Random and site-specific immobilization of catalytic antibodies

doi:10.1016/0141-0229(93)90166-Y

Enzyme and Microbial Technology

Volume 15, Issue 11, November 1993, Pages 916-921

https://doi.org/10.1016/0141-0229(93)90166-Y Get rights and content

Abstract

The effects of immobilization on the immunologic and catalytic activity of a catalytic antibody were compared for randomly immobilized (via glutaraldehyde) whole antibody and site-specifically immobilized (via the reactive sulfhydryl group at the base of the fragment) Fab' fragments. Upon immobilization, the specific binding capacity (n) and the catalytic activity decreased significantly for both systems. Increases in the Michaelis constant (K_M) were accompanied by corresponding decreases in the equilibrium binding constant determined through immunoassays. For the immobilized Fab', n decreased dramatically with increased protein loading, suggesting that, despite the site-specific attachment and favorable orientation, molecular crowding denatured the Fab' fragments. These results also show that there is an optimal surface coverage, not necessarily at the maximum loading, for both immunologic and catalytic activity. Finally, the combining/active site conformation was probed using electron paramagnetic resonance (EPR) spectroscopy. In all antibody samples, there was no spectral evidence of conformational changes in the antibody active site.

References (38)

K. Bowdish et al.
Yeast expression of a catalytic antibody with chorismate mutase activity
J. Biol. Chem.
(1991)
N. Endo et al.
A novel covalent modification of antibodies at their amino groups with retention of antigen-binding activity
J. Immunol. Methods
(1987)
V.S. Prisyazhnoy et al.
Synthesis of high-capacity immunoaffinity sorbents with oriented immobilized immunoglobulins or their Fab' fragments for isolation of proteins
J. Chromatogr.
(1988)
D. Inbar et al.
Crystallization with happen of the Fab' fragment from a mouse IgA myeloma protein with antidinitrophenyl activity
J. Biol. Chem.
(1971)
H.H. Weetall
Covalent coupling methods for inorganic support materials
Methods Enzymol.
(1976)
J.N. Lin et al.
The influence of adsorption of native and modified antibodies on their activity
J. Immunol. Methods.
(1989)
K. Ernst-Cabrera et al.
High-performance affinity chromatography
Trends Anal. Chem.
(1988)
J.W. Jacobs
New perspectives on catalytic antibodies
Bio/Technology
(1991)
R.A. Lerner et al.
At the crossroads of chemistry and immunology: Catalytic antibodies
Science
(1991)
C.E. Cook
Catalytic antibodies
Immunochemica
(1990)

D. Hilvert

Catalytic antibodies

K.M. Shokat et al.

Catalytic antibodies: A new class of transition-state analogues used to elicit hydrolytic antibodies

Angew. Chem. Int. Ed. Engl.

(1990)

P.G. Schultz

Catalytic antibodies

Angew. Chem. Int. Ed. Eng.

(1989)

S.J. Pollack et al.

Selective chemical catalysis by an antibody

Science

(1986)

A. Tramontano et al.

Catalytic antibodies

Science

(1986)

R.A. Gibbs et al.

Construction and characterization of a single-chain catalytic antibody

D.Y. Jackson et al.

A Mutagenesis Study of a Catalytic Antibody

G. Gallacher et al.

A polyclonal antibody preparation with Michaelian catalytic properties

Biochem J.

(1991)

D.Y. Jackson et al.

An antibody-catalyzed Claisen rearrangement

J. Am. Chem. Soc.

(1988)

Cited by (33)

