Competitive Antibody-binding Assay Research Articles

A Fluorescence-Based Competitive Antibody Binding Assay for Kynurenine, a Potential Biomarker of Kidney Transplant Failure.

Kynurenine is a tryptophan metabolite linked to several inflammatory processes including transplant failure, a significant challenge in transplant medicine. The detection of small molecules such as kynurenine, however, is often complex and time consuming. Herein, we report the successful synthesis of a fluorescently labelled kynurenine derivative, showing proper fluorescence and anti-kynurenine antibody binding behavior in a magnetic bead immunoassay (MIA). The fluorescent kynurenine–rhodamine B conjugate shows a KD-value of 5.9 µM as well as IC50 values of 4.0 µM in PBS and 10.2 µM in saliva. We thus introduce a rapid test for kynurenine as a potential biomarker for kidney transplant failure.

Ex Vivo Modulation of Mobilized Peripheral Blood: Characterization of HSC and T-Cell Responses to Prostaglandin E2

16,16 dimethyl prostaglandin E2 (FT1050) was previously identified to be a critical regulator of hematopoetic stem cell (HSC) homeostasis and we hypothesized that a brief ex vivo modulation could improve patient outcomes in umbilical cord blood (UCB) transplantation by increasing the “effective dose” of HSCs. Using preclinical models, we have demonstrated that pulse treating human HSCs with FT1050 significantly enhances the homing and engraftment of donor cells to the bone marrow niche, in part by upregulating CXCR4.To date, preclinical and clinical translation of this therapeutic paradigm has focused on UCB. In an initial clinical evaluation, ex vivo modulation of UCB with FT1050 (2 hours, on site, just prior to infusion) was found to accelerate neutrophil recovery (median 17.5 vs 21 days, p = 0.045) in adult patients undergoing double UCB transplantation (Cutler, 2013). In this Phase 1b study, we also observed low rates of viral reactivation (17% incidence of CMV viremia vs. 42-67% reported in the literature) and GvHD, suggesting that FT1050 may also be having a therapeutically relevant effect on the T-cell compartment of the treated UCB units. To explore the potential therapeutic application of ex vivo modulation of other allogeneic HSC sources, we conducted a series of in vitro studies characterizing the effect of FT1050 on HSCs and T-cells derived from mobilized peripheral blood (mPB).CD34+ cells isolated from mPB or UCB were treated with 10μM FT1050 for 2 hours at 37ºC. We showed that FT1050 binds to the EP2 and EP4 receptors on the cell surface, activating adenylate cyclase which results in a rapid increase in intracellular cAMP levels. Results of a competitive antibody binding assay demonstrated robust and comparable increases in cAMP levels in CD34+ cells from both UCB and mPB. Genome-wide expression analysis of FT1050 treated CD34+ cells showed a highly similar expression response in cells isolated from mPB and UCB, including a 20-fold increase in the key HSC homing receptor CXCR4. We also observed similar FT1050-dependent increases in the surface CXCR4 protein levels and significant improvements in transwell migration rates to gradients of SDF-1. Genome-wide expression analysis within the T-cell compartments from both UCB and mPB (e.g., CD8, CD4, NK, Tregs) revealed a highly similar set of FT1050 induced genes which are involved in cell cycle (e.g., CCND1, CCNE1), tolerance (e.g., DUSP5, FLT1) and anti-viral response (e.g., CD56, EFNB2). The results of this preclinical characterization study provides the basis upon which to explore the future use of FT1050 modulated mPB in a clinical setting. DisclosuresRobbins:Fate Therapeutics: Employment, Equity Ownership. Le:Fate Therapeutics: Employment, Equity Ownership. Troung:Fate Therapeutics: Employment, Equity Ownership. Foster:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics: Employment, Equity Ownership. Mitchell:Fate Therapeutics: Employment, Equity Ownership. Guerrettaz:Fate Therapeutics: Employment, Equity Ownership. Multani:Fate Therapeutics: Employment, Equity Ownership. Shoemaker:Fate Therapeutics: Employment, Equity Ownership.

