Antibody immobilization on the surface of a carrier is a critical step in the preparation of immunodiagnostic reagents. To enhance the detection limit and sensitivity of analytical methods, the application of appropriate site-specific conjugation techniques during antibody immobilization is particularly important. These techniques ensure that antibodies are immobilized in a specific orientation on the carrier surface, thereby maximizing the exposure of antigen-binding sites and improving the binding efficiency between the antibody and its target antigen. This approach not only helps reduce nonspecific binding but also enhances signal intensity, resulting in better detection limits and higher sensitivity. Furthermore, site-specific conjugation can improve the stability of antibodies, thereby optimizing the overall performance of immunodiagnostic reagents.
The following sections provide an in-depth analysis of three commonly used methods for antibody immobilization involving site-specific conjugation: physical adsorption, direct chemical coupling, and indirect coupling, with a discussion of their respective applications and effects.
Physical Adsorption
Physically adsorbing antibodies onto a carrier is the simplest method of immobilization and requires no prior modification of either the antibody or the surface. As such, it has become the most commonly used method for developing clinical immunoassays. However, this adsorption process is based on non-covalent interactions (such as hydrophobic interactions, van der Waals forces, and π–π interactions), leading to a random orientation of antibodies on solid supports like hydrophobic plastics. This orientation is influenced by the antibody’s dipole moment and the surface charge of the carrier.
To mitigate this randomness, various pretreatment techniques—such as UV irradiation, electric field treatment, electrochemical treatment, and plasma immersion ion implantation—can be used to promote directional adsorption. For example, passive adsorption of IgG1 and IgG2a antibodies on charged surfaces can achieve some degree of orientation. Under low surface charge density and high solution ionic strength, van der Waals forces dominate, leading to multi-orientation binding. In contrast, under high surface charge density and low ionic strength, antibody immobilization tends to occur through preferentially charged regions on the antibody, enabling a certain degree of orientation.
Direct Chemical Conjugation
Direct chemical conjugation typically involves covalently linking antibodies to the carrier surface using bifunctional crosslinkers. These crosslinkers can react with functional groups on the antibody (such as primary amines or carboxyl groups), enabling immobilization. However, this non-selective reaction often results in random antibody orientation and potential loss of antibody functionality.
To improve site specificity and orientation during immobilization, one can take advantage of specific functional groups generated by reducing antibody disulfide bonds (yielding free thiols) or by oxidizing carbohydrate moieties (generating aldehydes). These groups can then be selectively linked using thiol- or aldehyde-specific crosslinkers for more oriented immobilization.
Selective reduction of disulfide bonds in the antibody hinge region can generate site-specific free thiols. These thiols can react with carriers containing gold, maleimide, or pyridyldisulfide groups, achieving oriented antibody immobilization. However, this process may inadvertently reduce other disulfide bonds, compromising antibody activity. To avoid this, alternative methods such as the reaction of 2-iminothiolane with primary amines (without reducing disulfide bonds) can be used, or protein engineering strategies may be employed to precisely control the number and position of thiol groups within the antibody.
Oxidation of antibody glycans using periodate can generate aldehyde groups, which can be used to immobilize antibodies via hydrazide-derived crosslinkers or amine-functionalized surfaces. However, this method may oxidize critical amino acids, reducing site selectivity and impairing antigen-binding activity. Additionally, aldehydes can lead to antibody aggregation through crosslinking. As an alternative, boronic acids can form boronate esters with cis-diols in glycan structures, enabling site-specific immobilization. However, boronate ester formation is reversible at physiological pH and may be disrupted by glycoproteins in the sample. To overcome this, a photo-reactive crosslinker containing both a boronic acid and a diazirine moiety can be used: the boronic acid component first weakly captures the antibody via its glycans, and subsequent light activation of the diazirine forms a covalent bond with carboxyl groups on the antibody, achieving site-specific immobilization.
