猪流行性腹泻病毒单克隆抗体及其抗原表位的研究进展

猪流行性腹泻(Porcine epidemic diarrhea,PED)是由猪流行性腹泻病毒(PED virus,PEDV)引起的一种严重危害养猪生产的常见疫病。近年来,由于新的PEDV变异毒株的出现,许多国家的养猪业遭受了巨大的经济损失。PEDV也因此受到更多关注,关于PEDV的研究报道也日渐增多。基于国内外有关PEDV的最新研究进展,本文系统归纳和分析了PEDV结构蛋白和非结构蛋白单克隆抗体以及单克隆抗体识别的特异性抗原表位,以期为开发鉴别诊断方法和表位疫苗等提供信息。     

SARS-CoV单克隆抗体的制备及抗原表位的初步鉴定

参照已发表的SARS冠状病毒BJ01株基因序列 ,利用计算机软件预测并选取该病毒S、M、N三种主要结构蛋白部分抗原性优势区域 ,以编码Gly-Pro-Gly序列相连接合成两段嵌合基因A和B。并分别克隆于pGEX -6p- 1载体上用IPTG进行诱导表达 ,以纯化的嵌合蛋白A和B为抗原 ,分别免疫BALB c小鼠制备单克隆抗体。利用单克隆抗体亚型检测试剂盒和SARS CoV商品化ELISA检测试剂盒对其进行亚型和特异性鉴定。结果表明融合表达两段嵌合基因产物 ,其大小分别为 34kD和35kD ,Westernblot分析证实两种表达产物都能被SARS病人康复期血清所识别。获得了 6株能稳定分泌特异性抗体的阳性细胞克隆株。亚型鉴定结果除D3C5为IgG2a外其他单抗均为IgG1,而且所有单抗的轻链均为κ链。特异性鉴定发现除D3D1外 ,其余的 5株单抗均能与SARS CoV商品化ELISA检测试剂盒发生特异性反应。将D3D1与灭活后经超声波裂解的SARS CoV进行Westernblot分析 ,发现它能特异性识别 180kD的蛋白带。分别融合表达了 6个S蛋白的寡肽 (S1- S6 ) ,并对筛选出的单克隆…     

Analysis of the Epitopes Recognized by Mouse Monoclonal Antibodies Directed to Yersinia enterocolitica Heat-Shock Protein 60

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was perfomed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.     

Unique Epitopes Recognized by Antibodies Induced in Chikungunya Virus-Infected Non-Human Primates: Implications for the Study of Immunopathology and Vaccine Development

Chikungunya virus (CHIKV) is an Alphavirus that causes chronic and incapacitating arthralgia in humans. Although patient cohort studies have shown the production of CHIKV specific antibodies, the fine specificity of the antibody response against CHIKV is not completely defined. The macaque model of CHIKV infection was established due to limitations of clinical specimens. More importantly, its close relation to humans will allow the study of chronic infection and further identify important CHIKV targets. In this study, serum samples from CHIKV-infected macaques collected at different time-points post infection were used to characterize the antibody production pattern and kinetics. Results revealed that anti-CHIKV antibodies were neutralizing and the E2 glycoprotein, Capsid, nsP1, nsP3 and nsP4 proteins were targets of the anti-CHIKV antibody response in macaques. Furthermore, linear B-cell epitopes recognized by these anti-CHIKV antibodies were identified, and mapped to their structural localization. This characterizes the specificity of anti-CHIKV antibody response in macaques and further demonstrates the importance of the different regions in CHIKV-encoded proteins in the adaptive immune response. Information from this study provides critical knowledge that will aid in the understanding of CHIKV infection and immunity, vaccine design, and pre-clinical efficacy studies.     

