Structural requirements for high efficiency endocytosis of the human transferrin receptor.

Wild-type and mutant human transferrin receptors (TR) have been expressed in chicken embryo fibroblasts using a helper-independent retroviral vector. By functional studies of the mutant TRs, we have identified the tetrapeptide sequence, YXRF, in the cytoplasmic tail of the receptor as the internalization signal required for high efficiency endocytosis and shown that transplanted internalization signals from the low density lipoprotein receptor (LDLR) and the cation-independent mannose-6-phosphate receptor (Man-6-PR) are able to promote rapid internalization of the human TR. A six-residue LDLR signal, FDNPVY, is required for activity in TR, whereas a four-residue Man-6-PR signal, YSKV, is sufficient. These data indicate that internalization signals are interchangeable self-determined structural motifs and that signals from type I membrane proteins are active in a type II receptor. Putative internalization signals in the cytoplasmic tails of other receptors and membrane proteins can be identified based on the sequence patterns of the LDLR, Man-6-PR, and TR signals. Two such putative four-residue internalization signals, one from the poly-Ig receptor and one from the asialoglycoprotein receptor, were tested for activity by transplantation into TR and were found to promote high efficiency internalization. These results suggest that an exposed tight turn is the conformational motif for high efficiency endocytosis.     

Mapping of a dominant immunogenic region of synaptophysin, a major membrane protein of synaptic vesicles.

Synaptophysin is a major integral membrane protein of synaptic vesicles. Its transmembrane topology deduced from the cDNA sequence predicts 4 transmembrane regions and a carboxy-terminal cytoplasmic tail containing a characteristic pentapeptide repeat structure. The monoclonal antibody (mAb), SY38, binds to a cytoplasmic domain of synaptophysin. By using fusion proteins corresponding to truncated forms of the cytoplasmic tail, its epitope was located to a flexible segment in the center of the repeat structure. Four other mAbs (c7.1, c7.2, c7.3, c7.4) share the same epitope, which thus emerges as the major immunogenic region of this membrane protein.     

The tyrosine phosphorylation site of the acetylcholine receptor beta subunit is located in a highly immunogenic epitope implicated in channel function: antibody probes for beta subunit phosphorylation and function.

Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) seems to be involved in AChR desensitization and localization on the postsynaptic membrane. This study reveals a probable function of the single known beta subunit phosphorylation site (beta Tyr355) and provides suitable tools for its study. The epitopes for 15 monoclonal antibodies (mAbs) against the cytoplasmic side of the AChR beta subunit were precisely mapped using > 100 synthetic peptides attached on polyethylene rods. Eleven mAbs bound to a very immunogenic cytoplasmic epitope (VICE-beta) on Torpedo beta 352-359, which contains the beta Tyr355, and to the corresponding sequence of human AChR. The contribution of each VICE-beta residue to mAb binding was then studied by peptide analogues having single residue substitutions. Overall, each of the residues beta 354-359, including beta Tyr355, proved critical for mAb binding. Two of our four mAbs known to block the ion channel were found to bind at (mAb148) or close (mAb10) to VICE-beta. Tyrosine phosphorylation of Torpedo AChR by endogenous kinase(s) selectively reduced binding of some VICE-beta mAbs, including the channel blocking mAb148. We conclude that VICE-beta probably plays a key role in AChR function. Elucidation of this role should be facilitated by the identified mAb tools.     

Transplanted LDL and mannose-6-phosphate receptor internalization signals promote high-efficiency endocytosis of the transferrin receptor.

The internalization signals of several constitutively recycling receptors have recently been identified as regions of four or six amino acids that include an aromatic residue, usually tyrosine. Here, we show that transplanted signals from the low density lipoprotein receptor (LDLR) and cation-independent mannose-6-phosphate receptor (Man-6-PR) promote rapid internalization of the transferrin receptor (TR), directly establishing that recognition signals are interchangeable, self-determined structural motifs and that signals from type I membrane proteins are active in a type II receptor. We also show that the chemical and spatial patterns of critical residues in both four- and six-residue internalization motifs are consistent with a tight turn structure. A six-residue LDLR signal is needed for activity in TR, suggesting that an amino-terminal aromatic side chain is obligatory. In contrast, the carboxy-terminal aromatic side chain in the TR signal can be replaced by a large hydrophobic residue. Thus, internalization signals apparently require an aromatic amino-terminal residue and either an aromatic or large hydrophobic carboxy-terminal residue rather than a conserved tyrosine per se. Consistent with this conclusion, predicted internalization signals from the poly-Ig receptor, YSAF, and asialoglycoprotein receptor (ASGPR) subunit H1, YQDL, also promote internalization of TR.     