Confirmation of a false-positive result associated with a competition inhibition assay used for detecting antibodies to a protein epitope ofMycobacterium leprae

A competitive antibody-binding assay (CABA) was developed to detect antibodies in infected armadillos and leprosy patients which compete with an M. leprae-specific 125I monoclonal antibody IIIE9 for the species-specific M. leprae-IIIE9 epitope on the 65-kD protein. The results suggest armadillos and leprosy patients produce antibodies that inhibit the binding of 125I-IIIE9 monoclonal antibody to the IIIE9 epitope on crude, native 65-kD protein preparations. When purified, recombinant 65-kD protein was substituted for crude antigen, there was no evidence in the CABA of antibody to the IIIE9 epitope. False-positive results, possibly induced by steric hindrance, are likely to be associated with CABA which incorporate crude cell wall extracts as solid-phase antigen.

Relationship between JAK2 V617F Mutation Status, Granulocyte CD177 mRNA Expression and CD177 Soluble Protein Level in Patients with Polycythemia Vera.

A unique gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been recently described in patients with polycythemia vera (PV), essential thrombocythemia and chronic idiopathic myelofibrosis [N Engl J Med. 2005 Apr 28;352(17):1779–90]. Although the currently available data clearly demonstrate that the JAK2 V617F mutation participates in the pathogenesis of myeloproliferative disorders, the mutation's precise place in the hierarchical order of pathogenetic events remains to be established. We have recently reported that altered gene expression in myeloproliferative disorders correlates with activation of signaling by the V617F mutation of JAK2 (Blood. 2005 Aug 4; Epub ahead of print). Granulocyte CD177 (PRV1) mRNA overexpression has been initially reported as a potential marker of PV but later shown by us to rather be a marker of neutrophil activation [Br J Haematol. 2004 Sep;126(5):650–6]. In this study, we analyzed the relationship between JAK2 V617F mutation status, granulocyte CD177 mRNA expression and CD177 soluble protein level in 72 patients with PV. We also investigated the ontogeny of CD177 expression by hematopoietic cells with the aim of defining the stage of mRNA expression during myeloid, erythroid and megakaryocytic cell differentiation. Finally we studied the effect of soluble CD177 protein on hematopoietic cell proliferation and differentiation. Granulocyte CD177 mRNA expression and percentage of JAK2 V617F alleles were evaluated by quantitative Real Time PCR (qRT-PCR), while serum CD177 protein level was measured by a flow cytometry-based competitive antibody-binding assay. Liquid cultures were performed by culturing peripheral blood mononuclear cells obtained from healthy individuals and PV patients in the presence of high CD177-expressing, low CD177-expressing or CD177-depleted sera. After 12 days of culture, cells were collected, counted and evaluated for colony growth, and for flow cytometry analysis of myeloid, erythroid, megakaryocytic and CD34-positive cell subpopulations. qRT-PCR studies showed a close relationship between CD177 mRNA level and percentage of JAK2 V617F alleles (r=0.412, P<0.001). CD177 mRNA expression was almost undetectable in cell populations other than granulocytes. Studies of CFU-GM growth and differentiation indicated that CD177 mRNA expression is a late event restricted to the neutrophil stage of differentiation. Analysis of serum samples showed variable values for mean fluorescence intensity (MFI), indicating variable levels of the soluble CD177 protein in the patients studied. A very close relationship was found between granulocyte CD177 mRNA expression and soluble CD177 protein level (r=0.56, P=0.02). Incubation of mononuclear cells with serum samples showing high levels of soluble CD177 protein resulted in increased numbers of CD34-positive cells (P<0.02) and of erythroid progenitors (P<0.03). This effect was not detectable when low CD177-expressing or CD177-depleted sera were employed. These observations clearly indicate that the JAK2 V617F mutation is associated with enhanced granulocyte CD177 mRNA expression, and that this latter results in high levels of soluble CD177 protein. These elevated levels might contribute to the increased red cell production that characterizes polycythemia vera.

Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry.

Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme-linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell-bound antigen can be advantageous. Magnetic bead depletion of neutrophils in whole blood with CD16b antibody-conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand. Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1-50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen. The competitive antibody binding assay for shed and cell-bound CD16b antigen can be applied to similar GPI-linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available.

Localization and immunogenicity of a low molecular weight antigen of Eimeria tenella.

