Immunoaffinity System Research Articles

Tumor-Derived Exosomal Long Noncoding RNAs as Promising Diagnostic Biomarkers for Prostate Cancer

Background/Aims: Exosomal circulating long non-coding RNAs (lncRNAs) in blood are emerging as clinically useful and non-invasive biomarkers for tumor diagnosis. However, normal cells can also secrete exosomes, so it is a prerequisite to obtain tumor-derived exosomes for better understanding of their diagnostic impacts in cancer. In this study, a dual-antibody-functionalized immunoaffinity system was established to isolate exosomes and investigate their lncRNAs expression pattern and clinical significance in prostate cancer (PCa). Methods: A commercially available kit was used to isolate total exosomes, which were then purified by a dual-antibody-functionalized immunoaffinity system. RT-qPCR was performed to detect the expression of exosomal lncRNAs. Receiver operating characteristic (ROC) curves were plotted to assess the diagnostic value. Results: Expression levels of two lncRNAs in tumor-derived exosomes were significantly higher than those in total exosomes. The levels of SAP30L-AS1 were upregulated in benign prostatic hyperplasia (BPH), and SChLAP1 levels were significantly higher in PCa than in BPH and healthy individuals. The area under the ROC curve indicated that SAP30L-AS1 and SChLAP1 had adequate diagnostic value to distinguish PCa from controls. Two lncRNAs separately combined with prostate specific antigen (PSA) possessed a moderate ability for discrimination. SAP30L-AS1 expression level was related to PSA values and tumor invasion. SChLAP1 expression was significantly higher in patients with higher Gleason scores, and was also effective in differentiating between BPH and PCa when the concentration of PSA was in the gray zone. Conclusion: The isolation of tumor-derived exosomes by dual-antibody-functionalized immunoaffinity systems and detection of their lncRNAs in plasma may lead to the identification of suitable biomarkers, with potential diagnostic utility.

Detection of cerebral spinal fluid‐associated chemokines in birth traumatized premature babies by chip‐based immunoaffinity CE

A major concern in treating premature infants with birth-associated head trauma is the rapid determination of reliable biomarkers of neuroinflammation. To this end a chip-based immunoaffinity CE device has been applied to determine the concentrations of inflammation-associated chemokines in samples of cerebral spinal fluid collected from such subjects. The chip utilizes replaceable immunoaffinity disks, to which reactive antibody fragments (FAb) of six antichemokine-specific antibodies were immobilized. Following injection of a sample into the device, the analytes were captured by the immobilized FAbs, labeled in situ with a red laser dye, chemically released and separated by CE. Each resolved peak was measured on-line by LIF detection and the results compared to standard curves produced by running known chemokine standards through the immunoaffinity system. The complete processing of a sample took 10 min with separation of all six analytes being achieved in less than 2 min. The system compared well to commercial ELISA, analysis of the results by linear regression demonstrating r(2) values in the range of 0.903-0.978, and intra and interassay CV of the migration times and the measured peak areas being less than 2.3 and 5%, respectively. Application of the system to analysis of cerebrospinal fluid from head traumatized babies clearly indicated the group with mild trauma versus those with severe injury. Additionally, CE analysis demonstrated that the severe trauma group could be divided into individuals with good and poor prognosis, which correlated with the clinical finding for each patient.

Production and characterization of monoclonal antibodies (mAbs) against human serum albumin (HSA) for the development of an immunoaffinity system with oriented anti-HSA mAbs as immobilized ligand

Proteins present in human serum are of immense importance in the field of biomarker discovery. But, the presence of high-abundant proteins like albumin makes the analysis more challenging because of masking effect on low-abundant proteins. Therefore, removal of albumin using highly specific monoclonal antibodies (mAbs) can potentiate the discovery of low-abundant proteins. In the present study, mAbs against human serum albumin (HSA) were developed and integrated in to an immunoaffinity based system for specific removal of albumin from the serum. Hybridomas were obtained by fusion of Sp2/0 mouse myeloma cells with spleen cells from the mouse immunized with HSA. Five clones (AHSA1-5) producing mAbs specific to HSA were established and characterized by enzyme linked immunosorbent assay (ELISA) and immunoblotting for specificity, sensitivity and affinity in terms of antigen binding. The mAbs were able to bind to both native albumin as well as its glycated isoform. Reactivity of mAbs with different mammalian sera was tested. The affinity constant of the mAbs ranged from 108 to 109M−1. An approach based on oriented immobilization was followed to immobilize purified anti-HSA mAbs on hydrazine activated agarose gel and the dynamic binding capacity of the column was determined.

