Synthesis and chromatographic properties of an HPLC chiral stationary phase based upon human serum albumin

Summary A new HPLC stationary phase was synthesized by thein situ covalent immobilization of human serum albumin (HSA). The protein was immobilized on a commerically available diol column which had been activated with 1,1-carbonyldiimidazole. Initial chromatographic studies show that this phase can be used for chiral separations of enantiomeric solutes and that these separations may reflectin vitro binding to the HSA. The effects of mobile phase composition and temperature on the stereochemical resolutions are reported.     

利用液晶取向变化的光学免疫检测方法

利用共价固定方法将抗血清白蛋白固定到硅烷化玻璃基底上, 并通过摩擦其表面形成5CB液晶整齐均一取向排列的基底. 考察了不同浓度的人血清白蛋白与基底作用后液晶在基底上形成的偏光光学图像的差异, 并利用自行提出的“图像加权平均灰度值”定量分析了图像灰度与人血清白蛋白浓度的关系. 对比研究了基底上的特异性吸附与非特异性吸附引起的液晶偏光光学图像的差异以及调制偏振光能力, 结果表明, 该基底具有很高的特异性. 该方法可望发展成为一种灵敏、非标记的光学免疫检测方法.     

Effect of antigen size on optimal ligand density of immobilized antibodies for a high-performance liquid chromatographic support

The antigen binding capacities for purified polyclonal antibodies immobilized onto a silica-based high-performance liquid chromatographic (HPLC) affinity support are described for three serum proteins over a range of antibody ligand densities. The rate of decline in the specific activity of the immobilized antibodies with respect to increasing ligand density was found to increase with the molecular weights of the antigens. The antibodies used were purified from whole antiserum using high-performance affinity chromatography and were examined using HPLC on an SCX stationary phase. Conditions are also described for efficient coupling of the ligand to the support.     

Effect of borohydride reduction on antibodies

The effect of borohydride reducing reagents on monoclonal and polyclonal antibodies was examined by enzyme-linked immunosorbent assay (ELISA). Each antibody showed different stability characteristics to the reducing reagents. Sodium cyanoborohydride was at least five times milder toward immunological activity than sodium borohydride, however, sodium cyanoborohydride with a catalytic amount of metal ion (Zn²⁺ or Al³⁺ ) can be as harsh as sodium borohydride. Activated hydrophobic borohydrides, 9BBN-pyridine, did not have any advantages in respect to the stabilities of antibodies. Antibodies to be used for immunosorbent purification must be evaluated individually to determine whether their structure is stable to immobilization reagents and conditions prior to their linkage to the column support.     

Liquid crystal-based immunoassay for detecting human serum albumin

Human serum albumin (HSA) is the most abundant protein in human blood plasma. It is commonly used as a biomarker in urine samples to identify the chronic kidney disease caused by high blood pressure or diabetes. In our research, a thin layer of liquid crystals (LCs) is used as a readout system for developing an immunoassay that reports the presence of HSA in the aqueous solution with optical signals. The detection principle of this assay is based on the variation of surface density of protein upon the specific binding of HSA on an anti-HSA immobilized surface, which leads a dark-to-bright transition of LC images under cross-polarizers. Our results show that the LC-based immunoassay can detect HSA at a concentration of 50 μg/mL. By using the slide with immobilized anti-HSA in array format, the concentration level of HSA can be simply determined by the number of LC spot shown on the slide.     

Binding of caffeic acid to human serum albumin by the retention data and frontal analysis

A new mathematical model and frontal analysis were used to characterize the binding behavior of caffeic acid to human serum albumin (HSA) based on high‐performance affinity chromatography. The experiments were carried out by injecting various mole amounts of the drug onto an immobilized HSA column. They indicated that caffeic acid has only one type of binding site to HSA on which the association constant was 2.75 × 10⁴/m . The number of the binding site involving the interaction between caffeic acid and HSA was 69 nm . The data obtained by the frontal analysis appeared to present the same results for both the association constant and the number of binding sites. This new model based on the relationship between the mole amounts of injection and capacity factors assists understanding of drug–protein interaction. The proposed model also has the advantages of ligand saving and rapid operation. Copyright © 2014 John Wiley & Sons, Ltd.     

