Antibody Immobilization Methods Research Articles

Optimization of antibody immobilization for on‐line or off‐line immunoaffinity chromatography

The covalent immobilization of antibodies to solid supports for immunoaffinity (IA) purification is widely used in the biological sciences. However, relative immobilization yields, immobilization stability, and antigen-binding capacity vary significantly with the antibody and protocol used. A systematic study was conducted to determine the most versatile antibody immobilization method for use in on-line and off-line IA chromatography applications using commonly accessible immobilization methods. Four chemistries were examined using polyclonal and monoclonal antibodies and antibody fragments. We evaluated a method to survey optimal elution conditions and estimated immobilization yields, matrix stability, antigen binding capacities, and antigen recovery of different IA matrices. Some mAbs were sensitive to aminogroup-based immobilization, i.e., losing antigen binding capabilities after immobilization especially using epoxy chemistry. In general, the most optimal covalent antibody immobilization for on-line IA-LC-MS was achieved using aminogroup immobilization of intact antibodies by epoxy- or aldehyde-activated POROS R20-matrices and in some cases by chemical crosslinking to Protein G-POROS. Protein G-based matrices are very stable showing essentially no decline in performance after 50 application-wash-elution-reequilibration cycles and being easily prepared within 2-3 h of working time with a typical antibody coupling yield of above 80%. In off-line applications where constant flow conditions are not used, covalent crosslinking onto Protein G-POROS or Protein G-agarose is to be recommended.

An investigation of antibody immobilization methods employing organosilanes on planar ZnO surfaces for biosensor applications

One critical aspect for the development of label-free immunosensors is the employment of highly uniform and repeatable antibody immobilization techniques. In this study, we investigated the use of two different silane molecules (3-glycidyloxypropyl)trimethoxysilane (GPS), and (3-mercaptopropyl)trimethoxysilane (MTS) for the immobilization of fluorescently labeled IgG antibodies on planar ZnO surfaces. The chemical modification of the surfaces was investigated using water contact angle measurements, AFM, and fluorescence microscopy. The results of the water contact angle measurements indicate increased surface hydrophobicity after treatment with GPS and MTS as compared to the control. Surface modification was further verified through AFM measurements which demonstrate an increased surface roughness and particle height after treatment with antibodies. The results of the fluorescence studies indicate that the immobilization protocol employing MTS produced 21% higher fluorescence on average with greater uniformity than the GPS-based protocol, which indicates a higher overall density in antibody coverage on the surface of the ZnO. Acoustic sensor tests were employed to confirm the functionality of sensors treated with the MTS protocol. The results indicate that the immobilization protocol imparts sensitivity and specificity to the ZnO-based devices.

Recent advances in immobilization methods of antibodies on solid supports

Antibody immobilization on a solid support is an essential process for the development of most immune-based assay systems. The choice of the immobilization method greatly affects antibody-antigen interactions on the assay surface. For the past several years, numerous strategies have been reported to control antibody immobilization, mainly by directing the orientation, stability, and density of bound antibodies on different assay platforms. Here we discuss recent developments in antibody immobilization methods with a particular focus on the strengths and limitations of reported approaches, and thereby provide a useful guideline for the selection of suitable antibody coupling procedures.

Antibody Immobilization on Waveguides Using aFlow–Through System Shows Improved Listeria monocytogenesDetection in an Automated Fiber Optic Biosensor: RAPTORTM

Recent outbreaks of food borne illnesses continue to support the need for rapidand sensitive methods for detection of foodborne pathogens. A method for detecting Listeriamonocytogenes in food samples was developed using an automated fiber-optic-basedimmunosensor, RAPTORTM. Detection of L. monocytogenes in phosphate buffered saline(PBS) was performed to evaluate both static and flow through antibody immobilizationmethods for capture antibodies in a sandwich assay. Subsequent detection in frankfurtersamples was conducted using a flow through immobilization system. A two stage blockingusing biotinylated bovine serum albumin (b-BSA) and BSA was effectively employed toreduce the non-specific binding. The sandwich assay using static or flow through mode ofantibody immobilization could detect 1

Site-directed antibody immobilization on gold substrate for surface plasmon resonance sensors

A simple method for the site-directed immobilization of antibodies on gold substrates has been developed for surface plasmon resonance (SPR) applications. A freshly prepared gold surface on a glass slide was modified with Protein A via homobifunctional cross linker dithiobissuccinimide propionate (DSP) to achieve uniform, stable, and sterically accessible antibody coating. The density of the immobilized antibodies bound to Protein A was examined as a function of the protein concentration, buffer pH, buffer type and reaction time. The suggested scheme was also employed for the modification of gold-coated substrates for quartz crystal microbalance (QCM) applications. The modified gold surface was stable for several weeks and the reproducibility was satisfactory. The current fabrication technique could be applied to construct other immunosensors or biochips.

