Change in the accessibility of an epitope of the human granulocyte-colony stimulating factor after binding to receptors

In a previous work we demonstrated that monoclonal antibody (mAb) 8C2 recognized a human granulocyte-colony stimulating factor (hG-CSF) region left unmasked after binding to placenta receptors, whereas mAb 6E3 defined a receptor-buried epitope. Herein we examined the role of these antigenic regions on the proliferative response induced by hG-CSF on a myeloid leukaemia cell line. Both mAbs significantly inhibited the hG-CSF-induced cell growing, although epitope 8C2 but not 6E3 remained exposed in hG-CSF:cell receptor complexes. When cytokine:receptor complexes already formed at 4 degrees C were incubated 1 h at 37 degrees C under conditions preventing the internalization, a significant reduction in the amount of accessible 8C2 epitopes was evident. However, this effect was not observed when mAb 8C2:hG-CSF complexes previously bound to cells were incubated at 37 degrees C. Thus, results suggest that a receptor oligomerization process could account for the temperature-induced epitope 8C2 masking. The identification of epitope 8C2 accomplished by synthesis of overlapping octapeptides, revealed that it is formed by sequences 39-52 and 155-164, both in close proximity in the three-dimensional structure of the hG-CSF molecule. Since part of this region has been proposed as a second binding site to receptors, we infer that the change of epitope 8C2 accessibility could be the result of either receptor aggregation or epitope binding to another receptor. In addition, our data support the hypothesis that a ligand-induced receptor oligomerization is required for transduction of cytokine signals.     

Identification of a linear epitope of interferon-alpha2b recognized by neutralizing monoclonal antibodies.

Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-alpha2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.     

Mapping of conformational mAb epitopes to the C domain of human angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.     

Characteristics of monoclonal antibody binding with the C domain of human angiotensin converting enzyme

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied and the inhibition of the enzyme by the mAb 4E3 was found. The dissociation constants of complexes of two mAbs, IB8 and 2H9, with tACE were 2.3 +/- 0.4 and 2.5 +/- 0.4 nM, respectively, for recombinant tACE and 1.6 +/- 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) IB3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.     

N-succinimidyl 3-[(131)I]iodo-4-phosphonomethylbenzoate ([(131)I]SIPMB), a negatively charged substituent-bearing acylation agent for the radioiodination of peptides and mAbs

An important criterion in design of acylation agents for the radioiodination of internalizing monoclonal antibodies (mAbs) is to maximize the retention of radioiodine in the tumor following mAb intracellular processing. We have previously shown that labeling methods that generate positively charged catabolites have enhanced tumor retention. Herein we have extended this strategy to investigate the potential utility of labeling internalizing mAbs with an acylation agent that yielded labeled catabolites that would be negatively charged at lysosomal pH. The negatively charged acylation agent, N-succinimidyl 3-[(131)I]iodo-4-phosphonomethylbenzoate ([(131)I]SIPMB), was prepared from its tin precursor, N-succinimidyl 4-di-tert-butylphosphonomethyl-3-trimethylstannylbenzoate (tBu-SPMTB), in 40% radiochemical yield. The free acid, 3-[(131)I]iodo-4-phosphonomethylbenzoic acid ([(131)I]IPMBA), was also prepared from the corresponding precursor, 4-di-tert-butylphosphonomethyl-3-trimethylstannylbenzoic acid (tBu-PMTBA), in 80% radiochemical yield. The rapidly internalizing mAb L8A4 was conjugated to [(131)I]SIPMB in 25-40% yield with preservation of its immunoreactivity. Internalization and processing in the U87DeltaEGFR glioma cell line was studied in a paired label format with L8A4 labeled with (125)I using the Iodogen method. Retention of initially bound radioactivity in these cells at 24 h from [(131)I]SIPMB-labeled mAb was approximately 6-fold higher than that for directly labeled mAb. Catabolite analysis demonstrated that this difference reflected an order of magnitude higher retention of low molecular weight species in these cells. The [(131)I]SIPMB-L8A4 conjugate was intact over the first 2 h; thereafter, lysine-[(131)I]SIPMB was the predominant catabolite. In contrast, L8A4 labeled using Iodogen rapidly gave rise to mono-[(125)I]iodotyrosine within 2 h, which then cleared rapidly from the cells. These results suggest that SIPMB could be a potent candidate for labeling internalizing mAbs and warrant further study.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs). Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries, displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and 4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4 mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized a continoous topographic epitope.     

