Working with antibodies

Fast Shingles Cure

Fast Shingles Cure by Bob Carlton

Get Instant Access

The structure of antibody molecules Antibody molecules have a very specific structure that is often described as a "wine glass" shape. They are made up of two light and two heavy chains, and the two antigen-combining sites (made up of both heavy and light chains) are at the top of the wine glass (the Fab region). Antibody molecules directed against different antigens have different amino acid sequences in this variable region.

The stem of the wine glass (the Fc region) is made up of an amino acid sequence for all constant antibody molecules of a given class, no matter what the antigen with which they react is. This region serves as a signal to the cell that an antibody molecule is there. It is important to the immune reaction and can be used both diagnostically and in the laboratory. An antibody molecule is shown diagrammatically in Fig. 12.2.

Monoclonal antibodies The immune response is a result of proliferation of many different B- and T-cell types responsive to various antigenic determinants presented by the pathogen or by the antigen. Thus, each immature B cell stimulated by a specific epitope was stimulated into dividing into many daughter cells, all with identical genomes and all secreting identical antibody molecules. Such a clone of cells is short-lived in the body, but specific manipulations can be made to immortalize a single B cell so that a culture of clonally derived B cells, all secreting antibody molecules with identical sequence, can be isolated. The antibodies expressed by such a cell line are monoclonal antibodies and have a number of important uses in diagnostics, therapies, and research.

The generation of monoclonal antibodies involves a number of steps that are outlined in Fig. 12.3. These steps include immunizing the animal that is to be the source of the B cells (often a mouse), isolation of lymphocytes from the animal's spleen, transformation of cells to immortalize them, screening of specific populations, and selection of immortal cells that produce antibodies. Individual B cells that secrete only one antibody molecule reactive with only one determinant can be cloned by fusion of a mature B-lymphocyte population (each secreting a specific — and different — antibody) with immortal myeloma cells (tumor cells derived from lymphocytes that do not produce any antibody molecules).

If myeloma and B cells are induced to fuse with a very mild detergent, the cell culture contains short-lived parental B cells that will die, immortal myeloma cells, and fused cells. The key to the value of the method is that these fused cells (hybridoma cells) are also immortal. The job now is to get rid of the unfused cells, then screen the hybridoma cells for their ability to produce the desired antibody.

Getting rid of unfused B cells is no trick because they have a very short lifetime in culture and will die in a few days. Myeloma cells, however, offer a different problem because they are immortal and will continue to replicate, but they can be eliminated by using a mutant myeloma cell line that can be selected against. A convenient method uses a myeloma line that has been mutated so that it does not express hypoxanthine-guanine phosphoribosyltransferase (HGPRT negative), an essential enzyme in the biosynthesis of nucleotides. The advantage of this mutant

Antigen binding domains

Fab region

Antigen binding domains

Fab region

Variable region

Light chain Constant region

Glycosylation site

Constant region

Fig. 12.2 The structure of an antibody molecule, IgG. This molecule is made up of four chains: two heavy and two light. The antigen-combining domains are at the N-terminal of the four chains and are made up of variable amino acid sequences, a specific sequence for each specific antibody molecule. The C-terminal region has a constant amino acid sequence no matter what the antibody's specificity. This is the Fc region.

Variable region

Light chain Constant region

Glycosylation site

Constant region

Fig. 12.2 The structure of an antibody molecule, IgG. This molecule is made up of four chains: two heavy and two light. The antigen-combining domains are at the N-terminal of the four chains and are made up of variable amino acid sequences, a specific sequence for each specific antibody molecule. The C-terminal region has a constant amino acid sequence no matter what the antibody's specificity. This is the Fc region.

is that since the parental myeloma cells cannot synthesize nucleotides, they need to get the nucleotides from the medium using a salvage pathway. This salvage pathway can be blocked with the drug aminopterin, which blocks the myeloma cell's ability to pick up nucleosides from the outside medium.

To understand this, remember that the hybridoma cells are not just derived from myeloma; they also have the genetic background of B cells, and the B cells are HGPRT positive. This means that adding aminopterin to the mixture of hybridoma and myeloma cells will result in the death of only the myeloma cells. The fused hybridoma cells will grow. The mixed hybridoma then can be screened by taking individual cells, growing clones from them, and testing the produced antibody for its ability to react with the antigen of interest.

