Detection Of Intracellular Antigens Research Articles

Subcellular distribution of class II major histocompatibility antigens in enterocytes of the human and rat small intestine

Class II antigens encoded within the major histocompatibility complex (MHC) have been examined at the ultrastructural level in enterocytes from both man and rat. A protocol has been developed for fixation which, in conjunction with a pre-embedding indirect immunoperoxidase technique preserves antigenicity of the class II molecules and allows detection of intracellular antigen. Details of the technique are provided and discussed in relation to the general paucity of information available on ultrastructural localization of class II MHC molecules. Class II MHC antigens have been identified on the basolateral cell membranes of enterocytes in both species and they have also been found in association with intracellular organelles that have the appearance of multivesicular bodies and secondary lysosomes. These observations link class II molecules with the endocytic pathway in enterocytes and suggest a possible role in the handling of gut antigens. The findings may have a more general significance in relation to the site of engagement between processed antigen and MHC molecules in specialized antigen-presenting cells.

Kinetics of Infected Cell Appearance as a Determinant of Number of Human Immunodeficiency Virus-1 Infectious Units

In order to optimize detection of human immunodeficiency virus-1 (HIV-1)-infected cells, the temporal appearance of virus antigens in newly infected H9 cell cultures was examined. Analyses were accomplished by indirect immunofluorescence labeling with each of 10 monoclonal antibodies and evaluation by flow cytometry. Of the antibodies examined, those specific for HIV-1 capsid protein p24, matrix protein p17, or their precursor molecule p55 allowed the earliest and most sensitive detection in infected cells fixed to allow detection of intracellular antigen. Discrimination of infected cells from uninfected cells was much less sensitive when three antibodies specific for HIV-1 glycoproteins were used to detect intracellular or cell surface antigen. In several experiments involving the time course of infection, we observed no differences in cell numbers between infected and uninfected H9 cultures initiated at identical cell concentrations. We hypothesized that it might be possible to quantitate infectious HIV-1 virions from the kinetics of infected cell appearance. Straight-line relationships between the log p24-positive cells and the time after infection were observed. These quantitative observations were employed to calculate the number of infectious units originally added to the culture that were capable of infecting H9 cells. The production of infectious virus, but not of cytopathic effects, was required. The results of this novel approach to the titration of infectious HIV-1 particles agreed well with those from median cell culture infective dose determination. This method could be employed with other infectious agents for which detection of cell-associated antigens is possible in cell cultures not destroyed by infection.

Ultrastructural indirect immunolocalization of transferrin in cultured rat hepatocytes permeabilized with saponin.

Transferrin was localized in 48-hr cultured adult rat hepatocytes by indirect immunoperoxidase following paraformaldehyde--glutaraldehyde fixation and the use of saponin as a membrane permeabilizing agent. The protein, present in all the parenchymal cells in variable amounts, was found to be specifically located in the endoplasmic reticulum and Golgi apparatus. These results are consistent with recent reports claiming that all adult hepatocytes may synthesize a given liver plasma protein at a given time. The procedure used in this study should be particularly useful for the detection of intracellular antigens in various intact cell types.

Detection of intracytoplasmic antigens by flow cytometry

A new technique is described for the detection of intracellular antigens by immunofluorescence and flow cytometry. The technique utilizes lysolecithin (lysophosphatidylcholine), a naturally occuring phospholipid, to permeabilize cell membranes and allow antibodies to reach intracellular antigens. The technique is rapid and sensitive, and retains sufficient integrity of the cells being treated to enable differentiation of cell types on the basis of light scatter (e.g., lymphocytes from monocytes). Permeabilization of cells following lysolecithin was assessed using standard techniques including trypan blue exclusion, propidium iodide staining, and hydrolysis of fluorescein diacetate. Lysolecithin treatment was accompanied by only minimal increases in non-specific background fluorescence, and no increase in autofluorescence. Our studies have demonstrated that lysolecithin treatment of human mononuclear cell populations permits flow cytometric analysis of cytoskeletal structures, including intermediate filaments, as well as cytoplasmic immunoglobulin. Studies currently in progress in our laboratory have demonstrated broad intracellular reactivity with some monoclonal antibodies identifying leukocyte differentiation and tumor-associated antigens. These findings contrast with the more restricted expression of these antigens on the cell surface and demonstrate not only the value of the lysolecithin technique but also the importance of the study of intracellular antigens in our overall understanding of the specificity, distribution, synthesis, and function of cellular antigens.

Immunocytochemical localization of intracellular antigens with SEM.

A method for the localization of intracellular antigens with a scanning electron microscope using peroxidase-labelling antibodies is described. A search for a hydrogen donor which may be deposited at the sites of antigen by enzymatic action and emit secondary electrons or generate backscatter electrons was made. It was found that when 4-chloro-1-naphthol was used, the peroxidase deposited reaction product which resulted in a strong secondary electron emission at the site of antigen. With this method, the presence of luteinizing hormone in secretion granules and other cytoplasmic structures of gonadotropic cells was demonstrated. The level of detection of intracellular antigens with this method is not as high as that detectable with light microscopical examination of the same specimens, that is, more reaction product at the site of antigen is required to be detectable with scanning electron microscopy than with light microscopy. In spite of the lack of high sensitivity, the intracellular antigens may be localized with the method described.

The detection of intracellular antigens in human leucocytes by immunoperoxidase staining.

An immunoperoxidase procedure is described for staining intracellular leucocyte antigens in peripheral blood and bone marrow smears. Brief exposure of cell smears to a buffered formol acetone mixture was found to give optimal fixation, combining good cellular morphology with preservation of antigenic reactivity. The immunoperoxidase method is superior to immunofluorescence in that it provides a permanent preparation which can be counterstained with orthodox reagents and viewed by conventional light microscopy. In addition the technique is considerably more sensitive than immunofluorescence procedures. Immunoglobulin was demonstrated in plasma cells, Türk cells and a minority of peripheral blood lymphocytes. Lysozyme was found in cells of the neutrophil series from promyelocytes to mature granulocytes. Monocytes stained for lysozyme but the reaction was less intense than in neutrophils and some monocytes were devoid of activity. Lactoferrin stained strongly in mature neutrophil polymorphs and metamyelocytes, but was weak or absent in earlier myeloid cells. These reaction patterns are in keeping with previous reports on the distribution of these antigens in human leucocytes. In the case of immunoglobulin and lysozyme it was possible to abolish leucocyte staining by incubation of the specific antisera with the appropriate purified antigen, providing additional proof of the specificity of the reactions. Anti-ferritin antisera stained granulocytes and myeloid precursors strongly, and reached weakly with a minority of monocytes. These latter observations are not entirely in accordance with published data on the leucocyte distribution of ferritin and may be attributed to antibody activity of unknown specificity in the anti-ferritin antiserum.