which form stable complexes with lanthanides. The order of binding is EDTAβ-diketones have a bidentate capacity and are not as stable. However, they have been modified to form 3:1 complex with lanthanides and can accommodate antenna molecules. The β-diketones have been part of the DELFIA system of immunoassays where they serve as solution ligands after dissociation of lanthanide tags from a non-fluorescent chelate-containing immunocomplex [28].

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Figure 4. Chelating agents for Lanthanides.

Lanthanide chelates (especially those of Eu (III) and terbium Tb(III))] when coupled to antibodies with free amine and/or carboxyl groups through a stable isothiocyanate linker, detect tiny quantities of antigen, making the CLIA (chemiluminescence based immunoassay) the most sensitive immunoassay [33] These conjugates have been used in clinical diagnostics [34], in vitro DNA hybridizations [35], cytotoxicity assays [36], receptor-ligand binding assays [37] and as potential therapeutics for cancer [38].

Other transition metals have been explored for the detection of biological assay systems. Meso Scale Discovery (MSD) electrochemiluminescence platform uses ruthenium. Chaturvedi S et al labelled an IL-6 detection antibody through the SULFO‐TAG™ NHS‐Ester kit from MSD [39].

Haptens are small antigenic molecules that need a carrier protein to elicit an immune response. While fluorescent dyes qualify as haptens, antibodies have also been conjugated to haptens such as biotin, streptavidin and biomagnetic beads for use in different techniques.

The noncovalent interaction between biotin and avidin (or streptavidin) has a very high Kd (dissociation constant) making the binding between the two almost irreversible [40]. Hence, conjugation of one or both the molecules to antibodies makes them ideal for detection, quantification, and localization of biomolecules in ELISAs and IHC.

Biotin has a fused heterocyclic ring with an aliphatic valeryl tail while avidin and streptavidin are proteins. The conjugation of biotin to antibodies is usually through an NHS activated molecule and an amine group on the antibody. Some commercially available activated biotin preparations come with an additional linker which allows better positioning of the molecule (see www.thermoscientific.com/pierce ). Silva MC et al used Thermo Scientific EZ-LinkNHS-PEG4-Biotinylation Kit to label tau proteins for Bio-layer interferometry biosensor assay [41]. Chaturvedi S et al conjugated an IL‐6 MSD assay capture antibody with EZ‐Link Sulfo‐NHS‐LC‐Biotin reagent from Thermo‐Pierce [39]. Streptavidin/avidin is linked to antibodies using bifunctional agents that crosslink two proteins.

Magnetic beads are iron oxide particles embedded in an epoxy resin matrix with an approximate diameter of 0.5μm. These are commercially available with various activated groups such as amine (-NH₂), hydrazide (-N₂H), carboxyl (-COOH), iodoacetyl (-CHICOO), sulfhydryl (-SH) and aldehyde (-CHO) groups to enhance antibody attachment. To facilitate proper orientation of binding, ready-made magnetic beads conjugated to Protein A/Protein G are also available.

Recovery of the conjugate is done using a magnet, thus reducing losses during centrifugation. A single magnetic bead can bind approximately 50-70 μg of antibody. These beads have a neutral pH and allow for gentle separation of protein complexes. Amines, thiols, and urea should be avoided in the reaction buffers and antibody solutions. After conjugation BSA, glycerol and azide can be added to the storage buffer since these do not interfere with assays. Magnetic bead-conjugated antibodies are stable for up to 12-18 months at 0°C.

For example, Engle DD et al conjugated CA19-9 monoclonal antibodies 5B1 and NS19-9, EGFR clone D38B1 to Thermo Fisher M-270 Epoxy Dynabeads for immunoprecipitation [42].

The amalgamation of ELISA with the PCR resulted in the immune-PCR (iPCR) which was first devised by Sano et al in 1992 [43]. Here, an antibody to a target antigen is linked to a specific oligonucleotide. Upon binding of the antibody to the antigen, the DNA is amplified using the polymerase chain reaction (PCR), allowing for almost 100-10,000 fold amplification of the signals detected by normal ELISA. The level of antigen is then calculated according to the amount of oligonucleotide bound and amplified. iPCR has been used to detect viral and bacterial antigens as well as disease-specific IgG associated with mumps [44].

While the sandwich iPCR involves linker molecules such as biotin-avidin/streptavidin systems, direct linking of the specific oligonucleotide can also be conjugated to antibodies. Here, the oligo and the antibody are activated by heterobifunctional crosslinking agents that finally combine to link the former to an amine group on the antibody. A disadvantage of this method is the susceptibility of the linkage to conditions required for purification of the conjugate by chromatography. Cell Mosaic®, Innova ThunderLink and LightningLink from Expedeon supply amine-conjugated oligos and antibodies.