Monoclonal antibodies are very useful for precise diagnosis of specific viral infections, as even closely related viruses will encode some proteins with different antigenic determinants. Each different determinant will react with only a specific monoclonal antibody generated against it. The monoclonal antibodies are also valuable tools for localizing viral proteins within the infected cell or animal, and as reagents to isolate and analyze specific viral proteins for study.

Immunization

Immunization

HGPRT+ Ig+ B cells isolated from spleen

Myeloma cell culture

Myeloma cell culture

Myeloma cells

Myeloma cells

Plate cells in HAT selective media

B lymphocytes die (short lived)

Hybrid 1

Myeloma cells die (resulting from selection for HGPRT+ cells)

Grow hybridomas

Hybrid n

Hybrid n

Screen hybridomas for synthesis of desired antibody

Freeze hybridoma for future use

Antibody in culture medium

Positive culture Culture cells

Freeze hybridoma for future use

Grow as fluid tumor

Antibody in culture medium

Grow as fluid tumor

Antibody isolated from ascites

Wa WA

Monoclonal antibodies

Fig. 12.3 Generation of monoclonal antibodies by making hybridoma cells between mouse immune B lymphocytes and myeloma cells that are not able to grow in selective (HAT) medium. Antibody-secreting clones are screened by testing with an antigen. Once the hybridoma cell line is made, it can be stored frozen, and then either grown in culture or injected into the peritoneal cavity of a mouse where a tumor grows as a disorganized group of individual cells and fluid (an ascites). The ascites cells secrete the monoclonal antibody into the body cavity's fluid where it can be harvested. HGPRT = hypoxanthine-guanine phosphoribosyltransferase; HAT=hypoxanthine, aminopterin, and thymidine.

Detection of viral proteins using immunofluorescence

A number of methods to measure antibody reactions involve use of the antibody molecule's Fc region as a "handle." Figure 12.4 shows some examples using a fluorescent dye either attached directly to the antibody (direct) or attached to a second antibody that is reacted against the Fc region of the first (indirect). Methods using immunofluorescence are very important to localize viral antigens inside infected cells, and to generate easily measurable immune reactions.

Tissue section

Fluorescein-Unlabeled labeled antibody antibody jL

Antigen z

Slide

Slide

Direct test

Indirect test

Fig. 12.4 Outline of immunofluorescence as a means of detecting and localizing an antibody—antigen complex. The antibody specific against the antigen is allowed to react. If it has a fluorescent tag on its Fc region, it can be seen directly when illuminated with ultraviolet light since the tag emits visible light. For indirect immunofluorescence microscopy, a second antibody reactive with the Fc region of the first is used, and this antibody has the florescent tag. This method is somewhat more specific and allows the same tagged antibody preparation to be used with a number of different antibodies of differing specificities.

Direct test

Indirect test

Fig. 12.4 Outline of immunofluorescence as a means of detecting and localizing an antibody—antigen complex. The antibody specific against the antigen is allowed to react. If it has a fluorescent tag on its Fc region, it can be seen directly when illuminated with ultraviolet light since the tag emits visible light. For indirect immunofluorescence microscopy, a second antibody reactive with the Fc region of the first is used, and this antibody has the florescent tag. This method is somewhat more specific and allows the same tagged antibody preparation to be used with a number of different antibodies of differing specificities.

There are a number of micrographs of infected and uninfected cells in which antigens of interest are located with fluorescent antibodies in this text. A notable series is shown in Fig. 3.5 where the passage of rabies virus through an infected animal was traced. Another excellent example showing the effect of HSV infection on the cytoskeleton of an HSV-infected cell is provided in Fig. 10.4. Immunofluorescence can also be used with two (and even three) antibodies if each is tagged with a different chromophore. Two- and three-color immunofluores-cence can provide a tremendous amount of information about the colocalization of proteins and other antigens of interest. The availability of lasers and prisms (or mirrors) that can differentially allow the passage of one wavelength of light while excluding others is used in confocal microscopy to allow the precise measure of the cellular distribution of viral and other antigens.

