A Method for the Parallel and Sequential Generation of Single-Domain Antibodies for the Proteomic Analysis of Human Blood Plasma

A new efficient method for the parallel and sequential stepwise generation of single-domain antibodies to various high-abundance human-plasma proteins has been described. Single-domain antibodies have a number of features that favorably distinguish them from classical antibodies. In particular, they are able to recognize unusual unique conformational epitopes of native target proteins, small in size, and relatively easily produced and modified; have enhanced stability; and rapidly renature after denaturation. As a consequence, the immunosorbents that utilize these antibodies can be reused without any significant loss of activity. The principal novelty and universality of the described method is that it enables the sequential generation of antibodies to a number of high-abundance and yet unknown antigens of a complex protein mixture without the need for purified antigens. The effectiveness of the method is demonstrated by the example of generation of single-domain antibodies to a number of high-abundance proteins of the human blood plasma. The produced antibodies are promising biotechnological tools that can be used to develop prototypes for new diagnostic and therapeutic agents, as well as appropriate immunoaffinity-based methods for removal, enrichment, analysis, and/or targeting of specified proteins and their complexes from (in) the human blood. As we show, the generated single-domain antibodies can be efficiently used in designing new immunosorbents. As a rule, commercially available analogous immunosorbents that utilize classical antibodies remove many major proteins from the blood plasma immediately, while immunosorbents for many individual proteins are difficult to find and rather expensive. Single-domain antibodies generated by our method are unique new materials that allow for the development of more efficient and delicate approaches to pretreatment of plasma and the analysis of various blood plasma biomarkers.     

Use of cellulose carbonate for the preparation of immunosorbents: the radioimmunoassay of follicle-stimulating hormone

The use of cellulose 2,3-carbonate as a matrix for the insolubilisation of biologically active molecules has been extended to the preparation of insoluble antigen and antibody (immunosorbents). Both human pituitary follicle-stimulating hormone (FSH) and its homologous antibody have been covalently attached to cellulose carbonate by the nucleophilic attack of their primary amino groups on the cyclic carbonate groups. Antibody to FSH retains its immunological reactivity on insolubilisation, and is therefore suitable for use in the solid phase radioimmunoassay of unknown amounts of FSH by competitive binding of radioactively labelled and unlabelled FSH. Acceptable inhibition curves can be obtained, and the low, non-specific absorption characteristics have advantages over other systems. FSH also retains immunological reactivity on insolubilisation, and the derivative holds potential for the radioimmunoassay of FSH as it can be layered immunologically with anti-FSH and then FSH itself.     

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.     

Stirrer tank: an appropriate technology to immobilize the CB.Hep-1 monoclonal antibody for immunoaffinity purification

The CB.Hep-1 monoclonal antibody was coupled to CNBr-activated Sepharose CL 4B at three different immobilization scales for purification of recombinant hepatitis B surface antigen. Standard laboratory apparatus to obtain immunosorbents of 1 l (scale I) and 3 l (scale II) as well as a stirrer tank to prepare 6 l immunosorbents (scale III) were used. The binding capacity at scale III was 2- and 1.5-fold higher with respect to the scales II and I, while a reduction in the ligand leakage of 5- and 2-folds was observed. Immunosorbents from scale II showed a significantly reduced adsorption, and an increased ligand leakage. Differences in the coupling efficiency were not observed. Antigen purity eluted from the immunosorbents was always above 85%.     

Determination of antibody affinity by using antigenic and antibody immunosorbents

To determine the affinity of the active centers of antibodies, cellulose immunosorbents for antibodies and antigens have been used. The fixation of serum proteins on the sorbent, the interaction of fixed antibodies with a monovalent antigen and the graphic analysis of the results thus obtained allows one to assess not only the concentration of the effective active centers on the sorbent, but also all known characteristics of antibody affinity: the average association constant K0, the common association constant Kt, the geometric association constant Kg, the average association constants which determine the affinity of different antibody groups. The use of antigenic immunosorbent permits one to determine the value of the average internal association constant K0. The determination of antibody affinity in hyperimmune antiplague sera by means of immunosorbents and red blood cells coated with capsular antigen has resulted in obtaining similar values of affinity indices.     

Immunological cross reactions between polysaccharides of Streptococci groups A and L]

Antibodies on sepharose immunosorbents containing A-polysaccharide-sepharose or synthetic beta-N-acetylglucosamine, were isolated from the sera of rabbits immunized with streptococci, group A, by means of affinity chromatography. Antibodies obtained from some sera with both immunosorbents reacted with streptococcus, group A and L polysaccharides. Partial identity of these polysaccharides was revealed by the immunodiffusion test. Absorption of antibodies with polysaccharides, group A and L, showed their different specificities. These antibodies could apparently be directed against the end parts of molecules of streptococcus, group A polysaccharide.     

