Antibody Immobilization Methods Research Articles

Highly-Sensitive Polymer Optical Fiber SPR Sensor for Fast Immunoassay

A new type of human immunoglobulin G (IgG) sensors based on the surface plasmon resonance (SPR) in the low refractive index (RI) plastic optical fiber (POF) and an antibody immobilization method is presented. A 50-nm-thick gold film was formed on the polished D-shaped fiber surface by magnetron sputtering. The RI response of the POF sensor is 30 049.61 nm/RIU, which is 26.5 times higher than that of single mode fiber (SMF) SPR sensors. The proposed SPR biosensor can be developed by simple and rapid modification of the gold film with 11-mercapto undecanoic acid (MUA). Upon immobilization of the goat anti-human IgG antibody, the resonance wavelength shifts by 11.2 nm. The sensor can be used to specifically detect and quantify the human IgG at concentrations down to 245.4 ng/mL with the sensitivity of 1.327 7 nm per µg/mL, which offers an enhancement of 12.5-fold compared to that of the conventional SMF based SPR sensors. The proposed device may find the potential applications in the case of use at the point of care.

Development of a universal, oriented antibody immobilization method to functionalize vascular prostheses for enhanced endothelialization for potential clinical application

BackgroundThrombosis is a common cause of vascular prosthesis failure. Antibody coating of prostheses to capture circulating endothelial progenitor cells to aid endothelialization on the device surface appears a promising solution to prevent thrombus formation. Compared with random antibody immobilization, oriented antibody coating (OAC) increases antibody-antigen binding capacity and reduces antibody immunogenicity in vivo. Currently, few OAC methods have been documented, with none possessing clinical application potential.ResultsDopamine and the linker amino-PEG8-hydrazide-t-boc were successfully deposited on the surface of cobalt chromium (CC) discs, CC stents and expanded polytetrafluoroethylene (ePTFE) grafts under a slightly basic condition. CD34 antibodies were immobilized through the reaction between aldehydes in the Fc region created by oxidation and hydrazides in the linker after t-boc removal. CD34 antibody-coated surfaces were integral and smooth as shown by scanning electron microscopy (SEM), had significantly reduced or no substrate-specific signals as revealed by X-ray photoelectron spectroscopy, were hospitable for HUVEC growth as demonstrated by cell proliferation assay, and specifically bound CD34 + cells as shown by cell binding testing. CD34 antibody coating turned hydrophobic property of ePTFE grafts to hydrophilic. In a porcine carotid artery interposition model, a confluent monolayer of cobblestone-shaped CD31 + endothelial cells on the luminal surface of the CD34 antibody coated ePTFE graft were observed. In contrast, thrombi and fibrin fibers on the bare graft, and sporadic cells on the graft coated by chemicals without antibodies were seen.ConclusionA universal, OAC method was developed. Our in vitro and in vivo data suggest that the method can be potentially translated into clinical application, e.g., modifying ePTFE grafts to mitigate their thrombotic propensity and possibly provide for improved long-term patency for small-diameter grafts.

Rapid and sensitive detection of Staphylococcus aureus by using a long-period fiber grating immunosensor coated with egg yolk antibody

The rapid and reliable detection of bacteria plays an important role in clinical and veterinary practice. A stable, label free, compact, and sensitive long-period fiber grating (LPFG) sensor based on egg yolk antibody (IgY) was proposed for the detection of Staphylococcus aureus (S. aureus). LPFG was fabricated with laser writing technology, and specific IgY was further immobilized on the grating region and then combined with the corresponding bacteria. S. aureus was detected by tracking the change of resonance wavelength in the LPFG transmission spectrum caused by bacteria-antibody interaction induced by the increase of biological cover thickness and density after the immune reaction. The testing results showed that the proposed sensor was selective and sensitive to S. aureus measurement, and the detection limit is approximately 33CFU/ml. The proposed antibody immobilization method is very simple, and the optical fiber can be manufactured in batch to reduce the cost. The detection time of the sensor is around 20min, which is fast and suitable for detection. The assay was successfully applied for the quantitative analysis of S. aureus in natural waters and met the needs of on-site screening trace pathogenic bacteria in food safety control.

