Analyst, March 1996, Vol. 121 (29R-32R) 29R Tutorial Review Oriented Immobilization of Antibodies and Its Applications in lmmunoassays and I m m u nosensors Bin Lua, Malcolm R. Smytha* and Richard O’Kennedyb a School of Chemical Sciences, Dublin City University, Dublin 9, Ireland h School of Biological Sciences, Dublin City University, Dublin 9, Ireland The immobilization of antibodies on solid-phase materials has been used in many areas such as purification, diagnostic immunoassays and immunosensors. Problems associated with the loss of biological activity of the antibodies upon immobilization have been noticed in many cases. One of the main reasons for such loss is attributed to the random orientation of the asymmetric macromolecules on support surfaces. In this paper, the approaches for achieving oriented coupling of antibodies to increase the antigen binding capacity are reviewed.Some issues such as steric hindrance caused by neighbouring antibody molecules, the distance between an antibody and the support surface and the use of antibody fragments are dealt with. Some applications of the oriented immobilized antibodies in immunoassays and immunosensors are examined. Keywords: Orientation; antibody; immobilization; immunoassay; immunosensor Owing to their high specificity, antibodies immobilized on various supports have been widely used for different purposes. In immunoaffinity chromatography, antibodies immobilized on traditional affinity gels or porous particles are employed to separate proteins from dilute mixtures under mild conditions.’ The use of immobilized antibodies in diagnostic immunoassays has expanded considerably in recent years.2 Immunosensors, in which antibodies are immobilized on optical fibres, electrodes or semiconductor chips for the detection of antigen^,^-^ represent another promising application.Numerous coupling strategies have been developed for immobilizing antibodies on different solid surfaces through the formation of defined linkages in which glutaraldehyde, carbodiimide and other reagents such as succinimide ester, maleinimide and periodate are widely used. However, problems associated with the loss of biological activity upon immobilization of antibodies are noticeable in many cases. When antibodies are covalently attached to solid supports, their specific binding capacity is usually less than that of soluble antibodies.One of the main reasons for this reduction is attributed to the random orientation of the antibodies on support surfaces. The asymmetric macromolecule immunoglobulin G (IgG) is composed of two different fragments, F(ab’)2 and Fc (Fig. 1). The Fc fragment contains antibody effector functions, so it has no antigen binding affinity. The F(ab’)2 fragment contains two identical Fab’ fragments. The antigen binding site * To whom correspondence should be addressed. is positioned at the amino end of each fragment. In normal coupling schemes, coupling does not discriminate between possible attachment points near or removed from the specific binding site (or sites), which results in spatial orientation of antibodies on the supports that might prohibit formation of an antibody-antigen complex (Fig. 2).For instance, if multiple lysine groups are present on the surface of an antibody molecule, multiple attachment might occur. This may result in Fig. 1 Schematic diagram of an antibody molecule and its fragments. The molecule can be subdivided into two parts. The Fc fragment contains the antibody effector functions, such as complement activation, cell membrane receptor interaction and transplacental transfer.6 The F(ab’)z fragment contains two identical Fab’ fragments, which are held together by the disulfide linkages in the hinge (H) region. The Fab’ fragment (antigen- binding site-containing fragment) consists of the heavy (H) and light (L) variable (V) chains (VH and V,) and the constant ( C H I and CL) chains.Other segments are the Fv (variable fragment) consisting of the VH and VL chains and the Fd fragment which contains the VH and C H I chains. The carbohydrate moieties locate at the c H 2 domain and the binding sites for Fc receptors (protein A, protein G and recombinant protein A/G) locate between the domains of CH2 and cH3. Inactive Parttally active Fully active Fig. 2 coupling procedure. Ideal representation of IgG antibody immobilized by random30R Analyst, March 1996, Vol. 121 different orientations of the antibody on the support surface depending on which lysine group binds to the support. When immobilization occurs through the antigen binding sites on the Fab’ portions, the ability of that antibody to bind antigen may be severely impaired or eliminated entirely.To eliminate these drawbacks, several approaches for achieving oriented antibody coupling have been developed. In one