Monoclonal antibodies to Ebola virus: isolation, characteristics, and study of cross reactivity with Marburg virus

Thirteen hybridoma strains producing monoclonal antibodies (Mabs) to Ebola virus were prepared by fusion of NS-O mouse myeloma cells with splenocytes of BALB/c mice immunized with purified and inactivated Ebola virus (Mayinga strain). Mabs directed against viral proteins were selected and tested by ELISA. Protein specificity of 13 Mabs was determined by immunoblotting with SDS-PAGE proteins of Ebola virus. Of these, 11 hybridoma Mabs reacted with 116 kDa protein (NP) and 2 with Ebola virus VP35. Antigenic cross-reactivity between Ebola and Marburg viruses was examined in ELISA and immunoblotting with polyclonal and monoclonal antibodies. In ELISA, polyclonal antibodies of immune sera to Ebola or Marburg viruses reacted with heterologous filoviruses, and two anti-NP Ebola antibodies (Mabs 7E1 and 6G8) cross-reacted with both viruses. Target proteins for cross-reactivity, Ebola NP (116 kDa) and Marburg NP (96 kDa), and VP35 of both filoviruses were detected by immunoblotting with polyclonal and monoclonal antibodies (6G8) to Ebola virus.     

Generation and characterization of monoclonal antibodies against the flounder Paralichthys olivaceus rhabdovirus

Two MAbs (3C7 and 3C9) against flounder Paralichthys olivaceus rhabdovirus (PORV) were generated with hybridoma cell fusion technology and characterized by an indirect enzyme-linked immunosorbent assay, isotype test, Western blot and immunodot analysis and immunofluorescence assay. Isotyping tests demonstrated that both of the two MAbs belonged to IgM subclass. Western blot analysis showed the MAbs reacted with 42, 30, and 22 kDa viral proteins, which were localized within the cytoplasm of PORV-infected grass carp ovary (GCO) cells analyzed by indirect immunofluorescences tests. The MAb 3C7 was also selected at random for detecting virus antigens in the inoculated grass carp tissues by immunohistochemistry assay. Flow cytometry tests showed that at the 36 h postinfection (0.25 PFU/cell), the 23% PORV-infected GCO cells could be distinguished from the uninfected cells with the MAb 3C7. Such MAbs could be useful for diagnosis and potential treatment of viral infection.     

Identification of three distinct antigenic sites in parapoxviruses

Summary.  Monoclonal antibodies (Mabs) were generated in BALB/c mice immunized with gradient-purified particles, envelopes and cores of intracellular mature orf virus D-1701. Three distinct antigenic sites were identified in this virus strain. Their topographical relationships was determined by pairwise epitope specificity studies in competition ELISAs. One MAb (class IgM) neutralized virus infectivity. Four μg/ml purified IgM gave a 50% reduction of 100 PFU of orf virus D-1701. As shown by immunogold electron microscopy (ELMI), all MAbs reacted with epitopes localized on the virus surface. Western blotting analysis demonstrated that two proteins of a Mr of 39kDa and 22kDa were the main targets for the Mabs. Cross-reactivity studies of several parapoxviruses (PPV) differentiated stomatitis papulosa virus strains from orf virus and milker’s node virus (MNV) by a missing antigenic site. Received May 31, 1996 Accepted September 4, 1996     

Western and dot blotting analyses of Ehrlichia chaffeensis indirect fluorescent-antibody assay-positive and -negative human sera by using native and recombinant E. chaffeensis and E. canis antigens

Human monocytic ehrlichiosis is an emerging infectious disease caused by Ehrlichia chaffeensis, a gram-negative obligatory intracellular bacterium closely related to E. canis. The immunoreactive recombinant fusion proteins rP28 and rP30 have become available after cloning and expressing of the 28- and 30-kDa major outer membrane protein genes of E. chaffeensis and E. canis, respectively. Western immunoblotting was performed to analyze the antibody responses of the 37 E. chaffeensis indirect fluorescent-antibody assay (IFA)-positive and 20 IFA-negative serum specimens with purified whole organisms, rP28, and rP30. All IFA-negative sera were negative with purified whole organisms, rP28, or rP30 by Western immunoblot analysis (100% relative diagnostic specificity). Of 37 IFA-positive sera, 34 sera reacted with any native proteins of E. chaffeensis ranging from 44 to 110 kDa, and 30 sera reacted with 44- to 110-kDa native E. canis antigens. The 28-kDa E. chaffeensis and 30-kDa E. canis native proteins were recognized by 25 IFA-positive sera. Fifteen IFA-positive sera reacted with rP28 by Western blot analysis, whereas 34 IFA-positive sera reacted with rP30 (92% relative diagnostic specificity), indicating that rP30 is more sensitive than rP28 for detecting the antibodies in IFA-positive sera. These 34 IFA-positive sera were positive by the dot blot assay with rP30, distinguishing them from IFA-negative sera. Except for three rP30-negative but IFA-positive specimens that instead showed an E. ewingii infection-like profile by Western immunoblotting, the results of Western and dot blot assays with rP30 matched 100% with the IFA test results. Densitometric analysis of dot blot reactions showed a positive correlation between the dot density and the IFA titer. These results suggest that rP30 antigen would provide a simple, consistent, and rapid serodiagnosis for human monocytic ehrlichiosis.     

