Targeted Adsorption Of Protein And Its Characteristics At The Solid-liquid Interface

Adsorption of protein at a solid-liquid interface is necessary for antibody chips,biosensors,immunoassays,and other molecular recognition technologies.Retaining adsorbed protein,including antigens and antibodies,in an active state at high density is the critical success factor in such technologies,particularly in those miniaturized high-throughput platforms,such as micro-or nano-fluidic chips,which have become the leading technologies that allow the parallel detection of thousands of analytes in a single experiment.However,it has been found that the adsorbed molecules often lose their immunobinding ability because active sites located on their surface may be partially or totally blocked by the substrate surface,which makes them inaccessible to the analytes.Only a minority of immobilized molecules actively participating in the antigen-antibody reaction,high signal intensities and high throughput can hardly be achieved under these conditions.In this study,we mainly focused on the following methods of controlling and analyzing the orientation of proteins when they are adsorbed at the solid-liquid interface.1)In order to develop an affinity ligand for site-directed immobilization of target proteins on polystyrene(PS)surface,a linear 12-mer peptide phage display random library was screened.Phage clones that specifically bound to PS plate were sequenced after three rounds of biopanning.The obtained DNA sequences revealed that there were several aromatic and basic amino acid residues,which may be critical to binding with the aromatic moieties of PS via p-p stacking effect.One of the selected dodecapeptides,which appeared the most times in biopanning,was named Ligl(FKFWLYEHVIRG).2)Ligl was genetically fused to the N/C-terminus of recombinant antigen ENV which could be recognized by specific antibodies against human immunodeficiency virus type 1(HIV-1),to investigate its performance as an affinity ligand.The ligand-fused ENVs expressed in prokaryotic system were compared to the original one in terms of the adsorption characteristics on PS plate in enzyme-linked immunosorbent assay(ELISA).The results indicated that the ligand-fused antigens were preferentially adsorbed on PS plate in a site-directed model,in which all the epitopes pointed to one direction and ideally toward solution phase,suffering only scarcely from interference by coexisting protein molecules.Anti-HIV-1 ELISA system,which employed Lig1-ENV(Ligl fused to ENV N-terminus)as immobilization antigen also exhibited sufficiently higher sensitivity and specificity in serodiagnosis tests.3)Ligl was genetically fused to the N/C-terminus of chimeric antigen HCV which could be recognized by specific antibodies against hepatitis C virus(HCV).Immunoassay characteristics of ligl-fused HCVs immobilized on PS surface were compared to that of original HCV in both direct and indirect enzyme-linked immunosorbent assay(ELISA).The results indicated that the ligand-fused antigen HCV-Lig1 exhibited a considerable affinity to PS surface by specific interaction via its unique ligand(the affinity constants of HCV-Ligl and original HCV for PS surface determined to be 2.6×108 M-1 and 2.9×105 M-1,respectively).HCV-Ligl was preferentially adsorbed on PS surface in a site-oriented manner and would expose specific antibody-binding sites well,which resulted in a substantial enhancement of detection sensitivity.AFM(atomic force microscopy)images showed that,compared to the original one,HCV-Ligl was arranged on PS surface in an ordered state and its conformational and steric distortions induced during the interfacial binding process were much slighter.As long as the specific epitope of a coating antigen is not located on both its N and C-terminus,the ligand fusion approach could be an ideal strategy for site-oriented protein adsorption.4)By imitating the molecular mechanisms of immunomodulatory interference by Staphylococcus aures,a novel yet simple method for targeted adsorption of antibody at high density was developed.Two HisTag-SPA(polyhistidine-tagged Staphylococcal protein A),obtained from differentiated introduction of HisTag to SPA,were used for the assembly of five IgG(immunoglobulin G)antibodies in an anti-parallel model.Following the chelation of His-tag with Ni2+,antibodies were immobilized on a solid surface in a three-dimensional(3D)manner and exposed the analyte receptor sites well,which resulted in a substantial enhancement of the analytical signal with more than 64-fold increase in HBeAg(Hepatitis B early Antigen)detection sensitivity.Pull-down assays confirmed that IgG antibody can only bind to Ni2+ matrix indirectly via a SPA linkage,where the HisTag is responsible for binding Ni2+ and five homologous domains are responsible for binding IgG Fc.The chromogenic signals(quantitatively expressed as IODs)of five hybridizing bands,each more stronger than its predecessor,indicate the amount of IgG pulled down by Ni2+-NTA beads via SPA was gradually increased with the increasing domains of SPA.The immobilization approach proposed here may be an attractive strategy for the construction of high performance antibody arrays and biosensors as long as the antibody probe is of mammalian IgG.5)Polystyrene microtitre plates modified by 60Co y-ray irradiation were used in an indirect ELISA for detection of anti-HIV and anti-HCV.The plates with 8-9 kGy(optimum dose)irradiation showed 3-5-fold higher detection sensitivity in serodiagnosis tests compared to unmodified ones,and a more than 4-fold lower enzyme concentration than the control used was still detectable at the same sensitivity level.Adsorption/Desorption experiment results,AFM images and XPS(X-ray photoelectron spectroscopy)analysis provide the reason for this improvement.The surface oxidation during irradiation with a corresponding increase in hydrophilicity was probably the main cause of improved adsorption capacity of the modified plate,which could adsorb a larger amount(1.5-3-fold)of protein than the unmodified one.A direct ELISA system confirmed that the enhanced ELISA sensitivity was primarily owing to the increased adsorption ability of modified surface rather than the better stereo presentation of coated antigens.6)By measuring the adsorption amount,the adsorption rate,the time to reach adsorption equilibrium and the equilibrium concentration,the adsorption kinetics and thermodynamics of anti-HBeAg monoclonal antibody on PS surface,HisTag-SPA/anti-HBeAg on Ni2+surface were studied,respectively.The adsorption of anti-HBeAg from liquid phase on PS surface can be classified as a mono-layer and exothermic physical process.Adsorption data were found to fit Langmuir isotherms model successfully with significant high values of correlation coefficient(R2?0.995).The adsorption rate is controlled by liquid film diffusion and particle dispersion.Both pseudo-first and pseudo-second order models match well with the experimental kinetics data;The adsorption of HisTag-SPA/anti-HBeAg on Ni2+surface,whose capacity is 4-5 times higher than the one on PS surface,is of a multi-layer and endothermic process.The linear correlation coefficients(R2?0.991)show that the adsorption isotherm is fitted well with Freundlich model.The pseudo-second order equation was found to be more suitable than the pseudo-first order one for describing the adsorption kinetic behavior.Liquid film diffusion model and particle dispersion model are all not suitable for fitting the adsorption of HisTag-SPA/anti-HBeAg on Ni2+surface.The validity of Weber-Morris model reveals that the adsorption process can be divided into two stages:a rapid first stage,in which protein molecules diffuse to matrix surface from liquid phase and the boundary layer is not yet formed at this time,and a slower second stage that achieved equilibrium(a rate-controlling step),which may be a dynamic process coordinated by matching activities and binding capacities,involving the formation of boundary layer and isochronous interactions of HisTag-SPA,IgG antibody,and chelating surface.