Oligonucleotide-conjugated antibodies are also used in the CODEX imaging technique [45].

Nanoparticles of different molecules have improved solubility and activity profiles as compared to the native molecules. When conjugated to antibodies, these can be used for therapeutic or diagnostic purposes. Several conjugates have been developed by several companies including Merck & Co., Invitrogen Inc. and Miltenyi Biotech [46].

Semiconductor nanoparticles (quantum dots) conjugated to antibodies have been used in biomedical imaging. The conjugation of quantum dots to andies has been done using a popular zero-length crosslinker, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) [47]. EDC forms neutral amide bonds between carboxyl groups and primary amines of an antibody. EDC is a water-soluble reagent that can easily be added to buffers or removed by washing with water or dilute acid. One disadvantage of this method is that the antigen binding sites of an antibody may be blocked by the non-selective formation of amide bonds at the vicinity of Fab.

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Figure 5. Conjugation of a reporter system to an antibody using EDC.

Similarly, colloidal gold nanoparticles have been conjugated to antibodies using the carbodiimide method [48]. Different conjugation chemistry that creates an acid-labile bond using a citrate/citraconic moiety has been used in the conjugation of gold nanoparticles to antibodies for use in diagnostic imaging, for example, detecting SARS-CoV-2 virus [49], and as a drug delivery vehicle [50].

Dextran-conjugated IgD antibodies were first used to study B-cell and T-cell activation and Ig secretion. Being a large molecule, dextran can trigger cell division even when conjugated to (otherwise non-mitogenic) monovalent Fab fragments or bivalent anti-IgM antibodies.

Today, dex-monoclonal IgG are being used to study immunoglobulin secretion, class switching of Ig genes and B cell receptor signaling [51] and IgE expression [52].

To simplify antibody conjugation - especially to dyes, enzymes, and nanoparticles, there are several commercially available kits in the market. While some of these have been discussed in the context of specific labels, the most commonly used kits are those for hapten conjugation using NHS crosslinking chemistry (Table 5). Some of the kits tolerate BSA and other proteins, while others require PBS-only antibodies, which are often available from commercial suppliers. For example, Pastushok L et al labelled an Fab fragment or anti-IgG antibody in PBS buffer with Licor IRDye 800CW Protein Labeling Kit and removed free IRDye800CW from the conjugates using desalting spin columns from Thermo Scientific [53].

To ensure the efficient use of a conjugated antibody, it is necessary to purify the conjugate. When the label to be conjugated is much smaller in size than the antibody, it is possible to separate the unbound label using gel filtration chromatography. It is more difficult to separate the labeled antibody from the unlabelled antibody. To avoid this situation, an excess of the label can be used to ensure that the conjugation to the antibody is high and gel filtration chromatography can be performed.

The efficiency of conjugation is determined by calculating the ratio of the molecules of the reporter system that are stably attached to the antibody. This is done using spectroscopy or chromatography. To attain maximum efficiency, the concentration of the reporter system is generally ten to fifty times that of the antibody to be conjugated. The unconjugated label is then separated from the antibody conjugate by gel-filtration chromatography.

The ratio of the antibody conjugate to the free label is calculated based on the specific activity (for enzymes), fluorimetry (for fluorescent dyes) or UV-vis spectrophotometry (for oligonucleotides) [57]. The specific activity or absorbance of the conjugate is compared to that of the free label to determine the conjugation ratio (see Table 6).

The efficiency of conjugation can be calculated using the following equation (A₂₈₀ conjugated antibody / A₂₈₀ unconjugated antibody) X 100.

Most antibody conjugate preparations are generally stable up to six months at 4°C. Freeze-thaw of antibodies is not recommended. Should one want to store the conjugate at -20°C, 20% glycerol should be included in the buffer. Fluorescent dye or lanthanide-chelate-tagged antibodies should be stored in the dark. To prevent contamination of the conjugated antibody preparation, preservatives such as 0.1% azide or thiomersol may be added.

Antibody conjugates have come a long way since the first fluorophore was attached to antibodies in 1942. With better linkers and controlled reactions, it is now possible to design conjugates that can deliver specific results. The generation of conjugates has gathered momentum in the last two decades because of the successful production of at least two antibody-conjugated drugs. Most research in the field involves simple conjugation chemistry to generate antibody conjugates with greater flexibility and stability. The development of a single step protocol with commercially available derivatized reporter systems would be ideal for better results in research, diagnostics and therapy.