Structural analysis of chick ephrin-A2 by function-blocking and non-blocking monoclonal antibodies

Ephrins, ligands for the Eph family of receptor tyrosine kinases, play key roles in diverse biological processes. In this study, we determined the epitopes and kinetic parameters of function-blocking (B3) and non-blocking (IV) monoclonal antibodies (mAbs) recognizing chick ephrin-A2. We show that the epitope for the non-blocking mAb is the residue Asp(105) of chick ephrin-A2. However, the binding of the function-blocking mAb depends mostly on residue Ser(108) and its epitope may reside within residues 105-132, which appear crucial for the receptor interaction site. Kinetic studies suggest a possible mechanism why mAb IV, despite recognizing a region very close to the mAb B3 epitope, fails to block the ligand-receptor interaction.     

Effects of monoclonal antibodies on the function of acetylcholine receptors purified from Torpedo californica and reconstituted into vesicles

We tested the effects of 62 monoclonal antibodies (mAbs) to acetylcholine receptors from Torpedo californica on the function of receptor reconstituted into lipid vesicles. Two of these mAbs, mAbs 148 and 168, inhibited carbamylcholine-induced 22Na+ uptake into vesicles. The rate of 125I-labeled alpha-bungarotoxin (125I-alpha BGT) binding to the reconstituted liposomes was also reduced, although 125I-alpha BGT binding at equilibrium was not affected. Agonist-induced desensitization of the receptor was also affected by these mAbs. mAb 148 binds to the beta subunit of receptor, and mAb 168 binds to the gamma subunit. Both mAbs bind to the cytoplasmic surface of the receptor; correspondingly, both block function when added before reconstitution, and both were found to have no effect on function when added to preformed vesicles. Their effects were not due to interference with the reconstitution process. Both mAbs were capable of cross-linking receptors. In contrast to the bivalent mAbs, monovalent Fab fragments of these two mAbs had little effect on receptor function, which suggests that the effects of the bivalent mAbs resulted primarily from cross-linking receptors.     

Monoclonal antibodies to soluble human TNF receptor (TNF binding protein) enhance its ability to block TNF toxicity.

A soluble extracellular fragment of the human 55-60 kDa tumor necrosis factor receptor (sTNF-R I), originally isolated from urine, binds both TNF-alpha and TNF-beta and blocks the activity of these cytokines in biological assays. Three monoclonal antibodies (mAbs) raised against sTNF-R I (TBP-1, -2 and -6) as well as a mAb developed by immunization with the intact receptor (H398) were analysed for their epitope specificities in ELISAs and for biological activity in cytotoxicity assays on murine L-M cells. TBP-2 and H398 bind to related epitopes on sTNF-R I; they compete with TNF-alpha for binding and block the protective effect of sTNF-R I in the bioassay. MAbs TBP-1 and TBP-6 recognize two further, independent epitopes; both bind sTNF-R I in the presence of an excess of TNF-alpha. Both TBP-1 and TBP-6 markedly enhance the ability of sTNF-R I to protect cells against the cytotoxic activities of TNF-alpha and TNF-beta, but have no activity in the absence of sTNF-R I. Fab fragments show much lower activity. We propose that the ability of certain mAbs to enhance the protective activity of sTNF-R is due to a steric hindrance phenomenon.     

Casein kinase II activity is required for transferrin receptor endocytosis.

The effect of protein kinase inhibitors on transferrin receptor (TR) internalization was examined in HeLa, A431, 3T3-L1 cells, and primary chicken embryo fibroblasts. We show that TR endocytosis is not affected by tyrosine kinase or protein kinase C inhibitors, but is inhibited by one serine/threonine kinase inhibitor, H-89. Inhibition occurred within 15 min, was completely reversible after H-89 withdrawal, and was specific for endocytosis rather than pinocytosis since a TR mutant lacking an internalization signal was not affected. Interestingly, H-89 also inhibited the internalization of a TR chimera containing the major histocompatibility complex class II invariant chain cytoplasmic tail, indicating that the effect was not specific for the TR. Since H-89 inhibits a number of kinases, we employed a permeabilized cell endocytosis assay to further characterize the kinase. In permeabilized 3T3-L1 cells, addition of pseudosubstrate inhibitor peptides of casein kinase II (CKII) blocked TR internalization by more than 50%, whereas pseudosubstrates of cyclic AMP-dependent kinase A, protein kinase C, and casein kinase I had no effect. Furthermore, addition of purified CKII to the cell-free reactions containing CKII pseudosubstrates reversed the endocytosis block, suggesting that CKII or a CKII-like activity is required for constitutive endocytosis.     