Characteristics of monoclonal antibody binding with the <Emphasis Type="Italic">C</Emphasis> domain of human angiotensin converting enzyme

Binding of a panel of eight monoclonal antibodies (mAbs) with the C domain of angiotensin converting enzyme (ACE) to human testicular ACE (tACE) (corresponding to the C domain of the somatic enzyme) was studied, and the inhibition of the enzyme by the mAb 4A3 was found. The dissociation constants of complexes of two mAbs, 1B8 and 2H9, with tACE were 2.3 ± 0.4 and 2.5 ± 0.4 nM, respectively, for recombinant tACE and 4.7 ± 0.5 and 1.6 ± 0.3 nM for spermatozoid tACE. Competition parameters of mAb binding with tACE were obtained and analyzed. As a result, the eight mAbs were divided into three groups, whose binding epitopes did not overlap: (1) 1E10, 2B11, 2H9, 3F11, and 4E3; (2) 1B8 and 3F10; and (3) 1B3. A diagram demonstrating mAb competitive binding with tACE was proposed. Comparative analysis of mAb binding to human and chimpanzee ACE was carried out, which resulted in revealing of two amino acid residues, Lys677 and Pro730, responsible for binding of three antibodies, 1E10, 1B8, and 3F10. It was found by mutation of Asp616 located close to Lys677 for Leu that the mAb binding epitope 1E10 contains Asp616 and Lys677, whereas mAbs 1B8 and 3F10 contain Pro730.     

The role of antibody polyspecificity and lipid reactivity in binding of broadly neutralizing anti-HIV-1 envelope human monoclonal antibodies 2F5 and 4E10 to glycoprotein 41 membrane proximal envelope epitopes

Two neutralizing human mAbs, 2F5 and 4E10, that react with the HIV-1 envelope gp41 membrane proximal region are also polyspecific autoantibodies that bind to anionic phospholipids. To determine the autoantibody nature of these Abs, we have compared their reactivities with human anti-cardiolipin mAbs derived from a primary antiphospholipid syndrome patient. To define the role of lipid polyreactivity in binding of 2F5 and 4E10 mAbs to HIV-1 envelope membrane proximal epitopes, we determined the kinetics of binding of mAbs 2F5 and 4E10 to their nominal gp41 epitopes vs liposome-gp41 peptide conjugates. Both anti-HIV-1 mAbs 2F5 and 4E10 bound to cardiolipin with K(d) values similar to those of autoimmune anti-cardiolipin Abs, IS4 and IS6. Binding kinetics studies revealed that mAb 2F5 and 4E10 binding to their respective gp41 peptide-lipid conjugates could best be defined by a two-step (encounter-docking) conformational change model. In contrast, binding of 2F5 and 4E10 mAbs to linear peptide epitopes followed a simple Langmuir model. A mouse mAb, 13H11, that cross-blocks mAb 2F5 binding to the gp41 epitope did not cross-react with lipids nor did it neutralize HIV-1 viruses. Taken together, these data demonstrate the similarity of 2F5 and 4E10 mAbs to known anti-cardiolipin Abs and support the model that mAb 2F5 and 4E10 binding to HIV-1 involves both viral lipid membrane and gp41 membrane proximal epitopes.     

A monoclonal antibody to beta 1 integrin (CD29) stimulates VLA- dependent adherence of leukocytes to human umbilical vein endothelial cells and matrix components

The leukocyte beta 1 integrin receptor very late activation antigen-4 (VLA-4) (alpha 4 beta 1, CD49d/CD29) binds to vascular cell adhesion molecule-1 (VCAM-1) expressed on cytokine-activated endothelium. A mAb designated 8A2 was identified that stimulated the binding of U937 cells to CHO cells transfected with VCAM-1 cDNA but not endothelial-leukocyte adhesion molecule or CD4 cDNA. mAb 8A2 also rapidly stimulated the adherence of peripheral blood lymphocytes (PBLs) to VCAM-1-transfected CHO cells or recombinant human tumor necrosis factor-treated human umbilical vein endothelial cells. mAb 8A2-stimulated binding of PBL was inhibited by mAbs to VLA-4 or VCAM-1. Surface expression of VLA-4 was not altered by mAb 8A2 treatment and monovalent Fab fragments of mAb 8A2 were active. Immunoprecipitation studies reveal that mAb 8A2 recognizes beta 1-subunit (CD29) of integrin receptors. In contrast to mAbs directed to VLA-4 alpha-subunit (alpha 4, CD49d), mAb 8A2 did not induce homotypic aggregation of PBL. Additionally, mAb 8A2 stimulated adherence of PBL and hematopoietic cell lines to purified matrix components laminin and fibronectin. This binding was blocked by mAbs to the VLA alpha-subunits alpha 6 (CD49f), or alpha 5 (CD49e) and alpha 4 (CD49d), respectively. We conclude that mAb 8A2 modulates the affinity of VLA-4 and other leukocyte beta 1 integrins, and should prove useful in studying the regulation of beta 1 integrin function.     

