Intracellular antigens

Roth J, Bendayan M, Orci L (1978) Ultrastructural localization of intracellular antigens by the use of protein A gold complex. J Histochem Cytochem 26 1074 1081... 

Fluorescence Labeling of Intracellular Antigens of Attached or Suspended Tissue-Culture Cells... 

Fig. 1. Flowchart of an intracellular antigen labeling protocol. Abbreviations as in text.

Fluorescent Labeling of Surface or Intracellular Antigens in Whole-Mounts... 

Primary antibody (e.g., mouse monoclonal antibody to a surface or intracellular antigen) (see Note 2). 

Hydroxychloroquine is an antimalarial agent with immunosuppressant properties. It is thought to suppress intracellular antigen processing and loading of peptides onto MHC class II molecules by increasing the pH of lysosomal and endosomal compartments, thereby decreasing T-cell activation. 

Intracellular antigens could be modified internal proteins, which are continuously removed by the cell, the structure altered and attached to MHC I. This takes place in the rough endoplasmic reticulum. The protein/pep tide-hapten fragments are then presented to the external surface of the cell membrane as a complex with the MHC I. Then cytotoxic T cells (CD8+) accept the protein/peptide and destroy the cell. This mechanism gives rise to a type IV response. 

As described above, the haptenised protein or hapten protein complex has to be presented to the immune system for the system to be aware of it as an antigen. With intracellular antigens, this requires expression of fragment of the hap ten-protein complex on the cell surface via major his toco mpatibility complex (MHC) molecules. These can bind almost any peptide (Fig. 7.78). 

Some experiments require that antigenic information be obtained from the surface of the cell others are interested in intramembrane antigens or intracellular antigens, or indeed the relationship of one antigen-bearing cell to another. The specimen can be prepared by one of several different methods to expose antigens and, thus, obtain the desired antigenic information from the sample (I). 

The limitations of the application of conventional detergents mentioned above can be circumvented by replacing this approach with cell membrane permeabilization by microwave heating. Improved detection of intracellular antigens can be obtained with microwave heating used in combination with flow cytometry. This approach yields histogram patterns that show clear discrimination between intact cells and cell debris (Fig. 9.5). 

Millard, I., Degrave, E., Philippe, M., and Gala, J.-L. 1998. Detection of intracellular antigens by flow cytometry Comparison of two chemical methods and microwave heating. Clin. Chem. 44 2320-2330. 

C., and Janossy, G. 1994. Detection of membrane and intracellular antigens by flow cytometry following ORTHO PermeaFix fixation. Leukemia 8 672-676. 

Proteomic Fluorescence microscopy In situ visualisation of cellular/ECM protein with fluorescence-labelled antibody Multiplexing capability (typically 3 fluorophores). Extra- and intracellular antigen location on opaque biomaterials. Issues with bleaching and autofluorescence. Yes/no... 

In the previous chapters, I have discussed what may be thought of as mainstream applications of flow technology. Cells stained for surface and intracellular antigens and nuclei stained for DNA content together constitute a large majority of the particles that flow through the world s cytometers. As noted in the Preface, however, flow cytometry has continued to surprise everyone with its utility in unusual and unpredicted fields of endeavor. By the time this book appears in print, some new applications will almost certainly have progressed into the flow mainstream and other newer applications will have taken their place in the tributaries (is it time to kill this metaphor ). 

Natural killer cells express some antigens that are also expressed by cytotoxic T cells. NK cells are usually CD2-, cCD3-, CD16-, and CD56-positive and can express CD7, CD8, and CD57. Furthermore, NK cells and some cytotoxic T cells also express cytotoxic proteins such as perforin, granzyme B, and T cell intracellular antigen 1 (TIA-1) (B22, C12, F8, Jl, K21, Y2). 

One major problem area in immunofluorescence is the choice of the primary fixative. For intracellular antigens, unlike surface antigens, the access of a large protein, such as an antibody, requires permeabilizing the cell membrane, and the preservation of cell architecture and antigen distribution after membrane permeabilization requires precise structural... 

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