Measurement of antibody binding to protein immobilized on gold nanoparticles by localized surface plasmon spectroscopy

Antibody binding to bovine serum albumin (BSA) and human serum albumin (HSA) immobilized onto gold nanoparticles was studied by means of localized surface plasmon resonance (LSPR) spectroscopy. Amine-modified glass was prepared by self-assembly of amine-terminated silane on substrate, and gold (Au) nanoparticles were deposited on the amine-modified glass substrate. Au nanoparticles deposited on the glass surface were functionalized by BSA and HSA. BSA immobilization was confirmed by LSPR spectroscopy in conjunction with surface-enhanced Raman scattering spectroscopy. Then, LSPR response attributable to the binding of anti-BSA and anti-HSA to BSA- and HSA-functionalized Au nanoparticles, respectively, was examined. Anti-HSA at levels larger than ∼10 nM could be detected by HSA-immobilized chips with LSPR optical response, which was saturated at concentrations greater than ∼650 nM of anti-HSA. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorized users.     

Preparation and chromatographic behavior of acetate-fiber filter rods with dye affinity ligands

Summary The dyes cibacron blue F3GA and reactive red 120 have been immobilized on acetate fiber filter rods to produce potential affinity matrixes. The isothermal adsorption of bovine serum albumin or human serum albumin on these matrixes was investigated and proved to conform to the Freundlich equation. In the static adsorption of human plasma the adsorption capacity for human serum albumin was 12.5 mg g⁻¹ fiber filter and one band was observed in SDS-PAGE electrophoresis of human serum albumin isolated by the immobilized cibacron blue F3GA filter rod. Ligand utilization efficiencies and breakthrough volumes were obtained for adsorption of HSA on the cibacron blue F3GA filter rod when the feed-rates were from 0.5 to 6 mL min⁻¹. In the affinity chromatography of human plasma the yield of human serum albumin was 1.27 g per 100 mL plasma.     

Chromatographic separation of proteins on metal immobilized iminodiacetic acid-bound molded monolithic rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate)

Continuous rod of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) was prepared by a free radical polymerization within the confines of a stainless-steel column. The epoxide groups of the rod were modified by a reaction with iminodiacetic acid (IDA) that affords the active site to form metal IDA chelates used for immobilized metal affinity chromatography (IMAC). The efficiency of coupling of IDA to the epoxide-contained matrix was studied as a function of reaction time and temperature. High-performance separation of proteins, based on immobilized different metals on the column, were described. The influence of pH on the adsorption capacity of bovine serum albumin on the Cu2+-IDA continuous rod column was investigated in the range from 5.0 to 9.0. Purification of lysozyme from egg white and human serum albumin (HSA) on the commercially available HSA solution were performed on the naked IDA and Cu2+-IDA continuous rod columns, respectively; and the purity of the obtained fractions was detected by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry.     

Impedimetric immunosensor for human serum albumin detection on a direct aldehyde-functionalized silicon nitride surface

In this work we report the fabrication and characterization of a label-free impedimetric immunosensor based on a silicon nitride (Si₃N₄) surface for the specific detection of human serum albumin (HSA) proteins. Silicon nitride provides several advantages compared with other materials commonly used, such as gold, and in particular in solid-state physics for electronic-based biosensors. However, few Si₃N₄-based biosensors have been developed; the lack of an efficient and direct protocol for the integration of biological elements with silicon-based substrates is still one of its the main drawbacks. Here, we use a direct functionalization method for the direct covalent binding of monoclonal anti-HSA antibodies on an aldehyde-functionalized Si-p/SiO₂/Si₃N₄ structure. This methodology, in contrast with most of the protocols reported in literature, requires less chemical reagents, it is less time-consuming and it does not need any chemical activation. The detection capability of the immunosensor was tested by performing non-faradaic electrochemical impedance spectroscopy (EIS) measurements for the specific detection of HSA proteins. Protein concentrations within the linear range of 10⁻¹³–10⁻⁷ M were detected, showing a sensitivity of 0.128 Ω μM⁻¹ and a limit of detection of 10⁻¹⁴ M. The specificity of the sensor was also addressed by studying the interferences with a similar protein, bovine serum albumin. The results obtained show that the antibodies were efficiently immobilized and the proteins detected specifically, thus, establishing the basis and the potential applicability of the developed silicon nitride-based immunosensor for the detection of proteins in real and more complex samples.     

