Immunoaffinity chromatography of human thyroid peroxidase: The stability of the three-dimensional structure and immunoreactivity of antigen and antibodies under various elution conditions

Actions of various chemical agents modeling immunoaffinity chromatography elution conditions caused structural changes of the components of human thyroid peroxidase (TPO) complexes with monoclonal antibodies (MABs) F8 and A1 whose antigenic determinants have a conformational nature and are located in the immunodominant region and a peripheral region of TPO, respectively. These changes became apparent in the circular dichroism and fluorescence spectra of TPO and both MABs as well as in the immunoassay. The effectiveness of the chemical reagents with respect to TPO desorption from an immobilized MAB decreased in the following order: 0.2 M ammonia (pH 11.5) > 0.1 M lithium 3,5-diiodosalycilate > 0.1 M glycine-HCl (pH 2.5) > 1 M NaI > 30% propylene glycol + 1 M NaCl > 30% propylene glycol > 1 M NaCl. At pH 11.5, the three-dimensional structure and immunoreactivity of TPO retained completely and only minor alterations of MAB analogical parameters took place, thus providing a high yield of the functional active human TPO and favoring repeated use of the immobilized MABs in immunoaffinity chromatography. The results may be used as a strategy for the optimization of various protein antigens immunoaffinity chromatography.     

Simple method to assess stability of immobilized peptide ligands against proteases

Although peptides are used as affinity chromatography ligands, they could be digested by proteases. Usually, peptide stability is evaluated in solution, which differs from the resin‐bounded peptide behavior. Furthermore, the study of the degradation products requires purification steps before analysis. Here, we describe an easy method to assess immobilized peptide stability. Sample peptides were synthesized on hydroxymethylbenzamide‐ChemMatrix resin. Peptidyl‐resin beads were then incubated with solutions containing proteases. Peptides were detached from the solid support with ammonia vapor and analyzed by matrix‐assisted laser desorption/ionization and electrospray ionization mass spectrometry, allowing the detection of the whole peptides as well as their C‐terminal degradation products. The method allowed a fast evaluation of peptide ligand stability in solid phase towards proteases that may be present in the crude sample before their use as ligands in affinity chromatography. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Biomass estimation of Aspergillus niger growing on real and model supports in solid state fermentation

Summary Direct hydrolysis of Aspergillus niger mycelium growth on amberlite IRA-900 or sugar cane bagasse on solid state fermentation followed by the analysis of soluble protein by the dye binding method was carried out. Hydrolysis with phosphoric acid 0.25M during 7 min allowed maxima protein extraction available to be measured. Color interference of medium components was not observed, allowing the use of this method for biomass estimation when amberlite IRA-900 or sugar cane bagasse are used as support in solid state fermentation processes.     

Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.     

Preparation of no-carrier-added technetium-99m complexes via metal-assisted cleavage from a solid phase

A novel method for the preparation of no-carrier-added (nca) complexes [99mTc(CO)3L] (L = diethylenetriamine or picolylamine-N-acetic acid) is described. The ligands were covalently bound to a solid support of organic polymers via formation of a tertiary amine from the chelating unit. This C-N bond to the solid phase is selectively cleaved during the formation of the technetium complexes by intramolecular nucleophilic attack of a remaining hydroxy ligand to the alpha-carbon. The complex [99mTc(CO)3L] is released into solution while uncomplexed ligand and uncleaved complex remain solid-phase bound. High specific activity technetium complexes can then be isolated by simple filtration. Cleavage yield depends on temperature, pH, and ligand. Up to 50% release from the solid phase could be achieved under optimized conditions. Corresponding to the 99mTc concentration, free ligand is present in concentrations lower than 10(-7) M. If a targeting vector is conjugated to these ligands, no-carrier-added radiopharmaceuticals can be prepared in that way.     

