3D modeling and characterization of the human CD115 monoclonal antibody H27K15 epitope and design of a chimeric CD115 target

The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.     

3D modeling and characterization of the human CD115 monoclonal antibody H27K15 epitope and design of a chimeric CD115 target

The humanized monoclonal antibody H27K15 specifically targets human CD115, a type III tyrosine kinase receptor involved in multiple cancers and inflammatory diseases. Binding of H27K15 to hCD115 expressing cells inhibits the functional effect of colony-stimulating factor-1 (CSF-1), in a non-competitive manner. Both homology modeling and docking programs were used here to model the human CD115 extracellular domains, the H27K15 variable region and their interaction. The resulting predicted H27K15 epitope includes mainly the D1 domain in the N-terminal extracellular region of CD115 and some residues of the D2 domain. Sequence alignment with the non-binding murine CD115, enzyme-linked immunosorbent assay, nuclear magnetic resonance spectroscopy and affinity measurements by quartz crystal microbalance revealed critical residues of this epitope that are essential for H27K15 binding. A combination of computational simulations and biochemical experiments led to the design of a chimeric CD115 carrying the human epitope of H27K15 in a murine CD115 backbone that is able to bind both H27K15 as well as the murine ligands CSF-1 and IL-34. These results provide new possibilities to minutely study the functional effects of H27K15 in a transgenic mouse that would express this chimeric molecule.     

Expression and characterization of a recombinant Fab fragment derived from an anti-human alpha-fetoprotein monoclonal antibody

Alpha-fetoprotein (AFP) is a well-known molecular marker indicating the development of cancer as well as fetal abnormalities such as open neural tube defects. Accordingly the measurement of serum AFP is important for the diagnosis of hepatocellular carcinoma (HCC) and other abnormalities. Monoclonal antibodies (McAb) to AFP were produced to develop an immunoassay kit, and to study the possibility of an antibody (Ab) therapy. The immunoglobulin genes were cloned from hybridoma cells, and expressed in E. coli as an Fab soluble into a culture medium. The Fab of anti-AFP McAb exhibited binding to AFP and similar affinity compared to the original IgG. This recombinant antibody can be studied further for in vivo imaging and immunotherapeutics.     

Fine-epitope mapping of an antibody that binds the ectodomain of influenza matrix protein 2

Our previous work found that the monoclonal antibody 8C6, which recognized the epitope EVETPIRN on influenza A virus M2 protein, conferred protection against influenza virus challenge. In this study, 8C6 was used to screen the 7-mer phage peptide library in order to identify the crucial amino acid residues on the protective epitope EVETPIRN. Nine positive phage clones were selected by a test of dose-dependent binding activity to 8C6 after three rounds of panning. The phage clones exhibited a consensus motif (TXXR), which was found on the epitope EVETPIRN. Site-directed mutation analysis indicated that Thr and Arg on the epitope EVETPIRN played a key role in the recognition by 8C6. Furthermore, sequence alignment and analysis revealed that Thr and Arg on the epitope were highly conserved. Our results could provide useful information for influenza vaccine design based on M2 mimotope.     

Ultrastructural characterization of embryonic chick cartilage proteoglycan core protein and the mapping of a monoclonal antibody epitope.

The ultrastructure of embryonic chick cartilage proteoglycan core protein was investigated by electron microscopy of specimens prepared by low angle shadowing. The molecular images demonstrated a morphological substructural arrangement of three globular and two linear regions within each core protein. The internal globular region (G2) was separated from two terminally located globular regions (G1 and G3) by two elongated strands with lengths of 21 +/- 3 nm (E1) and 105 +/- 22 nm (E2). The two N-terminal globular regions, separated by the 21-nm segment, were consistently visualized in well spread molecules and showed little variation in the length of the linear segment connecting them. The E2 segment, however, was quite variable in length, and the C-terminal globular region (G3) was detected in only 53% of the molecules. The G1, G2, and G3 regions in chick core protein were 10.1 +/- 1.7 nm, 9.7 +/- 1.3 nm, and 8.3 +/- 1.3 nm in diameter, respectively. These results are similar to those described previously for proteoglycan core proteins isolated from rat chondrosarcoma, bovine nasal cartilage, and pig laryngeal cartilage (Paulsson, M., Morgelin, M., Wiedemann, H., Beardmore-Gray, M., Dunham, D., Hardingham, T., Heinegard, D., Timpl, R., and Engel, J. (1987) Biochem. J. 245, 763-772). However, a significant difference was detected between the length of the elongated strand (E2) of core proteins isolated from chick cartilage, E2 length = 105 +/- 22 nm, compared to bovine nasal cartilage, E2 length = 260 +/- 39 nm. The epitope of the proteoglycan core protein-specific monoclonal antibody, S103L, was visualized by electron microscopy, and the distance from the core protein N terminus to the S103L binding site was measured. The S103L binding site was localized to the E2 region, 111 +/- 20 nm from the G1 (N terminus) domain and 34 nm from the G3 (C terminus) domain. cDNA clones selected from an expression vector library of chicken cartilage mRNA also show this epitope to be located near the C-terminal region (R. C. Krueger, T. A. Fields, J. Mensch, and B. Schwartz (1990) J. Biol. Chem. 265, 12088-12097).     