The kinetic study on the interactions of IgG and antibody-binding proteins based on SPR sensor

We study the interactions between staphylococal protein A or streptococcus protein G and immunoglobulin IgG, respectively, based on the self-developed wavelength-based surface plasmon resonance (SPR) sensing system combined with the kinetic analysis. The experimental results show that the association rate constants k_{<italic>a</italic>} of protein A and protein G with IgG are 1.3×10⁵ L·mol^-1·s^-1 and 5.0×10⁴ L·mol^-1·s^-1, indicating that the protein A binds to IgG more efficiently. At the same time, the dissociation rate constants k_{<italic>d</italic>} of protein A and protein G from IgG are detected to be 2.1×10^-2 s^-1 and 5.0×10^-3 s^-1, indicating that the complex formed by protein G and IgG is more stable. In addition, the ratio of k_{<italic>d</italic>} to k_{<italic>a</italic>} indicates that protein G has higher affinity for IgG. This study may provide necessary reference and important theoretical support for the optimization of antibody immobilization methods.

Comparative study on antibody immobilization strategies for efficient circulating tumor cell capture

Methods for isolation and quantification of circulating tumor cells (CTCs) are attracting more attention every day, as the data for their unprecedented clinical utility continue to grow. However, the challenge is that CTCs are extremely rare (as low as 1 in a billion of blood cells) and a highly sensitive and specific technology is required to isolate CTCs from blood cells. Methods utilizing microfluidic systems for immunoaffinity-based CTC capture are preferred, especially when purity is the prime requirement. However, antibody immobilization strategy significantly affects the efficiency of such systems. In this study, two covalent and two bioaffinity antibody immobilization methods were assessed with respect to their CTC capture efficiency and selectivity, using an anti-epithelial cell adhesion molecule (EpCAM) as the capture antibody. Surface functionalization was realized on plain SiO2 surfaces, as well as in microfluidic channels. Surfaces functionalized with different antibody immobilization methods are physically and chemically characterized at each step of functionalization. MCF-7 breast cancer and CCRF-CEM acute lymphoblastic leukemia cell lines were used as EpCAM positive and negative cell models, respectively, to assess CTC capture efficiency and selectivity. Comparisons reveal that bioaffinity based antibody immobilization involving streptavidin attachment with glutaraldehyde linker gave the highest cell capture efficiency. On the other hand, a covalent antibody immobilization method involving direct antibody binding by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)-N-hydroxysuccinimide (NHS) reaction was found to be more time and cost efficient with a similar cell capture efficiency. All methods provided very high selectivity for CTCs with EpCAM expression. It was also demonstrated that antibody immobilization via EDC-NHS reaction in a microfluidic channel leads to high capture efficiency and selectivity.

Automatic microfluidic enzyme-linked immunosorbent assay based on CLOCK-controlled autonomous centrifugal microfluidics

This paper reports on the development of an autonomous centrifugal microfluidic device, which automatically executes an enzyme-linked immunosorbent assay (ELISA) on a steady rotational frequency. The device was designed on the principles of CLOCK (Control of Liquid Operation on Centrifugal hydroKinetics) to achieve autonomous centrifugal microfluidics. These demonstrations confirmed that the device achieves accurate flow control of an automated ELISA with fluctuations of the interval time for implementation of each unit (antigen-antibody reaction, wash and injection of detection substrate) being less than 5%. The most optimal method for the immobilization of primary antibodies was investigated for the developed platform. We compared three methods of antibody immobilization on the developed platform, by using polystyrene microbeads, a polyurethane foam structure, and direct immobilization to the reaction chamber. We chose the use of polyurethane foam as the best immobilization method based on reaction efficiency and the convenient handling of the immobilized antibody on the solid phase. Measurement of human albumin was demonstrated using the developed device, and a calibration curve of human albumin detection was successfully prepared with an overall assay operation time of 18 min. The limit of detection (LOD) was estimated to be 0.516 ng/ml, which is comparable with the LOD of 0.707 ng/ml obtained by the assay on a conventional titer plate assay platform.

Human neonatal Fc receptor as a new potential antibody binding protein for antibody immobilization.

A critical challenge in producing an antibody-based assay with the highest reproducibility and sensitivity is the strategy to immobilize antibodies to solid phase. To date, numerous methods of antibody immobilization were reported but each was subjected to its advantages and limitations. The current study proposes a new potential antibody binding protein, the human neonatal fragment crystallizable (Fc) receptor. This protein has shown its high affinity to the Fc of antibody either in vivo or in vitro. Human neonatal Fc receptor is a heterodimer constructed by p51 α-heavy chain and β2-microglobulin light chain; however, the binding sites toward the antibody are located in the p51 α-heavy chain. Hence, vector cloning and recombinant protein expression were carried out to express the p51 α-heavy chain of the human neonatal Fc receptor (hFcRn-α). The recombinant protein expressed, hFcRn-α, was adopted to pin rabbit IgG against hepatitis B virus surface antigen to a solid phase. A sandwich enzyme-linked immunosorbent assay was further developed to evaluate the efficiency of hFcRn-α-directed immobilization in antigen detection. The result was compared with the conventional physical adsorption method. The findings demonstrated that human neonatal Fc receptor was efficient in pinning antibodies and generating higher signals compared with the physical adsorption of antibody.