Purification of the putative alpha-latrotoxin receptor from bovine synaptosomal membranes in an active binding form.

alpha-Latrotoxin (alpha-LTx, apparent mol. wt. 130 000) is a presynaptically active neurotoxin purified from the venom of the black widow spider that causes massive exocytotic release of neurotransmitters, presumably via binding to presynaptic membrane protein(s). Solubilization and purification experiments were undertaken to identify and characterize this membrane component. An immunoaffinity matrix was prepared by sequentially binding anti-alpha-LTx antibodies and alpha-LTx to Protein A-Sepharose CL-4B. Beads were irreversibly cross-linked with dimethyl pimelimidate. These beads were capable of extracting alpha-LTx binding activity from Triton X-100 solubilized bovine synaptosomal membranes. Following extensive washing, bound material was eluted with 6 M urea. Analysis of silver stained and radiolabel-containing gels revealed one major band (apparent mol. wt. 200 000) under non-reducing conditions and two major bands (apparent mol. wts. 66 000 and 54 000) under reducing conditions. The purified material was still capable of specifically binding alpha-LTx as determined by solid phase assays on microtiter plates. The affinity for alpha-LTx of the purified preparation was similar to that of the native membrane (KA approximately 10(10) M). It is concluded that a putative alpha-LTx receptor protein can be purified from synaptosomal membranes using an immunoaffinity matrix in a form that retains its defined biological property (alpha-LTx binding).     

Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies.

A 190-kDa centrosomal protein interacts with microtubules when Drosophila embryo extracts are passed over microtubule-affinity columns. We have obtained a partial cDNA clone that encodes this protein. Using a fusion protein produced from the clone, we have developed a novel immunoaffinity chromatography procedure that allows both the 190-kDa protein and a complex of proteins that associates with it to be isolated in in a single step. For this procedure, the fusion protein is used as an antigen to prepare rabbit polyclonal antibodies, and those antibodies that recognize the 190-kDa protein with low affinity are selectively purified on a column containing immobilized antigen. These low-affinity antibodies are then used to construct an immunoaffinity column. When Drosophila embryo extracts are passed over this column, the 190-kDa protein is quantitatively retained and can be eluted in nearly pure form under nondenaturing conditions with 1.5 M MgCl2, pH 7.6. The immunoaffinity column is washed with 1.0 M KCl just before the elution with 1.5 M MgCl2. This wash elutes 10 major proteins, as well as a number of minor ones. We present evidence that these KCl-eluted proteins represent additional centrosomal components that interact with the 190-kDa protein to form a multiprotein complex within the cell.     

Solid-phase assembly of DNA duplexes from synthetic oligonucleotides

A new method of rapid and efficient assembly of extended DNA duplexes in solid phase was developed. Subassemblies of separately annealed oligonucleotides were stepwise hybridized to each other on a solid support. Two types of supports with anchor oligonucleotide were tested: Fractosil-1000 with oligo-dT sequence and Sephacryl S-500 with an oligonucleotide bound via CNBr-activation procedure. Sephacryl S-500 turned out to be the support of choice since all enzymatic reactions of the assembly procedure (phosphorylation, ligation, restriction enzyme digestion) could be efficiently performed with DNA immobilized on Sephacryl S-500 particles.     