The nucleotide binding site (NBS), a conserved region within the variable domain of immunoglobulins, exhibits affinity for nucleotides and aromatic amino acids, among which indole-3-butyric acid (IBA) binds most effectively to the NBS. Leveraging the high site specificity of the NBS allows for the orderly capture and photo-crosslinking covalent immobilization of antibodies on IBA-terminated surfaces, without compromising their antigen-binding capability. Additionally, antibodies possess other non-canonical binding sites for small molecules, offering new strategic options for conjugation and immobilization.
Indirect Conjugation
Indirect conjugation involves coupling antibodies to solid surfaces through affinity tags (ligand-receptor pairs), enzyme-substrate interactions, or proteins that specifically recognize antibodies. Compared with physical adsorption and direct chemical conjugation, indirect methods offer easier realization of antibody orientation on carrier surfaces.
Affinity tags such as biotin-streptavidin/avidin, polyhistidine/(metal)-nitrilotriacetic acid (NTA), and other peptide affinity tags can be easily conjugated or genetically fused to non-critical regions of the antibody without altering its conformation or immunoreactivity. Among them, the biotin-(strept)avidin system is widely used for antibody immobilization due to its high stability, efficiency, specificity, and binding affinity. Although polyhistidine tags (His-tags) exhibit affinity for certain transition metal ions, the binding strength is relatively weak and often enhanced by increasing the number of histidines, using multivalent NTA, or employing thioalkyl chelating agents. Other peptide affinity tags like FLAG tags are also employed, which rely on antigen-antibody interactions to offer greater affinity and specificity. These affinity tags provide a range of effective strategies for antibody immobilization, each with its own advantages and limitations.
Inspired by the mechanism through which enzyme inhibitors form covalent bonds with enzyme active sites to irreversibly inhibit enzyme activity, enzymes or their active domains can be genetically fused with target proteins to achieve highly site-specific immobilization. For example, the covalent linkage between keratinase and phosphonate inhibitors has been utilized to immobilize antibody fragment fusion proteins onto solid supports, demonstrating high affinity and specificity. Additionally, enzymatic methods that establish covalent bonds between two reactants—such as Sortase A-mediated conjugation—can be used to introduce one reactant into the antibody and graft the other onto a surface, enabling covalent bond formation under enzymatic catalysis for site-specific antibody labeling. These methods exploit the specificity of enzymes to enable effective antibody immobilization while preserving functional activity.
Proteins A and G, derived respectively from Staphylococcus aureus and Streptococcus, contain multiple binding domains specific for the Fc region of mammalian IgGs and are widely used for directional antibody immobilization, thereby improving assay performance. Compared with protein A, protein G offers broader Ig binding affinity, though native protein G also binds other molecules. As a result, engineered versions such as Fc-specific truncated protein G and recombinant protein A/G fusion proteins have been developed. Additionally, protein L binds to immunoglobulins via the κ light chain. The main challenge in using these Ig-binding proteins is achieving precise surface localization and orientation, which can be addressed through site-specific fusion, gold-binding peptides, His-tags, or enzymatic conjugation. Engineered analogs of protein A, such as the Z domain—especially the tandem ZZ domains—have shown more efficient antibody capture. Although the interactions between protein A or G and IgGs are non-covalent and reversible, which supports regeneration and reuse, their reversibility may affect stability. Therefore, chemical or photo-reactive crosslinkers are often employed to achieve covalent immobilization, enhancing the efficiency of antibody-antigen binding.
Fluorescence Labeling of Antibody
Gold Nanoparticles labeled Antibody
Magnetic Beads labeled Antibody
Silver Nanoparticles Labeled Antibody
Antibody Oligonucleotide Conjugation
Antibody-Antibiotic Conjugation
Antibody-siRNA Conjugation (ARC)
Antibody Cell Conjugation (ACC)
Immune-Stimulating Antibody Conjugation (ISAC)
Antibody-Exotoxin Conjugation (AExC)
Antibody-Photosensitizer Conjugation (APC)
Antibody-Biopolymer Conjugation (ABC)
Protein-Antibody Conjugation (PAC)
Degrader-Antibody Conjugation (DAC)
Molecular Glue–Antibody Conjugate