Antibody Immobilization Methods Research Articles

End-on Covalent Antibody Immobilization on Dual Polarization Interferometry Sensor Chip for Enhanced Immuno-sensing

End-on immobilization of antibody is an important technology to greatly improve the sensitivity of immuno-sensing. The end-on, or constant fragment (Fc) site-specifically oriented antibodies expose more active antigen binding sites (Fab) and tend to capture more analytes from the solution. In this study, dual polarization interferometry (DPI), which is the only available equipment that can concurrently obtain the mass, thickness, density, and refractive index in real time, was applied to investigate two end-on antibody immobilization methods, as exemplified by prostate-specific antigen (PSA) antibody. In the first method, antibody was immobilized via the saccharide chain linked to Fc portion of the antibody, by chelation to the surface-bound boronic acid. In the second method, antibody was partially reduced by tris(2-carboxyethyl) phosphine (TCEP) under mild conditions, followed by covalent conjugation to the surface via thiol-maleimide reaction. Time-resolved measurement from DPI verifies the end-on conformation of the antibody on the sensor surface. The second method shows better detection performance, with enhanced sensitivity and reproducibility than the first method, due to the optimal alignment of the antibody. Finally, these two methods were compared with the protein G-based antibody orientation control and random antibody immobilization in terms of the mass, thickness, density, refractive index, reproducibility, and stability. The detailed antibody immobilization parameters obtained in this paper are of great importance in the developing of solid–liquid phase immunosensors with enhanced detection sensitivity.

Surface Plasmon Resonance for C-Reactive Protein Detection in Human Plasma

In this work, we describe an approach of detecting biomarkers by Surface Plasmon Resonance imaging (SPRi) technique in real samples. Two C-Reactive Protein (CRP)-antibody immobilization methods were used: The first method was based on direct physisorption of CRP-antibody onto gold surface; the second one was based on oriented CRP-antibody with protein G intermediate layer. The two developed immunosensors were tested against CRP antigen in phosphate buffer saline solution with the SPRi technique. The response of the developed immunosensors was reproducible and stable. The detection limit of 10 pg&#183mL&#451 and 50 pg&#183mL&#451 CRP-antigen was observed with and without protein G respectively with this technique. Moreover, the developed SPRi immunosensor was used for CRP-antigen detection in human plasma. A detection limit of 5 ng&#183mL&#451 and 10 ng&#183mL&#451 was obtained with and without protein G respectively. These obtained results were compared to those obtained with QCM (Quartz Crystal Microbalance) and Enzyme-Linked Immunosorbent Assay (ELISA) techniques.

Label-free brain injury biomarker detection based on highly sensitive large area organic thin film transistor with hybrid coupling layer

We describe a sensitive, large-area thin film transistor (TFT) sensor platform for real time detection of low-concentration protein analytes in solution. The sensing area is 7 mm by 7 mm. p-channel (pentacene) and n-channel (a naphthalenetetracarboxylic diimide, NTCDI) organic molecules were each used as semiconductors in conjunction with a newly designed receptor–antibody-functionalized top dielectric layer. This layer, incorporating both a fluorinated polymer and vapor-deposited hydrocarbon, provided maximum capacitive coupling and minimal interference from the aqueous analyte solution, and allowed convenient solvent processing of the antibody coupling layer. Additionally, a new antibody immobilization method was introduced, which led to high immobilization yield and surface coverage. Using glial fibrillary acidic protein (GFAP) as a model protein analyte, this sensor platform demonstrated significant selectivity and recognition of target protein even in much more concentrated non-target protein backgrounds. The dose–response relationship yielded a Langmuir isotherm from which a reasonable affinity constant was calculated for the protein and antibody. A zeta potential measurement provided further evidence of the surface potential change being detected by the TFTs. We explicitly verified for the first time that the response is in fact predominantly from perturbations of TFT channel current. To the best of our knowledge, this is the most sensitive organic TFT (OTFT) protein sensor yet reported, and also the first demonstration of the expected opposite current responses by p- and n-channel semiconductors to the same protein.

