Peptide-based immunoadsorbents: molecular grafting of IgG-Fc-binding epitopes of Protein A onto a de novo-designed helix-loop-helix peptide.

The development of immunoadsorbents that have high specificity for immunoglobulin and no immunogenicity is essential for immunoadsorption treatment of autoimmune diseases. In this study, we designed peptide immunoadsorbents by molecular grafting of the IgG-Fc binding epitopes of Protein A onto a de novo-designed helix-loop-helix peptide. Linear (linG7A5) and cyclic (cyG7A5) grafted peptides were synthesized to test their binding affinity and specificity. Peptide cyG7A5 demonstrated high specificity for human IgG-Fc, with a K(D) of 19 µM, and demonstrated no affinity to other plasma proteins, human serum albumin, or fibrinogen. To evaluate their immunoadsorbance efficiency, the grafted peptides and Protein A were conjugated to polyvinyl acetate resin and tested in a batch-wise process for adsorption removal of IgG from human plasma. The IgG capture capacities of the peptides correlated well with their binding affinities. Interestingly, cyG7A5 showed a higher binding specificity for IgG than did Protein A.

[New bioaffine sorbents for selective elimination of autoantibodies against human thyroperoxidase in autoimmune thyroid diseases].

New bioaffine sorbents containing bioselective ligand, synthetic analog of the human thyroperoxidase antigenic determinant--tetrapeptide H-Glu-Gln-betaAla-Lys-OMe, immobilized on two polymeric matrixes--a polyacrylamide gel and CNBr-activated sepharose 4B were synthesized. The offered immunosorbents were shown have high selectivity in relation to autoantibodies against thyroperoxidase and can find an application for medicine and experimental biochemistry for selective elimination of autoantibodies from serum or plasma of the patients suffering from autoimmune thyroid diseases.

A novel affinity ligand for polystyrene surface from a phage display random library and its application in anti-HIV-1 ELISA system.

In order to develop an affinity ligand for site-directed immobilization of target proteins on polystyrene (PS) surface, a linear 12-mer peptide phage display random library was screened. Phage clones that specifically bound to PS plate were sequenced after three rounds of biopanning. The obtained DNA sequences revealed that there were several aromatic and basic amino acid residues, which may be critical to binding. One of the selected dodecapeptides, named Lig1 (FKFWLYEHVIRG), was genetically fused to the N/C-terminus of recombinant antigen ENV which could be recognized by specific antibodies against human immunodeficiency virus type 1 (HIV-1), to investigate its performance as an affinity ligand. The ligand-fused ENVs overexpressed in Escherichia coli were compared to the original one in terms of the immobilization characteristics on PS plate in enzyme-linked immunosorbent assay (ELISA). The results indicated that the ligand-fused proteins showed a considerably improved affinity to PS surface, and were preferentially adsorbed on PS plate suffering only scarcely from interference by coexisting protein molecules. Anti-HIV-1 ELISA system, which employed Lig1-ENV (Lig1 fused to ENV N-terminus) as immobilization antigen also exhibited sufficiently high sensitivity and specificity in serodiagnosis tests.

The reversible binding of anti-human serum albumin to poly beta-cyclodextrin-coated porous silica supports.

A supramolecular system involving host-guest interactions between immobilized beta-cyclodextrin (beta-CD) cavities and adamantyl groups was evaluated for the preparation of immunosorbents which can be regenerated after use. First a dextran layer bearing both adamantyl groups and carboxylic functions is immobilized onto beta-CD-modified porous silica particles (400 nm) by formation of inclusion complexes. Then, antibody molecules are grafted to the polymer layer. The stationary phases can be prepared in batch or directly in the column. They are stable in aqueous media and are able to trap specifically the corresponding antigen. In case of alteration of the antibody layer, it is possible to remove it by passing a SDS solution through the column. The feasibility of the procedure was evaluated, using the anti-HSA/HSA system.

Preparation of an immunoadsorbent coupled with a recombinant antigen to remove anti-acetylcholine receptor antibodies in abnormal serum.

An immunoadsorbent that removes anti-acetylcholine receptor antibodies (AChRAb) in abnormal serum of myasthenia gravis (MG) patient was efficiently prepared by an expression product, the functional fragment of AChR(alpha205) fused with maltose binding protein (MBP). The ligand can then covalently bind to amylose resin through MBP fusion protein. It was shown from the result of this study with anti-AChR mice sera that the removal rate of AChRAb on this immunoadsorbent reached 87+/-10% (mean value of 10 mice) and the maximally binding capacity of AChRAb was approximately 260 microg/g immunoadsorbent (wet weight). Moreover, the immunoadsorption test of sera in two MG patients indicated that about 90% and 96% of abnormal AChRAb could be eliminated, while other serum components such as albumin, IgG, IgM and IgA only dropped 18%, 35%, 22%, 15% and 24%, 27%, 15%, 12%, respectively, for two MG patient sera. It is anticipated from this study that the immunoadsorbent reported here could, with further development, find its clinical application for removal of AChRAb from patient serum.