Comprehensive Mapping Antigenic Epitopes of NS1 Protein of Japanese Encephalitis Virus with Monoclonal Antibodies

Japanese encephalitis virus (JEV) non-structural protein 1 (NS1) contributes to virus replication and elicits protective immune responses during infection. JEV NS1-specific antibody responses could be a target in the differential diagnosis of different flavivirus infections. However, the epitopes on JEV NS1 are poorly characterized. The present study describes the full mapping of linear B-cell epitopes in JEV NS1. We generated eleven NS1-specific monoclonal antibodies from mice immunized with recombinant NS1. For epitope mapping of monoclonal antibodies, a set of 51 partially-overlapping peptides covering the entire NS1 protein were expressed with a GST-tag and then screened using monoclonal antibodies. Through enzyme-linked immunosorbent assay (ELISA), five linear epitope-containing peptides were identified. By sequentially removing amino acid residues from the carboxy and amino terminal of peptides, the minimal units of the five linear epitopes were identified and confirmed using monoclonal antibodies. Five linear epitopes are located in amino acids residues ⁵AIDITRK¹¹, ⁷²RDELNVL⁷⁸, ²⁵¹KSKHNRREGY²⁶⁰, ²⁶⁹DENGIVLD²⁷⁶, and ³⁴¹DETTLVRS³⁴⁸. Furthermore, it was found that the epitopes are highly conserved among JEV strains through sequence alignment. Notably, none of the homologous regions on NS1 proteins from other flaviviruses reacted with the MAbs when they were tested for cross-reactivity, and all five epitope peptides were not recognized by sera against West Nile virus or Dengue virus. These novel virus-specific linear B-cell epitopes of JEV NS1 would benefit the development of new vaccines and diagnostic assays.     

Characterization of Antigenic Polypeptides Recognized by Monoclonal Antibodies Specific to the Myoplasm of Eggs of the Ascidian Ciona intestinalis

The determinants responsible for the differentiation of ascidian larval muscle cells are thought to be contained within the egg myoplasm. To analyze the macromolecules composing the myoplasm, several hybridoma cell lines which secrete monoclonal antibodies specific to myoplasmic components of Ciona eggs have been established (17). In the present investigation, seven of these myoplasm-specific antigens were characterized according to their molecular features and distribution patterns within the egg cytoplasm. Four of the seven antigenic polypeptides were shown to be components of the cortical cytoplasm, two were related to mitochondria, and one is likely to be a yolk protein. An antigen recognized by IIG6B2 antibody, which inhibited muscle development when injected into fertilized eggs, was a single polypeptide of relative molecular mass about 40,000 and isoelectric point about 5. The antigen was designated myoplasmin-C1 after its characteristic localization. The IIF9E9 antigen was a single 35-kDa polypeptide related to mitochondria and was thus designated myoplasmin-M1. The other five antibodies recognized two or more spots by immunoblotting analysis using two-dimensional gel electrophoresis. All of these myoplasm-specific antigens, except for the IIH10D6 antigen, are likely to be produced by the oocyte itself. Synthesis of IIH10D6 antigen seems to be associated with test cells.     

Subcellular Distribution of Distinct Nucleolin Subfractions Recognized by Two Monoclonal Antibodies

Monoclonal antibodies binding to different domains of nucleolin have been used to localize nucleolin in tissue culture cells ofXenopus laevis.The monoclonal antibody b6-6E7 binds to an epitope in the N-terminal domain, which contains arrays of phosphorylation consensus sites. This monoclonal antibody binds to nucleolin of oocytes and of eggs with high affinity. In contrast, the monoclonal antibody Nu-1H6 binds poorly to the modified forms of nucleolin arising during meiosis and mitosis. In interphase cells, monoclonal antibody b6-6E7 preferentially stains the periphery of the nucleoli, where most of the rRNA accumulates. Staining by monoclonal antibody Nu-1H6 complements this pattern by staining mainly the center of the nucleoli. The epitope of monoclonal antibody Nu-1H6 is within the central domain of nucleolin, which contains the first two RNA binding domains. RNase treatment of cells results in loss of nucleolin from nucleoli. In mitotic cells, both monoclonal antibodies decorate the surface of condensing chromosomes in prophase. The periphery of the condensed chromosomes in metaphase and anaphase is preferentially stained by monoclonal antibody b6-6E7.     