Solid-phase biotinylation of antibodies

Biotinylation is an established method of labeling antibody molecules for several applications in life science research. Antibody functional groups such as amines, cis hydroxyls in carbohydrates or sulfhydryls may be modified with a variety of biotinylation reagents. Solution-based biotinylation is accomplished by incubating antibody in an appropriate buffered solution with biotinylation reagent. Unreacted biotinylation reagent must be removed via dialysis, diafiltration or desalting. Disadvantages of the solution-based approach include dilution and loss of antibody during post-reaction purification steps, and difficulty in biotinylation and recovery of small amounts of antibody. Solid-phase antibody biotinylation exploits the affinity of mammalian IgG-class antibodies for nickel IMAC (immobilized metal affinity chromatography) supports. In this method, antibody is immobilized on a nickel-chelated chromatography support and derivitized on-column. Excess reagents are easily washed away following reaction, and biotinylated IgG molecule is recovered under mild elution conditions. Successful solid phase labeling of antibodies through both amine and sulfhydryl groups is reported, in both column and mini-spin column formats. Human or goat IgG was bound to a Ni-IDA support. For sulfhydryl labeling, native disulfide bonds were reduced with TCEP, and reduced IgG was biotinylated with maleimide-PEO(2) biotin. For amine labeling, immobilized human IgG was incubated with a solution of NHS-PEO(4) biotin. Biotinylated IgG was eluted from the columns using a buffered 0.2 M imidazole solution and characterized by ELISA, HABA/avidin assay, probing with a streptavidin-alkaline phosphatase conjugate, and binding to a monomeric avidin column. The solid phase protocol for sulfhydryl labeling is significantly shorter than the corresponding solution phase method. Biotinylation in solid phase is convenient, efficient and easily applicable to small amounts of antibody (e.g. 100 microg). Antibody biotinylated on-column was found to be equivalent in stability and antigen-recognition ability to antibody biotinylated in solution. Solid-phase methods utilizing Ni-IDA resin have potential for labeling nucleic acids, histidine-rich proteins and recombinant proteins containing polyhistidine purification tags, and may also be applicable for other affinity systems and labels.     

Surface-bound cytomimetic assembly based on chemoselective and biocompatible immobilization and further modification of intact liposome

A surface-bound cytomimetic assembly based on chemically selective and biocompatible immobilization and further modification of intact liposome is described. Liposomes carrying PEG-triphenylphosphine were chemoselectively immobilized onto azide-modified glass slides through Staudinger ligation, followed by modification with azide-modified lactose as a model biomolecule through Staudinger ligation to afford the surface-bound cytomimetic assembly. The intact liposome immobilized and modified and its protein binding activity were confirmed by fluorescence imaging, fluorescent dye releasing kinetics, and AFM techniques. The resultant surface-bound cytomimetic assembly showed sustained stability and fluorescent dye releasing kinetics and specific protein binding activity. The reported method provides a robust platform for preparation of a complex immobilized liposome system with multifunctional components, which mimics the cell surface in both geographical and content features and thus will find important biomedical applications.     

Preparation of bioconjugates by solid-phase conjugation to ion exchange matrix-adsorbed carrier proteins

A solid-phase conjugation method utilizing carrier protein bound to an ion exchange matrix was developed. Ovalbumin was adsorbed to an anion exchange matrix using a batch procedure, and the immobilized protein was then derivatized with iodoacetic acid N-hydroxysuccinimid ester. The activated protein was conjugated with glutathione, the conjugation ratio determined by acid hydrolysis, and amino acid analysis performed with quantification of carboxymethyl cysteine. Elution of conjugates from the resin by a salt gradient revealed considerable heterogeneity in the degree of derivatization, and immunization experiments with the eluted conjugates showed that the more substituted conjugates gave rise to the highest titers of glutathione antibodies. Direct immunization with the conjugates adsorbed to the ion exchange matrix was possible and gave rise to high titers of glutathione antibodies. Conjugates of ovalbumin and various peptides were prepared in a similar manner and used for production of peptide antisera by direct immunization with the conjugates bound to the ion exchanger. Advantages of the method are its solid-phase nature, allowing fast and efficient reactions and intermediate washings, and the ability to release conjugates from the solid phase under mild conditions.     

Immunoenrichment of urinary S-phenylmercapturic acid

A carefully designed hapten-protein conjugate has enabled the generation of a monoclonal antibody reactive with S-phenylmercapturic acid (S-PMA). The immobilized antibody retains immunoreactivity and can be used to enrich S-PMA from the urine of workers exposed to benzene. The performance of the immunoaffinity column has been validated by comparison with data obtained by GC/MS analysis from the urine of benzene-exposed workers (range 12-168 μgl^-1, corr. coeff. 0.98, n = 23). Furthermore immunoaffinity chrom atography facilitates the quantitative determination of urinary S-PMA by reversed phase HPLC. Bioconcentration of S-PMA from the urine of benzeneexposed workers has perm itted the quantification of S-PMA by HPLC at 8 h TWA (time weighted average) exposures of around 1 ppm. The potential application of immunoaffinity enrichment in biomonitoring studies is discussed.     