Although there are many variations on the method, confocal microscopy basically depends on the ability of a laser light source to be so coherent that it can be focused to a single focal plane within a cell. This, along with the use of appropriate prisms or filters and fluorescent dyes, can allow one to visualize only the fluorescence emanating from a single plane within the cell. Since fluorescent radiation, of physical necessity, must be emitted at a wavelength longer than the incident radiation, the light path in a microscope can be used for both illumination and viewing.

The technique is shown schematically in Fig. 12.5(a), and an example of the type of data that can be obtained is shown in Fig. 12.5(b). For the studies shown in Fig. 12.5(b), cells were infected with human cytomegalovirus (CMV), a herpesvirus with a very long replication period, and then the expression of two proteins that localize to different parts of the cell was examined. The first protein, IE72, was detected with an antibody that was tagged with Texas red, which fluoresces red under illumination with the appropriate laser beam. This protein is synthesized in the cytoplasm, but quickly migrates to the nucleus, where it remains and serves as a regulatory protein controlling expression of other CMV genes. The second protein, which fluoresces

Detector

Selective filter Pinhole

Confocal point Lens

Dichroic mirror (directional beam splitter)

-|— Scanning mirrors Lens

Fluorescent light emission

Objective lens

Sample

Fluorescence only from region illuminated by focused incident beam

Fig. 12.5 Confocal microscopy to detect colocalization of antigens. (a) The use of a laser beam and a specific filter to separate the incident laser light from the fluorescence that travels on the same light path. The ability to precisely focus the laser beam onto a single plane in the microscopic field allows one to observe fluorescence from proteins only in that plane. (b) Top: Confocal microscopic visualization of two human cytomegalovirus (HCMV) proteins, IE72 (red) and pp65 (green). Primary aortic endothelial cells were infected with a strain of HCMV isolated from a human patient. This high-magnification view of a cell shows nuclear and cytoplasmic staining of the two HCMV proteins at 8 days following infection. (Photograph courtesy of K. Fish and J. Nelson.) Bottom: A series of three photographs of the identical field viewed with three different filters to localize two specific proteins to the same region. The first panel shows the association of varicella-zoster virus (VZV) glycoprotein E (gE), tagged with a green fluorescent antibody, with the surface of an infected cell. This glycoprotein was expressed in transfected cells. The second panel shows the localization of the red fluorescence due to the transferrin receptor in the same cell, and the third panel shows that both fluorescent signals are located in the same sites on the cell, indicated by the yellow color, seen when a filter that allows both colors to pass is used for viewing. (Photographs courtesy of C. Grose.)

green due to a fluorescein isothiocyanate (FITC) tag, is pp65. This protein functions in the cytoplasm and is expressed later than IE72. The separation of the two proteins is clearly seen in the close view.

The lower photographs in Fig. 12.5(b) demonstrate that another herpesvirus glycoprotein, varicella-zoster virus (VZV) gE, localizes to the same region of the cell as does the transferrin receptor. This latter cellular protein is internalized into endocytotic vesicles of cells that are induced to take up iron borne by the carrier cellular protein transferrin. The fact that the VZV gE protein, which is expressed in transfected cells, colocalizes with the cellular receptor suggests that VZV may be internalized by endocytosis also. The specific glycoprotein for the virus (gE) was tagged with green fluorescent FITC-tagged antibody, while the transferrin receptor was tagged with Texas red fluorescent antibody. It is clearly evident that when both antibodies are observed, they are in the same precise location at the surface of the cell,

Fig. 12.5 Continued as indicated by the color of the fluorescent light being yellow, which is a mix of the two colors.

Was this article helpful?

0 0
How To Bolster Your Immune System

How To Bolster Your Immune System

All Natural Immune Boosters Proven To Fight Infection, Disease And More. Discover A Natural, Safe Effective Way To Boost Your Immune System Using Ingredients From Your Kitchen Cupboard. The only common sense, no holds barred guide to hit the market today no gimmicks, no pills, just old fashioned common sense remedies to cure colds, influenza, viral infections and more.

Get My Free Audio Book


Post a comment