Development of ovarian hyperstimulation syndrome: interrogation of key proteins and biological processes in human follicular fluid of women undergoing in vitro fertilization

Ovarian hyperstimulation syndrome (OHSS) is an iatrogenic complication and potentially life-threatening condition resulting from excessive ovarian stimulation during assisted reproductive technologies. Our aim was to identify candidate proteins in follicular fluid (FF) using various proteomic approaches which may help to identify patients at risk of OHSS. We analysed the proteome alterations in FF from patients suffering from severe forms of OHSS (OHSS+) compared with a control group of women without or with only mild signs of OHSS (OHSS-). The 12 abundant proteins of FF were removed using an immunoaffinity system. Pools of remaining depleted proteins were applied to the two-dimensional (2D) electrophoresis and 2D liquid chromatography and proteins in differentially expressed protein spots/fractions were identified by mass spectrometry. Among a total of 19 candidate proteins differentially expressed (P< 0.05) between OHSS+ and OHSS- FF samples, three proteins, namely ceruloplasmin, complement C3 and kininogen-1, were found using both 2D techniques. Computer modelling highlighted the important role of kininogen-1 as an anchor for mediated interactions with other identified proteins including ferritin light chain and ceruloplasmin, hepatocyte growth factor-like protein, as well as complement C3 and gelsolin, thus linking various biological processes including inflammation and angiogenesis, iron transport and storage, blood coagulation, innate immunity, cell adhesion and actin filament polymerization. The delineation of such processes may allow the development of informed corrective therapeutic intervention in patients at risk of OHSS and a set of key proteins of the FF may be helpful as potential biomarkers for monitoring IVF therapy.

Application of an ELISA-elution Assay to Dissociate Digoxin-antibody Complexes in Immunoaffinity Chromatography

In this study, we used a modified enzyme-linked immunosorbent assay (ELISA)-elution technique as a screening tool to select specific elution conditions. We examined 12 different elution conditions for the removal of antibodies from a complex on an ELISA plate; 0.2 mol/l glycine-HCl (pH 2.5), 1.0 mol/l acetic acid (pH 2.5), 25% methanol (pH 2.5) and 3 mol/l NaSCN showed a higher elution efficiency. We conducted affinity chromatography with these four conditions for the purification of anti-digoxin antibodies from hyperimmune sera with a digoxin-specific column using omega-aminoalkyl derivatives of Sepharose 4B, whose elution efficiency was similar to that of ELISA. We also monitored the relative specific activities during elution from the digoxin-specific column. The optimum, general-purpose dissociation reagent for this immunoaffinity system was identified as 25% methanol (pH 2.5) with an elution efficiency and relative specific activity of 88.40% and 62.25%, respectively. The high purity of the purified antibodies was demonstrated with sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Comparative Proteomic Analysis of Intra- and Interindividual Variation in Human Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a potential source of biomarkers for many disorders of the central nervous system, including Alzheimer disease (AD). Prior to comparing CSF samples between individuals to identify patterns of disease-associated proteins, it is important to examine variation within individuals over a short period of time so that one can better interpret potential changes in CSF between individuals as well as changes within a given individual over a longer time span. In this study, we analyzed 12 CSF samples, composed of pairs of samples from six individuals, obtained 2 weeks apart. Multiaffinity depletion, two-dimensional DIGE, and tandem mass spectrometry were used. A number of proteins whose abundance varied between the two time points was identified for each individual. Some of these proteins were commonly identified in multiple individuals. More importantly, despite the intraindividual variations, hierarchical clustering and multidimensional scaling analysis of the proteomic profiles revealed that two CSF samples from the same individual cluster the closest together and that the between-subject variability is much larger than the within-subject variability. Among the six subjects, comparison between the four cognitively normal and the two very mildly demented subjects also yielded some proteins that have been identified in previous AD biomarker studies. These results validate our method of identifying differences in proteomic profiles of CSF samples and have important implications for the design of CSF biomarker studies for AD and other central nervous system disorders.

