scholarly journals Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors.

1979 ◽  
Vol 150 (3) ◽  
pp. 580-596 ◽  
Author(s):  
J C Unkeless
Keyword(s):  
Monoclonal Antibody ◽  
Cell Lines ◽  
Fc Receptors ◽  
Inhibitory Effect ◽  
Fc Receptor ◽  
Antigenic Relationship ◽  
Mouse Macrophage ◽  
Spleen Cells ◽  
Binding Studies ◽  
Fab Fragment

To investigate the antigenic relationship between the macrophage and lymphocyte Fc receptors (FcR), a monoclonal antibody capable of blocking mouse macrophage Fc receptors was selected. Hybrids were formed by fusing the P3U1 mouse myeloma and spleen cells from a rat immunized with the mouse macrophage-like cell lines J774 and P388D1. The Fab fragment of the monoclonal IgG secreted by clone 2.4G2, inhibited the trypsin-resistant Fc receptor II (FcRII), which is specific for immune aggregates of mouse IgG1 and IgG2b, but had no inhibitory effect on the trypsin-sensitive Fc receptor I (FcRI), which binds monomeric IgG2a and erythrocytes coated with IgG2a. Thus, the monoclonal 2.4G2 IgG appeared to be specific for macrophage FcRII. Further evidence that the 2.4G2 IgG was directed against FcRII came from binding studies of the monoclonal antibody to J774 cells and a series of independently isolated variants which do not express FcRII. These variants of J774 bound 5% as much of the monoclonal antibody as the parent line, which bound 600,000 molecules of 2.4G2 IgG per cell. The antigenic relatedness of mouse lymphocyte FcR to mouse macrophage FcRII was demonstrated by the binding of 2.4G2 IgG to FcR-bearing lymphoid cell lines and the inhibition of the lymphocyte FcR by the monoclonal antibody. Preincubation of spleen cells and peritioneal cells with 2.3G2 IgG likewise inhibited rosette formation with ox erythrocytes coated with rabbit IgG. The ability of the hybridoma IgG to inhibit mouse FcRII was independent of the major histocompatibility complex. The 2.4G2 IgG antigenic determinant was not present on rat, guinea pig, rabbit, or human FcR-bearing cells.

scholarly journals Relationship between Fc receptors, antigen-binding sites on T and B cells, and H-2 complex-associated determinants.

1975 ◽  
Vol 141 (3) ◽  
pp. 547-560 ◽  
Author(s):  
A Basten ◽  
J F Miller ◽  
R Abraham
Keyword(s):  
T Cells ◽  
B Cells ◽  
Specific Antigen ◽  
Fc Receptors ◽  
Fc Receptor ◽  
Antigen Binding ◽  
T And B Cells ◽  
Spleen Cells ◽  
Fab Fragment ◽  
Thymus Cells

The relationship between H-2 complex-associated determinants, Fc receptors, and specific antigen-recognition sites on T and B cells was examined by binding and functional assays. The Fc receptor was detected by radiolabeled immune complexes or aggregated human IgG. Both these reagents selectively bound to B cells, not to T cells. When spleen cells, from mice primed to several antigens, were exposed to highly substituted radioactive aggregates, their capacity to transfer both a direct and indirect plaque-forming cell response to these antigens was abrogated. Addition of B cells, but not of T cells, restored responsiveness. Complexed Ig binding to Fc receptors was prevented by pretreatment of mixed lymphoid cell populations with antisera directed against membrane components on the same cell (e.g., H-2) and on other cells (e.g., theta). The lack of specificity of inhibition was thought to be due to the formation on cell surfaces of antigen-antibody complexes which would then attach to the Fc receptor during the incubation precedure. Specific blockade of the Fc receptor during the incubation procedure. Specific blockade of the Fc receptor however occurred when B cells were pretreated with the Fab fragments of anti-H-2 antibody. This was demonstrated autoradiographically and by inhibition of aggregate-induced suicide. The blocking activity of ante-H-2 Fab was removed by absorption with spleen cells from thymectomized irradiated mice but not with thymus cells of appropriate specificity. This suggested that the antibodies involved had specificity for determinants on the B-cell membrane distinct from those coded by the K or D end of the H-2 complex, and either absent from, or poorly represented on, thymus cells. Specific antigen-induced suicide of B cells was achieved simply by incubating the cells with radioactive antigen in the cold. T-cell suicide on the other hand required that the 125I-labeled antigen be presented to the T cells at 37 degrees-C on the surface of spleen cells from antigen-primed mice. Pretreatment of T cells with the Fab fragment of anti-H-2 antibody protected them from the suicide effect. By contrast no such protection of B cells could be achieved by this procedure. In other words H-2 (? Ir)-associated determinants may not only be in close proximity to the antigen-binding site on T cells but, in addition, may be involved in the effective operation of the receptor.