An ion-imprinted functionalized silica gel sorbent prepared by a surface imprinting technique combined with a sol-gel process for selective solid-phase extraction of cadmium(II).

A new ion-imprinted thiol-functionalized silica gel sorbent was synthesized by a surface imprinting technique in combination with a sol-gel process for selective on-line, solid-phase extraction of Cd(II). The Cd(II)-imprinted thiol-functionalized silica sorbent was characterized by FT-IR, the static adsorption-desorption experiment, and the dynamic adsorption-desorption method. The maximum static adsorption capacity of the ion-imprinted functionalized sorbent was 284 micromol g(-1). The largest selectivity coefficient for Cd(II) in the presence of Pb(II) was over 220. The static uptake capacity and selectivity coefficient of the ion-imprinted functionalized sorbent are higher than those of the nonimprinted sorbent. The breakthrough capacity and dynamic capacity of the imprinted functionalized silica gel sorbent for 4 mg L(-1) of Cd(II) at 5.2 mL min(-1) of sample flow rate were 11.7 and 64.3 micromol g(-1), respectively. No remarkable effect of sample flow rate on the dynamic capacity was observed as the sample flow rate increased from 1.7 to 6.8 mL min(-1). The imprinted functionalized silica gel sorbent offered a fast kinetics for the adsorption and desorption of Cd(II). The prepared ion-imprinted functionalized sorbent was shown to be promising for on-line, solid-phase extraction coupled with flame atomic absorption spectrometry for the determination of trace cadmium in environmental and biological samples. All competitive ions studied did not interfere with the determination of Cd(II). With a sample loading flow rate of 8.8 mL min(-1) for 45-s preconcentration, an enhancement factor of 56, and a detection limit (3sigma) of 0.07 microg L(-1) were achieved at a sampling frequency of 55 h(-1). The precision (RSD) for 11 replicate on-line sorbent extractions of 8 mug L(-1) Cd(II) was 0.9%. The sorbent also offered good linearity (r = 0.9997) for on-line, solid-phase extraction of trace Cd(II).

Differences between synthetic oligosaccharide immunoabsorbents in depletion capacity for xenoreactive anti-Galalpha1-3Gal antibodies from human serum.

Extracorporeal immunoabsorption for removal of anti-Galalpha1-3Gal (anti-Gal) antibodies in putative pig-to-human xenotransplantation is considered a major prophylactic measure to avoid hyperacute and acute vascular rejections. However, the efficacy of the procedure does depend on choosing the appropriate oligosaccharide epitopes for the binding of human anti-Gal. The synthetic oligosaccharides Galalpha1-3Gal (B-disaccharide, Bdi) and Galalpha1-3Galbeta1-4Glc ('type 6' trisaccharide, Tri6), covalently coupled to Sepharose via polyacrylamide (Sorbents Bdi and -Tri6, respectively), as well as a mixture thereof (Sorbent Mix), were examined for their efficacy to absorb anti-Gal from 20 human serum samples. Sorbent Bdi removed 81% of anti-Gal IgM and 85% of -IgG when assessed on Bdi by ELISA, but only 49% of IgG and 75% of IgM when assessed on Tri6. Sorbent Tri6 and -Mix showed significantly better absorption capacities in so far as Sorbent Tri6 removed 65% of anti-Gal IgM and 80% of -IgG as assessed on Bdi and 85% of IgM/87% of IgG when tested on Tri6, and Sorbent Mix absorbed > 90% anti-Gal of both isotypes of either specificity. Direct hemagglutination of rabbit erythrocytes (ER) was reduced by 75% (median value, range 0-94%) and the median cytotoxicity to PK15 target cells by > 94% after absorption on Sorbent Mix. Neither the decrease in ER agglutination titers nor the reduction of PK15 cytotoxicity revealed significant differences between the three immunoabsorbents tested. The large variation ranges of absorption efficacies within the 20 tested sera suggest that "tailor-made" immunoabsorption treatments may be needed for putative xenotransplant recipients.

Specific immunoadsorbent for myasthenia gravis treatment: development of synthetic peptide designed to remove antiacetylcholine receptor antibody.

