The use of antigen-coated protein beads as immunoadsorbent for the purification of antibodies.

A procedure is described using antigen-coated beads of calf serum as immunadsorbent for the purification of antibodies against ferritin and against leukemia-sarcoma virus-induced antigens.     

Reliable procedure for silica gel preparation

A simple and reliable method is described for preparation of silica gel media for Nitrosomonas and Nitrobacter species.     

Octadecyl silica: a solid phase for protein purification by immunoadsorption.

Immunoaffinity chromatography involves binding of an antigen or antibody to a solid matrix, usually agarose, frequently using the cyanogen bromide method. These methods are laborious, rather expensive, and their use has been mostly restricted to immunopurifications on the microscale. We propose here the use of octadecyl silica (SiCl8) beads, a matrix for HPLC, as an alternative solid phase for protein immunopurification and immunoadsorption. Antibodies or antigens are strongly bound to SiCl8 by a simple incubation; radiolabeled antibodies can only be eluted from SiCl8 by detergent-containing solutions. After the remaining free binding sites have been saturated with bovine serum albumin, SiCl8 is incubated with the antigen- or antibody-containing crude preparations and is then poured into a minicolumn. The nonspecifically bound proteins are removed by washing; specific proteins are eluted by disruption of the antigen-antibody complexes with a low pH buffer. With this methodology, we have obtained high purity preparations of proteins in single steps, even when these proteins are present in trace amounts (picograms) in a complex mixture such as human serum. Similarly, specific antibodies against an intracellular parasite (Trypanosoma cruzi) were completely absorbed from human serum with SiCl8 coated with parasite antigens.     

Immobilization of DNA onto a polymer support and its potentiality as immunoadsorbent

Immobilization of DNA to the surface of poly(ethylene terephthalate) (PET) microfibers with a high specific surface area of 0.83 m(2)/g was carried out to give the fiber surface an affinity for anti-DNA antibody. Following ozone oxidation, the microfibers were subjected to graft polymerization of monomers including acrylic acid, methacryloyloxyethyl phosphate, N,N-dimethylaminoethyl methacrylate, N-vinylformamide, and glycidyl methacrylate. Calf thymus DNA was immobilized to the grafted fiber surface through either covalent binding or polyion complexation with the grafted polymer chains. The highest surface density of DNA immobilized (0.6 mug/cm(2)) was obtained when DNA was immobilized through formation of phosphodiester linkage between the hydroxyl group of DNA and the phosphate group in grafted poly(methacryloyloxyethyl phosphate) using 1,1-carbonyldiimidazole, or through polyion complexation between the anionic DNA and the cationic grafted poly(N,N-dimethylaminoethyl methacrylate) chains. Batch adsorption of anti-DNA antibody to the grafted PET fibers with and without DNA immobilized on their surface was conducted with serum obtained from systemic lupus erythematosus model mice. The DNA-immobilized PET fibers exhibited a higher adsorption capacity and specificity than the others. In addition, the DNA-immobilized fibers effectively adsorbed human anti-DNA antibody.     

The preparation of silica gel thin layers with a new apparatus.

An apparatus for the preparation of uniform thin layer chromatography gel layers is described. The apparatus employs rubber cushion runners to compensate for differences in plate thickness and a gel applicator which functions independently of plate edge variability. Consistently uniform layers are prepared by careful establishment of the plate-to-applicator distance. Silica gel layers averaged 87% of the applied thickness with a variability of +/-2% within a single run and +/-8% between independent runs.     

Synthesis of oligodeoxyribonucleotides on silica gel support

A rapid solid phase method of oligonucleotide synthesis based on monomeric protected nucleosides has been developed.     

Large-scale purification of angiotensin I-converting enzyme from human plasma utilizing an immunoadsorbent affinity gel

Angiotensin I-converting enzyme was purified 1500-fold from human plasma utilizing an immunoadsorbent affinity gel prepared by coupling antibody to baboon lung angiotensin I-converting enzyme to CNBr-activated Sepharose 4B. The enzyme was eluted from the gel using 2 m magnesium chloride, pH 5.8. Subsequent hydroxylapatite and Sephadex G-200 chromatography yielded 2.6 mg of homogenous enzyme with a specific activity of 40 units/mg with hippuryl-l-histidyl-l-leucine as substrate from 48 liters of plasma. Use of the immunoadsorbent allowed the 48 liters of plasma to be processed in one-half the time it previously took to process 2 liters of plasma by other methods. This protocol enables us to obtain sufficient amounts of enzyme for structural studies that were previously impossible because of insufficient amounts of enzyme.     

Chemically modified porous silica gel as a bioadsorbent and a biocatalyst.