A novel piezoelectric quartz micro-array immunosensor based on self-assembled monolayer for determination of human chorionic gonadotropin

A novel multi-channel 2×5 model of piezoelectric quartz micro-array immunosensor has been developed for quantitative detection of human chorionic gonadotropin (hCG) in serum or urine samples. Every crystal unit of the fabricated piezoelectric hCG micro-array immunosensor can oscillate independently without interfering each other. A 2×5 model of micro-array immunosensor as compared with a one-channel immunosensor can provide eight times higher detection speeds for hCG assay. The anti-hCG antibody is deposited on the gold electrode’s surface of 10 MHz quartz AT-cut crystal by self-assembled technique using sulfosuccinimidyl 6-[3′-(2-pyridyldithio) propionamido] hexanoate (Sulfo-LC-SPDP), and serves as an antibody recognizing layer. The highly ordered self-assembled monolayers (SAM) ensure well-controlled surface structure and offer many advantages to the performance of the sensor. Compared with conventional antibody immobilization methods, the amount and the reaction activity of antibody monolayer coated by the SAM binding are bigger than those by the SPA method, and less non-specific binding caused by other analytes in sample is found. Under the optimized experimental conditions, the results showed that micro-array immunosensor quantitatively detected serum or urine hCG in the range of 2.5–500 mIU/ml with high precision (CV<5%); other hormones in human serum and urine did not interfere with the determination markedly. Serum and urine samples of 60 patients were detected by the micro-array immunosensor, and the results agreed well with those given by the commercial radioimmunoassay test kit, with correlation coefficient of 0.92. After regeneration with urea solution the coated immunosensor can be reused five times without appreciable loss of activity.

Evaluation of antibody immobilization methods for piezoelectric biosensor application

The immobilization of anti- Salmonella antibodies by two methods were studied and evaluated for their potential use in a piezoelectric biosensor. The optimum temperature–time combinations for the highest immobilization yields were determined for both methods. Protein A binding was found to be 67.4±3.8% on the gold surface which then allowed an immobilization of 42.1±2.09% antibody. The degree of antibody immobilization via surface aldehyde groups of glutaraldehyde (GA) on a precoated quartz crystal with polyethylenimine (PEI) was 31.6±0.3%. A piezoelectric probe was designed and used in dry assays to observe the frequency change due to addition of mass by the immobilization layers. The frequency changes recorded showed a better reproducibility and less added mass for the Protein A method. The frequency decrease due to μg of added antibodies was compared to frequency decrease calculated by the Sauerbrey equation. The experimental data was found to be only ∼8% of theoretical data. The functionality of the immobilized antibodies with the Protein A method was tested with S.typhimurium in a wet chamber and the frequency decrease was compared to results of a similar system activated with PEI–GA immobilization. The frequency decreases with S.typhimurium concentration of ∼1.5×10 9 CFU/ml were 50±2 Hz and 44±3 Hz for the Protein A method and PEI-GA method, respectively. It was concluded that although both methods resulted in comparable activities in terms of % immobilized protein and frequency decreases due to Salmonella binding, the Protein A method was favorable due to stability and better reproducibility of the immobilization layers.

Use of Immunomatrix Methods to Improve Protein-Protein Interaction Detection.

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized protein A or protein G to isolate antibody-bound target antigens. The main disadvantage of traditional immunoprecipitation and coimmunoprecipitation is that the conditions used to elute the precipitated antigen also release the antibody, contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized protein A or protein G, or by coupling it directly to the resin. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yields IP and co-IP results similar to traditional protocols but eliminates the antibody heavy and light chains contamination.

XPS, AFM and EIA Studies of IgG Molecules Site-Directed Immobilized on APTES Modified Silicon Wafer Surfaces

A convenient and efficient method for site-directed immobilization of antibodies to silicon wafer surfaces modified by 3-aminopropyltriethoxylsilane (APTES) is reported here. X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and enzyme immunoassay (EIA) were used to characterize the immobilized surfaces.