Characterization of Simian-Human Immunodeficiency Virus Envelope Glycoprotein Epitopes Recognized by Neutralizing Antibodies from Infected Monkeys

We characterized human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein epitopes recognized by neutralizing antibodies from monkeys recently infected by molecularly cloned simian-human immunodeficiency virus (SHIV) variants. The early neutralizing antibody response in each infected animal was directed mainly against a single epitope. This primary neutralizing epitope, however, differed among individual monkeys infected by identical viruses. Two such neutralization epitopes were determined by sequences in the V2 and V3 loops of the gp120 envelope glycoprotein, while a third neutralization epitope, apparently discontinuous, was determined by both V2 and V3 sequences. These results indicate that the early neutralizing antibody response in SHIV-infected monkeys is monospecific and directed against epitopes composed of the gp120 V2 and V3 variable loops.     

Antigenic Pattern of Human Brain Glycoproteins as Described by Monoclonal Antibodies

A battery of monoclonal antibodies was raised against a preparation of lentil lectin-binding membrane glycoproteins from human brain. Out of 26 established hybridomas, nine produced antibodies against the human Thy-1 antigen. For the remaining 17 lines, reactivity with at least six other antigens could be identified after immunoprecipitation and immunoblotting. Several of the antigens were di- or trimeric, mainly in the molecular weight range of 60-120 kDa. Two of the antibodies were reactive with high-molecular-weight aggregates and four targets for the antibody reactivity were not identifiable by immunoprecipitation of iodinated antigens. Three of the identified antigens were shown by quantitative enzyme-linked immunosorbent assay tests on various human tissues to be specifically expressed in the brain.     

Definition of Epitopes for Monoclonal Antibodies Developed Against Purified Sodium Channel Protein: Implications for Channel Structure

To test sodium channel structural models, we defined the epitopes for nineteen independently cloned monoclonal antibodies previously generated against purified, detergent-solubilized, adult rat skeletal muscle sodium channel protein using channel proteolysis, synthetic peptides, and fusion proteins. All identified epitopes were continuous and unique to the skeletal muscle subtype α-subunit. Of the nineteen independent clones, seventeen had epitopes located either in the origin of the amino-terminus or in the interdomain 2–3 region while only two antibodies had epitopes located in the mid-portion of the interdomain 1–2 region. No immunogenic regions were identified on the α-subunit's extracellular regions, interdomain 3–4 segment, or carboxyl-terminus or on channel β-subunits. While immune tolerance may explain the lack of immunogenicity of extracellular regions, the lack of immunogenicity of most of the channel's cytoplasmic mass may be due to segment inaccessibility from organization of these regions as globular domains, to insertion of parts of these regions into the membrane phase, or to interaction with other protein elements. The definition of monoclonal antibody epitopes allows us to reinterpret previously reported monoclonal antibody competition studies, providing independent support for our model of sodium channel cytoplasmic domain structure. In addition, these data suggest additional testable hypotheses concerning the interactions of the sodium channel amino- and carboxyl-termini with each other as well as with other protein elements. Received: 4 March 1998/Revised: 15 May 1998     

Specific and Common Epitopes in Mating Pheromones of Euplotes raikovi Revealed by Monoclonal Antibodies

Polypeptide mating pheromones Er-1 and Er-2, purified from the supernatant of Euplotes raikovi cultures of mating type I and mating type II, respectively, were used to immunize mice and obtain monoclonal antibodies. Five hybridoma clones producing antibodies specific to the mating pheromones were selected. They were analyzed for immunospecificity by immunoperoxidase assay, immunoblotting, and for their efficacy in inhibition of mating pheromone activity. Monoclonal antibodies from two hybridoma clones recognized only the mating pheromone used as antigen; those from the other three clones reacted, to comparable extents, with both mating pheromones. On the basis of these results it was assumed that two immunogenic sites exist in Er-1 and Er-2, one specific and the other common to both mating pheromones.     