Immunosensor for the Measurement of Human Serum Albumin in Urine Based on the Spreeta Surface Plasmon Resonance Sensor

The application of SPR for measurement of the concentration of human serum albumin (HSA) in urine was studied using the compact integrated SPR sensing system Spreeta. HSA was immobilized via cystamine and glutaraldehyde onto the gold sensing area and a competitive assay for HSA was developed using a limited amount of the monoclonal antibody AL-01 in solution. Measurements were carried out in the flow-through mode and the interaction between immobilized HSA and antibody was observed in real time. To obtain reproducible results, different conditions of the measurement (method of immobilization of HSA, data evaluation, concentration of antibody, regeneration procedure) were tested. The calibration curve for clinically relevant concentrations of HSA in urine samples was constructed using 300-times diluted antibody in the form of ascites fluid. The measuring range was between 0.1 and 5 mg/l of HSA, the sensing surface was successfully regenerated and suitable for more than 20 assays. The developed method was tested on real samples of urine; to overcome the non-specific adsorption of urine components, the differential approach was adopted and the measured signal was corrected by subtraction of the response observed in the absence of the antibody.     

中药小分子阿魏酸和丹皮酚与人血清白蛋白的竞争结合作用研究

用亲和色谱研究了两种中药小分子阿魏酸(FA)、丹皮酚(PAE)在人体生理条件缓冲溶液(pH7.4)条件下与人血清白蛋白(HSA)的相互作用.从药物分子在蛋白质分子上有多种类型相互独立的结合位点的假定出发,应用Langmuir吸附模型和竞争置换分析研究了FA,PAE与HSA的竞争性相互作用.结果表明,FA,PAE与HSA之间存在一类位点,且FA与PAE竞争HSA上的indole位点(siteⅡ).根据热力学参数推测出FA,PAE与HSA之间的作用力主要为氢键作用.从FA,PAE竞争HSA上同一位点的角度,对中医用药中常将含有FA与含有PAE的中药配伍使用,以提高疗效的临床用药现象进行了解释.     

Characterization of drug interactions with soluble beta-cyclodextrin by high-performance affinity chromatography

This study examined the use of an immobilized human serum albumin (HSA) column to study solution-phase reactions between drugs and beta-cyclodextrin (beta-CD). Chromatographic equations were developed to characterize the binding of chemicals to a soluble ligand (beta-CD) in the presence of an independent immobilized ligand (HSA). Situations considered included the presence of both a homogeneous and heterogeneous immobilized ligand, as well as complex interactions between the chemical of interest and soluble ligand. Three drugs (warfarin, tamoxifen, and phenytoin) were examined by this approach. This method involved injecting a small amount of each drug onto an HSA column in the presence of various concentrations of beta-CD in the mobile phase. By measuring the change in the drug's retention factor as the concentration of beta-CD was varied, it was possible to determine the stability constant between the injected drug and beta-CD. With this approach, warfarin and beta-CD were found to have 1:1 interactions with a stability constant of 5.2 x 10(2) M(-1) at 37 degrees C and pH 7.4, a result in close agreement with previous literature values. Tamoxifen and phenytoin were also found to have 1:1 interactions with beta-CD and had stability constants of 0.9-1.2 x 10(4) and 6-9 x 10(2) M(-1) respectively. With these latter solutes, the effects of secondary binding to the chromatographic support had to be considered. The theory and methods described in this report are not limited to these drugs and beta-CD but can be applied to other analytes and soluble ligands.     