Enzymic glycosphingolipid synthesis on polymer supports.

Two synthetic approaches leading toN-4-carboxymethyl-2-nitrobenzyloxycarbonyl derivatives of model compounds and of glycosylsphingosines are described. The carboxymethyl group can be converted into a hydrazido derivative and the resulting products react with an active ester polymer yielding light-sensitive polymeric products that may subsequently serve as acceptors in glycosyltransferase reactions.Author for correspondence. Address until September 1990; Graduate Department of Biochemistry, Brandeis University, 415 South Street, Waltham, MA 02254, USA     

Isolation of antilymphocytic antibodies with the aid of cellular immunosorbents]

The authors suggest a method of obtaining purified antilymphocytic antibodies by mean of a specific immunosorbent of human or mouse lymphocytes fixated with glutharic aldehyde Such immunosorbents subjected to special treatment could be used repeatedly; their sorptive capacity was retained in such case. Only from 5 to 12% of the activity could be obtained from immunosorbents sorbed from the serum with 77--93% activity. In comparison with the initial serum, purification was from 6- to 15-fold. Thus, the suggested method provided considerable purification of the antilymphocytic preparations and permitted to obtain highly active antilymphocytic antibodies.     

Nature of the inhibitory serum factor in mice injected with isologous antibodies conjugated with cellulose

The formation of antigen-specific serum inhibitory factor was induced by injection of covalently bound to cellulose syngeneic antibodies to sheep red blood cells into (CBA X C57BL/6)F1 mice. This factor was absorbed by cellulose immunosorbents immobilized with antibodies against sheep red blood cells and with rabbit antibodies against mouse gamma-globulin and was not absorbed by immunosorbents immobilized with immunoglobulins of intact mice or immunoglobulin containing antibodies against rat red blood cells. These data, and evidence obtained by the authors previously, indicate that inhibitory factor of the serum is likely to be due to idiotypic antibodies.     

A protective factor of ribosomal shigellosis vaccine

Shigella ribosomal vaccine was shown previously to possess protective properties in the keratoconjunctival test on guinea pigs and to be capable of preventing experimental infection in 90% of challenged monkeys. The presence of the O-specific component (OSC) constituting about 0.5% of the ribosomal preparation by serological activity suggested its importance for the protective effect. This was studied in experiments with two O-specific immunosorbents prepared by coupling anti-O rabbit antibodies with Staphylococcus aureus cells or with CNBr-Sepharose. Ribosomes treated with immunosorbents proved to be lacking the serologically active OSC and lost their ability to induce O-antibody response in rabbits and mice. After the removal of this component ribosomal preparations were incapable of ensuring protection from Shigella kerato-conjunctival infection. The isolated OSC was also inactive in this test. The data obtained in this investigation confirm the hypothesis stating that the protective activity of Shigella ribosomal vaccine is based on the combined action of ribosomes and O-specific factor whose nature and properties require further study.     

The use of monoclonal antibodies for purification of ribonuclease H from E. coli

Monoclonal antibodies to E. coli ribonuclease H were obtained from two hybrid clones. Using the monoclonal antibodies two immunosorbents were synthesized for RNase H which have a slight difference in the capacity and do not differ in the conditions of antigen elution. A homogeneous (according to electrophoresis in PAAG) preparation of the enzyme was obtained using the synthesized immunosorbents.     

Separation of antigens by immunological specificity: Use of cellulose-linked antibodies as immunosorbents

1. Immunosorbents were prepared by coupling activated aminocellulose with the γ-globulin concentrates of antisera prepared against ovalbumin and human serum albumin. 2. The immunosorbents were low in solubility, but high in capacity for homologous antigens. 3. The high specificity of these immunosorbents was demonstrated by their use in fractionating various mixtures of fluorescent ovalbumin, ¹³¹I-labelled human serum albumin, lysozyme and ribonuclease.     

Isolation and purification of Brucella antigens synthesized by Escherichia coli K12 cells]

The homogeneous preparations of the brucella protein antigens were isolated from the hybrid producer strains Escherichia coli 6SE579 and 6SE800 by the cold osmotic shock technique and further purification on immunosorbents. The 18 + 38 and 38 kDa antigens were obtained. The antiserum specific to brucella 38 kDa antigen was obtained and used for isolation of the 18 kDa antigen from the producer strain 6SE579 synthesizing two brucella antigens. The immunosorbent developed on the basis of BrCn-agarose conjugated with antibodies from the serum has permitted isolation of 18 kDa protein antigen preparation. Thus, the combined technique of cold osmotic shock and affinity chromatography on immunosorbents permits one to isolate highly purified individual antigens of brucella from Escherichia coli K12 producer cells.     

