Polymeric supports for affinity chromatography and high-performance affinity chromatography

Affinity chromatography is the most selective chromatographic method for the purification of biologically active materials. It is based on the biospecific interaction of the substrates with a ligand, which is chemically immobilized onto a suitable matrix (support). Different matrices provided by natural and synthetic polymers are used for the preparation of affinity supports. In this communication we describe and compare the properties of various supports based on polysaccharides, polyacrylamides and inorganic materials. In particular, we discuss the utility of different silica derivatives (especially primary hydroxyl silica) for the immobilization of ligands and high-performance affinity chromatography.     

Automated microsystem for electrochemical detection of cancer markers

The development of a fully automated microsystem housing an amperometric immunosensor is presented. The microfluidic cell integrates reagent storage and electrochemical immunodetection and was applied for the detection of breast cancer markers. The main advantage of this system is that no external fluidic storage is required and the instrumental setup is thus greatly simplified. The fluidics of the microsystem is computer controlled and requires minimal end-user intervention. The analytical performance of the device was compared with a manually driven system and applied for the amperometric detection of the carcinoembryonic antigen (CEA) and cancer antigen 15-3 (CA15-3). This automation methodology greatly improves the analytical performance of the immunosensor in terms of accuracy and reproducibility as evidenced by a reduction of LOD observed for CEA and CA15-3 with respect to a manually driven system. Finally, the automated microsystem was applied for the analysis of real patient serum samples, demonstrating excellent correlation with a commercial ELISA.     

Integrated microsystem for dielectrophoretic cell concentration and genetic detection

We have directly integrated a continuous-flow, electrokinetic method of bacterial cell concentration with room temperature, sequence-specific genetic detection. The system we have developed traps cells from a continuous-flow sample stream via dielectrophoresis, providing a 160-fold increase in cell concentration. PDMS microvalves then define a 100 nL volume around the trapped cells to which cell lysis buffer and genetic detection components are introduced. Direct, optical detection of Escherichia coli MC1061 cells is then accomplished via the sequence-specific hybridization of an rRNA-directed optical molecular beacon. This integrated microsystem is capable of sequence-specific genetic detection of 25 cells within 30 min.     

High-sensitivity nanosensors for biomarker detection

High sensitivity nanosensors utilize optical, mechanical, electrical, and magnetic relaxation properties to push detection limits of biomarkers below previously possible concentrations. The unique properties of nanomaterials and nanotechnology are exploited to design biomarker diagnostics. High-sensitivity recognition is achieved by signal and target amplification along with thorough pre-processing of samples. In this tutorial review, we introduce the type of detection signals read by nanosensors to detect extremely small concentrations of biomarkers and provide distinctive examples of high-sensitivity sensors. The use of such high-sensitivity nanosensors can offer earlier detection of disease than currently available to patients and create significant improvements in clinical outcomes.     

High-performance affinity chromatography

High-performance affinity chromatography is a new technique for the fast and efficient purification of biologically active molecules. It combines the biospecificity of affinity chromatography with the high speed and resolution obtained in high-performance liquid chromatography. In particular, the immobilization of ligands to different silica derivatives and their suitability for high-performance affinity chromatography are discussed.     

Immobilized urokinase column as part of a specific detection system for plasminogen species separated by high-performance affinity chromatography

Immobilized urokinase was used as part of a post-column reactor for the specific detection of human plasminogen species which were fractionated using a high-performance affinity column. After on-line activation of each peak, plasmin activity was measured by mixing the eluate with a specific fluorogenic substrate and the product was detected by a fluorescence monitor. This detection system gave linear calibration graphs for both purified plasminogens (0.1-50 micrograms) and plasminogens contained in plasma (25-100 microliters). Relative standard deviations for the determination of plasminogens in plasma were 6.1-6.6% (n = 12), showing good reproducibility. The detection limit was as low as 0.1 micrograms of plasminogen. Immobilized urokinase was very stable and no appreciable decrease in activity was found after 100 cycles of operation. In combination with an immobilized benzamidine column, this system made it possible to separate and detect Glu-plasminogen and Lys-plasminogen contained in human plasma samples as small as 100 microliters without any pretreatment.     