Characterization of the Binding Performance of Silica-Based Immunoaffinity Adsorbents Prepared via Online Coupling Reactions

Antibodies against a conjugate of daidzein and bovine serum albumin (BSA) were immobilized onto dihydrazide-activated silica beads via online coupling reactions. The binding performances of the obtained immunoaffinity adsorbents to the corresponding hapten and carrier protein were all optimized and characterized. It was found that online coupling reactions offered a convenient and practical way to generate the requisite immunoaffinity columns. The optimum binding buffers for BSA and daidzein were found to be pH 5.0, 0.01 mol L^?1 phosphate-buffered saline (PBS) and 0.05% Tween-20 in pure water, indicating different interactions between the immobilized antibodies with different parts of the antigen. Under the optimum operating conditions, the silica-based immunoaffinity adsorbents showed specific and selective binding properties to the target protein; while for small molecules, a mixed adsorption mechanism caused by both specific and non-specific interactions was observed. For comparison, offline coupled silica-antibody immunoaffinity adsorbents and Sepharose 4B support immobilized with the same antibody were also prepared and tested. The experimental results of this study may provide useful information for further development of other high-performance immunoaffinity chromatographic methods for different purposes.     

Capillary electrophoresis immunoassay based on an on-column immunological reaction

An on-column immunological reaction was employed to achieve simple and rapid analysis in an immunoassay based on capillary electrophoresis using semiconductor laser-induced fluorescence detection. Human serum albumin (HSA) labeled with sulfoindocyanine succinimidyl ester (Cy5), a fluorescent compound with an absorption maximum at 649 nm, was used as a fluorescent probe for the immunoassay. In a binding assay, with anti-HSA as the analyte molecule, Cy5-HSA was injected in a capillary column followed by the injection of anti-HSA so as to form individual zones. By applying a potential, the anti-HSA reacted with Cy5-HSA at the boundary between Cy5-HSA and anti-HSA zones, since anti-HSA has a higher electrophoretic mobility than Cy5-HSA. Furthermore, the on-column method enhances the sensitivity by injecting a large volume of the sample. Free Cy5-HSA and its immunocomplex with anti-HSA were separated with less degradation in resolution than that predicted from the injection time of anti-HSA, even when the injection time for anti-HSA was increased. The ratio of the peak area of the complex to that of the total Cy5-HSA (free Cy5-HSA and the complex) increased in proportion to the injection time of anti-HSA. As a result, the detection limit was improved up to eight-fold (the concentration detection limit, 0.007 mg mL(-1), for an injection time of 240 s, compared to that obtained using an off-column sample preparation. Furthermore, the on-column reaction method was applicable to an immunoassay to determine native HSA, in which native HSA and Cy5-HSA react with anti-HSA stepwise. The detection limit in the stepwise reaction immunoassay was 0.005 mg mL(-1), which is 14 times lower than that in an off-column method, with the analysis time less than 10 min as the result of increasing the injection time of native HSA. In addition, the present on-column immunoassay was applied to the sample containing a high concentration of salts for investigating the effect of salts in the sample solution.     

Chromatographic competitive binding immunoassays: a comparison of the sequential and simultaneous injection methods

Two approaches for performing competitive binding immunoassays by HPLC and other flow-based systems are the simultaneous and sequential injection methods. Both these techniques make use of a column with a limited amount of antibody, onto which is injected a sample and a fixed amount of a labeled analyte analog. An indirect measure of the unlabeled analyte in the sample is then obtained by looking at the amount of analog in either the nonretained or retained peaks. In the simultaneous injection mode, the sample and labeled analog are applied at the same time to the column, while in the sequential mode the sample is injected first, followed by the analog. This results in a difference in the analytical characteristics of these two approaches. This study used chromatographic theory and previous data obtained for injections of human serum albumin (HSA) onto an anti-HSA antibody column to compare the response, detection limits, range, and sensitivity of these methods. Under equivalent conditions, it was found that the sequential method always provided the best lower limit of detection and sensitivity. However, the simultaneous mode had a broader dynamic range and higher upper limit of detection. From these observations, several guidelines were developed regarding the use and selection of such assays for new applications.     