A comparative study on antibody immobilization strategies onto solid surface

Antibody immobilization onto solid surface has been studied extensively for a number of applications including immunoassays, biosensors, and affinity chromatography. For most applications, a critical consideration regarding immobilization of antibody is orientation of its antigen-binding site with respect to the surface. We compared two oriented antibody immobilization strategies which utilize thiolated-protein A/G and thiolated-secondary antibody as linker molecules with the case of direct surface immobilization of thiol-conjugated target antibody. Antibody immobilization degree and surface topography were evaluated by surface plasmon resonance and atomic force microscope, respectively. Protein A/G-mediated immobilization strategy showed the best result and secondary antibody-mediated immobilization was the worst for the total immobilization levels of target antibodies. However, when considering real-to-ideal ratio for antigen binding, total target antigen binding levels (oriented target antibody immobilization levels) had the following order: secondary antibody-mediated immobilization>protein A/G-mediated immobilization>direct thiol-conjugated immobilization. Thus, we confirmed that protein A/G- and secondary antibody-mediated strategies, which consider orientation of target antibody immobilization, showed significantly high antigen binding efficiencies compared to direct random immobilization method. Collectively, the oriented antibody immobilization methods using linker materials could be useful in diverse antibody-antigen interaction-involved application fields.

Application of oriented and random antibody immobilization methods in immunosensor design

An immunosensor for the determination of bovine leukemia virus (BLV) antigen (gp51) using surface plasmon resonance (SPR) equipment and SPR-chip modified by oriented and randomly immobilized antibodies (anti-gp51) was developed. Different conditions of anti-gp51 reduction by 2-mercaptoethanol (2-ME) and dithiothreitol (DTT) solutions were compared and explored. The best gp51 detection sensitivity was monitored using SPR-chip modified with oriented antibody fragments (frag-anti-gp51) obtained after reduction with DTT at 50mM concentration. The SPR sensor could detect gp51 antigen in a range from 0.01 to 0.5mg/mL. The developed SPR sensor offered the limit of detection (LOD) as low as 0.0028mg/mL, while the limit of quantification was as low as 0.0092mg/mL with very good repeatability during the three detection-regeneration cycles (1–4%). Oriented immobilization of frag-anti-gp51 was found to be more suitable for the design of SPR-immunosensor for the detection of gp51 if compared with random immobilization of anti-gp51. This method was successfully applied for the detection of gp51 in blood serum samples in a rapid, reliable and selective manner.

Dextran Modified Silicon Photonic Microring Resonator Sensors

We present a dextran modified silicon microring resonator sensor for high density antibody immobilization. An array of sensors consisting of three sensor rings and a reference ring was fabricated and its surface sensitivity and the limit of detection were obtained using polyelectrolyte multilayers. The mass sensitivity and the limit of detection of the fabricated sensor ring are 0.35 nm/ng mm and 42.8 pg/mm in air, respectively. Dextran modified sensor surface was successfully prepared by covalent grafting of oxidized dextran on 3-aminopropyltriethoxysilane (APTES) modified silicon sensor surface. The antibody immobilization on hydrogel dextran matrix improves 40% compared to traditional antibody immobilization method via APTES and glutaraldehyde linkage. Keywords—Antibody immobilization, Dextran, Immunosensor, Label-free detection, Silicon micro-ring resonator

Comparison of sensing strategies in SPR biosensor for rapid and sensitive enumeration of bacteria

Rapid and sensitive detections of microorganisms are very important for biodefence, food safety, medical diagnosis and pharmaceutics. The present study aims to find out the most proper bioactive surface preparation method to develop rapid, sensitive and selective bacteria biosensor, based on surface plasmon resonance (SPR) spectroscopy. Escherichia coli (E. coli) was used as a model bacterium and four sensing strategies in SPR were tested. Three of these strategies are antibody immobilization methods that are non-specific adsorption, specific adsorption via the avidin–biotin interaction, and immobilization of antibodies via self-assembled monolayer formation. The fourth strategy is a novel method for bacteria enumeration based on the combination of the SPR spectroscopy and immunomagnetic separation with using gold-coated magnetic nanoparticles. According to results, the most efficient SPR method is the one based on gold-coated magnetic nanoparticles. This method allows to specifically separate E. coli from the environment and to quantify rapidly without any labeling procedure. The developed method has a linear range between 30 and 3.0×104cfu/ml, and a detection limit of 3cfu/ml. The selectivity of the method was examined with Enterobacter aerogenes and Enterobacter dissolvens, which did not produce any significant response. The usefulness of the method to detect E. coli in real water samples was also investigated, and the results were compared with the results from plate-counting method. There was no significant difference between the methods (p>0.05).