Generation of Monoclonal Antibodies against Dengue Virus Type 4 and Identification of Enhancing Epitopes on Envelope Protein

The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.     

Partial Analysis of the Flagellar Antigenic Determinant Recognized by a Monoclonal Antibody to Clostridium tyrobutyricum

In order to count Clostridium tyrobutyricum spores in milk after membrane filtration, murine 21E7-B12 monoclonal antibody was produced. Elution of the monoclonal antibody from this antigen, the flagellar filament protein, by carbohydrate ligands was used to study the epitope structure. A competitive elution of an anti-dextran monoclonal antibody by carbohydrate ligands served as a control in order to validate the immunological tool applied to flagellin epitope study. The carbohydrate moiety of flagellin contained D -glucose and N-acetyl-glucosamine in a molar ratio of 11:1 as determined by gas-liquid chromatography and 2 low-abundancy unidentified compounds. In ELISA, D -glucose and N-acetyl-glucosamine did not dissociate the antibody-flagellin complex contrary to maltose, maltotriose, maltotetraose and maltopentaose. The efficiency of elution increased from the dimer to the pentamer and became nil for maltohexaose and maltoheptaose. The fact that the hexamer and heptamer could not react with the 21E7-B12 monoclonal antibody could be explained by a drastic conformational change. The overall stretched maltopentaose switch to a helical-shaped maltoheptaose which could not fit the 21E7-B12 monoclonal antibody antigen-combining site. Thus, flagellin epitope may contain α(1→4) linked glucose residues plus either N-actyl-glucosamine or an unidentified compound that maintain it in an extended shape.     

Identification of an Antigenic Determinant on Anionic Peanut Peroxidase by Monoclonal Antibodies

Two monoclonal antibodies (46–12-C12 and 23–6-C12)raised against anionic peanut peroxidase were found to haveindependent epitope sites. These topographic sites were foundto be located within a tryptic glycopeptide (Atgp) from theanionic isozyme by both indirect and non-competive ELISA andWestern blotting. The Atgp has a M_r equal to 11 000 of which 70% is carbohydrateand the peptide is probably highly hydrophobic as determinedby its high R_F (0.83) value and the amino acid composition.McAb 23–6-C12 recognized a contiguous epitope which encompassedalso the sole N-linked oligosaccharide on the anionic isozyme.That the monoclonal antibody also recognized the oligosaccharideon the -amylase, ß-glucosidase, acid phosphatase,and horse-radish peroxidase may be related to similarities insugars. Sugar removal from the Atgp or from the cross-reactivepeptide of enzymes caused loss of antibody affinity. The monoclonal antibody 46–12-C12 recognized specificallya conformational epitope near the region of the cysteine, tryptophaneand methionine residue on Atgp. Digestion of the anionic isozymeby trypsin resulted in a 40-fold loss of affinity with thismonoclonal antibody. Moreover, treatment of the Atgp with performicacid or trifluoromethane sulphonic acid caused a loss of affinitybetween the treated Atgp and this monoclonal antibody. Key words: Monoclonal antibodies, peanut, anionic peroxidase, glycopeptide, trypsin digest     

Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope ¹²³¹PTGSGKSTK¹²³⁹ (EP05) or core motif ¹³⁷³IPFYGKAI¹³⁸⁰ (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.     