A Crosslinked Carboxylic Acid Containing Cation Exchange Monolithic Cryogel for Human Serum Albumin Separation

For this work, we synthesized poly(N-isopropylacrylamide-acrylamide)-acrylic acid (poly(NIPAM-Am)-AAc) monolithic cryogel for a human serum albumin separation (HSA) from a protein mixture (human serum immunoglobulin, human serum albumin and lysozyme) and performed HSA adsorption studies using the cryogel to do continuous system experiments in a syringe column connected by a peristaltic pump. Poly(NIPAM-Am)-AAc with a pore size of 10–100 μm was produced by free radical polymerization that proceeded in an aqueous solution of monomers frozen inside a syringe column. The monolithic poly(NIPAM-Am)-AAc cryogel was characterized by performing swelling studies, FTIR and SEM that showed a swelling ratio of 6.2 g H₂O/g dry cryogel. The maximum HSA adsorption by the cryogel was 42.5 mg/g polymer at pH 4.0 in a 50 mM acetate buffer. We also studied the effect of two different temperatures (25 and 40°C). The higher temperature increased the adsorption capacity of the cryogel. HSA molecules could be reversibly adsorbed and desorbed five times with the same poly(NIPAM-Am)-AAc cryogel without a noticeable loss of their HSA adsorption capacity. The synthesized cryogel was used to separate albumin from the protein mixture. Adsorbed albumin was eluted by changing the pH of the buffer (pH 7.0 and 25°C). Poly(NIPAM-Am)-AAc monolithic cryogel behaved as a cation exchange column because of its functional carboxylic group.     

Towards biosensors based on conducting polymer nanowires

We report the electrochemical deposition of poly(pyrrolepropylic acid) nanowires, their covalent modification with antibodies and their conversion into potential functional sensor devices. The nanowires and the devices were characterised by optical microscopy, fluorescence microscopy, electron microscopy and electrical measurements. Fluorescence images, current–voltage (I–V) profiles and real-time sensing measurements demonstrated a rapid and highly sensitive and selective detection of human serum albumin (HSA), a substance that has been used to diagnose incipient renal disease. The detection is based on the selective binding of HSA onto anti-HSA that is covalently attached to the nanowires. The binding changes the electrical properties of the nanowires thus enabling the real-time detection. Whilst the utility of the research was demonstrated for protein binding/detection, the technology could easily be designed for the detection of other analytes by the modification of polymer nanowires with other analyte-specific molecules/biomolecules. Therefore, the technology has the potential to positively impact broad analytical applications in the biomedical, environmental and other sectors. Figure Real-time dynamic current response on sequential exposure of buffer, bovine serum albumin (BSA) and human serum albumin (HSA) onto anti-HSA modified poly (pyrrolepropylic acid) nanowires. Fluorescence images of poly(pyrrolepropylic acid) nanowire (top right) and polypyrrole nanowire control (bottom right) after sequential treatment with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), anti HSA and fluorophore-labeled HSA.     

Solid-phase luminescent catalyst immunoassay for human serum albumin with hemin as labeling catalyst

A solid-phase luminescent catalyst immunoassay is described for the determination of human serum albumin (HSA) in solution; hemin is used as a label which catalytically amplifies the sensitivity. The method is essentially a non-radioactive and non-enzymatic sandwich immunoassay. Anti-HSA antibody is covalently bound to a transparent plate, which then undergoes the immunochemical reaction with HSA in the test solution, and with the fixed amount of hemin-labeled anti-HSA antibody. After the two-step immunoreaction, the immunochemically-adsorbed hemin-antibody conjugate is quantified by means of the luminescence produced in a solution containing luminol and hydrogen peroxide. The luminescence intensity is correlated with the amount of HSA. The limit of detection for HSA is 1 ng ml^-1.     

Single-step biocompatible coating for sulfhydryl coupling of receptors using 2-(pyridinyldithio)ethylcarbamoyl dextran

2-(Pyridinyldithio)ethylcarbamoyl dextran (PDEC dextran) is developed herein as a novel biosensor coating material aimed for direct and facile fabrication of a sulfhydryl-specific capture surface on gold; the site-directed immobilization of a single free cysteine residue presented protein (human serum albumin, HSA) on PDEC dextran-coated surfaces without prior activation and subsequent specific binding of mouse monoclonal anti-HSA antibody to the resultant surface were demonstrated using surface plasmon resonance (SPR) assays.