A low molecular weight (LMW) antigen recognized by a murine monoclonal antibody (C(3)4F(1)) was localized within endogenous stages of Eimeria tenella (USDA strain 80). Using indirect fluorescent antibody assay and immunoelectron microscopy, the LMW antigen was found in: sporozoites, first, second and third generation meronts, gamonts, unsporulated oocysts, and sporocysts. The antigen was observed in the cytoplasm and pellicle of the parasite, and in the parasitophorous vacuole, sporocyst walls and cytoplasm of infected host cells. The immunogenicity of this LMW antigen was assessed by antigen-specific serum antibody responses in chickens orally inoculated with live oocysts or injected intramuscularly with dead sporozoites. LMW antigen-specific serum antibodies were detected using Western blots of E. tenella sporozoites as early as 4 days after sporozoite injection and 6 days after oocyst inoculation. Unusually, the monoclonal antibody C(3)4F(1) reduced the binding of immune chicken serum to the antigen in a competitive antibody binding assay, but not the reverse, suggesting that there is a single, immunodominant epitope on this antigen.

Antigenic epitope characterization of matrix protein of Newcastle disease virus using monoclonal antibody approach: Contrasting variability amongst NDV strains

A panel of 15 monoclonal antibodies (MABs) against matrix (M) protein of Newcastle disease virus (NDV) was obtained and the specificity towards the M protein was proven by radioimmunoprecipitation assay and antigen capture enzyme-linked immunosorbent assay (ELISA). Further studies were directed to antigenic epitope mapping of the M protein by means of this panel. The epitope characterization was performed by competitive antibody-binding assay by means of labelling each MAB with biotin [3]. At least three clear non-overlapping and two partially overlapping groups were determined, each including four, one, eight, one, and one MAB, respectively. All the above MABs appeared to be induced by structural epitopes formed in conditions of tertiary structure of the native M antigen. Twelve reference and 51 recently isolated local NDV strains have been studied by means of this MAB panel, several lineages having been revealed. The high stability of some epitopes and different variability of the others was demonstrated. No correlation between the above lineages and some other properties of the studied NDV strains (host specificity, date and place of isolation) has been found.

Control of β1 Integrin Function: LOCALIZATION OF STIMULATORY EPITOPES

The beta1 integrins can be expressed on the surface of cells in a latent form, which is activated by a variety of stimuli. As an approach to examining the transition to an active receptor, a panel of stimulatory antibodies to beta1 were produced and characterized. These antibodies induced adherence of the T-leukemic cell line Jurkat to collagen and fibronectin. Competitive antibody binding assays indicated the existence of at least three distinct epitope clusters A (B3B11, JB1B, 21C8), B (B44, 13B9), and C(N29) defined by the indicated antibodies. Two antibodies to the A site, JB1B and B3B11, were shown to localize to positions 671-703 and 657-670, respectively, of the beta1. This region is located in an area encompassing a predicted disulfide bond between linearly distant cysteines in beta1 (Cys415-Cys671). The homologous region of the beta3 integrin (490 690 and 602 690) has been shown to be one of the sites recognized by stimulatory antibodies to ligand-induced binding sites. The present results indicate the existence of multiple stimulatory regions and suggest considerable homology between the locations of beta1 and beta3 regulatory sites.

Strain variation and nuclear association of Newcastle disease virus matrix protein.

Five monoclonal antibodies to the matrix (M) protein of Newcastle disease virus (NDV) Australia-Victoria (AV) strain were generated and characterized. In competitive antibody-binding assays, the antibodies fell into three discrete groups. The antigenic sites described by these antibody groups were designated M1, M2, and M3. Each antibody reacted with a panel of NDV strains in a manner unique to its group, confirming the grouping by competitive antibody binding. Only site M1 was found on all 12 of the strains tested and may be a "pan-NDV" epitope. A large portion of the M protein of strain AV was detected in the nuclei of infected cells by all five monoclonal antibodies. In addition, the antibodies only stained the nuclei of cells infected with NDV strains expressing M protein containing the corresponding antigenic site. These results confirm that the immunoreactivity in the nucleus is actually caused by the M protein and not by a cross-reacting host protein induced by viral infection.

Characterization of monoclonal antibodies to a crystal protein of Bacillus thuringiensis subsp. kurstaki.

