Quantitative measurement of human immunoglobulin E using monoclonal antibodies to distinct epitopes

Monoclonal antibodies (MAb) which recognize distinct epitopes on human immunoglobulin E have been used to develop two-site sandwich radio- and enzyme-linked immunoassays for the quantitation of human IgE. In the first step, a purified anti-IgE MAb coated to polyvinyl or polystyrene microtiter plates specifically bound the IgE contained in the samples. In the second step, another anti-IgE MAb (either iodinated or conjugated to beta-galactosidase) directed to a different antigenic determinant was used to estimate the amount of bound IgE. This simple method permitted the determination of IgE concentrations of 10 ng/ml and greater in about 3 h. Coefficients of variation on a single day did not exceed 7.5% for IgE levels, covering a wide range of the standard curve. The values obtained on serum samples showed a good correlation with those obtained using the paper radioimmunosorbent test (PRIST).     

T cell activation by monoclonal antibodies directed to different epitopes on the human T cell receptor/CD3 complex: evidence for two different modes of activation

The mouse monoclonal antibody (mAb) BMA031 (IgG2b) has recently been described to be directed against a monomorphic part of the human T cell receptor (TcR) alpha/beta. In vitro analysis of its stimulatory potential for mononuclear cells revealed two patterns of responsiveness. Out of 35 tested individuals only 2 generated a proliferative response to low antibody concentrations (15 ng/ml; "high responders"), the others ("low responders") responded only to high antibody concentrations (1.5 micrograms/ml); the anti-CD3 mAb UCHT1 (IgG2b) stimulated only the two high responders. This response pattern to BMA031 was determined by the accessory cell compartment in the culture. Stimulation by BMA031 in low responders demonstrated some unusual features: (a) high antibody concentrations were required, (b) addition of autologous serum had no inhibitory effect and (c) vigorous depletion of macrophages reduced but did not abolish the proliferative response. These characteristics were shared by two other mAb, BMA032 and BW239/347, presumably directed against the TcR alpha/beta but not by several other antibodies to the TcR/CD3 complex. Thus, the results demonstrate unusual stimulatory properties of three anti-TcR alpha/beta mAb, inducing a proliferative response without antibody cross-linking. This suggests that the stimulatory effect of anti-TcR/CD3 complex mAb is not only determined by their isotype, but also strongly depends on their epitope specificity.     

具有显著反应性滤泡的T细胞淋巴瘤

目的 探讨伴有明显反应性淋巴滤泡的T细胞淋巴瘤的组织病理学特点和免疫表型。方法 报道2例少见的T细胞淋巴瘤类型，结合光镜特点及免疫表型，并与国外文献报道的相似病例一起进行研究。结果 患者表现为全身淋巴结病，间断发热、皮疹。其临床表现、组织学特点及免疫表型类似于血管免疫母细胞性T细胞淋巴瘤，但具有明显的反应性滤泡。结论 对这类临床少见的类型，组织学特点尤为重要，应注意与反应性增生及B细胞滤泡型淋巴瘤相鉴别。     

Mapping epitopes on the insulin molecule using monoclonal antibodies

A panel of 18 monoclonal antibodies (mAb) delta to insulin have been prepared and used to begin to map antigenic determinants on the insulin molecule. All 18 mAb were of the IgG class, with 14 IgG1, 2 IgG2a and 2 IgG2b. The affinities of these mAb for their immunizing insulin ranged from 1 X 10(6) to 3 X 10(8) 1/M. The epitope recognized by three of the mAb, 1, 7 and 16 involves the three residues of the A chain, A 8-10, the so called A chain-loop determinant. This A chain loop is one of the most evolutionarily diverse regions of insulins from different species. Another mAb, 10, has been hypothesized to recognize a nearby epitope composed of the A chain residues, A4 and A8 and a B chain residue, B29, that are adjacent on the surface of the insulin molecule. Four of the mAb bind to synthetic B chain. The epitopes recognized by these 4 mAb and the last 10 mAb are unknown but the mAb are grouped according to their ability to bind to different species of insulin or proinsulin. The results of an 18 X 18 matrix analysis of pairs of mAb binding simultaneously to insulin indicate that, despite the finding that some mAb see similar antigenic sites on the insulin molecule, each of the mAb recognizes a unique site on the insulin molecule. Finally, a lower estimate of the number of possible antibodies made to insulin has been calculated to be greater than or equal to 115, a number only 10-fold lower than the lower limit of antibodies made to dinitrophenyl (DNP) or (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP), following hapten protein immunization.     