Immobilization of Fab' fragments onto substrate surfaces: A survey of methods and applications
2015, Biosensors and Bioelectronics
Citation Excerpt :
The former explanation can be discarded as it was shown in the previous study (Spitznagel and Clark, 1993) that Fab’ fragments bound with this immobilization technique maintained specific orientations. It is possible that catalysis is more difficult to achieve with immobilized Fab’ fragments due to any conformational changes associated with surface binding (Spitznagel et al., 1993). Similar to certain immobilization techniques on gold, Vikholm et al. (1999) demonstrated the feasibility of immobilizing Fab’ fragments onto quartz surfaces using a mixed lipid monolayer.
Antibody immobilization onto surfaces has widespread applications in many different fields. It is desirable to bind antibodies such that their fragment-antigen-binding (Fab) units are oriented away from the surface in order to maximize analyte binding. The immobilization of only Fab’ fragments yields benefits over the more traditional whole antibody immobilization technique. Bound Fab’ fragments display higher surface densities, yielding a higher binding capacity for the analyte. The nucleophilic sulfide of the Fab’ fragments allows for specific orientations to be achieved. For biosensors, this indicates a higher sensitivity and lower detection limit for a target analyte. The last thirty years have shown tremendous progress in the immobilization of Fab’ fragments onto gold, Si-based, polysaccharide-based, plastic-based, magnetic, and inorganic surfaces. This review will show the current scope of Fab’ immobilization techniques available and illustrate methods employed to minimize non-specific adsorption of undesirables. Furthermore, a variety of examples will be given to show the versatility of immobilized Fab’ fragments in different applications and future directions of the field will be addressed, especially regarding biosensors.
Gold nanoparticle-antibody conjugates for specific extraction and subsequent analysis by liquid chromatography-tandem mass spectrometry of malondialdehyde-modified low density lipoprotein as biomarker for cardiovascular risk
2015, Analytica Chimica Acta
Citation Excerpt :
On the other hand, a higher surface coverage would only translate into enhanced adsorption capacities if the immobilized Abs are freely accessible for the antigens which could be a limiting factor for the large MDA-LDL particles as antigen (vide infra). Many factors such as number of immobilized antibodies, surface density, and orientation of Abs can strongly influence the binding parameters of immunosorbents and are strongly dependent on the immobilization strategy [77]. Thus, extraction efficiency, maximum binding capacity as well as non-specific binding were studied for each GNP-Ab conjugate.
Oxidized low-density lipoproteins (OxLDLs) like malondialdehyde-modified low-density lipoprotein (MDA-LDL) play a major role in atherosclerosis and have been proposed as useful biomarkers for oxidative stress. In this study, gold-nanoparticles (GNPs) were functionalized via distinct chemistries with anti-MDA-LDL antibodies (Abs) for selective recognition and capture of MDA-LDL from biological matrices. The study focused on optimization of binding affinities and saturation capacities of the antiMDA-LDL-Ab-GNP bioconjugate by exploring distinct random and oriented immobilization approaches, such as (i) direct adsorptive attachment of Abs on the GNP surface, (ii) covalent bonding by amide coupling of Abs to carboxy-terminated-pegylated GNPs, (iii) oriented immobilization via oxidized carbohydrate moiety of the Ab on hydrazide-derivatized GNPs and (iv) cysteine-tagged protein A (cProtA)-bonded GNPs. Depending on immobilization chemistry, up to 3 antibodies per GNP could be immobilized as determined by ELISA. The highest binding capacity was achieved with the GNP-cProtA-Ab bioconjugate which yielded a saturation capacity of 2.24 ± 0.04 μg mL⁻¹ GNP suspension for MDA-LDL with an affinity K_d of 5.25 ± 0.11 × 10⁻¹⁰ M. The GNP-cProtA-antiMDA-LDL bioconjugate revealed high specificity for MDA-LDL over copper(II)-oxidized LDL as well as native human LDL. This clearly demonstrates the usefulness of the new GNP-Ab bioconjugates for specific extraction of MDA-LDL from plasma samples as biomarkers of oxidative stress. Their combination as specific immunoextraction nanomaterials with analysis by LC–MS/MS allows sensitive and selective detection of MDA-LDL in complex samples.
Characterization of fully functional spray-on antibody thin films
2014, Applied Surface Science
Citation Excerpt :
These results suggest that the structural stability of the antibody is maintained during the formation of the film. These experimental results also demonstrate that there is apparently no significant denaturation due to “molecular crowding” interactions as was suggested to occur by Spitznagel et al. [61]. The capture activity of the antibody film created by pneumatic spray was calculated only on glass slides to be able to compare with the avidin–biotin bridge immobilization method which is performed in the same material.
The authors recently demonstrated that fully functional Escherichia coli O157:H7 antibody thin films can be prepared using a simple pneumatic nebulizer on glass surface [1]. This paper focuses on the investigation of the morphology and physical properties of these films with the aim to better understand their performance. A series of E. coli O157:H7 antibody spray-on thin films were investigated by ellipsometry, X-ray photoelectron spectroscopy (XPS), immunoassays, attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), fluorescence microscopy, atomic force microscope (AFM) and contact angle analysis. These data were compared to measurements on films prepared with the biotin–avidin covalent bonding scheme. The investigation showed that films created by a 2 min pneumatic spray deposition time can capture antigens similar as the avidin–biotin wet-chemical method. The results also suggests that an influential factor for the comparable capture cell ability between sprayed and covalent films is an increased antibody surface coverage for the sprayed films (non-equilibrium technique), which compensates for the lack of its antibody orientation. There was no significant antibody denaturation detected on any of the sprayed films. Both techniques led to the formation of cluster-aggregates, a factor that seems unavoidable due to the natural tendency of protein to cluster. The avidin–biotin bridge films generally had a higher roughness, which manifested itself in a higher wettability compared to the sprayed films.
