Antigens In Single Cells Research Articles

Wireless Electrochemical Visualization of Intracellular Antigens in Single Cells

Electrochemical microscopy has been developed in the past decades for imaging biomolecules in single cells; however, electrochemical visualization of an intracellular protein in one cell is difficu...

High-throughput determination of the antigen specificities of T cell receptors in single cells.

We present tetramer-associated T-cell receptor sequencing (TetTCR-Seq), a method to link T cell receptor (TCR) sequences to their cognate antigens in single cells at high throughput. Binding is determined using a library of DNA-barcoded antigen tetramers that is rapidly generated by in vitro transcription and translation. We applied TetTCR-Seq to identify patterns in TCR cross-reactivity with cancer neo-antigens and to rapidly isolate neo-antigen-specific TCRs with no cross-reactivity to the wild-type antigen.

Immunochemical demonstration of different antigens in single cells in paraffin-embedded histological sections.

A new modification of the double staining immunochemical technique is described, which is capable of demonstrating the presence of several antigens in single cells at the light microscopical level. This method was employed by the authors in order to detect the simultaneous occurrence of different types of cytoplasmic immunoglobulin chains in neoplastic plasma cell precursors in paraffin-embedded lymph-node specimens from patients with B-cell malignancies.

Enrichment of human bone marrow mononuclear phagocytes and characterization of macrophage subpopulations by immunoenzymatic double staining.

In order to isolate and enrich bone marrow mononuclear phagocytes, we performed magnetic-activated cell sorting using beads coupled to a monoclonal antibody directed against the monocyte/macrophage surface molecule CD14. Colocalization of antigens in single cells was achieved by combining an alkaline phosphatase-anti-alkaline phosphatase and an avidin-biotin complex immunoassay, avoiding the use of peroxidase. Bone marrow macrophages were first labelled by the monoclonal antibody PG-M1 (anti-CD68). Subsequently, cytoplasmic and/or surface double staining by the monoclonal antibodies against HLA-DR and Mac-2 antigen or the lectin GSA-I-B4 was carried out. Whereas HLA-DR was co-expressed by the great majority of PG-M1+ macrophages (84.9%+/-6.9%), only a subpopulation exhibited Mac-2 (69.9%+/-5.9%) antigen or galactoside structures detected by GSA-I-B4 (65.0%+/-6.7%). The latter result differed only slightly from the percentage of GSA-I-B4+ macrophages determined in a previous comparative immunomorphometrical study. Therefore, using our method of isolation and enrichment by magnetic-activated cell sorting, only a negligible portion of macrophages is apparently stimulated, as shown by GSA-I-B4 staining. This methodology seems to be a valuable tool for further studies on the monocyte-macrophage system.

12 Flow Cytometric Analysis of Whole Organs and Embryos

Publisher Summary This chapter discusses the specific technical considerations for performing flow cytometric analysis when the original materials are whole tissues and embryos, and present some selected applications. The general technical basis and a wide review of applications of flow cytometry are beyond the scope of this chapter. Flow cytometry's main achievement, the rapid quantification of large numbers of single events (particles or cells), has generally limited its application to cell lines or primary cells growing in isolation, such as blood cells. It is currently one of the essential techniques in the field of immunology. However, whole organs and organisms, constituted by various heterogeneous populations of cells tightly adherent to each other, present the particular problem of having to be dissociated into a single-cell suspension representative of the tissue or organism of origin. This is requisite to apply the fruitful flow cytometry technique to other fields of biology, such as developmental biology. Flow cytometric analysis permits an accurate and rapid quantification of the level of expression of antigens in single cells. In this way, it is possible to define cell populations with different sets of markers.