Analysis of rat hemopoietic cells using monoclonal antibodies

Rat hemopoietic cells were analyzed with immunohistochemical technique, binding inhibition assay and flow cytometer using a monoclonal antibody (UB-12) to rat fetal liver hemopoietic cells. UB-12 positive cells were recognized in only red pulp but not in white pulp of spleen. The number and fluorescence intensity of UB-12 positive cells in spleen appeared to reach to peak at 6 weeks old occupying about 60 to 70% of total cells in red pulp. On the other hand, OX-7 (anti-Thy-1) positive and W3/13 (anti-leuko-sialoglycoprotein) positive cells were found in both red and white pulp, but not in marginal zone of spleen. UB-12 antigen was found on the surface of the cells only in the early stages of hemopoiesis: relatively large nuclei of UB-12 positive cells were rich in heterochromatin. There were a large number of free-ribosomes and some mitochondria in cytoplasm, and a centriole was observed in cytoplasm at some sections of UB-12 positive cells. From the EPICS analysis of adult rat bone marrow cells using UB-12, OX-7 and W3/13 monoclonal antibodies, the percent of UB-12, OX-7 and W3/13 positive cells was 82%, i.e., 18% was negative from these monoclonal antibodies. UB-12 single positive, OX-7 single positive and W3/13 single positive cells were 7%, 7% and 47%, respectively. The percent of triple positive cells with these antibodies was about 2%.     

Induction of T cell activation by monoclonal antibodies specific for the transferrin receptor

The effect of the monoclonal antibodies (mAb), FG 1/5, FG 1/6 and FG 2/12, specific for different epitopes of the transferrin receptor (TfR) on T cell activation was studied. mAb FG 1/6 but not FG 2/12 or FG 1/5 was able to induce T cell proliferation in presence of submitogenic doses of phorbol esters. The costimulatory effect of FG 1/6 was seen only with phorbol esters known to be activators of protein kinase C. This proliferation occurred at low concentration (0.5 micrograms/ml) of antibody, required the simultaneous presence of both stimuli, phorbol esters and FG 1/6, and was independent of the presence of accessory cells. Furthermore, FG 1/6 mAb was able to increase the rate of modulation of CD3 surface expression induced by phorbol esters. FG 1/6 induced interleukin (IL) 2 synthesis by normal and transformed T lymphocytes. In addition, anti-IL2 receptor antibodies inhibited FG 1/6 plus phorbol ester-induced proliferation. Our results indicate that FG 1/6 mAb may provide to the T cells complementary signals to protein kinase C and that this activation is mediated by the IL2/IL 2R pathway.     

Characterization of two epitopes on insulin using monoclonal antibodies

Three monoclonal antibodies (MAb), 21 (IgG1), I10 (IgG1) and H38 (IgG2b), to insulin have been tested for cross-reactivity with 11 species variants of insulin and three of proinsulin. Correlations of differences of reactivities between the MAb and the species variants of insulin with the respective amino acid sequences of the latter have permitted the identification of two epitopes recognized by the MAb which encompass the regions in the A- and B-chains of insulin subject to frequent evolutionary amino acid substitutions. MAb 21 and H38 are directed to an epitope which includes residues B27-30 and A1 or A4 and can discriminate between human and pig insulins which differ only at B30. MAb 21 reacts with human (B30 thr) but not with pig (B30 ala) insulins, whereas MAb H38 exhibits a reciprocal specificity. Neither MAb 21 nor MAb H38 react with human or pig proinsulins respectively indicating that the presence of the C-peptide joining A1 to B30 masks the epitope. MAb 21 reacts with human insulin 125I-labeled at tyr A14 but not B26 suggesting that incorporation of the I atom at B26 also masks the epitope. MAb I10 is directed to an epitope which includes A8-10 and A4 or B3 with a specificity for the human A8-10 sequence. MAb I10 reacts with human proinsulin and human insulin 125I-labeled at either tyr A14 or B26.     