Evidence that a nuclear matrix protein participates in premessenger RNA splicing

The role of nuclear matrix proteins in premessenger RNA splicing has been investigated using antibodies raised against isolated rat liver nuclear matrix and cross-reactive with a 65-kDa HeLa cell nuclear matrix protein (IGA-65). IGA-65 is an internal nuclear matrix component which can be solubilized as a component of nuclear splicing extracts, by the action of endogenous ribonucleases, EDTA, and DTT during extract preparation. Preincubation of splicing extract with antibodies against IGA-65 (anti-IGA-65) inhibited in vitro splicing of exogenous adenovirus precursor RNA. Furthermore, assembly of precursor RNA into active spliceosome complexes was inhibited by pretreatment of extracts with anti-IGA-65, suggesting a role for IGA-65 during early spliceosome assembly. The IGA-65 present in splicing extracts was distinguishable from known U-snRNP and hnRNP proteins on protein gels. Furthermore, electrophoresis of splicing extract on native gels indicated that IGA-65 was present in protein complexes different from those containing U-snRNPs or hnRNP C protein. The data support identification of complexes containing IGA-65 as nuclear factors involved in pre-mRNA splicing and, by extension, suggest a role for the nuclear matrix during processing in vivo.     

Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain

Neurotransmitter receptors in vivo are linked to intracellular adaptor proteins and signalling molecules driving downstream pathways. Methods for physical isolation are essential to answer fundamental questions about the size, structure and composition of in vivo complexes and complement the widely used yeast 2-hybrid method. The N-methyl-D-aspartate receptor (NMDAR) binds postsynaptic density 95 (PSD-95) protein; both are required for synaptic plasticity and learning and participate in other important pathophysiological functions. Here we describe the development and optimization of novel methods for large-scale isolation of NMDAR--PSD-95 complexes from mouse brain including immunoaffinity, immunoprecipitation, ligand-affinity and immobilized PSD-95 binding peptides. Short PDZ binding peptides modelled on NMDAR subunits were shown to isolate NMDAR complexes. Gel filtration indicated the native NMDAR--PSD-95 complexes were 2000 kDa, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a complexity suggesting a huge network of both structural components and signalling enzymes. These methods can be used to define the structure of the complexes at different synapses and in mice carrying gene mutations as well as new tools for drug discovery.     

Isolation of high-density lipoproteins by immunoaffinity column chromatography from hog plasma

High density lipoprotein (HDL) was isolated from hog plasma by a simple immunoaffinity column chromatography procedure using immobilized anti-apolipoprotein AI. The composition of HDL isolated by immunoaffinity chromatography was nearly identical to that of a control sample that was isolated by an alternate method utilizing ultracentrifugation and gel chromatography. The HDL isolated by immunoaffinity chromatography had a larger number of polypeptide components that the control as indicated by acrylamide gel electrophoresis in the presence of urea. When the HDL isolated by immunoaffinity chromatography was applied to a heparin-agarose column the amount of protein retained was approximately twice that of the control. These findings indicate that the ultracentrifugation procedure probably induced the loss of apolipoprotein E containing components from the HDL complex.     