Docking of Antibodies into the Cavities of DNA Origami Structures.

Immobilized antibodies are extensively employed for medical diagnostics, such as in enzyme-linked immunosorbent assays. Despite their widespread use, the ability to control the orientation of immobilized antibodies on surfaces is very limited. Herein, we report a method for the covalent and orientation-selective immobilization of antibodies in designed cavities in 2D and 3D DNA origami structures. Two tris(NTA)-modified strands are inserted into the cavity to form NTA-metal complexes with histidine clusters on the Fc domain. Subsequent covalent linkage to the antibody was achieved by coupling to lysine residues. Atomic force microscopy (AFM) and transmission electron microscopy (TEM) confirmed the efficient immobilization of the antibodies in the origami structures. This increased control over the orientation of antibodies in nanostructures and on surfaces has the potential to direct the interactions between antibodies and targets and to provide more regular surface assemblies of antibodies.

Structure of the receptor layer in electrochemical immunosensors. Modern trends and prospects of development

Literature data on the modern state and prospects for the development of research in the area of receptor layer immobilization in electrochemical immunosensors were examined and systematized. The first section of the review is devoted to structural features and functions of biological immunoreceptors, viz., antibodies or their fragments. The main methods of antibody immobilization on the working electrode surface are considered in detail in the second section. The prospects for the development of electrochemical immunosensors are discussed in the third section of the review.

A comparison of nanoparticle-antibody conjugation strategies in sandwichimmunoassays

ABSTRACTPoint-of-care (POC) diagnostics such as lateral flow and dipstick immunoassays use gold nanoparticle (NP)-antibody conjugates for visual readout. We investigated the effects of NP conjugation, surface chemistries, and antibody immobilization methods on dipstick performance. We compared orientational, covalent conjugation, electrostatic adsorption, and a commercial conjugation kit for dipstick assays to detect dengue virus NS1 protein. Assay performance depended significantly on their conjugate properties. We also tested arrangements of multiple test lines within strips. Results show that orientational, covalent conjugation with PEG shield could improve NS1 detection. These approaches can be used to optimize immunochromatographic detection for a range of biomarkers.

A highly sensitive impedimetric label free immunosensor for Ochratoxin measurement in cocoa beans

In this work the development and optimization of an impedimetric label free immunosensor for the detection of Ochratoxin A (OTA) is reported.Two antibody immobilization methods (oriented and not oriented) were compared highlighting a lower limit of detection (5pg/ml) for the not oriented immobilization but a closer linear range in contrast to oriented anti-OTA immunosensors which showed linearity in the range of 0.01–5ng/mL OTA. The analysis of the Atomic Force Microscopy (AFM) images showed two different nanostructures indicating that the use of oriented immobilization created a more ordered and highly dense antibody surface. Finally the oriented immunosensor was used to quantify OTA in spiked cocoa bean samples and the results were compared with those registered with competitive ELISA kit. The immunosensor was sensitive to OTA lower than 2μg/kg that represents the lower acceptable limit of OTA established by European legislation for the common food products.

Detection of vibrio cholerae O1 by using cerium oxide nanowires - based immunosensor with different antibody immobilization methods

In this work, we evaluated the effects of different antibody immobilization strategies on the response of a CeO2-nanowires (NWs)-based immunosensor for Vibrio cholerae O1 detection. Accordingly, the changes in the electron-transfer resistance (Ret) from before to after cells bind to an antibody-modified electrode prepared by using three different methods of antibody immobilization were determined. The values were 16.2%, 8.3%, and 6.65% for the method that utilized protein A, antibodies activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS), and absorption, respectively. Cyclic voltammetry confirmed that the change in the current was highest for the immunosensors prepared using protein A (11%), followed by those prepared with EDC/NHS-activated antibodies (9%), and finally, those prepared through absorption (7.5%). The order of the antibody immobilization strategies in terms of resulting immunosensor detection limit and sensitivity was as follows order: absorption (3.2 × 103 CFU/mL; 45.1 Ω/CFU·mL−1) < EDC/NHS-activated antibody (1.0 × 103 CFU/mL; 50.6 Ω/CFU·mL−1) < protein A (1.0 × 102 CFU/mL; 65.8 Ω/CFU·mL−1). Thus, we confirmed that the protein A - mediated method showed significantly high cell binding efficiencies compared to the random immobilization method.