Immunoregulatory T cell subpopulations in patients with scleroderma using monoclonal antibodies.

Twenty-eight patients with scleroderma were compared with 22 healthy age-matched subjects. Monoclonal antibodies were used to detect the whole T cell population (OKT3), T helper cells (OKT4), and T suppressor/cytotoxic cells (OKT8) by indirect immunofluorescence on isolated peripheral blood mononuclear cells. A subset of scleroderma patients (i.e. 30% or eight of 28 patients) exhibited an elevated ratio of OKT4/OKT8 cells which could be accounted for, mainly by a reduction in OKT8 cells compared with controls. The scleroderma patients with an elevated OKT4/OKT8 ratio tended to be younger, have a shorter disease duration and more extensive skin involvement than patients with a normal OKT4/OKT8 ratio. There was no correlation with the presence of autoantibodies, drug therapy, or HLA-DR type. In order to further determine whether this imbalance in immunoregulatory cell subpopulations was specific for scleroderma, we further studied 16 patients with psoriatic arthritis but without manifest autoimmunity and delineated a similar subset of patients with an elevated OKT4/OKT8 cell ratio (i.e. 38% or six of 16 patients). The results demonstrate similar immunoregulatory T cell imbalances in patients with scleroderma and psoriatic arthritis. These findings suggest that numerical imbalances in lymphocyte subpopulations may not be specific for autoimmune disorders.     

Selectivity of Thy-1 monoclonal antibodies in enhancing neurite outgrowth.

Thy-1 monoclonal antibodies (MAbs) have previously been shown to promote neurite outgrowth from retinal ganglion cells and a variety of other neurons. We have studied the effect on neurite outgrowth of several Thy-1 MAbs with quantitatively similar binding properties and found that only certain Thy-1 MAbs promote neurite outgrowth. This finding suggests that the antibody effects depend on specific interactions with one or more active sites on the Thy-1 glycoprotein.     

Detection of Thy-1 on cell surface of human T lymphoid cell lines by a monoclonal antibody

A mouse IgG2b(kappa) monoclonal antibody (MAb) HB-2S-1 against human brain Thy-1 was secreted by a hybridoma clone after fusion of mouse myeloma cells with spleen cells from a mouse that went through a prolonged immunization procedure before fusion. When tested against isolated human Thy-1 by the enzyme-linked immunosorbent assay (ELISA), MAb HB-2S-1 in culture supernatant showed a titer of over 100,000, and a titer of over 10 million in ascites of a mouse injected with the hybrid clone. By immunoblotting, this antibody was found to bind a doublet of protein bands of approximately 25,000 daltons among all proteins solubilized by deoxycholate (DOC) from membrane of human brain cells. When tested on human lymphoid cell lines by immunofluorescence, MAb HB-2S-1 strongly stained four T lymphoma cell lines, C91-Pl, HUT-102, HUT-78, and C5-MJ; and weakly two leukemia cell lines, MOLT-3 and Jurkat(clone E6-1). It did not stain a third T leukemia cell line, CCRF-CEM; a human B cell line, Raji; a plasmacytoma cell line, HMy2; or a myelomonocytic cell line, HL-60. Peripheral blood lymphocytes from ten normal human adults and the viable T cells isolated from another normal individual were also negative.     

Enumeration of T cell subsets in atopic dermatitis using monoclonal antibodies

Peripheral blood lymphocytes from 22 patients with atopic dermatitis, 17 age-matched healthy controls, 10 patients with other skin diseases, and 14 patients with either asthma or allergic rhinitis were characterized by reactivity with monoclonal antibodies to the surface antigens of helper-inducer (T4) and suppressor-cytotoxic (T8) T cell subsets and to a common T cell antigen (T3). In contrast to healthy controls and controls with other skin diseases or respiratory allergic disease, patients with atopic dermatitis had a reduced percentage of T3-positive (T3+) cells (p < 0.01) and T8-positive (T8+) cells (p < 0.001) but not of T4-positive cells (T4+) (p > 0.05). A selective increase in the ratio of T4+ cells over T8+ cells was observed in 17 of 22 patients with atopic dermatitis but not in any of the controls. Thus there is a loss of circulating suppressor-cytotoxic T cells in the majority of patients with active atopic dermatitis.     