Comparative studies of the antigens recognized by sperm-immobilizing monoclonal antibodies.

Characteristic properties of the antigens recognized by sperm-immobilizing monoclonal antibodies (SI-mAbs) from different sources were compared by ELISA competitive inhibition assay, Western blot analysis, chromatographic analysis, and enzymatic digestion studies. Among 9 SI-mAbs, human mAb H6-3C4 and three mouse mAbs--2C6, 2B6, and 2E5--also possessed strong sperm-agglutinating activity. Binding of human mAb H6-3C4 to sperm was strongly inhibited by the three mouse mAbs (2C6, 2B6, and 2E5), but not by the rat or the other four mouse mAbs. SDS-PAGE revealed that mAb H6-3C4 and three mouse mAbs recognized the same antigen molecules of 15-25 kDa present in both sperm extracts and seminal plasma. Chemical treatments with trifluoromethanesulfonic acid and sodium metaperiodate destroyed the antigen determinants recognized by the above four mAbs, as detected by both ELISA and antibody absorption tests. Western blot analysis revealed that the antigens were susceptible to treatments with papain, proteinase K, and N-glycanase, but resistant to trypsin, V8 protease, and thermolysin. These results indicate that one of the major antigens recognized by mAbs with sperm-immobilizing action may be a sperm membrane-associated glycoprotein of 15-25 kDa and the epitope may involve N-linked oligosaccharides.     

A specific l-tri-iodothyronine-binding protein in the cytosol fraction of human breast adipose tissue

1. Binding of l-tri-[(125)I]iodothyronine to the cytosol fraction of normal human female breast adipose tissue was investigated by the charcoal adsorption method. Equilibrium of binding was reached after 120s at 25 degrees C. 2. The l-tri-[(125)I]iodothyronine-binding component is a protein; this was confirmed by experiments in which binding was totally lost after heating the cytosol fraction for 10min at 100 degrees C and in which binding was diminished after treatment with proteolytic enzymes and with thiol-group-blocking reagents. The binding protein was stable at -38 degrees C for several months. 3. It displayed saturability, high affinity (apparent K(d) 3.28nm) and a single class of binding sites. 4. High specificity for l-tri-iodothyronine and l-3,5-di-iodo-3'-isopropylthyronine was observed, whereas other iodothyronines were less effective in displacing l-tri-[(125)I]-iodothyronine from its binding site. 5. The binding of the hormone by the cytosol fraction did not show a pH optimum. 6. When cytosol fractions of adipose tissue from different females were subjected to radioimmunoassay for the determination of thyroxine-binding globulin a value of 0.304+/-0.11mug/mg of cytosol protein (mean+/-s.d., n=4) was obtained; the mean concentration in plasma was 0.309+/-0.07mug/mg of plasma protein (mean+/-s.d., n=3). 7. The K(a) value of 6.3x10(8)m(-1) of l-tri-[(125)I]iodothyronine for binding to plasma, the similar thermalinactivation profiles of binding and the reactivity to thiol-group-blocking reagents were some properties common between the binding components from the cytosol fraction and plasma. 8. These results suggest that the cytosol fraction of human female breast adipose tissue contains thyroxine-binding globulin; the protein that binds l-tri-[(125)I]iodothyronine with high affinity and specificity appears to be similar to thyroxine-binding globulin.     

Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.     

Distinct immunohistochemical localization in Kuru plaques using novel anti-prion protein antibodies

By immunizing Prnp-knockout mice with synthetic polypeptides, a panel of mAbs directed to bovine PrP(C) was obtained. The mAb panel was characterized by the ELISA method, where synthetic polypeptides were used for epitope mapping. Different reactivity patterns were identified. The ability of these mAbs to detect abnormal PrP(Sc) in CJD cases was studied by immunohistochemistry. All mAbs were tested for PrP(Sc) in murine, bovine, monkey and human brain tissues. Three mAbs recognized the fragmented PrP epitope in our ELISA. Antibody 1D12 was strongly reactive to ovine and squirrel monkey tissues infected with a scrapie agent, although non-reactive to scrapie-infected mouse tissues. Antibody 2D8 was clearly reactive to type-2 but not type-1 CJD human tissues. Of particular interest was the reactivity of mAb 6C4 with the inner structure of Kuru plaques (peripheral pattern) in a type-2 CJD case and mAb T2, 1D12, 2B11, 2D8, 4B5 and 6G3-2 with the central area (central pattern). The fact that different anti-PrP mAbs possess distinct staining properties suggests that the PrP(c) to PrP(Sc) conversion might involve a multiple-step process.     