IMMUNOAFFINITY ANALYSIS OF SUBSTANCE P IN COMPLEX BIOLOGICAL FLUIDS: ANALYSIS OF SUB-MICROLITER SAMPLES

ABSTRACT There is a growing need for systems capable of measuring specific analytes in biological samples, especially complex biological fluids. Additionally, this need extends to applying such analyses to precious clinical or research samples, for many of which only micro quantities are still in existence. Immunoaffinity separations provide a useful approach to isolating specific analytes from complex matrices, as well as being capable of miniaturization. In the present communication, we have designed an immunoaffinity system capable of measuring a single analyte in 50 nL samples. We have chosen the clinically important neuropeptide, substance P (SP), as our model analyte and applied it to studying the ability of a micro-chromatography system to measure SP in a number of different biological matrices. The system is capable of isolating SP from biological fluids, including cell cytosols, with reasonable efficiency and reproducibility. The incorporation of laser-induced fluorescence detection enabled the system to achieve a lower limit of detection around 500 femtograms/mL and could isolate the specific analyte in under 5 min, thus, making it potentially useful for high throughput clinical studies.

Direct Resolution of Enantiomers in High-Performance Immunoaffinity Chromatography under Isocratic Conditions

This paper describes the application of stereoselective antibodies as tailor-made chiral selectors for the separation of enantiomers in HPLC under isocratic conditions. Stereoselective monoclonal antibodies to D- and L-alpha-amino acids, raised against protein conjugates of p-amino-D- and L-phenylalanine, were immobilized on a synthetic high-flow-through support material and used for rapid enantiomer separation of a number of amino acids at flow rates between 0.1 and 10 mL/min. Since separations could be performed in a mild buffer, column lifetime considerably exceeded that of classical immunoaffinity systems. Using an anti-D-amino acid antibody as chiral selector, the L-enantiomers eluted with the void volume, while the D-enantiomers eluted second. Inverted elution orders were obtained on chiral stationary phases prepared from an anti-L-amino acid antibody. These results demonstrate, for the first time, that antibody-based chiral stationary phases are useful for routine enantiomer separation under true high-performance chromatographic conditions.

Efficient recovery of recombinant proteins using membrane‐based immunoaffinity chromatography (MIC)

A systematic approach to the design and development of membrane-based immunoaffinity systems for the purification of recombinant proteins is presented. The preparation and characterization of immunoaffinity membranes are described. The immunoaffinity purification process for recombinant interferon-alpha2a is used as a model system to determine the operational parameters in membrane-based immunoaffinity chromatography. The high volumetric throughput of membranes, combined with the typically fastbinding kinetics of antigen-antibody interactions, enable the purification of recombinant proteins from dilute feed stream in less time, using less antibody than conventional systems. Three recombinant proteins, human interferon-alpha2a, interleukin-2, and interleukin-2 receptor, have been purified efficiently employing membrane-based immunoaffinity chromatography. Overall, membrane-based immunoaffinity chromatography is shown to be a viable and scalable method, ideal for the industrial-scale production of recombinant proteins.

Optimum dissociating condition for immunoaffinity and preferential isolation of antibodies with high specific activity

Using an immunoaffinity model consisting of a high performance matrix (Affi-prep-10) with normal human IgG as ligand, and hyperimmune goat anti-human IgG (heavy and light chain active) antibodies, we compared the efficacies of 13 elution reagents. Efficacy was considered in terms of specific activity and total quantitative recovery of the eluted antibody. The optimum, general-purpose dissociation reagent for this immunoaffinity system is 3.0 M MgCl 2·6H 2O, 0.075 M Hepes/NaOH, with 25% ethylene glycol pH 7.20. The antibodies recovered from diluted ( 1 2 ) goat serum with this dissociation reagent have a SpAct of 1.87 times and a total recovery of 6.33 times that of a comparable experiment using the ususal eluant of 1.0 M glycine/HCl, pH 2.00. We also demonstrated that the SpSct of antibodies recovered from immunoaffinity procedures performed under antibody excess and antigen limiting conditions is 2.36–8.00 times higher than that produced by antigen excess and antibody limiting configurations.