scheme, antibodies were bound to Fc receptors on solid supports, such as protein A, protein G or recombinant protein A/ G [Fig. 3(a)]. These Fc receptors bind to the Fc portion of many IgG subclasses, leaving the antigen-specific sites free. Another strategy for oriented coupling relies on the chemical or enzymic oxidation of the IgG carbohydrate moiety [Fig. 3(h)]. Im- munoglobulins contain carbohydrate moieties linked mainly to the CH2 domain of the Fc fragments. Under mild conditions, the hydroxy groups of the carbohydrates can be oxidized to aldehyde groups without significantly impairing the active sites of the antibody. The oxidized antibodies can then be im- mobilized to hydrazide-activated supports via their oxidized groups by forming covalent hydrazone bonds.The third method is to utilize the sulfhydryl group of the Fab’ fragment to create an oriented antibody fragment [Fig. 3(c)]. This can be achieved by first making F(ab’):! with pepsin digestion, followed by reduction of the sulfide bond between monovalent Fab’ fragments, or by using artificial Fab’ fragments produced by molecular genetic techniques. The sulfhydryl group formed in the C-terminal region of the fragment can be used to couple the fragment to an insoluble support in such a way that its antigen binding site is available for interaction with antigen. The approach to binding antibodies to immobilize the Fc receptors on solid surfaces is by no means a new concept.The first Fc receptor immobilized on a solid support was protein A. Protein A was found in the cell wall of Staphylococcus aureus. In 1978, Gersten and Marchalonis7 linked antibody to protein A-Sepharose to improve its antigen-binding capacity. The procedure circumvented the problem of random antibody- matrix coupling inherent in the cyanogen bromide technique. Subsequently, Schneider et a1.8 modified the technique by generating the affinity cross-linked matrices with dimethyl pimelimidate. The immunoaffinity columns on which antibod- ies were cross-linked were stable and had larger antigen-binding capacities. This scheme was further modified by Sisson and Castor,g the linkage between the protein A-immobilized matrix Fig. 3 Representation of approaches for achieving oriented antibody immobilization. (a) Antibody binds to Fc receptors on solid supports; (b) antibody is coupled to the solid support via an oxidized carbohydrate moiety on its CH2 domain of the Fc fragment; (c) monovalent Fab’ fragment is bound to insoluble support via a sulfhydryl group in the C-terminal region of the fragment.and the bound immunoglobulin being enhanced. After the modification, almost all bound antibody was coupled to the matrix and full antigen-binding capacity of the antibody was reserved, which indicated that the cross-linker does not interfere with the variable region of the IgG molecules, and the antibody is positioned on the gel matrix in an orientation that allows maximum antigen binding.In this scheme, the Fc receptor is the key element for the oriented immobilization of the IgG antibody. It is expected that the receptor has the same affinity to all IgG isotypes to make the receptor-coupled support a universal inducing matrix for oriented IgG immobilization. Protein A has been used success- fully to bind the Fc portion of IgG from many mammalian species. However, it fails to react with IgG from several other species such as goat, sheep, cow and horse.10 A more versatile and efficient alternative to protein A for binding IgG isotypes is protein G, which is found in the cell wall of Streptococcus human pathogenic strains of the Lancefield group G. Protein G has some advantage over protein A because it reacts with more IgG isotypes and it reacts less with other immunoglobulins such as human IgM and IgA.ll However, it does not bind strongly to several IgGs with which protein A reacts well.The third generation of IgG-binding Fc receptor, recombinant protein A/ G, has recently been developed. Recombinant protein A/G is a gene fusion product secreted from a non-pathogenic form of Bacillus. The secreted protein A/G is designed to contain four Fc binding domains from protein A and two from protein G.12 This makes it combine the IgG-binding profiles of both protein A and protein G. Recently, all three kinds of Fc receptors have been successfully immobilized on different affinity gels to bind IgG antibodies. Several commercial products for preparing oriented IgG columns by using these immobilized Fc receptors have appeared recently.13 The concept of oriented immobilization of antibodies relying on the oxidation of IgG carbohydrate moieties appeared about 10 years ago.O’Shannessy and Quarles14 demonstrated in a preliminary experiment that periodate-oxidized goat antibody retained its biological activity after binding to a