Improved antibody immobilization using aniline‐catalyzed aldehyde‐hydrazide chemistry for the study of protein‐protein interactions

Covalent attachment of an antibody to a beaded support before immunoprecipitation prevents antibody contamination upon antigen elution and allows for the detection of antigens with similar molecular weights as antibody heavy and light chains. Common methods of antibody immobilization include direct coupling of antibody amino groups to aldehyde supports followed by reduction and indirect coupling of antibodies to Protein A/G‐coated beads followed by amine‐reactive crosslinking. Drawbacks of these methods include the loss of antibody functionality due to chemical modification or obstruction of the antigen binding site. An alternative approach for the covalent attachment of antibody to a beaded support utilizes oxidization of sugars in the antibody Fc region to aldehydes, which react with a hydrazide‐activated support. This method allows for unobstructed and oriented antibody conjugation without adverse modifications to antigen binding sites. However, compared to amine‐reactive antibody crosslinking, aldehyde‐hydrazide coupling often has lower efficiency and requires significantly longer incubation times. In order to improve this conjugation chemistry, we developed new aldehyde‐hydrazide immobilization and immunoprecipitation kits. These kits include aniline as a catalyst in a buffer with optimized pH for increased antibody coupling efficiency. The addition of aniline results in an increase of coupling efficiency from 50% to 90% for most antibody subtypes, and shortens reaction times to less than 4 hours. In addition, we demonstrate improved immunoprecipitation of Epitheleal Growth Factor Receptor (EGFR) and Signal Transducers and Activator of Transcription 1 Protein (STAT1), as well as the co‐immunoprecipitation of endogenous Cyclin B1/Cyclin Dependant Kinase 1 (Cdk1) complex from cell lysates using 2–10μg of immobilized antibody.

Development of electrochemical immunosensors based on different serum antibody immobilization methods for detection of Japanese encephalitis virus

This paper describes the development of electrochemical immunosensors based on human serum antibodies with different immobilization methods for detection of Japanese encephalitis virus (JEV). Human serum containing anti-JEV antibodies was used to immobilize onto the surface of silanized interdigitated electrodes by four methods: direct adsorption (APTES-serum), covalent binding with a cross linker of glutaraldehyde (APTES-GA-serum), covalent binding with a cross linker of glutaraldehyde combined with anti-human IgG (APTES-GA-anti-HIgG-serum) and covalent binding with a cross linker of glutaraldehyde combined with a bioaffinity of protein A (APTES-GA-PrA-serum). Atomic force microscopy was used to verify surface characteristics of the interdigitated electrodes before and after treatment with serum antibodies. The output signal of the immunosensors was measured by the change of conductivity resulting from the specific binding of JEV antigens and serum antibodies immobilized on the electrodes, with the help of horseradish peroxidase (HRP)-labeled secondary antibody against JEV. The results showed that the APTES-GA-PrA-serum method provided the highest signal of the electrochemical immunosensor for detection of JEV antigens, with the linear range from 25 ng ml −1 to 1 μg ml −1, and the limit of detection was about 10 ng ml −1. This study shows a potential development of novel electrochemical immunosensors applied for virus detection in clinical samples in case of possible outbreaks.

Determination of Molecular Configuration by Debye Length Modulation

Silicon nanowire field effect transistors (FETs) have emerged as ultrasensitive, label-free biodetectors that operate by sensing bound surface charge. However, the ionic strength of the environment (i.e., the Debye length of the solution) dictates the effective magnitude of the surface charge. Here, we show that control of the Debye length determines the spatial extent of sensed bound surface charge on the sensor. We apply this technique to different methods of antibody immobilization, demonstrating different effective distances of induced charge from the sensor surface.