Epitope mapping using the X-ray crystallographic structure of complement receptor type 2 (CR2)/CD21: identification of a highly inhibitory monoclonal antibody that directly recognizes the CR2-C3d interface.

Complement receptor type 2 (CR2)/CD21 is a B lymphocyte cell membrane C3d/iC3b receptor that plays a central role in the immune response. Human CR2 is also the receptor for the EBV viral membrane glycoprotein gp350/220. Both C3d and gp350/220 bind CR2 within the first two of 15-16 repetitive domains that have been designated short consensus/complement repeats. Many mAbs react with human CR2; however, only one currently available mAb is known to block both C3d/iC3b and gp350/220 binding. We have used a recombinant form of human CR2 containing the short consensus/complement repeat 1-2 ligand-binding fragment to immunize Cr2(-/-) mice. Following fusion, we identified and further characterized four new anti-CR2 mAbs that recognize this fragment. Three of these inhibited binding of CR2 to C3d and gp350/220 in different forms. We have determined the relative inhibitory ability of the four mAbs to block ligand binding, and we have used overlapping peptide-based approaches to identify linear epitopes recognized by the inhibitory mAbs. Placement of these epitopes on the recently solved crystal structure of the CR2-C3d complex reveals that each inhibitory mAb recognizes a site either within or adjacent to the CR2-C3d contact site. One new mAb, designated 171, blocks CR2 receptor-ligand interactions with the greatest efficiency and recognizes a portion of the C3d contact site on CR2. Thus, we have created an anti-human CR2 mAb that blocks the C3d ligand by direct contact with its interaction site, and we have provided confirmatory evidence that the C3d binding site seen in its crystal structure exists in solution.     

Epitope mapping of conformational monoclonal antibodies specific to NhaA Na+/H+ antiporter: structural and functional implications

The recently determined crystal structure of NhaA, the Na ⁺/H ⁺ antiporter of Escherichia coli, showed that the previously constructed series of NhaA-alkaline phosphatase (PhoA) fusions correctly predicted the topology of NhaA's 12 transmembrane segments (TMS), with the C- and N-termini pointing to the cytoplasm. Here, we show that these NhaA-PhoA fusions provide an excellent tool for mapping the epitopes of three NhaA-specific conformational monoclonal antibodies (mAbs), of which two drastically inhibit the antiporter. By identifying which of the NhaA fusions is bound by the respective mAb, the epitopes were localized to small stretches of NhaA. Then precise mapping was conducted by targeted Cys scanning mutagenesis combined with chemical modifications. Most interestingly, the epitopes of the inhibitory mAbs, 5H4 and 2C5, were identified in loop X-XI (cytoplasmic) and loop XI-XII (periplasmic), which are connected by TMS XI on the cytoplasmic and periplasmic sides of the membrane, respectively. The revealed location of the mAbs suggests that mAb binding distorts the unique NhaA TMS IV/XI assembly and thus inhibits the activity of NhaA. The noninhibitory mAb 6F9 binds to the functionally dispensable C-terminus of NhaA.     

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.     

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity

Background

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity.

Methods and Principal Findings

Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity.

Conclusions

The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.     

Epitope mapping of novel monoclonal antibodies to human angiotensin I‐converting enzyme

Angiotensin I‐converting enzyme (ACE, CD143) plays a crucial role in blood pressure regulation, vascular remodeling, and immunity. A wide spectrum of mAbs to different epitopes on the N and C domains of human ACE have been generated and used to study different aspects of ACE biology, including establishing a novel approach–conformational fingerprinting. Here we characterized a novel set of 14 mAbs, developed against human seminal fluid ACE. The epitopes for these novel mAbs were defined using recombinant ACE constructs with truncated N and C domains, species cross‐reactivity, ACE mutagenesis, and competition with the previously mapped anti‐ACE mAbs. Nine mAbs recognized regions on the N domain, and 5 mAbs–on the C domain of ACE. The epitopes for most of these novel mAbs partially overlap with epitopes mapped onto ACE by the previously generated mAbs, whereas mAb 8H1 recognized yet unmapped region on the C domain where three ACE mutations associated with Alzheimer''s disease are localized and is a marker for ACE mutation T877M. mAb 2H4 could be considered as a specific marker for ACE in dendritic cells. This novel set of mAbs can identify even subtle changes in human ACE conformation caused by tissue‐specific glycosylation of ACE or mutations, and can detect human somatic and testicular ACE in biological fluids and tissues. Furthermore, the high reactivity of these novel mAbs provides an opportunity to study changes in the pattern of ACE expression or glycosylation in different tissues, cells, and diseases, such as sarcoidosis and Alzheimer''s disease.     