scholarly journals Structural analysis of free and liganded forms of the Fab fragment of a high-affinity anti-cocaine antibody, h2E2

2019 ◽  
Vol 75 (11) ◽  
pp. 697-706
Author(s):  
Kemin Tan ◽  
Min Zhou ◽  
Angela J. Ahrendt ◽  
Norma E. C. Duke ◽  
Nassif Tabaja ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cocaine Abuse ◽  
Phase 1 ◽  
Complex Structure ◽  
Van Der Waals Interactions ◽  
Binding Studies ◽  
Fab Fragment ◽  
Aromatic Residues ◽  
High Affinity ◽  
Insight Into

A high-affinity anti-cocaine monoclonal antibody, designated h2E2, is entering phase 1 clinical trials for cocaine abuse therapy. To gain insight into the molecular details of its structure that are important for binding cocaine and cocaine metabolites, the Fab fragment was generated and crystallized with and without ligand. Structures of the unliganded Fab and the Fab fragment bound to benzoylecgonine were determined, and were compared with each other and with other crystallized anti-cocaine antibodies. The affinity of the h2E2 antibody for cocaine is 4 nM, while that of the cocaine metabolite benzoylecgonine is 20 nM. Both are higher than the reported affinity for cocaine of the two previously crystallized anti-cocaine antibodies. Consistent with cocaine fluorescent quenching binding studies for the h2E2 mAb, four aromatic residues in the CDR regions of the Fab (TyrL32, TyrL96, TrpL91 and TrpH33) were found to be involved in ligand binding. The aromatic side chains surround and trap the tropane moiety of the ligand in the complex structure, forming significant van der Waals interactions which may account for the higher affinity observed for the h2E2 antibody. A water molecule mediates hydrogen bonding between the antibody and the carbonyl group of the benzoyl ester. The affinity of binding to h2E2 of benzoylecgonine differs only by a factor of five compared with that of cocaine; therefore, it is suggested that h2E2 would bind cocaine in the same way as observed in the Fab–benzoylecgonine complex, with minor rearrangements of some hypervariable segments of the antibody.

scholarly journals Purificaton of a functional mouse Fc receptor through the use of a monoclonal antibody.

1980 ◽  
Vol 152 (4) ◽  
pp. 1048-1069 ◽  
Author(s):  
I S Mellman ◽  
J C Unkeless
Keyword(s):  
Monoclonal Antibody ◽  
Fc Receptor ◽  
Galactose Oxidase ◽  
Fab Fragment ◽  
Electrophoretic Mobilities ◽  
Apparent Homogeneity ◽  
Rabbit Igg ◽  
J774 Cells ◽  
Immune Aggregates ◽  
Phosphate Buffered Saline

We recently reported the isolation of a rat monoclonal antibody designated 2.4G2 (9) that is directed against the mouse trypsin-resistant Fc receptor (FcR) for IgG2b and IgG1 immune aggregates. We have now utilized the Fab fragment of 2.4G2 as an affinity reagent to purify FcR from the macrophage cell line J774 to apparent homogeneity. The antigen isolated from J774 cells consisted of two general types of polypeptides with broad electrophoretic mobilities of approximately 60,000 and 47,000 mol wt. Similar broad bands ranging from 47,000 to 70,000 mol wt were isolated from various FcR-bearing cell lines of B, T, and null lymphocyte, as well as of macrophage origin. J774 FcR was judged to be a glycoprotein based on the sensitivity of its isoelectric point to neuraminidase digestion, its labeling with galactose oxidase/NaB[3H4], and its binding to concanavalin A-Sepharose. In phosphate-buffered saline, the isolated protein formed large aggregates that were shown to retain FcR activity, albeit with a somewhat altered IgG subclass specificity. The FcR aglutinated erythrocytes that were coated with both IgG2b and IgG2a that did not otherwise hemagglutinate. In addition, iodinated FcR bound to Sephadex beads coated with rabbit IgG, mouse IgG1, IgG2b, and IgG2a, but not to beads coated with mouse IgG3 or rabbit F(ab')2 fragments. The binding of the purified receptor to all IgG classes was inhibited by the Fab fragments of 2.4G2. In contrast, the binding of IgG2a to intact macrophages was inhibited by 2.4G2 Fab by only 15%, whereas rabbit IgG immune aggregate binding was almost completely abolished.

scholarly journals A new Fc receptor on mouse macrophages binding IgG3.