Enhancing immunoassay detection of antigens with multimeric protein Gs

This paper describes a method for the effective and self-oriented immobilization of antibodies on magnetic silica-nanoparticles using a multimeric protein G. Cysteine-tagged recombinant dimers and trimers of protein G were produced in Escherichia coli BL21 by repeated linking of protein G monomers with a flexible (GGGGS)3 linker. Amino-functionalized silica-coated magnetic nanoparticles (SiO2-MNPs, Fe3O4@SiO2) were prepared and coupled to the protein G multimers, giving the final magnetic immunosensor. The optimal conditions for the reaction between the protein Gs and the SiO2-MNPs was a time of 60min and a concentration of 100μg/mL, resulting in coupling efficiencies of 77%, 67% and 55% for the monomeric, dimeric and trimeric protein Gs, respectively. Subsequently, anti-hepatitis B surface antigen (HBsAg) was immobilized onto protein G-coupled SiO2-MNPs. The quantitative efficiency of antibody immobilization found the trimeric protein G to be the best, followed by the dimeric and monomeric proteins, which differs from the coupling efficiencies. Using all three protein constructs in an HBsAg fluoroimmunoassay, the lowest detectable concentrations were 500, 250 and 50ng/mL for the monomeric, dimeric and trimeric protein G-coupled SiO2-MNPs, respectively. Therefore, multimeric protein Gs, particularly the trimeric form, can be employed to improve antibody immobilization and, ultimately, enhance the sensitivity of immunoassays. In addition, the multimeric protein Gs devised in this study can be utilized in other immunosensors to bind the antibodies at a high efficiency and in the proper orientation.

Direct-binding quartz crystal microbalance immunosensor to detect carp metallothionein

Metallothionein (MT) is a valuable biomarker against xenobiotic heavy metal contamination in organs and blood of fish. In this study, MT was induced in carp blood by cadmium injection and was purified to homogeneity by DEAE-cellulose ion exchange column chromatography to be used dually as a standard and immunogen. A sensitive detection on carp MT was then followed using a batch-type direct-binding quartz crystal microbalance immunosensor system installed with a well cell. Among antibody immobilization methods tested, two methods that were carried out via a heterobifunctional thiolation cross-linker, and via the cross-linker and protein G showed their respective advantages in simplicity and sensitivity. When analyzed using the immunosensor for carp MT in the concentration range of 50–4000 ng/mL, the limit of detection around 250 ng/mL was obtained. Based on its reasonable sensitivity and real-time presentation for the sensor signal, the immunosensor of this study was presumed as a screening tool to monitor possible heavy metal contamination in a fish farm and imported fish.

CMOS and MEMS based micro hemoglobin-A1c biosensors fabricated by various antibody immobilization methods

This paper presents a micro multichannel immunosensor for hemoglobin-A1c level determination rapidly and conveniently. The micro immunosensor is composed of an integrated FET sensor chip and a disposable microelectrode-array chip based on CMOS and MEMS technique, respectively. Antibody immobilization methods, such as mixed self-assembled monolayers (SAMs), seed mediated growth nano-gold and nano-gold wrapped with mixed SAMs are studied. The immunosensor fabricated by nano-gold wrapped with mixed SAMs immobilization method shows the highest sensitivity due to the uniform distribution of the nano-golds without aggregation, and the increase of biomolecules amount on the sensitive area of the micro electrode chip. Whole blood samples obtained from hospital were examined using this immunosensor. Relative standard deviation of 2.63% and recovery of 102.54% were achieved.

Evaluation of zero-length cross-linking procedure for immuno-magnetic separation of Leptospira

Leptospirosis constitutes a major health problem in tropical and subtropical countries and is caused by pathogenic Leptospira. Immuno-magnetic separation (IMS) is considered to be an effective pre-enrichment method to isolate Leptospira from liquid specimen. We applied an inexpensive and simple IMS protocol using zero-length cross-linkers to immobilize polyclonal anti-leptospiral antibodies onto magnetic particles. The IMS-system has been optimized and evaluated by the assessment of the capture efficiency (CE). Main parameters that influence the conjugation procedure were optimized, including the amount of protein per milligram of magnetic particles, the pH and ionic strength of the conjugation buffer. The bead-bound leptospiral fraction was identified by using acridine orange fluorescence dye. The highest value for CE occurred when using high molar phosphate saline buffer at a pH around the isoelectric point of the antibodies. Finally, up to 3×108 leptospiral cells per mL could have been captured with approximately 50 μg of antibody-labelled particles. Strong particle agglutination could be observed during incubation for leptospiral concentrations in the range of 107–108 cells per mL. Despite covalent binding, we show that the physical adsorption parameters pH and ionic strength of the conjugation buffer greatly affect the entire immobilization process with regard to the CE, thus being able to increase the reactivity of the particles. We therefore conclude that a well-adjusted conjugation buffer for the used chemistry could possibly replace expensive and more complicated antibody immobilization methods.