Ten monoclonal antibodies were produced against a k-1-type crystal protein of Bacillus thuringiensis subsp. kurstaki. Eight of the antibodies belong to the immunoglobulin G1 (IgG1) subclass, with pI values ranging from 5.5 to 8.6, one could be assigned to the IgG2b subclass, and one could be assigned to the IgM class. Competitive antibody-binding assays and analysis of antibody specificity indicated that the 10 antibodies recognized at least nine distinct antigenic determinants. Eight antibodies bound to both protoxin and toxin, whereas the other two interacted with protoxin only. One antibody completely inhibited the biological activity of the delta-endotoxin, five antibodies reduced it by 15 to 82%, and four antibodies did not affect it at all. Based on cross-reaction studies, homologies and differences in the crystal protein structures of different B. thuringiensis subspecies were revealed. All of the monoclonal antibodies strongly cross-reacted with crystal proteins from strains of B. thuringiensis subsp. tolworthi, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. dendrolimus, B. thuringiensis subsp. sotto, and B. thuringiensis subsp. subtoxicus. Some antibodies interacted only weakly with crystal proteins from strains of B. thuringiensis subsp. morrisoni and B. thuringiensis subsp. entomocidus, and some of these did not interact with B. thuringiensis subsp. kenyae and B. thuringiensis subsp. darmstadiensis. No cross-reaction was found with the parasporal inclusion protein of B. thuringiensis subsp. israelensis. Studies with the monoclonal antibodies also disclosed that crystal proteins from strains of the same subspecies can exhibit substantial differences in antigenic structure. In particular, striking strain-specific differences in the protoxins of B. thuringiensis subsp. kurstaki and B. thuringiensis subsp. thuringiensis were observed.

New Monoclonal Antibodies That Define Multiple Epitopes and a Human‐Specific Marker on the Interleukin 2 Receptor Molecules of Primates

Twelve hybridoma cell lines producing monoclonal antibodies to the human interleukin 2 (IL-2) receptor (IL-2R) molecule were prepared. These antibodies were characterized by competitive antibody-binding assay and sequential immunoprecipitation assay with four known monoclonal antibodies to the human IL-2R molecule. The twelve new monoclonal antibodies were divided among the four known antibody types, the HIEI-, H-A26-, H-31-, and anti-Tac-type, and an additional new type, the H-48-type. The H-48 antibody did not compete with any other antibodies in the competitive binding assay. The binding of 125I-IL-2 to MT-2 cells and the IL-2-dependent growth of normal activated T-cells were both strongly inhibited by all the H-31- and anti-Tac-type antibodies, and partially or slightly inhibited by HIEI- and H-A26-type antibodies, but were not inhibited by the H-48 antibody. Thus, the same type of monoclonal antibodies had a similar effect on the function of IL-2R. These results suggest that epitopes for the same type of antibodies could be single identical epitopes or epitopes closely associated with each other. On the other hand, these antibodies also reacted variously with a panel of various human and simian lymphoid cell lines immortalized with human T-cell leukemia virus type-I (HTLV-I): the H-45 antibody reacted only with the human cell lines, the H-C1 and H-44 and H-47 antibodies reacted with human and ape cell lines, and the other antibodies reacted with cell lines of humans, apes and Old and New World monkeys. These differences in the reactivity of the antibodies with the primate cell lines suggest that the antigenic structure of the IL-2R molecule changed during evolutionary divergence of the primates.

Topographical analysis of bovine herpesvirus type-1 glycoproteins: Use of monoclonal antibodies to identify and characterize functional epitopes