Aminopropyl silica gel was prepared from porous silica gel by reaction with γ-aminopropyltrimethoxysilane in toluene and was used for immobilizing chymotrypsin (EC 3.4.4.5) and human serum albumin. Immobilized chymotrypsin was used for the resolution of N-acetyl-dl-phenylalanine and immobilized human serum albumin was used for the purification of goat anti-human serum albumin. Epoxy silica gel, prepared by reaction of porous silica gel with γ-glycidoxypropyltriethoxysilane, was coupled with m-aminobenzamidine and the resulting matrix was used for trypsin purification.     

Synthesis of oxytocin using iodine for oxidative cyclization and silica gel adsorption chromatography for purification.

Oxytocin (OT) was synthesized employing the solid phase method. Resins made of copolymers of polystyrene-1%-crosslinked with divinylbenzene gave better yields (73-95%) of Z-Cys(Bzl)-Tyr(Bzl)-Ile-Gln-Asn-Cys(Bzl)-Pro-Leu-Gly-NH2 (I) than 2%-crosslinked resins (10--56%). Reduction of I with Na-liq.NH3 and oxidation with I2-MeOH at -40 degrees minimized dimer and polymer formation, and resulted in good yields (49--54%) of OT. The large volumes of MeOH required when several grams of I are reduced and then oxidized were rapidly evaporated in vacuo, and the residue was desalted by dissolving the peptide in a small volume of glacial acetic acid and filtering to remove the salt. OT was purified by adsorption chromatography on a silica gel column with combinations of MeOH-CHCl3 of graded polarity. Oxytocin elutes with 33% MeOH-CHCl3. After two purification steps by adsorption chromatography, the resulting OT was found to be homogeneous. The hormone was characterized chemically and found to be active biologically.     

Angiostrongylus cantonensis: preparation of a specific antigen using immunoadsorbent columns.

Antigenic components which reacted specifically with sera of rats infected with Angiostrongylus cantonensis were prepared from adult worm extract by the use of immunoadsorbent columns. Components common to rat serum were removed from a whole worm extract by passing it through a column of cyanogen bromide-activated Sepharose coupled to rabbit antirat immunoglobulin. Consequently, components common to Ascaris suum were similarly removed using Sepharose linked to immunoglobulin to A. suum. Finally, the sample was applied to a column of Sepharose coupled with immunoglobulin from A. cantonensis infected rats and eluted with glycine-hydrochloric acid buffer. Intradermal skin tests using this preparation gave sensitive and specific reactions.     

Reverse-phase purification and silica gel thin-layer chromatography of porphyrin carboxylic acids

Thin-layer chromatography on silica gel 60 plates in the solvent N,N-dimethylformamide/methanol/ethylene glycol/glacial acetic acid/1-chlorobutane/chloroform (4/35/6/0.4/18/20 by volume) separates porphyrin carboxylic acids by the number of free carboxyl groups. Coproporphyrins I and III and isocoproporphyrin are separated in 30 min, other porphyrins in 15 min. The N,N-dimethylformamide and acetic acid in the solvent strongly increase porphyrin fluorescence on the plates. Fading and diffusion of the fluorescent patterns is prevented by storage of the plates in the cold and dark without oxygen and with desiccant. In a preliminary step, porphyrins are purified in high yields, concentrated, and deacidified rapidly (2 min) by reversephase chromatography on cartridges containing a C₁₈ spacer or on Amberlite XAD-2 columns. The methods are applied to urines of porphyria patients and for following porphyrin ester hydrolysis.     

Characterization of monoclonal antidigoxin antibodies immobilized to a solid support

A high-affinity monoclonal antidigoxin antibody, produced by somatic cell fusion, was amplified by the formation of ascites. Purification from ascites was accomplished by affinity chromatography by passing the ascites over a digitoxin-amine-agarose column. Affinity-purified antidigoxin antibody was coupled to a pellicular microbead at concentrations of 10, 25, 50, and 100 mg/g bead. The immobilized antibody was characterized for binding affinity, for specificity to other cardiac glycosides, and for binding capacity. There were no changes in the binding affinity observed for the immobilized antibody when compared to that of the antibody grown in culture media. Binding capacities for the immobilized antibody were decreased from calculated theoretical values. Saturating the microbead with increasing concentrations of antibody lowered the binding efficiency of the antibody from 32 to 22% of theoretical values. Attempts to improve the binding capacity by immobilizing antibodies to the microbead at the immunoglobulin carbohydrate by periodate oxidation were unsuccessful. These data demonstrate that antidrug antibodies immobilized on solid supports remain functional and may have the capability of removing drug from biological fluids passed over the support.     

Bone as a solid support for the immobilization of enzymes

Summary Poultry bone residue was found to serve as a solid support matrix to which catalase, pepsin, pectinase, lactase and invertase could be insolubilized by covalent attachment and adsorption. Bone has great potential for enzyme immobilization since it is inexpensive, abundant, chemically functional, porous, non-toxic and mechanically strong.