1981 ◽  
Vol 153 (3) ◽  
pp. 514-519 ◽  
Author(s):  
B Diamond ◽  
D E Yelton
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Lines ◽  
Cytochalasin B ◽  
Fc Receptors ◽  
Peritoneal Macrophages ◽  
Fc Receptor ◽  
Igg Subclasses ◽  
Macrophage Cell ◽  
Sheep Erythrocytes ◽  
Mouse Macrophages

Monoclonal antibodies to sheep erythrocytes (SRBC) have proved useful in identifying two Fc receptors on mouse macrophages, one for IgG2a, and one for IgG1 and IgG2b. We have used monoclonal IgG3 anti-SRBC to identify a third Fc receptor on mouse macrophages which binds IgG3 uniquely. This receptor is present on primary resident and thioglycolate-induced peritoneal macrophages and on some macrophage cell lines. The binding of IgG3-coated SRBC is inhibited by aggregated byt not monomeric IgG3, and not by IgG1, IgG2a, and IgG2b aggregates. It is unaffected by treating the macrophages with trypsin or cytochalasin B and occurs at both 4 degrees and 37 degrees C. IgG3, like all other IgG subclasses, mediates phagocytosis. We have also generated a variant macrophage line which bears the receptors for IgG1 and IgG2b and for IgG2a, but not for IgG3.

B-Cell Receptors Of Follicular Lymphoma Patients Recognize Themselves

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 633-633 ◽  
Author(s):  
Angela Schulz ◽  
Debra K Czerwinski ◽  
Ronald Levy
Keyword(s):  
T Cells ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Lines ◽  
Protein Modification ◽  
Conflicts Of Interest ◽  
Fc Receptors ◽  
Fc Receptor ◽  
Surface Binding

Abstract Follicular Lymphoma (FL) is the most common indolent lymphoma and is characterized by retained surface B-cell receptor (BCR) expression despite ongoing V region somatic mutation. Furthermore, every tumor has a unique BCR. Together, this suggests that the BCR is important for the survival of the malignant B cell. Recognition of a target antigen could lead to a constant stimulation of the malignant cells and serve as a driving force. Recombinant BCRs from a series of FL patients were expressed in the form of assembled proteins and all of IgG3 isotype. Recently we reported that 25% of these BCRs have binding activity against a human epithelial cell line1. In the current study we aimed to broaden the search for putative auto-antigens. As FL cells are in close contact with various peripheral blood cells (PBMC), an epitope on the surface of these cells might serve as a putative auto-antigen. Therefore, we incubated PBMCs from healthy donors with the lymphoma derived BCRs and quantified surface binding by FACS. We gated separately on B-cells, CD4 T-cells, CD8 T-cells, monocytes and natural killer (NK) cells. Thereby, we identified 7 out of 25 tumor-derived BCRs which bound to at least one cell type. Four of these strongly bound to NK, B-cells and monocytes from multiple different donors. None of the tested antibodies bound to T-cells. We tested the four reactive BCRs against B-cell lines (Daudi, Raji, Ramos, DHL-4, FL-18 and Reh) and one T-cell line (MOLT-4). We found that Daudi and to a lesser extent Raji but none of the other cell lines were positive for the same lymphoma derived BCRs as PBMCs. Because of the observed binding pattern we hypothesized that a common protein modification might be the target. We therefore treated the cell lines with tunicamycin, an inhibitor for N-linked glycosylation, in order to test if sugars might be the targets of the lymphoma derived BCRs. Surprisingly, this led to stronger binding of the same BCRs. it is known that de-glycosylation of Fc receptors increases Fc binding, suggesting that Fc receptors might be the target. In line with this hypothesis Daudi cells have Fc receptors but Ramos cells do not. Moreover, T-cells are the only PBMCs which do not express Fc receptors. Therefore, we tested Fc receptors directly as targets. Recombinant Fcγ1 (CD64) and Fcγ3 (CD16) were positive in ELISA tests with the four recombinant lymphoma-derived BCRs. In addition, blocking Fcγ2 (CD32) and Fcγ3 receptors on PBMCs before staining with lymphoma derived BCRs resulted in a signal reduction. In order to define if the Fc part of these BCRs is passively bound by Fc receptors or if the Fc receptor is recognized as specific target by the variable BCR regions we produced F(ab)2 fragments. Staining PBMCs with these abolished binding, suggesting that the Fc region of the lymphoma derived BCRs is bound necessary for cellular binding. As all BCRs have the same IgG3 constant region they should all bind to Fc receptors through their Fc regions with the same affinity. In vivo Fc receptor bearing cells are not activated by the binding of a single antibody but only when an antibody cluster is formed e.g. the antibody-coated surface of a pathogen. We therefore hypothesized that the BCRs which bound Fc receptors exist as clusters. This would be conceivable if the BCRs recognized a part of them-selves as target which subsequently would lead to dimer or oligomerization. Indeed, a Fc binding BCR which was coated on an ELISA plate could be detected with a biotinylated counterpart of itself. This same phenomenon was recently described to be the case for chronic lymphocytic leukemia2. Experiments are ongoing to elucidate which region of the BCRs are recognized as targets and for which percentage of them for which this is true. In conclusion these findings suggest a new target for the BCR of FL cells whose constant presence in its microenvironment might represent a novel mechanism of chronic BCR stimulation. 1Sachen KL et al., Blood 2012, 2Dühren von Minden M et al., Nature 2012 Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of a monoclonal antibody having selective reactivity with normal and neoplastic plasma cells