Mechanism of cholesteryl ester transfer protein inhibition by a neutralizing monoclonal antibody and mapping of the monoclonal antibody epitope

The plasma cholesteryl ester transfer protein (CETP, Mr 74,000) has a binding site for neutral lipid which can readily equilibrate with lipoprotein cholesteryl esters or triglycerides. Recently, a monoclonal antibody (TP2) was obtained which neutralizes the cholesteryl ester (CE) and triglyceride (TG) transfer activities of the CETP. In this report, the epitope of the inhibitory monoclonal antibody has been localized to a hydrophobic 26-amino acid sequence at the COOH terminus of CETP. The Fab fragments of TP2 caused partial (50%) inhibition of CE transfer and complete inhibition of TG transfer by the CETP. Similarly, the Fab fragments inhibited (37%) the binding of CE to the CETP and abolished the binding of TG to the CETP. Surprisingly, the TP2 Fab was also found to enhance the binding of CETP to plasma lipoproteins and to phospholipid vesicles. In conclusion, the TP2 monoclonal antibody inhibits lipid transfer by blocking the uptake of lipid by CETP. The COOH-terminal epitope may be in or near the neutral lipid binding site. Occupancy of this site by TP2 Fab fragments or by neutral lipid may result in a conformational change of CETP causing enhanced binding to lipoproteins or vesicles.     

Biochemical characterization of the molecules reactive with Qa-6 antiserum and the monoclonal antibody 20-8-4: evidence for structural similarity with the Qa-2 molecule

The Qa-6 alloantigen and the molecule that crossreacts with the monoclonal antibody (mAb) 20-8-4 have been shown to be serologically distinct from the Qa-2 alloantigen by strain distribution and tissue distribution, respectively. In this report, we address the biochemical relationships among Qa-2, Qa-6, and the 20-8-4 cross-reactive molecule by using immunoprecipitation and polyacrylamide gel electrophoresis. Each of these molecules had an apparent m.w. of approximately 41K and was associated on the cell surface with beta 2-microglobulin. Removal of N-linked oligosaccharides with endoglycosidase F reduced their apparent m.w. to approximately 33K to 34K. The determinants recognized by anti-Qa-6 and mAb 20-8-4 were shown to reside on the same molecule(s) precipitated by anti-Qa-2 sera by immunodepletion experiments. The mAb 20-8-4 was also shown to preclear the molecules detected by the Qa-6 and Qa-2 antisera. Two-dimensional gel electrophoresis analysis demonstrated complete co-migration of the approximately 41K molecules detected by the three antibodies. By peptide map analysis with V8 protease, all three molecules appeared identical. Also, the determinant recognized by Qa-6 antiserum co-modulated with that recognized by the anti-Qa-2 mAb D3.262. Taken together, these results demonstrate that the molecules recognized by these three antisera and/or mAb are biochemically indistinguishable. These data, in conjunction with the serologic and genetic findings suggest that mAb 20-8-4 recognizes a molecule that is biochemically similar and possibly identical to the Qa-2 antigen. Moreover, although the genetic, serologic, and biochemical data demonstrate that Qa-6 is not controlled by the Qa-2 locus, but rather by a gene telomeric to Qa-2, the molecule bearing the Qa-6 determinant is very similar, if not identical, to the Qa-2 molecule. Several possible explanations for these discrepancies are discussed.     

