Cellular Enzyme-linked Immunosorbent Assay Research Articles

A simplified cellular ELISA (CELISA) for the detection of antibodies reacting with cell-surface antigens

This paper describes the adaptation of a cellular enzyme-linked immunosorbent assay (CELISA) for the detection of antibodies to cell-surface antigens. This CELISA has the advantages of convenience and rapidity and is therefore ideally suited for the screening of a large number of hybridoma culture supernatants. The basic procedure involves the direct drying of cell suspensions onto the wells of enzyme immunoassay (EIA) plates and a subsequent EIA with appropriate blocking reagents. In order to overcome high background binding of primary antibodies to Fc receptors and of secondary antibodies to surface Ig (sIg), this method involves a blocking step consisting of unlabelled secondary antibodies. Once CELISA plates are prepared, they can be stored for a period of at least 6 months and hence this assay does not rely on the availability of fresh, viable cells for each assay. This assay is simple, reproducible and sensitive. The results can be assessed in an objective manner and can also be adapted for the detection of cellular antigens. This paper describes a CELISA for the detection of antibodies to blood group antigens and human leukocyte (HLA) antigens.

Lymphocyte activation as measured by interleukin‐2 receptor expression to gluten fraction 111 in coeliac disease

Lymphocyte activation was examined by interleukin-2 (IL-2) receptor expression on peripheral blood mononuclear cells from coeliac and control subjects. Purified T cells were incubated with gluten fraction 111 (a known toxic peptide for coeliac subjects), soyabean hydrolysate (an unrelated hydrolysed food antigen), and Concanavalin-A (Con-A, a non-specific mitogen). After 1-5 days incubation, expression of IL-2 receptors was assessed using a cellular enzyme-linked immunosorbent assay (ELISA). Gluten fraction 111 induced expression of IL-2 receptors on T lymphocytes from coeliac but not from normal subjects (P = 0.0005), whereas soyabean hydrolysate did not induce IL-2 receptor expression. Lymphocytes from both coeliac and normal subjects had similar increased IL-2 receptor expression after incubation with Con-A. Flow cytometry was also used to confirm specific expression of IL-2 receptor expression of lymphocytes from coeliac subjects. Interleukin-2 receptor expression increased from 0 to 5.4% of cultured mononuclear cells after 7 days incubation with gluten fraction III. These cells were CD3-positive and CD4-positive. We conclude that peripheral blood lymphocytes from coeliac subjects are sensitized specifically to gluten fraction III.

IgG and IgM anti-endothelial cell antibodies in patients with collagen-vascular disorders

A cellular enzyme-linked immunosorbent assay (ELISA) was used to detect circulating anti-endothelial cell antibodies (AECA) in sera of patients suffering from rheumatoid arthritis (RA). Felty's syndrome (FS), systemic lupus erythematosus (SLE), progressive systemic sclerosis (PSS) and those with a "lupus anticoagulant" (LA). IgG AECA were detected in RA, FS, SLE and LA sera, while IgM AECA were only detected in RA and FS. AECA were not specific for endothelial cells. However, IgG binding to endothelial cells and dermal fibroblasts was F(ab) mediated while in all other cell types tested, nonspecific binding most likely via the Fc region occurred. In RA and FS rheumatoid factor was shown to augment immunoglobulin binding to endothelial cells. Additional studies revealed that some of these pathological sera also contained cytotoxic IgG autoantibodies which fixed complement and damaged human umbilical vein endothelial cells in vitro. These studies suggest that a group of autoantibodies, present in a variety of collagen vascular disorders, react with endothelial cells and thus may be important aetiopathogenic factors in the vasculopathies associated with these disorders.