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 238-245 ◽  
Author(s):  
AW Tong ◽  
JC Lee ◽  
MJ Stone
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Pokeweed Mitogen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunoperoxidase Technique ◽  
Binding Studies ◽  
Bone Marrow Biopsies

A myeloma cell-reactive monoclonal antibody (MoAb), MM4, was generated from BALB/c mice immunized with alternate injections of cells from two human multiple myeloma (MM) cell lines. Screening by the enzyme-linked immunosorbent assay (ELISA) technique showed that MM4 reacted with human MM cell lines (7 of 7 positive), as well as bone marrow aspirates from MM patients (4 of 4 cases positive). MM4 did not react with marrow aspirates from control patients (3 cases), or with peripheral blood mononuclear (PBM) cells from normal subjects, lymphocytic (12 cases) and myelogenous (8 cases) leukemia patients. In addition, MM4 was negative with polymorphonuclear leukocytes and RBCs from normal donors. By means of the immunoperoxidase technique, the MM4-reactive antigen was detected in paraffin-embedded, Zenker formalin-fixed bone marrow biopsies of MM (12 of 12 cases positive), Waldenstrom's macroglobulinemia (2 of 2 cases positive), asymptomatic plasma cell dyscrasia (4 of 4 cases positive), and certain lymphomas (2 of 5 cases positive). Marrow biopsies from lymphocytic (5 cases) and myelogenous (5 cases) leukemias were uniformly negative. The MM4-reactive antigen also was expressed on plasma cells generated from pokeweed mitogen (PWM)-stimulated normal PBM cultures. The pattern of reactivity of MM4 with lymphocytes of B origin was similar to that of the plasma cell MoAb PCA-1. Competitive binding studies showed, however, that these two MoAbs recognized distinct antigenic determinants. These observations suggest that MM4 may be useful for the study of human plasma cell dyscrasias.

An Algorithm for “Indeterminate” Test Results in the Platelet Serotonin Release Assay for Heparin-Induced Thrombocytopenia (HIT).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1048-1048
Author(s):  
Carol A. Smith ◽  
Andrew E. Warkentin ◽  
Theodore E. Warkentin ◽  
Donald M. Arnold ◽  
Jane C. Moore ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Activation ◽  
Immune Complexes ◽  
Fc Receptors ◽  
Fc Receptor ◽  
Serotonin Release ◽  
Indeterminate Result ◽  
Receptor Blocking ◽  
Release Assay ◽  
Test Result