Insulin-like growth factor (IGF)-I binding to a cell membrane associated IGF binding protein-3 acid-labile subunit complex in human anterior pituitary gland

The binding characteristics of [(125) I]insulin-like growth factor (IGF)-I were studied in human brain and pituitary gland. Competition binding studies with DES(1-3)IGF-I and R(3) -IGF-I, which display high affinity for the IGF-I receptor and low affinity for IGF binding proteins (IGFBPs), were performed to distinguish [(125) I]IGF-I binding to IGF-I receptors and IGFBPs. Specific [(125) I]IGF-I binding in brain regions and the posterior pituitary was completely displaced by DES(1-3)IGF-I and R(3) -IGF-I, indicating binding to IGF-I receptors. In contrast, [(125) I]IGF-I binding in the anterior pituitary was not displaced by DES(1-3)IGF-I and R(3) -IGF-I, suggesting binding to an IGF-binding site that is different from the IGF-I receptor. Binding affinity of IGF-I to this site was about 10-fold lower than for the IGF-I receptor. Using western immunoblotting we were also unable to detect IGF-I receptors in human anterior pituitary. Instead, western immunoblotting and immunoprecipitation experiments showed a 150-kDa IGFBP-3-acid labile subunit (ALS) complex in the anterior pituitary and not in the posterior pituitary and other brain regions. RT-PCR experiments showed the expression of ALS mRNA in human anterior pituitary indicating that the anterior pituitary synthesizes ALS. In the brain regions and posterior pituitary, IGFBP-3 was easily washed away during pre-incubation procedures as used in the [(125) I]IGF-I binding experiments. In contrast, the IGFBP-3 complex in the anterior pituitary could not be removed by these washing procedures. Our results indicate that the human anterior pituitary contains a not previously described tightly cell membrane-bound 150-kDa IGFBP-3-ALS complex that is absent in brain and posterior pituitary.     

IL-12: monoclonal antibodies specific for the 40-kDa subunit block receptor binding and biologic activity on activated human lymphoblasts.

IL-12, formerly known as cytotoxic lymphocyte maturation factor, is a cytokine that stimulates proliferation of PHA-activated human peripheral blood lymphoblasts and synergizes with low concentrations of IL-2 in the induction of lymphokine-activated killer cells. IL-12 is a 75-kDa heterodimer composed of disulfide-bonded 40-kDa and 35-kDa subunits. mAb prepared against a partially purified preparation of natural IL-12 have been characterized by 1) immunoprecipitation of 125I-labeled IL-12, 2) immunodepletion of IL-12 bioactivity, 3) Western blotting of IL-12, 4) inhibition of [125I]IL-12 binding to its cellular receptor, and 5) neutralization of IL-12 bioactivity. Twenty antibodies immunoprecipitate 125I-labeled IL-12 and immunodeplete IL-12 bioactivity as assessed in the T cell proliferation and lymphokine-activated killer cell induction assays. Western blot analysis demonstrated that each antibody binds to the 75-kDa heterodimer and to the 40-kDa subunit. An IL-12R-binding assay identified 12 individual antibodies that inhibited the binding of [125I]IL-12 to its cellular receptor. Two inhibitory antibodies, 4A1 and 7B2, were tested in the neutralization assay and found to block IL-12 bioactivity whereas one noninhibitory antibody, 8E3, was shown not to neutralize IL-12 bioactivity. Antibodies 4A1 and 8E3 can simultaneously bind to the 75-kDa heterodimer demonstrating that inhibitory and noninhibitory epitopes are spatially distinct on the 40-kDa protein. The ability of antibodies specific for the 40-kDa subunit of IL-12 to block receptor binding of [125I]IL-12 and to neutralize IL-12 bioactivity suggests that localized determinants on the 40-kDa subunit may be necessary for binding to the IL-12 cellular receptor.     

Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

ABSTRACT: BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20--57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.     

Non-isopeptide-selective endothelin receptors in human Girardi heart cells.