Antibody immobilization using pneumatic spray: Comparison with the avidin-biotin bridge immobilization method
2012, Journal of Immunological Methods
Citation Excerpt :
Studies suggest that random orientation reduces the ability of the antibody to react with the antigen due to the impact of steric hindrance generated by the arbitrary alignment of the antibodies (Lu et al., 1996; Xu and Zhao et al., 2006b). However, a reduction on antigen binding activity is not just related to randomly oriented films, because (Spitznagel et al. (1993) suggested that, despite the favorable orientation of the antibody at the surface of covalently attached films, molecular crowding can denature the fragment antigen binding (Fab) region making it necessary to find an optimal maximal coverage, which is not necessarily achieved by the avidin–biotin bridge method. Peluso et al. (2003) found an average increase of 33% in antigen binding activity for specifically oriented antibodies when compared with randomly oriented antibodies with the same antibody density at the surface.
The formation of a thin antibody film on a glass surface using pneumatic spray was investigated as a potential immobilization technique for capturing pathogenic targets. Goat-Escherichia coli O157:H7 IgG films were made by pneumatic spray and compared against the avidin–biotin bridge immobilized films by assaying with green fluorescent protein (GFP) transformed E. coli O157:H7 cells and fluorescent reporter antibodies. Functionality, stability, and immobilization of the films were tested. The pneumatic spray films had lower fluorescence intensity values than the avidin–biotin bridge films but resulted in similar detection for E. coli O157:H7 at 10⁵–10⁷ cells/ml sample concentrations with no detection of non-E. coli O157:H7 strains. Both methods also resulted in similar percent capture efficiencies. The results demonstrated that immobilization of antibody via pneumatic spray did not render the antibody non-functional and produced stable antibody films. The amount of time necessary for immobilization of the antibody was reduced significantly from 24 h for the avidin–biotin bridge to 7 min using the pneumatic spray technique, with additional benefits of greatly reduced use of materials and chemicals. The pneumatic spray technique promises to be an alternative for the immobilization of antibodies on glass slides for capturing pathogenic targets and use in biosensor type devices.
Study of the epoxydized magnetic hydroxyl particles as a carrier for immobilizing penicillin G acylase
2007, Enzyme and Microbial Technology
Magnetic hydroxyl microspheres were synthesized and activated by using epoxyl chloropropane by means of suspension polymerization. The effects of amount of crosslink agent on the immobilization yield and activity yield of immobilized enzyme were investigated. The results showed that the magnetic polymer with crosslink density 30% could be used as an optimized immobilized penicillin G acylase (PGA) support. The apparent Michaelis constant K_m and V_max of the immobilized enzyme were 8.312 × 10⁻⁵ mol/l and 21.64 μmol/min, respectively. The protecting immobilization was achieved by employing the interaction between the phenyl acetic acid (PAA) and PGA. It was found that activity yield of immobilized PGA was 1.0834-fold of the randomly immobilized enzyme. The optimal pH and temperature of the immobilized enzyme were 8.5 and 45 °C, respectively, for hydrolysis of penicillin G. Being applied for 80 times, the remained activity of the immobilized enzyme was about 94.2%.
Enhancing the synthetic utility of aldolase antibody 38C2
2006, Bioorganic and Medicinal Chemistry Letters
Three-phase partitioning (TPP) treated aldolase antibody 38C2 was evaluated for aldol reaction between p-nitrobenzaldehyde and acetone to give 4-(4′-nitrophenyl)-4-hydroxy-2-butanone. While TPP-treated 38C2 transformed 65% of p-nitrobenzaldehyde, the untreated 38C2 gave only 24% transformation in 18 h at 25 °C. However, since TPP-treated 38C2 also gave an additional (unidentified) product, its synthetic utility was limited. Crosslinked aggregate of 38C2, however, gave the biocatalyst which gave a single product and could be reused at 40 °C five times without loss of activity.

View all citing articles on Scopus

View full text

PaperRandom and site-specific immobilization of catalytic antibodies

Abstract

J. Biol. Chem.

J. Immunol. Methods

J. Chromatogr.

J. Biol. Chem.

Methods Enzymol.

J. Immunol. Methods.

Trends Anal. Chem.

New perspectives on catalytic antibodies

Bio/Technology

At the crossroads of chemistry and immunology: Catalytic antibodies

Science

Catalytic antibodies

Immunochemica

Catalytic antibodies

Catalytic antibodies: A new class of transition-state analogues used to elicit hydrolytic antibodies

Angew. Chem. Int. Ed. Engl.

Catalytic antibodies

Angew. Chem. Int. Ed. Eng.

Selective chemical catalysis by an antibody

Science

Catalytic antibodies

Science

Construction and characterization of a single-chain catalytic antibody

A Mutagenesis Study of a Catalytic Antibody

A polyclonal antibody preparation with Michaelian catalytic properties

Biochem J.

An antibody-catalyzed Claisen rearrangement

J. Am. Chem. Soc.

Paper
Random and site-specific immobilization of catalytic antibodies