Abstract HIT is a prothrombotic complication of heparin caused by antibodies that recognize complexes of platelet factor 4 (PF4) bound to heparin or certain other polyanions. These antibodies produce thrombocytopenia by activating platelets via their Fc receptors. A “functional” (platelet activation) assay that utilizes washed platelets, known as the 14C-serotonin release assay (SRA), has the highest reported sensitivity-specificity tradeoff for detecting clinically-significant antibodies that recognize PF4/heparin complexes (“HIT antibodies”). Platelet activation (% serotonin release) induced by patient (or control) serum is assessed under several different reaction conditions, including absence of heparin, therapeutic concentrations of unfractionated heparin (UFH, 0.1 to 0.3 U/mL) and low-molecular-weight heparin (LMWH, 0.2 U/mL), supratherapeutic concentrations of UFH (100 U/mL), and with 0.1 U/mL UFH in the presence of a platelet Fc receptor-blocking monoclonal antibody. Various technical aspects of the assay optimize test sensitivity and specificity (e.g., using heat-inactivated patient serum; washing the platelets with apyrase; resuspending the platelets in albumin-free Tyrode’s buffer; using platelets from healthy volunteers known to react well to IgG platelet agonists, etc.). The classic HIT platelet activation profile is strong platelet activation (>50% serotonin release) in the presence of therapeutic UFH or LMWH that is inhibited by supratherapeutic heparin and the platelet Fc receptor-blocking monoclonal antibody. One drawback to the assay is that some patient sera activate platelets via the Fc receptors in a heparin-independent fashion, i.e., the platelets are activated at all heparin concentrations. This is known as an “indeterminate” reaction profile, since the presence of in vitro immune complexes (generated by the heat-inactivation process) or in vivo immune complexes or other platelet-activating factors could “mask” the presence of a true HIT antibody. We developed an algorithm for dealing with such indeterminate reaction profiles. First, we repeat the SRA using another aliquot of patient serum that is newly heat-inactivated, and also use different platelet donors to perform the assay. Often, this results in an interpretable test result. However, if the repeat SRA also gives an indeterminate result, we then use an in-house anti-PF4/heparin ELISA (that detects only IgG class antibodies) to determine whether HIT antibodies could be present. From 2091 patient serum samples tested for HIT antibodies using the SRA, we identified 199 (9.5%) samples that gave an initial indeterminate result. Using our algorithm, 81 samples subsequently gave clearly negative results, and 35 samples gave clearly positive results. However, 83 samples (representing 41.7% of the retested samples, or 4.0% of the total samples) gave a repeat indeterminate test result. When this last group of samples was tested using the anti-PF4/heparin-IgG ELISA, 53 of the samples tested negative (OD<0.45) and 30 tested positive (OD >0.45). With this algorithmic approach, about 96% of patients can be classified as negative or positive using the SRA. However, 4% of patients require the use of a complementary assay- the anti-PF4/heparin-IgG ELISA, to evaluate for the presence of HIT antibodies. Further studies are required to determine the causes of persistent indeterminate results in the SRA, which may lead to new approaches to further optimize this assay.

scholarly journals Characterization of a monoclonal antibody having selective reactivity with normal and neoplastic plasma cells

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 238-245 ◽  
Author(s):  
AW Tong ◽  
JC Lee ◽  
MJ Stone
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Cell Lines ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Pokeweed Mitogen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Immunoperoxidase Technique ◽  
Binding Studies ◽  
Bone Marrow Biopsies

Abstract A myeloma cell-reactive monoclonal antibody (MoAb), MM4, was generated from BALB/c mice immunized with alternate injections of cells from two human multiple myeloma (MM) cell lines. Screening by the enzyme-linked immunosorbent assay (ELISA) technique showed that MM4 reacted with human MM cell lines (7 of 7 positive), as well as bone marrow aspirates from MM patients (4 of 4 cases positive). MM4 did not react with marrow aspirates from control patients (3 cases), or with peripheral blood mononuclear (PBM) cells from normal subjects, lymphocytic (12 cases) and myelogenous (8 cases) leukemia patients. In addition, MM4 was negative with polymorphonuclear leukocytes and RBCs from normal donors. By means of the immunoperoxidase technique, the MM4-reactive antigen was detected in paraffin-embedded, Zenker formalin-fixed bone marrow biopsies of MM (12 of 12 cases positive), Waldenstrom's macroglobulinemia (2 of 2 cases positive), asymptomatic plasma cell dyscrasia (4 of 4 cases positive), and certain lymphomas (2 of 5 cases positive). Marrow biopsies from lymphocytic (5 cases) and myelogenous (5 cases) leukemias were uniformly negative. The MM4-reactive antigen also was expressed on plasma cells generated from pokeweed mitogen (PWM)-stimulated normal PBM cultures. The pattern of reactivity of MM4 with lymphocytes of B origin was similar to that of the plasma cell MoAb PCA-1. Competitive binding studies showed, however, that these two MoAbs recognized distinct antigenic determinants. These observations suggest that MM4 may be useful for the study of human plasma cell dyscrasias.

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