Selective improvement of tumor necrosis factor capture in a cytokine hemoadsorption device using immobilized anti-tumor necrosis factor.

Sepsis is a harmful hyper-inflammatory state characterized by overproduction of cytokines. Removal of these cytokines using an extracorporeal device is a potential therapy for sepsis. We are developing a cytokine adsorption device (CAD) filled with porous polymer beads which efficiently depletes middle-molecular weight cytokines from a circulating solution. However, removal of one of our targeted cytokines, tumor necrosis factor (TNF), has been significantly lower than other smaller cytokines. We addressed this issue by incorporating anti-TNF antibodies on the outer surface of the beads. We demonstrated that covalent immobilization of anti-TNF increases overall TNF capture from 55% (using unmodified beads) to 69%. Passive adsorption increases TNF capture to over 99%. Beads containing adsorbed anti-TNF showed no significant loss in their ability to remove smaller cytokines, as tested using interleukin-6 (IL-6) and interleukin-10 (IL-10). We also detail a novel method for quantifying surface-bound ligand on a solid substrate. This assay enabled us to rapidly test several methods of antibody immobilization and their appropriate controls using dramatically fewer resources. These new adsorbed anti-TNF beads provide an additional level of control over a device which previously was restricted to nonspecific cytokine adsorption. This combined approach will continue to be optimized as more information becomes available about which cytokines play the most important role in sepsis.

A novel antibody immobilization and its application in immunoaffinity chromatography

A novel antibody immobilization and its application in immunoaffinity chromatography (IAC) were presented. Using acrylamide (AM) as monomer, ethylene glycoldimethacrylate (EGDMA) as cross-linker and bulk polymerization as synthetic method, we prepared a polymer in which the Cu(II) was embedded. The Cu(II)-embedded polymer was tested for its binding with protein. It was found that Cu(II)-embedded polymer displayed a strong binding with bovine serum albumin (BSA). At 80% of methanol, no BSA was released from Cu(II)-embedded polymer. The Cu(II)-embedded polymer was then used as a novel solid support for antibody immobilization. IAC column was prepared by immobilizing polyclonal antibody (pAb) against clenbuterol (CL) on Cu(II)-embedded polymer and packing the Cu(II)-embedded polymer-pAb into a common solid phase extraction (SPE) cartridge. Under optimal extraction conditions, the IAC column was characterized in terms of maximum binding capacity for target analyte, extraction efficiency and reusability. It was revealed that, for IAC column packed with 0.1 g of solid support immobilized with antibody, the maximum capacity for CL was 616 ng; the extraction recoveries of the column for CL from three spiked food samples were 84.4-95.2% with relative standard deviation (RSD) of 9.3-15.5%; after more than 30 times repeated usage, there was not significant loss of specific recognition. The results demonstrated the feasibility of the prepared IAC column for CL extraction. The proposed antibody immobilization method exhibiting the properties of simplicity, low cost, strong binding for target analyte, no leaching of antibody, etc., would be a very useful tool applied in the field of IAC.

Quantification of Interleukin-6 in cell culture medium using surface plasmon resonance biosensors

Interleukin (IL)-6, a multifunctional cytokine, is widely used as an index for illnesses such as inflammatory and autoimmune disorders, coronary artery disease, neurological disease, and gestational problems. It is thus very important to be able to precisely quantify the level of IL-6 for disease diagnosis and any subsequent therapy. Surface plasmon resonance (SPR) biosensors are sensitive in detecting the interaction between biomolecules by sensing the changes in the refractive index on the sensor chip. This study investigated the SPR technique to determine the IL-6 secretion of human fibroblast MRC5-CVI cells induced by lipopolysaccharide (LPS). To reduce non-specific binding, a mixed self-assembled monolayer of mercaptoundecanoic acid (MUA) and mercaptohexanol (MCH) was attached to the sensor, and then used for IL-6 determination using a sandwich type immunoassay. In addition, two antibody immobilization methods were applied to the sensor surface—direct immobilization and indirect immobilization via protein G affinity. The results demonstrated that the direct immobilization method had a better antibody binding capacity on the sensor surface. The level of cellular IL-6 secretion detected by the SPR biosensor showed a consistent correlation with the commercial kit of IL-6 enzyme-linked immunosorbent assay.