Inhibition of angiogenesis by a novel neutralizing antibody targeting human VEGFR-3

Vascular endothelial growth factor receptor 3 (VEGFR-3) is a receptor for the vascular endothelial growth factor C and D (VEGF-C and D) and plays a critical role in the development of embryonic vascular system and regulation of tumor lymphangiogenesis. In this report, we generated a novel panel of 17 monoclonal antibodies (mAbs) against human VEGFR-3 and determined their ability to inhibit the proliferation of human erythroleukemia (HEL) cells and angiogenesis of chick embryo chorioallantoic membrane (CAM). Among these mAbs, BDD073 was demonstrated to inhibit the interaction of soluble VEGFR-3 with VEGF-D and the proliferation of HEL cells. Furthermore, in chick embryo CAM angiogenesis experiments, the angiogenesis induced by recombinant glutathione-S-transferase-VEGF-D was decreased in the presence of antibody BDD073. These data suggest that this novel neutralizing antibody against human VEGFR-3 could be a tool for the investigations into the biology of VEGFR-3, and potentially a reagent for blocking VEGF-D-induced angiogenesis and lymphogenesis.     

Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.     

Therapeutic potential and challenges of targeting receptor tyrosine kinase ROR1 with monoclonal antibodies in B-cell malignancies

Background

Based on its selective cell surface expression in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), receptor tyrosine kinase ROR1 has recently emerged as a promising target for therapeutic monoclonal antibodies (mAbs). To further assess the suitability of ROR1 for targeted therapy of CLL and MCL, a panel of mAbs was generated and its therapeutic utility was investigated.

Methodology and Principal Findings

A chimeric rabbit/human Fab library was generated from immunized rabbits and selected by phage display. Chimeric rabbit/human Fab and IgG1 were investigated for their capability to bind to human and mouse ROR1, to mediate antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and internalization, and to agonize or antagonize apoptosis using primary CLL cells from untreated patients as well as MCL cell lines. A panel of mAbs demonstrated high affinity and specificity for a diverse set of epitopes that involve all three extracellular domains of ROR1, are accessible on the cell surface, and mediate internalization. The mAb with the highest affinity and slowest rate of internalization was found to be the only mAb that mediated significant, albeit weak, ADCC. None of the mAbs mediated CDC. Alone, they did not enhance or inhibit apoptosis.

Conclusions and Significance

Owing to its relatively low cell surface density, ROR1 may be a preferred target for armed rather than naked mAbs. Provided is a panel of fully sequenced and thoroughly characterized anti-ROR1 mAbs suitable for conversion to antibody-drug conjugates, immunotoxins, chimeric antigen receptors, and other armed mAb entities for preclinical and clinical studies.     

CD84 functions as a homophilic adhesion molecule and enhances IFN-gamma secretion: adhesion is mediated by Ig-like domain 1

CD84 is a member of the CD2 subset of the Ig superfamily of cell surface molecules. Its cytoplasmic tail binds to Src homology 2 domain-containing protein 1A (signaling lymphocytic activation molecule-associated protein), a protein encoded by the X-linked lymphoproliferative disease gene. It is preferentially expressed on B lymphocytes, monocytes, and platelets. We show that it is also expressed on thymocytes and T cells. CD84 was positive on CD4-CD8- thymocytes, and its expression decreased with cell maturation. It is expressed on mature T cells preferentially on CD45RO+. To identify the CD84 ligand, we generated a soluble Ig fusion protein containing the human CD84 extracellular domains (CD84-Ig). Because receptor-ligand interactions occur between several members of this subfamily, we assayed CD84-Ig binding with all members of the CD2 family. CD84-Ig bound to CD84-transfected cells, whereas no binding was detected with cells expressing other CD2 subfamily receptors, showing that CD84 binds to itself. Anti-CD84 mAbs recognizing epitopes wholly within domain 1 of CD84 blocked the binding of the CD84-Ig fusion protein to CD84-transfected cells and platelets. Data from CD84 domain human/mouse chimeras further revealed that only the first extracellular domain of the molecule is involved in the ligand receptor recognition. The CD84-CD84 interaction was independent of its cytoplasmic tail. Finally, concurrent ligation of human CD84 with mAbs or CD84-Ig and CD3 enhanced IFN-gamma secretion in human lymphocytes. Thus, CD84 is its own ligand and acts as a costimulatory molecule.     