Single nucleotide polymorphism detection method by temperature-gradient affinity chromatography using a single-stranded oligo-DNA coupled column

We developed an affinity chromatographic method for simple single nucleotide polymorphism (SNP) detection by use of a single-stranded DNA-coupled column and temperature gradient elution, utilizing the difference in thermal stability between hybridized double-stranded DNAs with and without mismatched base-pairs in the course of temperature gradient elution. We studied experimentally and theoretically the elution behavior of DNAs with and without SNPs in this chromatography and proposed a numerical calculation method based on a thermodynamic dissociation model. The effects of the column volume, flow rate of eluent and heating rate of the column on elution profiles were clarified. For designing DNA ligands, mismatched base-pair positions favorable for detection of SNPs were also explored by use of hybridized DNAs coding a part of the human TP53 gene.     

Microarray methods for protein biomarker detection

The application of protein biomarkers as an aid for the detection and treatment of diseases has been subject to intensified interest in recent years. The quantitative assaying of protein biomarkers in easily obtainable biological fluids such as serum and urine offers the opportunity to improve patient care via earlier and more accurate diagnoses in a convenient, non-invasive manner as well as providing a potential route towards more individually targeted treatment. Essential to achieving progress in biomarker technology is the ability to screen large numbers of proteins simultaneously in a single experiment with high sensitivity and selectivity. In this article, we highlight recent progress in the use of microarrays for high-throughput biomarker profiling and discuss some of the challenges associated with these efforts.     

High-performance affinity chromatography: a powerful separation technique

Analyses of complex biochemical samples can be performed within one minute using a method which combines the selectivity of affinity chromatography with the speed of high-performance liquid chromatography.     

核酸适配体亲和色谱的研究进展

核酸适配体亲和色谱是将核酸适配体作为色谱固定相上的亲和配体的一种新型色谱技术。核酸适配体是一种可以特异性地识别目标物的寡聚核苷酸,与免疫抗体相比,核酸适配体在筛选制备、稳定性及应用等方面都显示出独特的优点。本文介绍了核酸适配体亲和色谱在小分子、蛋白质和细胞的分离和分析中的应用,对核酸适配体亲和色谱的研究现状和发展前景进行了综述。     

Boronate functionalised polymer monoliths for microscale affinity chromatography

Novel macroporous monolithic stationary phase materials suitable for microscale boronate affinity chromatography were developed.     

混合抗体的亲和色谱分离策略

乳腺生物反应器可以高效表达重组人单克隆抗体,但是目标产品与乳液原料中的牛抗体性质、结构非常类似,分离难度很大。本文对牛抗体和重组人抗体的种属差异进行了分析,并在此基础上制定了新型分离策略,采取Protein A亲和色谱和免疫亲和色谱来解决混合抗体的分离问题,并讨论了色谱洗脱模式对分离效果的影响。结果表明,Protein A亲和色谱结合梯度洗脱可以有效地纯化得到混合抗体,但是难以彻底分离重组人抗体和牛抗体;相比之下,使用Protein A亲和色谱结合置换色谱模式可以更加高效地分离混合抗体,最终可以得到纯度高达95%以上的重组人抗体,回收率可达95%以上。免疫亲和色谱同样可以有效地分离纯化重组单克隆抗体,且其通用性更强,可以应用于任何动物乳腺表达重组人抗体的分离纯化中。     

Frontal affinity chromatography for the screening of mixtures

A protein stationary phase for frontal affinity chromatography was prepared, containing biotinylated beta-galactosidase immobilized to controlled pore glass beads via covalently bonded streptavidin. Single microaffinity columns of approximately 30 pmol of active beta-galactosidase were prepared from this material and characterized with a known ligand by frontal analysis. These columns were used to measure the specific interactions between the bound beta-galactosidase and a library of modified beta-galactopyranosides using electrospray mass spectrometry as the means of detection. The library contained 89 entries, each representing 4 diastereomers for a total of 356 library members. A single entry was analysed revealing differential activity among the 4 isomers. The library was grouped into 10 mixtures of 24-40 members each with each mixture infused under frontal chromatographic conditions. This deconvolution procedure led to the identification of 34 entries containing isomers with K(d) values better than 10 microM. A method based on a displacement principle was implemented as a rapid prescreen which served as the basis for a parallel column high throughput screening assay.     