日本血吸虫重组信号蛋白14—3—3的纯化及单克隆抗体的制备

纯化日本血吸虫（中国大陆株）重组信号蛋白（rSj14—3—3）,并制备其单克隆抗体。以纯化后的rsj14-3—3蛋白为抗原免疫Balb／c小鼠,用杂交瘤技术制备抗rSj14-3-3的单克隆抗体,并通过ELISA方法和Westernblotting测定抗体的效价与特异性。获得了大量高纯度的rSj14-3-3蛋白：筛选出了能够稳定分泌抗rSj14．3．3单抗的杂交瘤细胞株3H6。单抗亚型为IgG1。实验依靠大肠杆菌表达系统高效表达了rSj14—3—3蛋白,并利用该蛋白制备了单克隆抗体．可用于今后血吸虫病免疫诊断的实验研究。     

Characterization of a macaque recombinant monoclonal antibody that binds to a CD4-induced epitope and neutralizes simian immunodeficiency virus

A potent neutralizing Fab fragment from a long-term survivor of simian immunodeficiency virus (SIVsm) infection was used to construct a recombinant macaque immunoglobulin G1kappa (IgG1kappa) molecule, designated IgG1-201. A Chinese hamster ovary cell line expressing IgG1-201 was derived by stable transfection and optimized for antibody secretion by methotrexate selection and dihydrofolate reductase gene amplification. IgG1-201 effectively neutralized the homologous, molecularly cloned SIVsmH4 virus but had no activity against the heterologous SIVmac251/BK28 virus. The previously characterized, neutralization-resistant SIVsmE543-3 virus was also not neutralized by IgG1-201. Binding to SIVsmH4 gp120 was enhanced in the presence of recombinant soluble CD4, suggesting that IgG1-201 bound a CD4-induced epitope. IgG1-201 immunoprecipitated the SIVsmH4 but not the SIVsmE543-3 envelope despite a close relationship between these two clones. Immunoprecipitation of a panel of SIVsmH4/SIVsmE543-3 chimeric viruses tentatively assigned the neutralization epitope to the third constant domain, immediately C terminal to the V3 loop. These findings suggest the presence of at least one CD4-induced neutralization epitope on SIV, as is the case with human immunodeficiency virus type 1.     

Use of human CD3 monoclonal antibody for accurate CD4+ and CD8+ lymphocyte determinations in macaques: phenotypic characterization of the CD3- CD8+ cell subset

Macaque monkeys are frequently used in models for studies of infectious diseases, immunity, transplantation and vaccine development. Such use is largely due to the conservation of functionally important cell surface molecules and the phylogenetic proximity of their immune systems to that of humans. Some monoclonal antibodies (mAb) raised against human leukocyte antigens can be utilized in the monkey. Until recently, many primate centers have utilized the CD2 monoclonal antibody to enumerate T lymphocytes. We have evaluated the anti-human CD3 mAb in macaques and sooty mangabeys. Using this monoclonal antibody, pigtailed macaques were found to have a much higher proportion of CD2+ CD3- CD8+ cells as compared with rhesus macaques and sooty mangabeys. Such cells comprised approximately one-half of all CD8+ cells in the pigtailed macaque, but only one-quarter of CD8+ cells in the rhesus, and one-fifth in the sooty mangabey. Use of the CD2 monoclonal antibody as the T-cell marker resulted in underestimating CD4/CD8 ratios compared with using the CD3 mAb in pigtailed macaques. Phenotypic characterization of this subset of CD3- CD8+ cells indicated that they are CD16+, CD45RA+, CD11b+, CD69+ and CD28-. This would indicate that these cells represent an activated natural killer cell subset.     

Fine mapping of an epitope recognized by an invasion-inhibitory monoclonal antibody on the malaria vaccine candidate apical membrane antigen 1

Antibodies that inhibit red blood cell invasion by the Plasmodium merozoite block the erythrocytic cycle responsible for clinical malaria. The invasion-inhibitory monoclonal antibody (mAb) 4G2 recognizes a conserved epitope in the ectodomain of the essential Plasmodium falciparum microneme protein and vaccine candidate, apical membrane antigen 1 (PfAMA1). Here we demonstrate that purified Fab fragments of 4G2 inhibit invasion markedly more efficiently than the intact mAb, suggesting that the invasion-inhibitory activity of this mAb is not due solely to steric effects and that the epitope lies within a functionally critical region of the molecule. We have taken advantage of a synthetic gene encoding a modified form of PfAMA1, and existing x-ray crystal structure data, to fully characterize this epitope. We first validate the gene by demonstrating that it fully complements the function of the authentic gene in P. falciparum.We then use it to identify a group of residues within the previously described domain II loop of PfAMA1 that are critical for recognition by mAb 4G2 and demonstrate that the epitope lies exclusively within this loop with no contributions from residues in other domains of the molecule. This is the first complete characterization of a conserved invasion-inhibitory epitope on PfAMA1. Our results will aid in the design of subunit vaccines designed to generate a broadly effective, focused anti-PfAMA1 protective immune response and may help elucidate the function of PfAMA1.     