Preparation of Solid-Phase Immunosorbents by Coupling Human Serum Proteins to Cyanogen Bromide-Activated Agarose

The method of preparing solid-phase immunosorbents by covalently attaching proteins from whole human serum to cyanogen bromide-activated agarose has been investigated to determine optimum concentrations of cyanogen bromide and protein, and the optimum pH for the maximum attachment of proteins from serum. Two systems in which the above immunosorbents have proved useful are described: the removal of antibodies to normal serum proteins from anti-hepatitis B serum and the removal of light chain antibodies from anti-human immunoglobulin M serum.     

Studies on the purification of chlamydial agents grown in yolk sacs of embryonated eggs using disulphide-linked immunosorbents and enzymes.

Immunosorbents were derived from avid and non-avid sera raised in rabbits to multiple or single injections of chlamydiae passaged once or three times in HeLa cells after routine passage in eggs. Egg-derived suspensions of chlamydiae required pretreatment before application to immunosorbent columns; this was most conveniently done by fractionation on Sepharose 4B. Immunosorbents derived from avid serum had greater capacity than those from non-avid sera. However, organisms were desorbed in low yield from an avid immunosorbent, but in higher yields from non-avid immunosorbents. Even under the best conditions, the immunosorbents yielded suspensions of organisms still contaminated with egg antigens. Partially purified suspensions of chlamydiae were also treated with phospholipase C to yield suspensions of high purity.     

Separation of antigens by immunological specificity: Use of disulphide-linked antibodies as immunosorbents

1. γ-Globulin concentrates of antisera prepared against ovalbumin and human serum albumin were thiolated and cross-linked to form insoluble polymers. 2. These immunosorbents were of low solubility and of high capacity for homologous antigen. 3. The high specificity of these immunosorbents was demonstrated by fractionation of various binary mixtures of fluorescent ovalbumin, ¹³¹I-labelled human serum albumin, lysozyme and ribonuclease.     

Optimization of methods for immobilizing antibodies against the carcino-embryonic antigen on insoluble matrices. Equilibrium parameters of the interaction of the immobilized antibodies with the antigen

To obtain highly efficient immunosorbents for solid-phase immunoassay and affinity chromatography, methods for immobilization of antibodies against the carcino-embryonic antigen (CEA) on insoluble matrices were optimized. The immunosorbents obtained were characterized by equilibrium parameters of the reaction between immobilized anti-CEA and CEA calculated from rather a simple kinetic model. This model describes the interaction of the monovalent antigen with two independent types of binding sites. The role of some amino acid residues of anti-CEA in the interaction with CEA was investigated. The effects of immobilization density and the spacer arm length on the functional properties of the immobilized antibodies were studied. The optimal immunosorbent was used to purify 125I-CEA by immunoaffinity chromatography.     

Use of immunochemical methods for characterization and purification of tissue-specific inhibitors of mitotic activity (chalones)]

The 81% ethanol-precipitable fraction of water-soluble proteins from skin inhibiting the proliferation of epidermal tissues was shown to contain 9 protein components according to acrylamide gel disk-electrophoresis. Seven of these were identical to homologous serum proteins and could be adsorbed on the corresponding immunosorbents. Two proteins remained in the solution after immunoadsorption. Their electrophoretic properties were the same as those in the total 81% ethanol fractions. These proteins like the 81% ethanol fraction inhibited the entering of cells into mitosis and DNA synthesis phase.     

A comparison of some activated matrices for preparation of immunosorbents

The adsorption characteristics of monoclonal anti-(β-galactosidase) immobilised to a number of commercially available pre-activated matrices have been investigated in a series of small scale experiments. Binding characteristics were determined by batch isotherm techniques and estimates were obtained of the rate constants governing adsorption to the immobilised antibodies. The capacity of the different matrices for binding antibody and the specific activity of immunosorbents were measured.There was little effect of support matrix on the dissociation constant, K_d, for the interaction between β-galactosidase and immobilised anti-(β-galactosidase). However, the maximum amounts of antibody that could be immobilised, rates of adsorption and desorption of the enzyme to the immobilised antibody and the specific activity of immunosorbents were affected by the choice of support matrix. The importance of the relative sizes of the antigen and immobilised antibody and the influence of the nature of the support matrix on the properties of the resulting immunosorbent when used in large scale applications are discussed.