Polymer-supported biopolymer synthesis: 2. Phenolic poly(acryloylmorpholine)-based preparation of protected arginyl acylpeptide segments and derived arginyl peptides

A new poly(acryloylmorpholine)-based phenolic support matrix, Koch-Light Peptide Resin A, has been utilized for the solid (gel) phase assembly of protected or ginyl acylpeptide segments. Two methods, selective hydrazimolysis and autocatalysed transesterification with 2-dimethylaminoethanol, have been used to detach the peptide segments from the resin. Selective hydrazinolysis illustrates the use of the phenolic support matrix in the preparation of arginyl acylpeptide hydrazides bearing hydrazine-labile nitroguanidino and benylaxycarbonyl side chain protecting groups. Autocatalysed hydrolysis in trifluoroethanol/sodium trifluoroethanoate buffer has been used to convert the protected arginyl acylpeptide 2-dimethylaminoethyl esters to the corresponding protected arginyl acylpeptide acids. Total deprotection of the latter was effected by catalytic transfer hydrogenation in formic acid.     

Real time analysis of intact organelles using surface plasmon resonance

Membrane proteins remain refractory to standard protein chip analysis. They are typically expressed at low densities in distinct subcellular compartments, their biological activity can depend on assembly into macromolecular complexes in a specific lipid environment. We report here a real-time, label-free method to analyze membrane proteins inserted in isolated native synaptic vesicles. Using surface plasmon resonance-based biomolecular interaction analysis (Biacore), organelle capture from minute quantities of 10,000 g brain supernatant (1-10 microg) was monitored. Immunological and morphological characterization indicated that pure intact synaptic vesicles were immobilized on sensor chips. Vesicle chips were stable for days, allowing repetitive use with multiple analytes. This method provides an efficient way in which to characterize organelle membrane components in their native context. Organelle chips allow a broad range of measurements, including interactions of exogenous ligands with the organelle surface (kinetics, Kd), and protein profiling.     

Determination of immunoreactive gonadotropin-releasing hormone in serum and urine by on-line immunoaffinity capillary electrophoresis coupled to mass spectrometry

The need for urgent diagnoses has propelled the development of automated analyses that can be performed in a short time at reasonable cost. One such method is immunoaffinity capillary electrophoresis. This emerging hybrid technology employs two powerful techniques coupled on-line for the direct and rapid determination of analytes present in biological fluids. The first technique, immunoaffinity, is used for the selective extraction of a molecule present in a complex matrix, utilizing a microscale-format chamber affinity device. An analyte (affinity target) present in serum or urine is captured by an immobilized molecular recognition antibody molecule (affinity ligand) bound to a solid support constituent (glass beads or an appropriate porous structure) of a microchamber affinity device. The second technique, capillary electrophoresis, is used for the high-resolution analytical separation of the purified and concentrated affinity target material after elution from the microchamber affinity device. In this work, immunoaffinity capillary electrophoresis was developed for the identification and characterization of a single constituent of a complex matrix. Immunoreactive gonadotropin-releasing hormone was determined in serum and urine specimens derived from a normal individual and from a patient suffering from benign prostatic hyperplasia. Furthermore, the on-line immuno-separation system was coupled in tandem to mass spectrometry to obtain molecular mass information of the affinity isolated and CE separated neuropeptide. This hybrid immuno-analytical technology is simple, rapid, selective and sensitive. In addition, an attempt was also made to characterize other urinary constituents by CE–MS that may lead to marker activity in the urine of the diseased subject. The hyphenation of analytical techniques has proved valuable in enhancing their individual features. The future of bioanalysis using miniaturized affinity systems is discussed in this paper.     

Biointeraction analysis of immobilized antibodies and related agents by high-performance immunoaffinity chromatography

A method is described based on high-performance immunoaffinity chromatography for examining the interactions of immobilized antibodies or related binding agents with their targets. It is shown how this method can be used to obtain information on the binding, elution and regeneration kinetics of immobilized binding agents, such as those used with immunoaffinity supports. The theory behind this approach is briefly described and it is demonstrated how both the kinetic and thermodynamic properties of a biointeraction can be determined experimentally through this method. Several applications are used to illustrate this technique, including antibody-antigen interactions and the binding of aptamers with their targets in the presence of silica-based supports. The same approach can be adapted for use with other types of targets, binding agents and support materials.