We have developed Medisorba MG, a new immunoadsorbent column for myasthenia gravis (MG). The alpha 183-200 segment of the Torpedo Californica acetylcholine receptor (AChR) is recognized as the acetylcholine binding site by the blocking antibody, which is one of the anti-AChR antibodies involved in the pathogenesis of MG. As a specific affinity ligand to remove the blocking antibody, Torpedo alpha 183-200 was synthesized and immobilized covalently to porous cellulose beads. This immunoadsorbent showed specific removal of the blocking antibody without reducing IgG and albumin levels significantly in clinical evaluation and in vitro study. Clinical improvement was found in 78% of the cases, and no adverse effects were observed in any case. The Medisorba MG column has been confirmed as a useful device for the treatment of MG.

Purification of anti-chymotrypsin antibodies for the preparation of a bioaffinity matrix with oriented chymotrypsin as immobilized ligand.

Polyclonal antibodies suitable for the oriented immobilization of chymotrypsin were prepared by chromatography on a bioaffinity matrix which had the enzyme immobilized through its active site to antilysin, covalently linked to bead cellulose. After periodate oxidation of their carbohydrate moieties, the isolated antibodies were coupled to a hydrazide derivative of bead cellulose. The periodate oxidation step, which led to greater efficiency and stability of the immunosorbent, had no deleterious effect on antibody activity as assessed by ELISA. Addition of chymotrypsin to the immunosorbent yielded an enzymically active bioaffinity matrix with the optimum molar enzyme/antibody ratio of 2.

Synthesis of thiophilic adsorbents, containing amine spacer arms, and adsorption of immunoglobulins from adult bovine serum.

In order to remove immunoglobulins from adult bovine serum (ABS), adsorptions were carried out in suspension with thiophilic adsorbents without (T-gel) and with (ST-gels) short amine spacer. The developed ST-gels could be cleaned in sodium hydroxide and specifically adsorbed immunoglobulins from ABS at a lower ionic strength of the adsorption buffer than a T-gel. The ST-gels are suited for the performance of adsorptions in a suspension with a high concentration of the adsorbent. For an almost complete removal of immunoglobulins from ABS in 0.5 M potassium sulfate and 0.1 M potassium phosphate (pH 7), up to 75 g ST-gel per 1 suspension may process up to 100 ml ABS. The synthesized ST-gels contained an equal amount of amine spacers and sulfone groups. The ratio between coupled 2-mercaptoethanol molecules and sulfone groups was about 0.4. In comparison with the T-gel, the chemical stability of the ST-gels in solutions of 0.1 to 1 M sodium hydroxide was higher. The capacity for adsorbing pure immunoglobulins of the T-gel was 0.329 g g-1 (10.9 g l-1) and of the ST2-gel, 0.677 g g1 (52.3 g l-1). The capacity for adsorbing IgG from ABS of the T-gel was 0.177 g g1 (5.88 g l-1) and of the ST2-gel, 0.152 g g-1 (11.7 g l-1). This reduced capacity was attributed to a low affinity binding to the adsorbents of other proteins from ABS. When an incubation of the adsorbent with ABS was performed in 0.5 M potassium sulfate and 0.1 M potassium phosphate (pH 7), the affinity constant (Ka) for IgG of the T-gel was 2.5 x 10(6) M-1 and of the ST-gel, 3.1 x 10(7) M-1. In this adsorption buffer at a protein concentration above 5 g l-1, salt-promoted precipitation of IgG took place. When an adsorption was performed at the minimal ionic strength of the adsorption buffer, the Ka of the T-gel for IgG was about 9 x 10(3) M-1 and of the ST-gel, about 2.3 x 10(5) M-1. In this case, the removal of Igs from ABS was achieved by means of a partition equilibrium, rather than by high affinity adsorption.

Compared stability of Sepharose-based immunoadsorbents prepared by various activation methods.

During the use of chromatographic supports for the purification of proteins or the selective removal of substances by immunoaffinity, leakage of the antibodies immobilized on the matrix is systematically observed. When the cleansing of blood plasma by extracorporeal circulation is concerned, it is of prime importance that the immunoadsorbents exhibit an extensive chemical stability over the whole range of experimental conditions. To study and minimize this leakage, a matrix, Sepharose CL-4B, was activated by various chemical reagents and coupled to goat anti-apolipoprotein B polyclonal antibodies. Immunoadsorbents thus prepared were compared with those obtained earlier by cyanogen bromide activation. It turns out that divinyl sulphone- and tresyl chloride-activated supports lead to similar results in terms of coupling yield and adsorption capacity, but to a significant reduction in released antibodies.

[Synthesis of sorbent from cross-linked starch for affinity purification of glucose(mannose)-specific lectins]. / Syntez sorbentu na osnovi poperechno-zshitoho krokhmaliu dlia afinnoho ochyshchennia gliukozo(mannozo) spetsyfichnykh lektyniv.