Optimizing immobilization on two-dimensional carboxyl surface: pH dependence of antibody orientation and antigen binding capacity

The performance of immunosensors is highly dependent on the amount of immobilized antibodies and their remaining antigen binding capacity. In this work, a method for immobilization of antibodies on a two-dimensional carboxyl surface has been optimized using quartz crystal microbalance biosensors. We show that successful immobilization is highly dependent on surface p K a, antibody p I, and pH of immobilization buffer. By the use of EDC/sulfo-NHS (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/ N-hydroxysulfosuccinimide) activation reagents, the effect of the intrinsic surface p K a is avoided and immobilization at very low pH is therefore possible, and this is important for immobilization of acidic proteins. Antigen binding capacity as a function of immobilization pH was studied. In most cases, the antigen binding capacity followed the immobilization response. However, the antigen-to-antibody binding ratio differed between the antibodies investigated, and for one of the antibodies the antigen binding capacity was significantly lower than expected from immobilization in a certain pH range. Tests with anti-Fc and anti-Fab 2 antibodies on different antibody surfaces indicated that the orientation of the antibodies on the surface had a profound effect on the antigen binding capacity of the immobilized antibodies.

Fabrication of Oriented Antibody-Conjugated Magnetic Nanoprobes and Their Immunoaffinity Application

In an attempt to fabricate highly active immunoprobes for serum biomarker detection, we report a simple and effective method for site-specific and self-oriented immobilization of antibodies on magnetic nanoparticles (MNPs). Through boronate formation, the carbohydrate moiety within the constant domain, Fc, of the antibody can be specifically and covalently linked to a boronic acid-functionalized MNP (BA@MNP) without hindering the antigen binding domain, Fab. The performance was evaluated by immunoaffinity extraction of multiple serum antigens. Compared with the random immobilization of antibody on a MNP, the antibody self-oriented immunoprobe provides long-term stability (>2 months) and 5-fold extraction efficiency. It also provides 5-fold improved sensitivity at a low nM range (0.4 nM), presumably through enhanced antibody@MNP activity. In addition, false-positive detections arising from nonspecific binding can be completely minimized by effective surface protection using concentration-dependent dextran blocking. Compared with conventional antibody site-specific immobilization through protein G, this new BA-mediated covalent antibody immobilization provides interference-free extraction resulting from noncovalent immobilization of antibody by protein G. The new immunoassay was applied in comparative profiling of serum amyloid P (SAP), serum amyloid A (SAA), and C-reactive protein (CRP) in human serum. Our triple immunoassay revealed a distinct pattern among normal patients, patients with cancer, and patients with cardiovascular disease. Using the previously reported quantization capability of the MALDI MS readout, we expect that this site-specific immunonanoprobe-based immunoassay can be highly active, rapid, and accurate in nanodiagnosis.

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Conventional methods for pathogen detection and identification are labor-intensive and take several days to complete. Recently developed biosensors have shown potential for the rapid detection of foodborne pathogens. In this study, an impedimetric biosensor was developed for rapid detection of Salmonella typhimurium. To develop the biosensor, an interdigitated microelectrode (IME) was fabricated by using semiconductor fabrication process. Anti-Salmonella antibodies were immobilized based on either avidin-biotin binding or self assembled monolayer (SAM) on the surface of the IME to form an active sensing layer. To evaluate effect of antibody immobilization methods on sensitivity of the sensor, detection limit of the biosensor was analyzed with Salmonella samples innoculated in phosphate buffered saline (PBS) or food extract. The impedimetric biosensor based on SAM immobilization method produced better detection limit. The biosensor could detect 107 CFU/mL of Salmonella in pork meat extract. This method may provide a simple, rapid, and sensitive method to detect foodborne pathogens.