We characterized the endothelin (ET) receptor in Girardi heart (GH) cells derived from human atrium. The ET isopeptides ET-1, ET-2 and ET-3 induced the monotonous and long-lasting rise in cytosolic free Ca2+ concentration [( Ca2+]i) with almost the same potency in GH cells. Scatchard analysis of [125I]ET-1 and [125I]ET-3 binding revealed that GH cells have almost the same number of binding sites for either labeled ligand. All ET isopeptides displaced either [125I]ET-1 or [125I]ET-3 binding in GH cells almost equipotently. These results reveal that the functional ET receptors in GH cells are of the ETB-type. GH cells are the first cell line to be found to express the functional ETB-receptor.     

Synthesis and evaluation of C-11, F-18 and I-125 small molecule radioligands for detecting oxytocin receptors

Compounds 1-4 were synthesized and investigated for selectivity and potency for the oxytocin receptor (OTR) to determine their viability as radioactive ligands. Binding assays determined 1-4 to have high binding affinity for both the human and rodent OTR and also have high selectivity for the human OTR over human vasopressin V1a receptors (V1aR). Inadequate selectivity for OTR over V1aR was found for rodent receptors in all four compounds. The radioactive (C-11, F-18, and I-125) derivatives of 1-4 were synthesized and investigated for use as autoradiography and positron emission tomography (PET) ligands. Receptor autoradiography performed with [(125)I]1 and [(125)I]2 on rodent brain slices provided the first small molecule radioligand images of the OTR and V1aR. Biodistribution studies determined [(125)I]1 and [(125)I]2 were adequate for in vivo peripheral investigations, but not for central investigations due to low uptake within the brain. A biodistribution study with [(18)F]3 suggested brain uptake occurred slowly over time. PET imaging studies with [(18)F]3 and [(11)C]4 using a rat model provided insufficient uptake in the brain over a 90 and 45 min scan times respectively to merit further investigations in non-human primates.     

Generation of anti-idiotypic and anti-anti-idiotypic monoclonal antibodies in the same fusion. Support of Jerne's Network Theory

Upon immunization of mice with a mAb (290A-167) directed against an epitope of Lol p I (the major allergenic determinant of Lolium perenne), both anti-idiotypic (aId) mAb (Ab2) and anti-aId mAb (Ab3) were produced. The Ab2 displayed the following internal image properties of Lol p I: it can be affinity-purified on an immobilized Id column; its binding to the anti-Lol p I mAb (290A-167) is inhibited by Lol p I; it inhibits in a dose-response fashion the binding of the specific Id to Ag. It is recognized by anti-Lol p I antisera from different species such as mouse, human, and goat. The Ab3 which binds to Lol p I was also produced from the same fusion. This binding was inhibited significantly by aId mAb (Ab2), anti-Lol p I mAb (290A-167) and Lol p I. These data indicate that the two mAb with specificity for Lol p I (290A-167 and Ab3) share similar reactivity to the Ag and that aId mAb is the internal image of the epitope recognized by the Id. We showed also that the capacity of rabbit aId Ab directed against the 290A-167 Id to inhibit the binding of Ab1 and Ab3 to Ag was almost abolished by passage over a Ab3-coated Sepharose column. This would suggest that not only are the two mAb with reactivity to Lol p I (Ab1 and Ab3) directed against identical epitopes, but that they in fact shared identical idiotopes as well. The production of identical mAb upon immunization with either the Ag or the aId mAb supports that the conceptual framework proposed by Jerne finds its biologic application in the course of an immune response.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Phage peptide libraries constitute powerful tools for themapping of epitopes recognized by monoclonal antibodies (mAbs).Using screening of phage displayed random peptide libraries wehave characterized the binding epitopes of three mAbs directedagainst the surface envelope glycoprotein (gp46) of the humanT-cell leukemia virus type I (HTLV-I). Two phage libraries,displaying random heptapeptides with or without flankingcysteine residues, were screened for binding to mAbs 7G5D8, DB4and 4F5F6. The SSSSTPL consensus sequence isolated fromconstrained heptapeptide library defines the epitope recognizedby DB4 mAb and corresponds to the exact region 249–252 of thevirus sequence. The APPMLPH consensus sequence isolated fromnon constrained heptapeptide library defines the epitoperecognized by 7G5D8 mAb and corresponds to the region 187–193with a single amino acid substitution, methionine to leucine atposition 190. The third consensus sequence LYWPHD isolated fromconstrained heptapeptide library defines the epitope recognizedby 4F5F6 mAb. It corresponds to an epitope without directequivalence with the virus sequence. The data presented hereshowed that 7G5D8 and DB4 mAbs are raised against linearepitopes while 4F5F6 mAb recognized a continuous topographic epitope.