Detection of One Milliattomole of Ferritin by Novel and Ultrasensitive Enzyme Immunoassay

A novel and ultrasensitive enzyme immunoassay (immune complex transfer two-site enzyme immunoassay) for ferritin is described. Ferritin was reacted simultaneously with affinity-purified dinitrophenyl biotinyl anti-ferritin IgG and affinity-purified anti-ferritin Fab'-beta-D-galactosidase conjugate. The complex formed of the three components was trapped onto affinity-purified (anti-dinitrophenyl group) IgG-coated polystyrene balls. After eliminating excess conjugate by washing, the complex was eluted from the polystyrene balls with an excess of epsilon N-dinitrophenyl-L-lysine and transferred to streptavidin-coated polystyrene balls. The beta-D-galactosidase activity bound to streptavidin-coated polystyrene balls was assayed by fluorometry. Nonspecifically bound beta-D-galactosidase activity was remarkably lowered but there was much less decrease in specifically bound beta-D-galactosidase activity. As a result, the detection limit of ferritin was lowered to 1 milliattomole (1 x 10(-21) mol, 600 molecules as calculated from Avogadro's number). This technique will be useful for measuring, for example, antigens in single cells.

Divalent binding of monoclonal antibody to a cell surface antigen. Modelling of equilibrium data

The principles of the binding of a monoclonal Ab to a single epitope of a cell surface antigen were deduced. The equilibrium between the Ab and the antigen was regarded as a two-step reaction, involving free Ab and antigen as well as Ab, monovalently or divalently complexed to its antigen. By a few simple equations the concn of bound Ab was calculated from the values of free Ab concn, cell concn, single cell antigen density and the two equilibrium constants. This cell surface binding was compared to the binding of an ordinary one-step equilibrium reaction. It was shown that a bonus effect due to cell surface divalent binding is obtained when the product of the first association constant and the free Ab concn is less than one and, simultaneously, the product of the second equilibrium constant and the single cell antigen density is greater than one. In addition, when these conditions are fulfilled, an increase in the single cell antigen density may cause an increase in Ab binding that is much more than proportional to the change in antigen density. The implications of these results to tumor immunolocalization and immunotherapy are discussed.

Lateral and transmembrane redistribution of tissue-specific antigens in single cells and monolayers.

Tissue-specific antigens in the membranes of corneal endothelial cells react with anti-tissue antibodies only in metabolically active monolayers and dispersed cells. After metabolic inhibition by exposure of these preparations to cold, the antigen-antibody complexes, like free antigens, undergo transmembrane redistribution leading to their internalization by the cells. This transmembrane redistribution is reversible and can be followed by using fluorescein-labeled antibodies. Reexpression of the complexes on the cell surfaces occurs after return from metabolic inhibition to metabolic activity. Dispersed corneal endothelial cells are also capable of lateral redistribution (capping) of the complexes although cells in monolayers do not share this capability. Capping in the dispersed cells occurs only at ambient temperatures and, because it results in shedding of the complexes, is irreversible. The data indicate that macromolecules in the membranes of cells organized in tissues are restricted in their movement as compared to the macromolecules of cells functioning in a dispersed state.

Detection and Localization of Epstein-Barr Virus-Associated Early Antigens in Single Cells by Autoradiography Using 125I-Labeled Antibodies

A characteristic feature of virus-transformed cells is the expression of virus-specific antigens usually detectable by immunological or radioimmune procedures. We report here an autoradiographic method for the detection and localization of such antigens in individual cells which combines the cellular specificity and radiolabeling sensitivity of the above procedures. As a test system we have studied the reaction between virus antigen in some Epstein-Barr virus (EBV) DNA-containing cell lines and anti-virus antibody specificities in certain human sera. EBV DNA-containing cell lines express an EBV-determined nuclear antigen (EBNA) and some lines also express the EBV-associated antigens, early antigen (EA) and virus capsid antigen (VCA), in a minority of cells in the population. By employing appropriate 125I-IgG-labeled human sera, we clearly show that EA can be detected without difficulty in cell lines known to spontaneously express this antigen. Moreover, the specificity and sensitivity of the present method is such that low levels of EA cand be detected at cellular concentrations which remain undetectable by conventional immunofluorescence.