Photometric assays for FcR I-dependent binding, phagocytosis, and antibody-dependent cellular cytotoxicity mediated by monomeric IgG γ2a in murine peritoneal macrophages

Mouse peritoneal macrophages possess distinct Fc receptors (FcR) for binding the various murine IgG isotypes. FcR I binds monomeric IgG γ2a, but not monomeric IgG γ2b or IgG γ1 with high affinity at 4°C and is sensitive to trypsin degradation. We have assessed the functional consequences of the cytophilic binding at 4°C of monomeric IgG γ2a to FcR I of mouse peritoneal macrophages using newly developed photometric microassays for quantification of binding, phagocytosis, and antibody dependent cellular cytotoxicity (ADCC) of post-opsonized sheep red blood cell (SRBC) targets. Dose-dependent binding specificity of monomeric IgG γ2a, but not IgG γ2b or IgG γ1 to FcR I of oil-elicited mouse peritoneal macrophages at 4°C for 2 h was confirmed to display typical saturation kinetics both by the photometric assay and by a cellular enzyme-linked immunosorbent assay (CELISA). Binding of monomeric IgG γ2a to macrophage FcR I promoted highly efficient phagocytosis of opsonized SRBC in that most cells that were bound were also rapidly internalized by the phagocytic process during a 1 h incubation at 37°C. Upregulation of FcR I-dependent binding and phagocytosis occurred during 24–48 h in vitro culture of macrophages as shown both by the photometric assays and CELISA. Trypsin treatment of macrophages abrogated FcR I-dependent binding and phagocytosis by monomeric IgG γ2a, but had little effect on FcR II-dependent functions. Cytophilic binding of monomeric IgG γ2a to FcR I failed to trigger ADCC activation. Thus functional characterization of macrophage FcR I-dependent effector functions confirmed the fidelity of binding specificity of monomeric IgG γ2a to a trypsin degradable receptor which mediates highly efficient phagocytosis but fails to initiate the signal for ADCC activation. It appears that passively bound immune monomeric IgG γ2a could provide an efficient mechanism by macrophages in vivo for FcR I-dependent immune clearance of soluble or particulate cellular antigens without elicitation of potentially harmful cytolytic factors associated with ADCC activation.

Human Placenta—An Antibody Sponge?

Maternal IgG antibodies in sera and placenta eluates were studied by a cellular enzyme-linked immunosorbent assay (CELISA) method. Antibodies were not detectable in any of the serum samples obtained before or after delivery from nine normal primigravid women. Antibody activity was, however, present in five of nine placental eluates and two of nine neonatal sera tested in CELISA. Lymphocytotoxic antibodies were not detected in any of the samples tested. These results support the concept that the absence of antibody activity in maternal sera may be caused by the immunosorbent effect of the placenta.

Decreased reactivity of allosera against target lymphocytes obtained following thermal injury or long-term cyclosporine treatment

We speculated that two diverse causes of potent cell-mediated immune suppression, cyclosporine (CsA) and thermal trauma, may demonstrate some similar actions, and thus tested whether either could alter antisera reactivity against allogeneic target lymphocytes. Target splenocytes from 40% body surface area full-thickness burned Brown-Norway (BN) rats demonstrated significant ( P = 0.004) decreased reactivity (agglutination) with antisera produced across a full allogeneic barrier (RT1 major histocompatibility complex (MHC) and non-MHC) compared to control splenocytes. Depression of allogeneic splenic target cell reactivity against Lewis (LEW)-anti-BN allosera was similarly observed using lymphocytes from long-term CsA-treated rats ( P = 0.004). The decreased reactivity induced by burn trauma was transferable to pooled normal splenocytes or blood lymphocytes by preincubation with burn plasma ( P < 0.001), and was confirmed by a cellular enzyme-linked immunosorbent assay (CELISA) ( P = 0.003). In summary, a similarity consisting of decreased antibody reactivity against lymphocytes from either burned or long-term CsA-treated animals was demonstrated. These results suggested that lymphocyte cell surface allogeneic determinants and their expression and/or availability were altered by either regimen.

Anti-endothelial cell antibodies: detection and characterization using a cellular enzyme-linked immunosorbent assay.