A novel method to chemically immobilize antibody on nylon and its application to the rapid and differential detection of two Vibrio parahaemolyticus toxins in a modified enzyme-linked immunosorbent assay

A new method of chemically immobilizing antibody on nylon was developed. The method consists of serial treatments with HCl, polyethylene imine, and maleic anhydride methylvinyl ether copolymer, which resulted in the stable immobilization of sufficient amounts of antibodies on nylon. This principle was used to differentially detect two immunologically related but nonidentical hemolysins (thermostable direct hemolysin [TDH] and TDH-related hemolysin [TRH]) of Vibrio parahaemolyticus in a modified enzyme-linked immunosorbent assay with antibodies immobilized on nylon slips (NSIT). The results (dark purple color on nylon slips) were easily evaluated by the naked eye. The results with NSIT were compatible with those obtained by using DNA probes or a conventional bacterial culture test, not only with cultured specimens but also with clinical specimens (diarrheal stool samples). Furthermore, the NSIT differentially detected TDH and TRH in a single test. The antibody immobilization method developed here is applicable to various immunological detection methods and may improve their sensitivity and specificity.

Recent developments in piezoelectric immunosensors. A review.

The development of piezoelectric (PZ) devices as immunosensors is reviewed. The recent advances in sensor design have stimulated great interest in PZ technology and facilitated diverse applications in a variety of matrices. Methods of antibody immobilization on crystals and several applications are reported including microgravimetric immunoassays, microbial toxins and other contaminants.

A Comparison of an Enzyme Immunoassay, DNA Hybridization, Antibody Immobilization, and Conventional Methods for Recovery of Naturally Occurring Salmonellae from Processed Broiler Carcasses

Three hundred and ninety raw processed broiler carcasses were obtained from four processing plants and evaluated for the presence of salmonellae by the whole bird rinse procedure. Enzyme immunoassay (Salmonella-Tek™), colorimetric DNA hybridization (GENE-TRAKR), and antibody immobilization (1–2 Test™) methods were compared to the Food Safety Inspection Service (FSIS) culture method for detection of salmonellae.All samples were preenriched in buffered peptone water incubated at 37°C then transferred to and enriched in TT broth (Difco, Detroit, MI) incubated at 42°C for 24 h. After enrichment, manufacturer's instructions were then followed for “rapid” procedures and the FSIS recovery and confirmation scheme was followed for the cultural method. For each sample, if discrepant results were observed between the four procedures, selective plates were streaked from both the TT selective broth and the GN postenrichment broth (Difco). All samples which were positive after initial sampling of TT broth plus additional positive samples from TT selective and GN postenrichment broths were called confirmed cultural positives.Salmonellae were detected on 71% of the carcasses using confirmed cultural procedures and 65% with FSIS culture. Presumptive salmonellae positive samples were found 66, 71, and 76% of the time with the 1–2 Test, GENE-TRAK, and Salmonella-Tek, respectively. As compared to confirmed culture, only 1.4% false positives were observed with the 1–2 Test and GENE-TRAK, whereas 6% false positives were obtained using Salmonella-Tek. When the false negatives for the four methods were compared to confirmed culture, Salmonella-Tek, GENE-TRAK, 1–2 Test, and FSIS culture had 0.4, 2.5, 7.2, and 8.3%, respectively. The close correlation of these rapid procedures to the conventional procedures warrants consideration of these procedures for detection of salmonellae from broiler chickens.

Site-specific immobilization of antibodies by their oligosaccharide moieties to new hydrazide derivatized solid supports

This report describes a new method for immobilization of antibodies to solid supports. Antibodies are bound to the solid supports by covalent bonds between aldehydes generated on the carbohydrate side chains of the antibody and hydrazide groups on the solid support. The hydrazone bonds that are formed are stable at least from pH 2–10, permitting the acid elution of antigens from the affinity column. Over 25 mg of affinity-purified rabbit IgG binds per ml of solid support, with most of the bound antibodies retaining biological activity. Advantages of this new affinity support over existing technology are discussed along with procedures for the preparation and use of affinity columns containing monoclonal or polyclonal antibodies.

Reversed immunosorbents: A simple method for specific antibody immobilization

A method is presented for permanently converting an antigen immunosorbent into a purified antibody immunosorbent which retains specific reactivity for antigen. Following immunological reaction of the antibodies in an antiserum with the antigen adsorbent, the specific immunoglobulin is chemically bound to the antigen by means of a divalent cross-linking agent. Various such agents, including glutaraldehyde, suberimidate, a diazide, and isocyanates, were employed. The conditions of preparation using the first two were optimized, and the characteristics of the resulting immobilized antibodies were investigated. Applications in radioimmunoassay, preparative chromatography, and affinity-constant studies are discussed in view of the unique attributes of these reagents.