Evaluation of factors which affect column performance wiht immobilized monoclonal antibodies : Model studies with a lysozyme—antilysozyme system

Methods are described for the automated evaluation of affinity columns by frontal bounday analysis. These methods were used to evaluate the performance of immunoaffinity columns based on antilysozyme monoclonal antibody—lysozyme immunoaffinity system. This model system enabled the effect of (i) matrix activation and (ii) the density of immobilized antibody on the change in specific activity of immobilized antibody to be quantitatively assessed. Experimental data were accumulated with carbonyldiimidazole-activated Fractogel HW65F and Trisacryl (GF2000) resins and cyanogen bromide-activated Sepharose 4B. An increase in the molar ratio between the concentration of the active groups on the activated matrix and the concentration of immobilized antibody ligands did not result in significant change in the specific activity of the immobilized antibody in the immunochromatographic system. However, increased antibody density with the Fractogel HW65F resin resulted in an increase in the apparent heterogeneity of antibody binding sites for lysozyme and a significant decrease in the specific activity of the immobilized antibody. Furthermore, data from size-exclusion studies with these immunoaffinity matrices demonstrated that a high antibody densities, the accessibility of the immobilized antibody was further decreased due to steric resistance as the antigen size increased.

Immunoaffinity adsorption: process-scale isolation of therapeutic-grade biochemicals.

This review describes immunoaffinity purification as a tool for the process-scale isolation of high-value, therapeutic-grade biochemicals from complex media. To this end, practicalities of activation, antigen coupling and operation in anti-human IgG monoclonal antibody recovery have been studied using cyanogen-bromide activated Sepharose CL-4B and composite Macrosorb K4AX immobilised with polyclonal human IgG as a model for general immunoaffinity systems. The characteristics required of solid phases are discussed in the context of the efficient application of bioselective processes early in the purification sequences. The importance of the technique for isolating biologically active biomolecules for in-vivo administration has been discussed in the light of current, stringent regulatory requirements. Finally, the prospect of scale-up and automation of the technique has been evaluated.

A Simple Method to Optimize Elution from Affinity Chromatography

Abstract A rapid and convenient screening method to optimize elution from affinity chromatography is described. An immunoaffinity system employing antibody bound human thyroglobulin (hTg) served as a model. Anti - hTg was complexed to polystyrene microplates adsorbed hTg. Immune complex (IC) dissociation and elution of antigen by various agents was determined by enzyme linked immunosorbent assay (ELISA). The method permits simul taneous monitoring of residual antigenicity and of possible desorption of antigen from its solid support.

The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesvirus hydrophobic proteins or glycoproteins

A streptavidin/biotin-based immunoaffinity system was optimized to isolate herpesvirus (human cytomegalovirus) immediate early proteins or late glycoproteins from crude infected cell lysates. Biotinylation of the primary antibody by biotin substitution of the ϵ amino groups was superior to biotin substitution of sugar residues. Biotinylation of the primary antibody was superior to that of a secondary antibody. A biotin substitution of approximately 8 m biotin/ m antibody allowed for maximal recovery of viral antigens. The streptavidin/biotin-based immunoaffinity system can allow for relatively pure preparations of viral antigens that may be used for functional, immunological, or structural studies.

A serologic approach to the definition of human prostatic carcinoma antigens.

We have used an immunoaffinity system whereby immunoglobulin from patients with prostatic carcinoma was coupled to solid phase protein A; the immobilized IgG was subsequently exposed to radiolabeled antigen from autochthonous or allogeneic primary tumor extracts or to radiolabeled antigen from a nude mouse-supported prostatic carcinoma cell line. Material specifically bound by prostatic carcinoma patient immune IgG was quantitatively eluted from the immunoadsorbent and characterized with regard to molecular weight. Sequential adsorption analyses of patient sera with normal human tissue pools, normal prostatic tissue, prostatic carcinoma tissue, and tissue from other urogenital malignancies has allowed a definition of those antigenic specificities relevant in the immunobiology of prostatic carcinoma. The patient humoral response was observed to be directed primarily toward a complex array of antigens representing normal human tissue components; serorecognition of prostate tumor associated antigens was discernible from that of common tissue antigens only after rigorous adsorption analyses. Preliminary results indicate that the prostatic cancer patient humoral antibody response may be directed toward either altered histocompatibility complex antigens or toward antigens physically associated with histocompatibility complex antigens.