hydrazide- containing solid support. Subsequently, a general method for binding glycoproteins with specific sites to hydrazide supports was described by O’Shannessy and Hoffman.15 Subsequently, antibodies from several species were investigated by the same workers16 to determine their ability to bind to hydrazide gels and to determine the activity of the bound antibody. Parameters such as the pH of the oxidation, the pH of the binding and coupling time was optimized.The antigen-binding capacity of the antibody coupled to the supports (mol Ag bound/mol Ab coupled) ranged from 0.6 to 1.35, depending on the size of the bound antigen. The activity was three times larger than that of the same antibody bound to the same support through their E- amino groups. Prisyazhnoy et aZ.17 used a similar method for coupling oxidized rabbit anti-mouse IgG to two different gel supports. They suggested that the antigen-binding capacities ranged from 1.1 to 1.6, depending on the support used. Note that several workers have succeeded in detecting the dramatic increase in the antigen-binding capacity of periodate- oxidized polyclonal antibodies, but failed to detect any significant alteration in the capacity of periodate-oxidized monoclonal antibodies.Matson and Little18 suggested that both the antigen-binding site and the carbohydrate moiety of the monoclonal antibody could have been damaged during the affinity purification and excessive oxidation, so milder condi- tions for the purification and the conversion into aldo-IgG should be examined. Highsmith et al. 19 attributed the situation to the presence of carbohydrate moieties in the Fab’ regions of some monoclonal antibodies. However, the data from Solomon et al.’s report20 indicated that enzyme oxidation of monoclonal antibodies before the oriented immobilization improved the specific capacity of the antibodies. They used neuraminidaseAnalyst, March 1996, Vol. 121 31R and galactose oxidase, instead of periodate, to oxidize the carbohydrate moieties on the monoclonal antibodies. The treatments produced a maximum of two aldehyde groups per mole of antibody, compared with 3.2-4 aldehyde groups obtained after periodate oxidation.These enzyme-oxidized antibodies possessed a higher antigen binding activity than the corresponding chemically oxidized antibody immobilized on the same matrix. An example of utilizing the sulfhydryl group of Fab’ fragments to create an oriented antibody fragment on solid surfaces is that developed by Prisyazhnoy et al.17 Fab’ fragments from rabbit IgG were immobilized on to a Sepharose matrix derivatized with the maleimide group and approximately half of the coupled Fab’ fragments retained their antigen- binding capacity.Jimbo and Saitos have developed a technique for the fabrication of oriented Fab’ fragments immobilized in organic films on semiconductor substrates and suggested that a fairly high density of immobilized Fab fragments (6 X 10” cm-2) was obtained. Recently, Lu et ~1.21 studied the effects of the orientation of the Fab’ fragments on the antigen-binding capacity. The Fab’ fragments of rabbit anti-human IgG were immobilized in an oriented form on derivatized silica slide surfaces containing pyridyl disulfide groups, and in random form to glutaraldehyde-bound silanized silica slide surfaces. After immobilization, a higher surface density of the oriented fragment (1.6 X 10l2 cm-2) was obtained. The changes in orientation of the immobilized fragments drastically influenced the antigen-binding capacity of the fragments.The activity of the fragments in oriented form was about three times higher than that in random form. Such immobilized fragments could find extensive applicability in the design of immunosensors for monitoring soluble antigens. Although efforts have been made to develop new schemes to increase the biological activities of immobilized antibodies, little work has been done to compare the properties of the antibodies immobilized by the different techniques. Alarie and Sepaniak22 compared two of the above coupling schemes by immobilizing Fab’ fragments or intact antibody on silica beads via the cross-linker 2-fluoro- 1 -methylpyridinium toluene-4-sul- fonate (FMP) or immobilized protein A, respectively. Although the loadings of the antibody on two surfaces were nearly the same, the immobilized Fab’ fragments exhibited only half the activity of the intact antibody coupled via protein A.They attributed the loss in the activity to the reduction of the disulfide linkages in Fab’ fragments. However, the random adsorption of the fragments on the silica surfaces might be another reason for the loss.2’,24 In addition to the orientation of surface-coupled antibodies, several other factors, such as steric hindrance caused by neighbouring antibodies at high surface