Structure and Function Analysis of Therapeutic Monoclonal Antibodies against Dengue Virus Type 2

Dengue virus (DENV) is the most prevalent insect-transmitted viral disease in humans globally, and currently no specific therapy or vaccine is available. Protection against DENV and other related flaviviruses is associated with the development of antibodies against the viral envelope (E) protein. Although prior studies have characterized the neutralizing activity of monoclonal antibodies (MAbs) against DENV type 2 (DENV-2), none have compared simultaneously the inhibitory activity against a genetically diverse range of strains in vitro, the protective capacity in animals, and the localization of epitopes. Here, with the goal of identifying MAbs that can serve as postexposure therapy, we investigated in detail the functional activity of a large panel of new anti-DENV-2 mouse MAbs. Binding sites were mapped by yeast surface display and neutralization escape, cell culture inhibition assays were performed with homologous and heterologous strains, and prophylactic and therapeutic activity was evaluated with two mouse models. Protective MAbs localized to epitopes on the lateral ridge of domain I (DI), the dimer interface, lateral ridge, and fusion loop of DII, and the lateral ridge, C-C′ loop, and A strand of DIII. Several MAbs inefficiently inhibited at least one DENV-2 strain of a distinct genotype, suggesting that recognition of neutralizing epitopes varies with strain diversity. Moreover, antibody potency generally correlated with a narrowed genotype and serotype specificity. Five MAbs functioned efficiently as postexposure therapy when administered as a single dose, even 3 days after intracranial infection of BALB/c mice. Overall, these studies define the structural and functional complexity of antibodies against DENV-2 with protective potential.Dengue virus (DENV), a member of the Flaviviridae family of RNA viruses, is related to several other human pathogens of global concern, including yellow fever and tick-borne, West Nile, and Japanese encephalitis viruses. DENV infection in humans occurs after Aedes aegypti or Aedes albopictus mosquito inoculation and results in clinical disease, ranging from a febrile illness (dengue fever [DF]) to a life-threatening hemorrhagic and capillary leak syndrome (dengue hemorrhagic fever [DHF]/dengue shock syndrome [DSS]). Globally, there is significant diversity among DENV strains, including four distinct serotypes (DENV type 1 [DENV-1], DENV-2, DENV-3, and DENV-4) that differ at the amino acid level by 25 to 40%. Additional complexity occurs within each serotype, as genotypes vary from one another by up to 3% at the amino acid level (21, 49). No approved antiviral treatment is currently available, and several candidate tetravalent vaccines remain in clinical development (reviewed in reference 11). Because of the increased geographic range of its mosquito vectors, urbanization, and international travel, DENV continues to spread worldwide and now causes an estimated 50 to 100 million infections and 250,000 to 500,000 cases of DHF/DSS per year, with 2.5 billion people at risk (68).DENV is an enveloped icosahedral virus with a single-stranded, positive-polarity RNA genome. The 10.7-kb genome is translated as a single polyprotein, which is cleaved into three structural proteins (capsid [C], premembrane/membrane [prM/M], and envelope [E]) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) by host and viral proteases. The mature DENV virion is ∼500 Å in diameter, with a highly organized outer protein shell, a 50-Å lipid membrane bilayer, and a nucleocapsid core (26). Mature DENV virions are covered by 90 anti-parallel E protein homodimers, arranged flat along the surface with quasi-icosahedral symmetry. The immature virion, which lacks cleavage of the prM protein, has a rough surface with 60 spikes each composed of three prM-E heterodimers (7, 73). Exposure to mildly acidic conditions in the trans-Golgi network promotes virus maturation through a structural rearrangement of the flavivirus E proteins and cleavage of prM to M by a furin-like protease (29, 66, 69, 70). The ectodomain of DENV E protein is comprised of three discrete domains (34-36, 39). Domain I (DI) is a central, eight-stranded β-barrel, which contains a single N-linked glycan in most DENV strains. DII is a long, finger-like protrusion from DI, with the highly conserved fusion peptide at its distal end and a second N-linked glycan that recognizes DC-SIGN (37, 38, 46, 59). DIII, which adopts an immunoglobulin-like fold, has been suggested to contain cell surface receptor recognition sites (5, 64, 71). Several groups have recently defined contact residues for type-specific, subcomplex-specific, and cross-reactive monoclonal antibodies (MAbs) that recognize DIII of DENV-2 (16, 17, 31, 47, 57, 61). Type-specific MAbs with neutralizing activity against DENV-2 localized to the BC, DE, and FG loops on the lateral ridge of DIII, whereas subcomplex-specific MAbs recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310.To date, no study has compared the in vitro inhibitory activity of MAbs in cells against a genetically diverse range of DENV-2 strains and their protective capacity in animals. Here, we had the goal of generating strongly neutralizing MAbs that would recognize virtually all DENV-2 strains and function as a possible postexposure therapy. Twenty-four new anti-DENV-2 mouse MAbs were generated with moderate or strong neutralizing activity against the homologous virus in cell culture assays. Binding sites were mapped for the majority of these by yeast surface display, identifying distinct epitopes in regions in DI (lateral ridge), DII (dimer interface, lateral ridge, and fusion loop), and DIII (lateral ridge, C-C′ loop, and A strand). Several MAbs failed to neutralize efficiently at least one DENV-2 strain of a distinct genotype, suggesting that antibody recognition of neutralizing epitopes varies among DENV-2 genotypes.To begin to assess the utility of this new panel of inhibitory MAbs as possible therapeutics against DENV-2, we evaluated their protective capacity in a stringent intracranial challenge model in BALB/c mice. Among the 16 neutralizing MAbs tested in mice, most were protective when given as prophylaxis. Seven of these had postexposure therapeutic activity when administered as a single dose by intraperitoneal route even 3 days after intracranial infection. For the MAbs with the greatest therapeutic potential, protection was confirmed with an antibody-enhanced vascular leakage mouse model (2, 72) of DENV-2 infection.     