Localization of conserved B-cell epitopes among encapsulated and non-encapsulated Haemophilus influenzae P2 porin proteins using synthetic peptides.

The P2 outer membrane protein of Haemophilus influenzae belongs to a class of apparently ubiquitous proteins in Gram-negative bacteria that function as porins. Murine hybridomas raised to the P2 protein and synthetic peptides were used to investigate the structural and antigenic relationships among P2 proteins of encapsulated and non-encapsulated H. influenzae. Three monoclonal antibodies (mAbs), P2-17, P2-18 and P2-19, recognizing epitopes on the P2 protein, as shown by Western immunoblotting of outer membrane preparations, and purified and recombinant P2 proteins are described. The epitopes reactive with the mAbs were widely distributed among H. influenzae strains since 70-100% of strains of encapsulated and non-encapsulated isolates collected worldwide were recognized by individual mAbs. None of the mAbs reacted with H. parainfluenzae or other bacterial species. The peptide composition of P2 epitopes was determined by analysis of mAb reactivity with a series of overlapping synthetic peptides that covered the amino acid sequences of H. influenzae type b. The domains recognized by these mAbs were completely distinct. mAb P2-18, reactive with an epitope conserved among all H. influenzae P2 porin molecules which were screened, recognized a peptide corresponding to the N-terminal segment (residues 1-14). The P2-17- and P2-19-specific epitopes were located between residues 28 and 55, and 101 and 129, respectively. None of the epitopes were exposed on the cell surface since no mAbs bound to intact live bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation of cytoplasmic segments of acetylcholine receptor alpha- and beta-subunits probed by monoclonal antibodies: sensitivity of the antibody competition approach.

The conformation of the cytoplasmic side of Torpedo marmorata acetylcholine receptor (AChR) was investigated by 22 monoclonal antibodies (mAbs) binding to known sites on the amino acid sequences 339-378 and 336-469 of the AChR alpha- and beta-subunits respectively. Competitions among these mAbs for binding on the intact AChR were compared with their competition for binding on the SDS-denatured subunits and with their corresponding epitopes previously determined on the primary structure of the subunits. We found the following: The three approaches correlated very well suggesting that these mAbs bind on the intact AChR at the same sequences determined by synthetic peptides and not on irrelevant discontinuous epitopes; this finding supports conclusions of Ratnam et al. (1986a) that the amphipathic helix M5 is exposed on the cytoplasmic side of the AChR. The subunit segments alpha 339-378 and beta 336-469 seem to be extended over large distances on the cytoplasmic surface of the AChR. The cytoplasmic surface of beta-subunit has a very immunogenic region. The mAb-competition technique is very sensitive since mAbs to epitopes separated by only about seven residues did not exclude each other, and mAbs to overlapping epitopes exhibited differential competitions with other mAbs.     

In vitro expression and monoclonal antibody of RNA-dependent RNA polymerase for infectious bursal disease virus

VP1, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV), has been suggested to play an essential role in the replication and translation of viral RNAs. In this study, we first expressed the complete VP1 protein gene in Escherichia coli (E. coli), and then the produced polyclonal antibody and four monoclonal antibodies (mAbs) to recombinant VP1 protein (rVP1) were shown to bind the IBDV particles in chicken embryo fibroblast and Vero cells. The epitopic analysis showed that mAbs 1D4 and 3C7 recognized respectively two distinct antigenic epitopes on the rVP1 protein, but two pair of mAbs 1A2/2A12 and 1E1/1H3 potentially recognized another two topologically related epitopes. Immunocytochemical stainings showed that VP1 protein formed irregularly shaped particles in the cytoplasm of the IBDV-infected cells. These results demonstrated that the mAbs to rVP1 protein could bind the epitopes of IBDV particles, indicating that the rVP1 protein expressed in E. coli was suitable for producing the mAb to VP1 protein of IBDV, and that the cytoplasm could be the crucial site for viral genome replication of IBDV.