concentration, the distance between coupled antibodies and the support surface and the modified conformation, also influence antigen-binding capacity. Matson and Little’s and other workers25326 have studied the relationship between the activity and surface antibody concentration.Their results showed that steric hin- drance created by crowding of adjacent antibody molecules immobilized via oriented or random coupling mechanisms lead to the same net effect, a decrease in antigen-binding efficiency. Spitznagel and co-~orkers2~,~8 studied the effects of surface density and orientation on the immobilized antibody and antibody fragments. For all immunosorbents, a high protein loading led to relatively low specific activities. At higher loading, the activity of the immobilized whole antibody was lower for the large antigen than for the small hapten, whereas no effect of the hapten size on the activity was observed for either immobilized Fab’ or Fv fragments. No significant changes in the conformation of active immobilized antibodies were observed.The most interesting results in the study were that immobilized Fv fragments had the highest binding capacities, which indicated that removing unnecessary protein domains can be beneficial for improving the total capacity of immunosor- bents. As mentioned above, antibody IgGs are multi-fragment glycoproteins that may be dissected into individual fragments using proteases. The preparation of homogeneous antibody fragments by enzymic digestion can be difficult, and often conditions have to be optimized for each antibody. By contrast, molecular genetic techniques can be used to produce defined fragments of any immunoglobulin. Current molecular genetic techniques do not allow for the correct functional expression of whole antibody molecules.However, most expression vectors which are from plasmids that propagate in Escherichia (E. coli) could be used to express correctly recombinant light- and heavy-chain genes. Antibody fragments are easily manipulated using the expression vector available. Various fragments, which include Fab’, F(ab’)2, Fd and Fv, have been manipulated in numerous protocols.29-32 The resulting fragments have been shown to retain the antigen-binding activity of the parent molecule. Furthermore, this technology is not limited to the production of conventional immunoglobulin fragments and has been extended to the generation of novel fragments which have been produced by either isolating variable-region gene seg- ments or by inserting stop codons into heavy- and light-chain genes.33.34 The relatively inexpensive production of the anti- body derivatives opens up the possibility of developing new immunoassay agents.The schemes for oriented immobilization of solid antibodies on supports have been applied in the fabrication of different immunosensors. An immunosensor based on microelectronic techniques in which an antibody was immobilized on to the gate surface by means of immunochemical membranes containing protein A has been reported.35 Flow injection electrochemical enzyme immunoassays for theophylline using protein A immunoreactors have been developed.36.37 Fluorescence flow injection immunoassay for testosterone using protein A and for insulin using protein G immunoreactors have also been developed.38.39 Kaku et al .40 described an enzyme immunoelec- trode in which anti-insulin IgG was anchored on partial hydrophilic polypropylene membranes containing protein A, and suggested that the formation of the oriented antibody improved both the sensitivity and dynamic range of the sensor.Konig and Gratzel4’43 have studied piezoelectric immuno- sensors for the detection of the human herpes viruses and T- lymphocytes, in which three different methods of immobiliza- tion (via polyethylenimine, silane or protein A) were evaluated. The best results in terms of sensitivity, reproducibility and re- usability were obtained with the protein A immobilization method. Recently, Owaku et al.44 developed an optical immunosensor in which the antibody binds to a double monolayer protein A film formed by Langmuir-Blodgett film techniques.Several immunosensors based on the immobiliza- tion of the Fab’ fragments have been produced.5345-47 Betts and co-~orkers45,~7 have demonstrated that dansylated Fab’ frag- ments were useful in the fabrication of selective, sensitive, self- contained and re-usable fibre-optic-based immunosensors for the antigens of human serum albumin and several haptens. In conclusion, an examination of the literature has revealed that oriented coupling techniques offer several potential advantages over random coupling methods, which have resulted in the better separation in immunoaffinity chromatography and higher sensitivity in immunoassays. 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