IMMUNOLOGY - Roit I. - Mir 2000

Chapter 29. Immunological Methods

PRODUCTION OF PURIFIED ANTIBODIES

In immunological research, it is often necessary to obtain purified antibody preparations, i.e., either antigen-specific or non-specific IMMUNOGLOBULINS. The isolation of non-specific immunoglobulins from serum is typically carried out through sequential protein fractionation, which includes the following steps.

✵ Precipitation of gamma globulins in a 30–50% ammonium sulfate solution.

✵ Gel filtration to obtain molecules of appropriate size.

✵ Ion-exchange chromatography to isolate molecules carrying a net positive charge at neutral pH.

✵ Affinity Chromatography using natural immunoglobulin ligands, such as staphylococcal protein A (a Cell wall component of staphylococci that binds to the Cγ2 and Cγ3 domains of most IgG subclasses, i.e., IgG1, IgG2, and IgG3).

The isolation of antigen-specific immunoglobulins is performed using affinity chromatography. The antigen is "coupled" to Sepharose particles, and the "pure" Antibodies bound to it are eluted from the immunosorbent using chaotropic agents (e.g., sodium thiocyanate) or a buffer solution (Glycine–HCl or diethylamine). Affinity chromatography is also used to obtain purified antigen preparations (Fig. 29.15).

Class="center">

Fig. 29.15. A pure antibody population can be isolated using affinity chromatography. 1. A solid-phase immunosorbent (spAg) is prepared, consisting of an antigen covalently linked to an inert matrix (e.g., cross-linked dextran particles). The immunosorbent is packed into a Column through which an antibody mixture is passed under physiological conditions. Antibodies specific to the given antigen bind to the immunosorbent, whereas unbound Proteins pass freely through the column. 2. In the next step, the bound antibodies are eluted from the sorbent surface using a buffer solution [e.g., acetate buffer (pH 3.0), diethylamine (pH 11.5), or 3 M guanidine-HCl], which disrupts the bonds in the antigen-antibody complex. This method can also be used to obtain purified antigen preparations if an immunosorbent containing antibodies is used. Affinity chromatography is likewise employed to isolate Other types of molecules. For instance, all molecules possessing specific saccharide residues will be adsorbed onto The surface of particles with "coupled" lectin; these molecules are then eluted with a buffer solution containing free sugars that compete with the adsorbed proteins for the lectin-binding sites.

Production of Monoclonal Antibodies

Another approach for obtaining individual antibodies of defined Specificity is hybridoma technology—the creation of an immortalized cell line producing antibodies of a single specificity only, i.e., monoclonal antibodies (Fig. 29.16). Antibody production can be maintained in such a culture indefinitely. Monoclonal antibodies correspond incomparably better to the goals of immunoassay than heterogeneous antisera obtained from immunized animals, and therefore have found wide application in various fields of biology as highly specific probes.

Fig. 29.16. Animals (usually mice or rats) are immunized with an antigen. When antibody production reaches a high level, a cell suspension is prepared from the animals' spleens (Lymph Nodes may also be used). Splenocytes are then fused with myeloma line Cells using polyethylene glycol (PEG), a cell membrane fusion-promoting agent. This process succeeds only in a small number of cells. The Cell mixture containing the fused cells is cultured in HAT medium containing hypoxanthine, aminopterin, and thymidine. Aminopterin is a highly toxic agent that blocks a metabolic pathway—purine synthesis. Cells can utilize a salvage pathway if its intermediates, hypoxanthine and thymidine, are present in the medium. Splenocytes are able to grow in HAT medium, whereas myeloma cells die in it because they have a metabolic defect preventing them from utilizing the salvage pathway for purine synthesis. The cell suspension introduced into the HAT medium contains splenocytes, myeloma cells, and fused cells. Splenocytes die naturally in culture after 1–2 weeks, myeloma cells do not survive in HAT, while the fused cells remain viable because they combine The properties of the "immortal" myeloma and the Spleen cells utilizing the salvage pathway. Some of the fused cells also retain The ability to produce antibodies, just like the parental splenocytes. Culture medium from all wells of the plate showing cell growth is screened for the presence of antibodies of the desired specificity (often using enzyme-linked immunosorbent assay). Antibody-producing cultures are cloned by diluting the cell suspension during seeding so that only 1 cell falls into each well. This progenitor cell gives rise to an "immortal" clone producing monoclonal antibodies.

Any B cells can efficiently produce monoclonal antibodies provided they are made immortal and proliferating. Most commonly, hybrid cells are generated for this purpose by fusing mouse splenocytes with myeloma B cells from mice of the same strain that do not secrete their own antibodies. Interstrain or interspecies hybrids can also be obtained, but they are often unstable. Another method of immortalization is cell transformation, for instance, in the case of human B cells, by infection with Epstein-Barr virus.

A novel method for producing antibodies based on The Use of Bacteriophages has also been developed. This interesting technique allows the expression On the surface of filamentous bacteriophage M13 of variable regions (Vh and Vl) as antibody fragments (Fv) that bind antigen with defined specificity and avidity. Having a library of such phage-expressed fragments, one can select (based on interaction with a specific antigen) phage particles producing a particular Fv fragment. Furthermore, if appropriate Bacteria are infected with this bacteriophage, they begin to secrete Fv protein in large quantities into the culture medium. This approach does not require mandatory immunization of animals or humans (Fig. 29.17).

Monoclonal antibodies represent a well-defined reagent, but they do not possess higher specificity compared to a polyclonal antiserum that recognizes an ANTIGEN AS A result of the interaction of immunoglobulins with its various epitopes.

Fig. 29.17. To obtain antibody Fv fragments using a bacteriophage, cDNA for the Vh and Vl regions, synthesized using B-cell mRNA, is first amplified by Polymerase Chain Reaction. These genes are then joined by a linker to obtain the Gene for the Fv fragment. Such a gene is transfected into bacteria (E. coli) using a phagemid vector containing a leader sequence and a fragment of the gene encoding the M13 phage coat protein. Next, the bacteria are infected with M13 phage. The phage replicates and expresses Fv on one of its poles. Phages of the desired specificity are isolated using panning on antigen-coated surfaces and amplified. Antigen-specific phages can be used to infect bacterial strains that ensure the secretion of Fv protein into the culture medium.