Monoclonal Antibodies against Pectin: Recognition of a Conformation Induced by Calcium

Monoclonal antibodies have been produced that recognize a conformation of homopolygalacturonic acid (pectic acid) induced by an optimum concentration of calcium and sodium of about 1 and 150 millinormal, respectively. The epitope recognized is probably part of the dimers of pectin chains associated according to the `egg box' model.     

Antigenic and Functional Analyses of Glycoprotein of Rabies Virus Using Monoclonal Antibodies

Thirty-five monoclonal antibodies (MAbs) against glycoprotein (G protein) of the RC-HL strain of the rabies virus have been established. Using these MAbs, two antigenic sites (I and II) were delineated on the G protein of the RC-HL strain in a competitive binding assay. Of these, 34 MAbs recognized the epitopes on site IL Site II was further categorized into 10 subsites according to their patterns in a competitive binding assay. Each site II-specific MAb showed 5 to 23 nonreciprocal competitions. The reactivities of 35 MAbs to rabies and rabies-related viruses in an indirect immunofluorescent antibody test showed that six MAbs in group A binded to rabies and rabies-related viruses and eight MAbs in group E reacted only with rabies viruses, considering that the former represent the genus-specific of Lyssavirus and the latter are rabies virus-specific. From biological assays, 28 of the 35 MAbs showed neutralization activity, 31 showed hemagglutination inhibition (HI) activity, and 18 showed immunolysis (IL) activity. The MAbs recognizing neutralization epitopes fell into at least three groups: those exhibiting both HI and IL activity, those showing only HI activity, and those showing neither HI nor IL activity. All IL epitopes overlap with HA epitopes. Five of the nine MAbs which reacted with the antigen treated by sodium dodecyl sulfate in ELISA were not reduced, or reduced only slightly, in the titer. None of the MAbs reacted with 2-mercaptoethanol-treated antigen. Only one MAb that recognized site I reacted with the denatured G protein in a Western blotting assay, indicating that its epitope is linear. These results suggest that almost all of the epitopes on the G protein of the rabies virus are conformation-dependent and the G protein forms a complicated antigenic structure.