Expression and characterization of soluble and membrane-bound human nucleoside triphosphate diphosphohydrolase 6 (CD39L2)

Ecto-nucleoside-triphosphate diphosphohydrolase-6 (eNTPDase6(1), also known as CD39L2) cDNA was expressed in mammalian COS-1 cells and characterized using nucleotidase assays as well as size exclusion, anion exchange, and cation exchange chromatography. The deduced amino acid sequence of eNTPDase6 is more homologous with the soluble E-type ATPase, eNTPDase5, than other E-type ATPases, suggesting it may also be soluble. To test this possibility, both the cell membranes and the growth media from eNTPDase6-transfected COS-1 cells were assayed for nucleotidase activities. Activity was found in both the membranes and the media. Soluble eNTPDase6 preferentially exhibits nucleoside diphosphatase activity, which is dependent on the presence of divalent cations. Western blot analysis of eNTPDase6 treated with PNGase-F indicated both soluble and membrane-bound forms are glycosylated. However, unlike some membrane-bound ecto-nucleotidases, the eNTPDase6 activity was not specifically inhibited by deglycosylation with peptide N-glycosidase F. Soluble eNTPDase6 hydrolyzed nucleoside triphosphates poorly and nucleoside monophosphates not at all. Analysis of the relative rates of hydrolysis of nucleoside diphosphates (GDP = IDP > UDP > CDP > ADP) suggests that soluble eNTPDase6 is a diphosphatase most likely not involved in regulation of ADP levels important for circulatory hemostasis.     

Identification of functional regions on the tail of Acanthamoeba myosin- II using recombinant fusion proteins. I. High resolution epitope mapping and characterization of monoclonal antibody binding sites

We used a series of COOH-terminally deleted recombinant myosin molecules to map precisely the binding sites of 22 monoclonal antibodies along the tail of Acanthamoeba myosin-II. These antibodies bind to 14 distinguishable epitopes, some separated by less than 10 amino acids. The positions of the binding sites visualized by electron microscopy agree only approximately with the physical positions of these sites on the alpha-helical coiled-coil tail. On the other hand, the epitope map agrees precisely with competitive binding studies: all antibodies that share an epitope compete with each other for binding to myosin. Antibodies with adjacent epitopes can compete with each other at linear distances up to 5 or 6 nm, and many antibodies that bind 3-7- nm apart can enhance the binding of each other to myosin. Most of the antibodies that bind to the distal 37 nm of the tail disrupt assembly of octameric minifilaments and, depending upon the exact location of the binding site, stop assembly at specific steps yielding, for example, monomers, antiparallel dimers, parallel dimers or antiparallel tetramers. The effects of these antibodies on assembly identify sites on the tail that are required for individual steps in minifilament assembly. Experiments on the assembly of truncated myosin-II tails have revealed a complementary group of sites that participate in the assembly reactions (Sinard, J.H., D.L. Rimm, and T.D. Pollard. 1990. J. Cell Biol. 111:2417-2426). Antibodies that bind to the distal tail but do not affect assembly appear to have a low affinity for myosin-II. Antibodies that bind to the proximal 50 nm of the tail do not inhibit the assembly of minifilaments. Many antibodies that bind to the tail of myosin-II, even some that have no obvious effect on minifilament assembly, can inhibit the actomyosin ATPase activity and the contraction of an actin gel formed in crude extracts. An antibody that binds between amino acids 1447 and 1467 inhibits the phosphorylation of serine residues distal to residue 1483.     

Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope.

A monoclonal antibody was produced to the exterior envelope glycoprotein (gp120) of the human T-cell lymphotropic virus (HTLV)-IIIB isolate of the human immunodeficiency virus (HIV). This antibody binds to gp120 of HTLV-IIIB and lymphadenopathy-associated virus type 1 (LAV-1) and to the surface of HTLV-IIIB- and LAV-1-infected cells, neutralizes infection by cell-free virus, and prevents fusion of virus-infected cells. In contrast, it does not bind, or weakly binds, the envelope of four heterologous HIV isolates and does not neutralize heterologous isolates HTLV-IIIRF and HTLV-IIIMN. The antibody-binding site was mapped to a 24-amino-acid segment, using recombinant and synthetic segments of HTLV-IIIB gp120. This site is within a segment of amino acid variability known to contain the major neutralizing epitopes (S. D. Putney, T. J. Matthews, W. G. Robey, D. L. Lynn, M. Robert-Guroff, W. T. Mueller, A. J. Langlois, J. Ghrayeb, S. R. Petteway, K. J. Weinhold, P. J. Fischinger, F. Wong-Staal, R. C. Gallo, and D. P. Bolognesi, Science 234:1392-1395, 1986). These results localize an epitope of HIV type-specific neutralization and suggest that neutralizing antibodies may be effective in controlling cell-associated, as well as cell-free, virus infection.     