Biochemical dissection of DRw6 related epitopes using monoclonal antibodies

Three monoclonal antibodies (MoAbs) directed against polymorphic epitopes of Ia antigens were used as tools for a serological and biochemical dissection of the class II products encoded by the HLA-DRw6 haplotype. MoAb 16.23 defines an epitope common to DRw13 and DR3 haplotypes, MoAb S5 defines an epitope common to DRw13 and DR2 haplotypes, and MoAb S2 defines an epitope apparently restricted to DRw13. Not all DRw13 cells express these epitopes. Analysis of 16 DRw6 homozygous typing cells showed that expression of all three epitopes was restricted to those DRw13 cells that carried the Dw18 antigen, the DRw13 Dw19 cells being negative. The relationships among the molecules bearing these epitopes were investigated using sequential immunoprecipitation in lymphoblastoid cell lines derived from genetically characterized individuals. In both DRw13 and DR2 bearing cells, the DR2 + w13 epitope was localized to a population of DQ molecules which also carried the DQw1 specificity defined by MoAb Genox 3.53. The S5 epitope is therefore a private specificity that distinguishes the DQw1 antigens encoded by the DR2 and DRw13 haplotypes from the DQw1 antigens encoded by other haplotypes. The DRw13 and the DR3 + w13 epitopes were both shown to be expressed on DR molecules that also carried a DRw52-like specificity. In a DRw13 haplotype encoding both the S2 and 16.23 epitopes, the epitopes appeared to be located on separate molecules. The antibodies described here can distinguish between DRw13 cells which carry different Dw antigens, identify a private specificity on the DQw1 antigen, and define two distinct DR molecules encoded by some DRw13 haplotypes.     

Structural characterization of CD6: properties of two distinct epitopes involved in T cell activation

Studies from our laboratory have shown that anti-T12, a mAb which recognizes CD6, is a macrophage-dependent mitogen for human T cells and can augment T cell autoreactivity in vitro. To obtain additional information regarding the potential biological role of CD6 we sought to further characterize its biochemical properties. The CD6 molecule on 125I-surface-labeled T cells and by Western blot analysis was a monomer of mol. wt 130,000 under reducing conditions and mol. wt 117,000 under non-reducing conditions, suggesting the presence of intrachain disulfide bonds. The polypeptide contains a protease sensitive site. In activated T cells, the protein was serine phosphorylated. Analysis of biosynthetically labeled CD6 in the presence of tunicamycin revealed a reduction in mol. wt from 130,000 to 100,000, indicating that the polypeptide is extensively N-glycosylated. The mAb, anti-2H1, had been shown to activate T cells in combination with PMA or the anti-T11(3) mAb but, unlike anti-T12, not in the presence of macrophages alone. The present studies demonstrate by sequential immunoprecipitation that these two mAbs recognize the same polypeptide. However, Western blot analysis and indirect immunofluorescence cross-blocking studies demonstrate that the two mAbs recognize different determinants on CD6. Anti-T12 recognizes an epitope that is present only under non-reducing/non-denaturing conditions, while anti-2H1 recognizes an epitope that is also preserved under reducing/denaturing conditions. A direct comparison of activation properties of the mAbs confirmed that anti-T12 was optimally mitogenic in the presence of macrophages but not PMA, while, conversely, anti-2H1 was optimally mitogenic in combination with PMA but not macrophages, suggesting that the differences in epitope specificity may account for the distinct activation properties of each mAb. Taken together, the structural and functional data strongly suggest that the CD6 membrane glycoprotein may function as a physiologically important receptor structure on human T lymphocytes.     