Anti-endothelial cell antibodies (AECA) have been detected in autoimmune diseases such as systemic lupus erythematosus (SLE) and scleroderma (PSS) but their role in pathogenesis is unknown. Immunofluorescence, immunohistochemistry, complement-dependent antibody lysis, and radioimmunoassay have been used in the past to detect AECA. We have developed a rapid, sensitive, and quantitative cellular enzyme-linked immunosorbent assay (ELISA) to detect and characterize AECA. Sera were obtained from 28 normal volunteers, 28 patients with SLE, and 14 patients with PSS. We also performed studies in 47 patients with various monoclonal gammopathies. Endothelial cells (EC) were obtained from human umbilical veins by standard methods and subcultured on 96-well tissue culture plates without fixation. EC were then sequentially incubated with sera, peroxidase-conjugated goat anti-human Ig (IgG, IgM, or IgA), and substrate. Optical density readings were converted to arbitrary units by developing a standard curve. Heavy-chain specific antibodies were used to determine the class of AECA binding to EC. IgG was purified by using protein A columns and digested with pepsin to obtain F(ab')2 fragments. The mean units of AECA from normals were 19.3 for IgG and 12.5 for IgM. SLE sera showed significant levels of IgM AECA (37 units, P less than 0.001) but not IgG (29 units, P less than 0.1). PSS sera showed significant levels of both IgM AECA (38 units, P = 0.001) and IgG AECA (42.7 units, P less than 0.005). IgA AECA were not detected in normal, SLE, or PSS sera. Blocking Fc receptors with rabbit IgG did not affect the titer of IgG or IgM AECA.(ABSTRACT TRUNCATED AT 250 WORDS)

A monoclonal antibody to cellular transglutaminase.

A cellular enzyme-linked immunosorbent assay was developed for estimating cellular transglutaminase in situ using a monoclonal antibody produced to tissue transglutaminase. The minimum level of detection of TGase was 2-5 ng. The enzyme was present in greater amounts in WI-38 and IMR90 cells than in their simian virus-transformed counterparts. The levels of TGase in the virus-transformed cells increased significantly when the cells were grown in the presence of sodium butyrate to induce enzyme activity. Staining of confluent WI-38 cells by indirect immunofluorescence using the monoclonal antibody showed microscopic fibers suggesting that the enzyme may be associated with detergent-insoluble components.

Antigenic Heterogeneity of Human Colorectal Cancer Cell Lines Analyzed by a Panel of Monoclonal Antibodies. I. Heterogeneous Expression of la-Like and HLA-Like Antigenic Determinants&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;

The pattern of reactivity of 10 monoclonal antibodies (MCA) developed against human lymphoid leukemia cells and tested with human T-cells, B-cells, as well T-lymphoma and B-lymphoma cell lines suggested that six of them detect la-like determinants, three detect HLA-like determinants, and the remaining one detects a non-Ia B-cell antigen. With the use of three binding assays, the six MCA that appeared to detect Ia-like determinants reacted strongly with the human colorectal cancer cell (CCC) line LoVo, and none of the three MCA that reacted with HLA-like determinants reacted with this cell line. Immunoprecipitation and polyacrylamide gel electrophoresis analysis confirmed the apparent specificities of the MCA for Ia-like and HLA molecules, demonstrated the presence of Ia-like molecules in LoVo, and failed to detect HLA in these cells. The cellular enzyme-linked immunosorbent assay was used for the testing of our anti-Ia and anti-HLA MCA with 15 other CCC lines. Marked heterogeneity was found in the expression of different Ia-like and HLA determinants defined by different or overlapping subsets of MCA, which suggested that these determinants might be present on different molecules or that different conformations of the same molecules exist in various CCC lines. Analysis of the surface phenotype of subclones of LoVo cells revealed the presence of stable variant cell subpopulations, which lost reactivity with four out of six of the anti-Ia-like MCA but retained at least one Ia-like molecule recognized by two of our MCA. All of the subclones maintained the HLA-negative phenotype. The possible immunologic and diagnostic consequences of the presence or absence of Ia-like or HLA markers on nonlymphoid tumor cells are discussed.

Detection of cell surface antigens by the cellular enzyme-linked immunosorbent assay (CELISA): [formula omitted], Depts. of Surg. & Peds, Univ. of WIS.

Detection of cell surface antigens by the cellular enzyme-linked immunosorbent assay (CELISA): [formula omitted], Depts. of Surg. & Peds, Univ. of WIS.