One-step immobilization antibodies using ferrocene-containing thiol aromatic aldehyde for the fabrication of a label-free electrochemical immunosensor

This work focuses on a facile method for antibody immobilization to fabricate a label-free electrochemical immunosensor using ferrocene-containing thiol aromatic aldehyde (FcSA) synthesized by us.

Plasma treatment of paper for protein immobilization on paper-based chemiluminescence immunodevice

A novel protein immobilization method based on plasma treatment of paper on the low-cost paper-based immunodevice was established in this work. By using a benchtop plasma cleaner, the paper microzone was treated by oxygen plasma treatment for 4min and then the antibody can be directly immobilized on the paper surface. Aldehyde group was produced after the plasma treatment, which can be verified from the fourier transform infrared spectroscopy (FT-IR) spectra and x-ray photoelectron spectroscopy (XPS) spectra. By linked to aldehyde group, the antibody can be immobilized on the paper surface without any other pretreatment. A paper-based immunodevice was introduced here through this antibody immobilization method. With sandwich chemiluminescence (CL) immunoassay method, the paper-based immunodevice was successfully performed for carcinoembryonic antigen (CEA) detection in human serum with a linear range of 0.1–80.0ng/mL. The detection limit was 0.03ng/mL, which was 30 times lower than the clinical CEA level. Comparing to the other protein immobilization methods on paper-based device, this strategy was faster and simpler and had potential applications in point-of-care testing, public health and environmental monitoring.

A Novel Optical Biosensing System Using Mach-Zehnder-Type Optical Waveguide for Influenza Virus Detection.

In order to minimize the damage from viral epidemics, early detection of the causative agent of a viral epidemic and prevention of its immediate spread are urgent social demands. Therefore, in this study, we evaluated the utility of a Mach-Zehnder-type optical waveguide as a sensing device for influenza virus detection. However, it is impossible to detect a 100-nm-size virus using a sol-gel optical biosensor because sol-gel glass has a pore size of only a few nanometers, which makes it impossible for the virus to diffuse into the silica thin film. In order to construct the influenza-specific Mach-Zehnder optical biosensor for influenza detection, a stable antibody immobilization method with resulting high density on the sol-gel surface is strongly required. In this study, the sol-gel glass surface was modified with amino and carboxyl groups, and an anti-H1N1/HA1 antibody was covalently immobilized using a cross-linking agent. We successfully prepared a carboxyl-modified sol-gel surface, using NHS/EDC as the cross-linker, for antibody immobilization, and confirmed the detection of influenza virus using the antibody-immobilized sol-gel glass. After treatment with a 100μg/mL influenza virus solution for 15min, a peak wavelength shift (~24nm) was observed in the output light spectrum.

Development of effective QCM biosensors by cyclopropylamine plasma polymerization and antibody immobilization using cross-linking reactions

Biosensors have been extensively developed and applied for biomedical and environmental studies. Although there are many different types of biosensing techniques, immobilization of the biorecognition biomolecules onto the sensor surface is always required. Cyclopropylamine pulsed plasma polymerization in radio frequency (RF) capacitive discharges was employed to deposit amine thin films on the gold electrode of quartz crystal microbalance (QCM) biosensors and the monoclonal antibody AL-01, specific to human serum albumin (HSA), was immobilized onto the plasma polymer surface. Two different amine plasma polymers were studied. They had a similar amount of primary amine groups, 1.3–1.5 at.%, as determined by chemical derivatization but their stability in water was different. The 1st type, deposited at the floating potential and 120 Pa, exhibited 16% thickness loss after 24 h in water whereas the 2nd type deposited at RF electrode and 50 Pa was completely stable (2% thickness loss). Glutaraldehyde (GA) coupling of AL-01 was employed for both types of the amine films and the performance of QCM immunosensors was evaluated by the immunoassay flow test. A high-stability of the frequency signal was obtained in both the cases but the 2nd type of the film provided low response to HSA. It was explained by its highly cross-linked structure and steric hindrance of active sites. Three different antibody immobilization methods were explored for the 1st type of the film. Stable baseline, selective and high response were recorded for GA and sulfo-SMCC methods. The results confirmed that the presented methodologies for the grafting of biomolecules on the gold are highly efficient and very promising for future use in biosensing.