Cross-linked starch gel for the affinity chromatography of D-glucose (D-mannose)-specific lectins is suggested. In order to optimize hydrodynamic properties of gel 30% starch has been hydrolysed by HCI at 70 degrees C during 60 min and then cross-linked by epichlorohydrin under alkaline conditions. Every 100 g of starch require 18 ml of epichlorohydrin and 36 ml of 8 N KOH. The gel obtained has been successfully used for the purification of lectins from Pisum sativum L., Lens culinaris L., Vicia sativa L., and Vicia faba L. seeds. These lectins, purified on starch gel do not differ from sephadex-purified samples.

Immunoadsorbents with synthetic oligosaccharide hapten representing blood group A substances.

Immunoadsorbents with a synthetic oligosaccharide hapten representing human blood group A specific substances are prepared. The synthetic hapten, known as A-trisaccharide, which carries a space arm, is chemically attached to various solid supports, either directly through a suitable functional group at the end of the spacer arm or indirectly via a protein conjugated to the hapten. The preparation involves simple and mild procedures for the activation and/or derivatization of the supports. The latter includes naturally occurring polyhydroxy materials such as agarose, cellulose, or cellulose derivatives, and other particulate materials such as inorganic diatomites and a synthetic organic copolymer. The methods used for the coupling concern specifically the preparation of controlled-capacity and high-efficiency immunoadsorbents, with limited incorporations, which may be prepared easily and used for the selective removal, or affinity chromatographic separation, of specific antibodies from plasma environment or blood. It has been found that while hapten incorporation to the support may be varied rather easily, the physical nature of the support as well as the form of the hapten is important in determining the efficiency of an immunoadsorbent.

Immunosorbents for LDL-apheresis.

Low density lipoprotein (LDL) (1,03-1,05 g/ml) was utilized as antigen to obtain polyclonal (PcAb) and monoclonal (McAb) antibodies. Immunosorbents capable of selective removing LDL from human plasma were developed on the basis of the antibodies preparation. The sorbents called "Immunoliposorber PcAb" and "Immunoliposorber McAb" are currently undergoing clinical trials in the USSR Cardiology Research Center, Acad. of Med. Sciences.

Preparation and use of synthetic blood group specific immunoadsorbents.

The carbohydrate structures determining the ABO blood group system have been almost completely characterized. Recent advances in the stereoselective synthesis of such complex structures have made it possible to prepare pure oligosaccharides in large quantities and to study their possible uses for diagnostics and pharmaceutical processing techniques. In the experiments described here A and B blood group specific determinants were synthesized and bound to solid phases by means of suitable spacers in order to study their use as immunoadsorbents. The objective was to adsorb blood group specific antibodies from positive sera and from immunoglobulin preparations. It was shown that the anti-A and anti-B antibodies bound to the immunoadsorbents with high affinity could be removed effectively. The effects were achieved both using affinity chromatography and "batch processing".

Novel effective immunoadsorbents based on agarose-polyaldehyde microsphere beads: synthesis and affinity chromatography.

Agarose-polyaldehyde microsphere beads were produced by encapsulating polyacrolein microspheres or polyglutaraldehyde microspheres with agarose. Magnetic beads were formed by carrying out the encapsulation procedure in the presence of ferrofluidic particles. Proteins were bound covalently, at physiological pH, to the beads through their aldehyde groups to produce the Schiff base products. The conjugates, beads-proteins, were used successfully in affinity chromatography for specific purification of antibodies. Leaching of the proteins bound to the beads under physiological conditions and eluting conditions was not detected. The agarose-polyaldehyde microsphere beads are suggested as alternatives to the supports currently used in affinity chromatography.

[Immunosorbent based on porous cellulose sphere]. / Immunosorbent na osnove poristykh tselluloznykh sharikov.

The synthesis of an immunosorbent in the form of porous beads is described. The beads were prepared by emulsification of cuprammonium glucose solution in organic solvents, followed by precipitation of beads with a mixture of acetone and sulfuric acid. The product was oxidized with NaIO4 and conjugated with protein antigen or antibodies to obtain an immunosorbent fit for column chromatography. The capacity of the immunosorbent thus obtained was found to be 300-1000 mg of antibodies or 50-70 mg of antigen per 1 g of the sorbent.

[Synthesis of a high-capacity immunosorbent based on a cellulose suspension]. / Sintez vysokoemkogo immunosorbenta na osnove suspenzii tselliulozy.

A method is suggested for preparation of an immunosorbent on the basis of cellulose suspension. Antigen was coupled to periodate-oxidized cellulose via aldehyde groups. Optimal conditions (time of oxidation, amount of an oxidant, quantity of protein antigen added) for immunosorbent synthesis were determined. The amount of antibodies bound to the immunosorbent was approximately equal to the weight of the immunosorbent (up to 950 mg of antibodies per 1 g sorbent). Thus each molecule of antigen coupled to cellulose bound 5-8 molecules of antibodies.