Difference in cell binding patterns of two monoclonal antibodies recognizing distinct epitopes on a human melanoma-associated oncofetal antigen

Two monoclonal antibodies (MAbs), 140.240 and 96.5, generated independently in different laboratories, have been shown to detect the target structures of 87,000 (gp87) and 97,000 (p97) glycoproteins, respectively, both strongly expressed by melanoma cells and fetal small intestine. To determine whether MAb 140.240 and MAb 696.5 recognized a same target structure, they were tested in immunoprecipitation/SDS-PAGE using NP-40 lysates of melanoma cells labelled with [35S]methionine for 18 hr. Both antibodies precipitated a single band with Mr = 87,000. Reciprocal immunodepletion studies showed that neither of the two antibodies detected the 87,000 band in the lysate immuno depleted by either antibody, suggesting that these two antibodies recognize the same or extremely similar molecules. Two-dimensional tryptic peptide mapping analysis showed that the two identified molecules shared the same finger-printing pattern. A 40,000 fragment of the 87,000 molecule produced by protease digestion was precipitated by MAb 96.5 but not MAb 140.240, indicating that the epitopes recognized by the two antibodies are localized at discrete sites on the molecule. Serological studies on these two antibodies revealed slightly different binding patterns in the MAb 140.240 exhibited a more melanoma-restricted specificity, while MAb 96.5 had a specificity to melanoma and to some other cell types. The observed difference in epitope specificity may be important in the clinical applications of these antibodies.     

Thy-1-mediated activation of rat mast cells: the role of Thy-1 membrane microdomains.

The glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia (RBL) cells. The finding that Thy-1 from detergent-solubilized RBL-2H3 cells forms complexes with src-related protein-tyrosine kinase p56/p53lyn suggested that this kinase may play a key role in Thy-1-mediated mast-cell activation. The molecular mechanism of this activation is, however, unknown. Here we show that in RBL-2H3-derived cells extracted by the standard procedure with several non-ionic detergents, the majority of Thy-1 and p56/p53lyn were not released into postnuclear supernatant but remained associated with the detergent-resistant cytoskeletal/nuclear fraction. Pretreatment of the cells with the cholesterol-complexing agents, saponin or digitonin, resulted in complete solubilization of Thy-1 and p56/p53lyn in non-ionic detergents and dissociation of the complexes; this implies that cholesterol plays a crucial role in stabilization of the complexes. This conclusion was supported by double immunofluorescence colocalization experiments which also allowed us to estimate the size of the insoluble complexes to be about 0.1 micron. Sequential treatment with saponin and Nonidet P-40 was used to fractionate tyrosine-phosphorylated proteins during Thy-1-mediated activation of RBL-2H3 cells. Among the soluble cytoplasmic proteins the most dramatic change in tyrosine phosphorylation was found in pp72, whereas pp40 and pp33 were found mainly in the membrane fraction. Our data suggest that surface aggregation of GPI-anchored Thy-1 molecules leads to aggregation of p56/p53lyn kinase located in the same membrane microdomain, followed by transphosphorylation of both soluble and membrane-bound substrates.     

Evaluation of T cell subsets in myasthenia gravis using anti-T cell monoclonal antibodies

Immunoglobulin (Ig) secreting cells occur in all lymphoid tissues, including the bone marrow (BM). There are important differences between the various organs with respect to their number of Ig-secreting cells and the heavy chain isotype distribution of the secreted Igs. Furthermore, both distribution patterns depend on age. Early in life most Ig-secreting cells are localized in spleen and lymph nodes. In adults, however, the majority of all Ig-secreting cells of the individual are localized in the BM. Immunization can lead to the appearance of substantial numbers of antibody-forming cells in BM. The kinetics of the BM response are different from the response in the peripheral lymphoid tissues. Shortly after immunization most antibody-forming cells occur in the peripheral lymphoid tissues, but later on, especially during secondary type responses, most antibody-forming cells are localized in the BM. Apparently, antibody formation is regulated in such a way that peripheral lymphoid tissues respond rapidly, but only for a short period, whereas the BM response starts slowly, but takes care of a long-lasting massive production of antibodies to antigens which repeatedly challenge the organism.     

Characterization of T cell subsets in patients with atopic dermatitis using OKT monoclonal antibodies

The distribution of T cell subsets has been studied by means of OKT monoclonal antibodies in 19 children with atopic dermatitis. In these patients a decreased percentage of circulating OKT4+ cells has been observed, while no difference has been found between atopic and normal subjects, regarding the percentages of circulating OKT8+ and OKT11+ cells. An increased OKT4+/OKT8+ ratio has been detected only in three children with a particularly severe and extensive atopic eczema.