Fasciola gigantica: Production and characterization of a monoclonal antibody against recombinant cathepsin B3

A number of monoclonal antibodies (MoAbs) against a recombinant cathepsin B3 (rCatB3) of Fasciola gigantica were produced in BALB/c mice. Reactivity and specificity of these MoAbs were assessed by indirect ELISA and immunoblotting techniques. Six stable clones, namely 1C4, 1E9, 2E5, 2F9, 5B4, 5D7 were obtained. All MoAbs reacted with rCatB3 at molecular weight (MW) 37 kDa as well as the glycosylated peptide at 55–75 kDa and with the native CatB3 at MW 37 kDa in WB extracts of metacercariae (Met) and newly excysted juveniles (NEJ). It was found to be IgG₁ and λ light chain isotypes. Immunolocalization of CatB3 in metacercariae, NEJ, 4-week-old juvenile and adult F. gigantica performed by immunoperoxidase technique by using these MoAbs as probes indicated that CatB3 was present in high concentration in the caecal epithelium and caecal lumen of the Met and NEJ, but not in the 4-week-old juvenile and adult fluke. The MoAbs show no cross-reactions with antigens of other parasites including Gigantocotyl explanatum, Eurytrema pancreaticum, Paramphistomum cervi, Schistosoma spindale, S. mansoni, Haemonchus placei and Setaria labiato-papillosa. Thus, it is possible that these MoAbs could be a good candidate for immunodiagnosis of fasciolosis.     

Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody.

The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success. We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E. coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4). HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+. We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade. Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody. The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place. This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins. Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself. Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor.     

A monoclonal antibody recognizing an epitope shared by receptors for growth hormone,prolactin, interleukin 2 and interleukin 6

Many membrane proteins are implicated in the regulation of cell functions by triggering specific signaling pathways. Porins are known potential modulators of cell proliferation and differentiation. We explored the possible involvement of this protein in signal transduction pathways in mouse gut macrophages. In the present work we have shown that porins can trigger signal transduction in mouse macrophages infected with S. typhimurium. Activation of macrophages by porins results in an increase in inositol trisphosphate and intracellular Ca2+ mobilization. There is a translocation of protein kinase C to the membrane which is accompanied by nitric oxide release within the macrophages. This effect is the outcome of the expression of nitric oxide synthase, which is dependent on Protein kinase C. Further, we observed that there is an increased binding of the porins on macrophages infected with S. typhimurium which results in activation of macrophages and triggering of specific signaling pathways. These results indicate that porins induce the production of nitric oxide via a protein kinase C dependent pathway. Nitric oxide plays a fundamental role in macrophage effector function where it has both communication and defensive function.     

Immunochemical characterization of six monoclonal antibodies to human apolipoprotein A-I: epitope mapping and expression.

We have produced and characterized six murine monoclonal antibodies to human apolipoprotein A-I named A-I-9, A-I-12, A-I-15, A-I-16, A-I-19, and A-I-57. All monoclonal antibodies were specific for apolipoprotein A-I and bound between 55% and 100% of 125I-labeled high density lipoproteins (HDL) in a fluid phase radioimmunoassay. All antibodies possessed a higher affinity to apoA-I in HDL than to free, delipidated apoA-I. Two of them, particularly A-I-12 and A-I-15, which were directed to the same or very close epitopes on the molecule, recognized very poorly the delipidated protein. Binding of apoA-I to phospholipid restored the immunoreactivity of the monoclonal antibodies to the protein suggesting that lipids play an important role in determining the immunochemical structure of apoA-I. Using CNBr fragments and synthetic peptides, the epitopes for the antibodies were mapped as follows: A-I-19, CNBr fragment 1; A-I-12 and 15, CNBr fragment 2; A-I-9 and A-I-16, CNBr fragment 3; A-I-57, CNBr fragment 4. Antibody A-I-57 failed to recognized a mutant form of apoA-I, A-IMilano (Arg173----Cys) by immunoblotting and by competitive radioimmunoassay demonstrating that substitution of a single amino acid in human apoA-I may cause the loss of an antigenic determinant.