Novel affinity ligands for chromatography using combinatorial chemistry

Spatially addressable combinatorial libraries were synthesized by solution phase chemistry and screened for binding to human serum albumin. Members of arylidene diamide libraries were among the best hits found, having submicromolar binding affinities. The results were analyzed by the frequency with which particular substituents appeared among the most potent compounds. After immobilization of the ligands either through the oxazolone or the amine substituent, characterization by surface plasmon resonance showed that ibuprofen affected the binding kinetics, but phenylbutazone did not. It is therefore likely that these compounds bind to Site 2 in sub domain IIIA of human serum albumin (HSA).     

Immobilization of monoclonal antibodies for affinity chromatography using a chelating peptide.

A procedure was developed for oriented immobilization of monoclonal antibodies on a solid support. The technique involves the specific oligosaccharide-directed covalent modification of the monoclonal antibody (mAb) with the chelating peptide, Lys-Gly-(His)6, in conjunction with immobilized metal ion affinity chromatography. Chelating peptide-mAb conjugates with a molar ratio of 2.2 retained full antigen binding activity. On immobilization of the modified antibodies on a nickel affinity resin, the molar antigen binding ratio was 1.4. The high antigen binding capacity is indicative of oriented immobilization providing maximum access for the antigen. The described method can be used for the preparation of high-capacity immunosorbents for affinity chromatography and it is applicable for all immunoglobulin classes.     

Aptamer stationary phase for protein capture in affinity capillary chromatography

The thrombin-binding DNA aptamer was used with thrombin as a model system to investigate protein capture using aptamer stationary phases in affinity capillary chromatography. The aptamer was covalently attached to the inner surface of a bare fused-silica glass capillary to serve as the stationary phase. Proteins were loaded onto the capillary via an applied pressure. The capillary was then washed to remove unbound and non-specifically associated proteins. Finally, the bound protein was released and eluted using 20 mM Tris buffer containing 8 M urea, pH 7.3, at 50 degrees C. Eluate was collected after each step (load, wash and elute) and relative amounts of protein each were compared using fluorescence spectroscopy. The identity of the protein in the collections was confirmed using matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. The experiment was repeated for thrombin on a bare (unmodified) capillary and a capillary coated with a scrambled-sequence, non-G-quartet forming oligonucleotide that does not bind with thrombin. The results show that the aptamer stationary phase captures approximately three times as much thrombin as the control columns. The experiment was also repeated using human serum albumin (HSA) alone and in an equimolar mixture with thrombin. HSA was not retained on the aptamer capillary, nor did it affect the capture of thrombin from the mixture.     

Bead injection for biomolecular assays: Affinity chromatography enhanced by bead injection spectroscopy

Selective capture of target biomolecules by ligands immobilized on a solid support is a cornerstone of two seemingly unrelated techniques: micro-Affinity Chromatography (microAC) and micro-Bead Injection Spectroscopy (microBIS). This work shows, for the first time, how these techniques can be carried out using the same instrument and how the data obtained this way complement each other, yielding complete information on retention and elution of target biomolecules. Biomolecular association and dissociation were investigated by microAC and microBIS, using computer-controlled programmable flow and the same instrument for automated bead transport, packing of a micro-column, assay of the analyte, and bead disposal. The absorbance of the analyte was monitored within the fiber optic flow cell configured either for monitoring directly on the beads or post-column after elution. The separation, binding, and elution of immunoglobulins (human IgG, rabbit IgG, and horse IgG) on protein G-coated Sepharose beads were studied as model systems. The limit of detection of the microAC technique was determined to be 5 ng microL(-1) IgG, and that of the microBIS technique was 50 ng microL(-1) IgG.