The p16INK4a Antibody Immobilization Method for Immonosensor Application.

The p16INK4a is a protein that expressed in Liquid-based cervical cytology specimens and has been proved link to cervical cancer. The p16INK4a could be detection by piezoelectric immunosensor and the immobilization of the p16INK4a antibody influence the sensitivity of the piezoelectric immunosensor. 5μL mouse polyclonal antibody against p16INK4a was bound onto the surface of immonosensor through two methods. (directly immobilized method; protein A method). Absorb of the p16INK4a antibody on the surface of immonosensor caused a shift in the resonant frequency of the immunosensor and The frequency changes recorded showed a better reproducibility. The activity of the immobilization antibody with the directly method and protein A method was tested with p16INK4a antigen. The resonant frequency for different antibody immobilization methods were different, and the sensitivity for p16INK4a detection also different. The protein A method was found to be much more better than the directly method for the immobilization of the p16INK4A antibody on the gold electrode of the quartz crystal for cervical lesion detection. The Protein A method created more reproducible and stable immobilization antibody layers with p16INK4A antigen.

Sensitive Progesterone Determination Using a Magnetic Particle-Based Enzyme-Linked Immunosorbent Assay

A highly sensitive magnetic nanoparticle enzyme immunoassay of progesterone was established using horseradish peroxidase as a label. The “enzyme label” was prepared by coupling of progesterone-3-(O-carboxymethyl)oxime to horseradish peroxidase. The antiprogesterone antibody was immobilized on the aminomodified magnetic nanoparticles by glutaraldehydе. The typical standard curve for progesterone in buffer by the magnetic nanoparticles enzyme immunoassay was obtained with a detection limit of 0.04 ng mL−1. These results were compared to the data obtained for progesterone in milk. It has been shown that the progesterone magnetic particle-based enzyme-linked immunosorbent assay in milk had a minor depression in sensitivity (detection limit of 0.09 ng mL−1). The progesterone calibration curves obtained by the random antibody immobilization were compared with results by protein A oriented immobilization of the antibodies. The sensitivity of progesterone magnetic particle-based enzyme-linked immunosorbent assay in milk using random antibody immobilization method was lower than sensitivity of the progesterone magnetic particle-based enzyme-linked immunosorbent assay using protein A oriented immobilization method (detection limit of 0.08 ng mL−1). The influences of milk type and the fat content in milk on the immunoassay were investigated. With an improved sensitivity and simple operation, the magnetic particle-linked antibody for immunoassay of progesterone has great potential to supersede the traditional enzyme-linked immunosorbent assay for progesterone determination.

Rapid flow through immunoassay for CRP determination based on polyethylene filters modified with ω-aminocellulose carbamate.

A novel method for antibody immobilization is discussed and applied in a column based flow through immunoassay system (ABICAP). It is shown that porous polyethylene material can be modified with different ω-aminocellulose carbamates yielding an amino group containing biocompatible support for antibody immobilization. Anti-h CRP antibodies can be bound covalently to the surface via various homobifunctional cross-linkers. The antibody modified filters are validated for the CRP determination in a sandwich ABICAP system. In a 10 min test procedure based on colloidal dye particles, a limit of detection of 5 ng CRP mL(-1) and coefficients of variation of <9.1% are obtained.

Reflectometric interference spectroscopy-based immunosensing using immobilized antibody via His-tagged recombinant protein A

The proposed approach demonstrated in this study provides an immunosensing system based on reflectometric interference spectroscopy (RIfS) in combination with an antibody immobilization method using histidine-tagged recombinant protein A. Carboxymethyldextran (CMD) was immobilized on a 3-aminopropyltriethoxysilane-treated a silicon nitride-coated silicon wafer, followed by chelating histidine-tagged recombinant protein A with copper (II) ions. The CMD-layer was found to be advantageous in terms of not only immobilization of histidine-tagged recombinant protein A-mediated an antibody against myoglobin (anti-Myo) but also prevention of non-specific binding of myoglobin. Myoglobin was repeatedly detected, and the apparent detection limit was 0.1μgmL(-1). The proposed RIfS-based protein sensing system, in conjunction with the easy preparation of silicon-based inexpensive immunosensing chips, is expected to be applicable for label-free optical detection for other proteins in various fields.