Monoclonal antibodies, specific for two of the major bovine herpesvirus type-1 (BHV-1) glycoproteins, GVP 3 9 and GVP 6/11a/16, have been previously produced and characterized. The reactivity of each monoclonal antibody was determined by enzyme-linked immunosorbent assay (ELISA), the ability to neutralize viral infectivity, and the capacity to mediate complement-dependent (AbC′) lysis of virus-infected cells. The topography of epitopes on GVP 3 9 and GVP 6/11a/16 was analyzed, using selected monoclonal antibodies in a competitive antibody binding assay (CBA). Nine epitopes were identified on GVP 3 9 . Comparison of the biological activities and epitope specificities of the monoclonal antibodies against GVP 3 9 showed that one epitope was involved in virus neutralization, whereas six epitopes mediated AbC′ lysis of the virus-infected cell. Of the seven epitopes identified on GVP 6/11a/16, six were involved in neutralization and three participated in AbC′ lysis. In some cases, partial competition between monoclonal antibodies was observed, indicating that they were directed against overlapping or closely adjacent epitopes. In other instances, asymmetrical competition between monoclonal antibodies suggested conformational changes in the glycoprotein molecule induced by antibody binding. Mixtures of two monoclonal antibodies with different epitope specificities resulted in higher neutralizing activity than either antibody alone, suggesting that multiple monoclonal antibodies can exert a synergistic effect on virus neutralization.

Discrimination of epitopes identified by monoclonal antibodies by competitive binding to nitrocellulose bound antigens

A new method is described for determining the distribution of epitopes identified by monoclonal antibodies. The method utilizes nitrocellulose membranes as a solid support for antigens which are rapidly adsorbed to nitrocellulose by vacuum-blotting and then used in competitive antibody binding assays. The distribution of epitopes is established by the reciprocal cross-blocking of radiolabeled antibody by increasing concentrations of unlabeled antibody. When unlabeled antibody does not block the binding of labeled antibody to antigen, the 2 antibodies recognize distinct epitopes. When unlabeled antibody blocks the binding of labeled antibody to antigen, the 2 antibodies recognize the same epitope. The method is rapid, sensitive and should be applicable to screening monoclonal antibodies to any epitope.

Epitopic analysis of antigenic determinants on the surface of dengue-2 virions using monoclonal antibodies.

The relative binding sites of dengue serotype-specific, dengue subcomplex-specific, dengue complex-specific, flavivirus subgroup-reactive, and flavivirus group-reactive monoclonal antibody preparations were identified by using competitive antibody binding assays. A dengue complex-specific epitope, capable of mediating infection enhancement, was identified on a 20,000 dalton protein found on intracellular virions. The other epitopes were assigned relative positions on the E glycoprotein by competitive antibody binding. These could be grouped into 3 linkage groups based on the ability of some monoclonal antibodies to block contiguous binding sites. Some antibodies were able to increase or "promote" the binding of antibodies from other linkage groups. These results suggest that a continuum of antigenic reactivities exist on the E glycoprotein of the dengue viruses, and that the conformation of this glycoprotein may be altered after antibody binding.

Topologic mapping of protective and nonprotective epitopes on the variant surface glycoprotein of the WRATat 1 clone of Trypanosoma brucei rhodesiense.

Monoclonal antibodies were used in competitive antibody binding assays to define and map epitopes on the variant surface glycoprotein of the WRATat 1 clone of T. b. rhodesiense. By using a panel of 30 WRATat 1-specific monoclonal antibodies, 16 epitopes were defined that fall into four clusters, having 1, 1, 3, and 11 distinct epitopes respectively. All epitopes were easily classified as being 1) exposed uniformly on the surface of the trypanosome, 2) exposed only in the region of the flagellar pocket, or 3) "buried", based on the ability or inability of the monoclonal antibodies to bind living trypanosomes in a fluid phase immunofluorescence assay. Monoclonal antibodies that bind exposed surface epitopes are protective, whereas only three of seven that bind exclusively to flagellar pocket epitopes are protective. None of the nine monoclonal antibodies that recognize buried epitopes are protective. Also, antibody-mediated immunity to WRATat 1 trypanosomes is not associated with any particular subclass of antibody. The IgM, IgG1, IgG2a, IgG2b, IgG3, and IgA subclasses each contain examples of protective monoclonal antibodies.

Antigenic characterization of influenza A virus matrix protein with monoclonal antibodies.

Monoclonal antibodies were used to study antigenic variation in three distinct epitopes on the matrix protein of influenza A viruses. We found that two of these epitopes underwent antigenic variation, but in a very limited number of virus strains. A third epitope appeared to be an invariant type-specific determinant for influenza A viruses. Competitive antibody binding assays and Western blot analysis of proteolytically digested matrix protein indicated that at least two of the three epitopes are located in nonoverlapping domains on the matrix protein molecule.