Production of monoclonal antibodies to Naegleria fowleri, agent of primary amebic meningoencephalitis.

Monoclonal antibodies (MAbs) to Naegleria fowleri, the etiologic agent of primary amebic meningoencephalitis (PAM), have been produced and used as probes to identify N. fowleri amebae in brain sections of patients who died of that disease. These MAbs were characterized for their specificity by the indirect immunofluorescence assay (IIF), dot immunobinding assay (DIBA), and enzyme-linked immunotransfer blot technique (EITB). The MAbs reacted intensely with all strains of N. fowleri tested originating from different geographic areas in the IIF and DIBA tests, but showed no reactivity with four other species of Naegleria, N. gruberi, N. jadini, N. lovaniensis, and N. australiensis, or a strain of Acanthamoeba castellanii. In the EITB assay the MAbs reacted with the antigens of N. fowleri and produced intensely staining bands at the 160-, 104-, 93-, and 66-kilodalton (kDa) regions and several minor bands at the 30- and 50-kDa regions. The MAbs also reacted with the antigens of N. lovaniensis and produced a darkly staining band at 160 kDa and a diffusely staining band at 116 kDa, indicating that these antigens were shared by the two species. The MAbs, however, showed no reactivity with N. jadini and N. gruberi in the EITB assay.     

Production and characterization of monoclonal antibodies to a grouper iridovirus

A panel of six monoclonal antibodies (mAbs) against a grouper iridovirus (SGIV) were produced by immunization of Balb/c mice with purified virus preparations. Isotyping test revealed all the mAbs were IgG1. None of the mAbs possessed the ability to neutralize SGIV in cell cultures. Western blot showed that 4 mAbs reacted with 2 SGIV proteins at molecular mass of approximately 100 and 117 kDa in gradient-purified virus. Immunofluorescent studies showed that the two specific viral proteins VP100 and VP117 were localized within virus assembly sites in the cytoplasm of SGIV-infected grouper cells (GP). Fractionations of the iridovirus in a 20-60% sucrose gradient were detected successfully by all the six mAbs using immunodot blot. An antigen-capture enzyme-linked immunosorbent assay (ELISA) system, based on the use of mAb 7E11 for capture and a rabbit polyclonal antibody to SGIV for detection was developed. SGIV antigen was detected in gradient-purified virus and virus-infected grouper blood. These novel mAbs will facilitate the development of more specific and standardized diagnostic techniques for marine fish iridovirus.     

Monoclonal antibodies against Der s 1, a major allergen of Dermatophagoides siboney

Six stable clones secreting murine monoclonal antibodies (Mabs) against Der s 1 were obtained. The binding of Mabs showed cross-reactivity with Dermatophagoides farinae, as determined by enzyme-linked immunosorbent assay (ELISA). In a Western blot assay, antibodies reacted with a 24-kD protein considered to represent the major allergen Der s 1. The repertoire of antigenic sites on Der s 1 was studied using a panel of Mabs. Epitope specificity of the Mabs was determined by both competitive inhibition and sandwich ELISA assays. The results defined six different, non-overlapping and non-repeated antigenic sites on the allergen molecule. Der s 1 allergen from Dermatophagoides siboney extracts was purified by Mab affinity chromatography, this procedure gave 43% recovery of >90% pure allergen. The purified allergen had capacity to bind specific human IgE and demonstrated an allergenic activity of up to 77% of total D. siboney extract. An Mab-ELISA was developed using Mabs directed against different epitopes on Der s 1. This assay could detect up to 1 ng/ml of Der s 1 and Der f 1 in allergen preparations.     

Production, characterization, and application of monoclonal antibodies specific to recombinant (E2) structural protein in antigen-capture ELISA for clinical diagnosis of Chikungunya virus

The resurgence of Chikungunya (CHIK) virus in the form of an explosive, unprecedented epidemic with high virulence and unusual numbers of fatalities has created an immense public health concern in recent years. In the absence of an effective vaccine and specific antiviral therapy, early accurate diagnosis is essential for the best patient management. The present study describes the production and characterization of high-affinity and selective monoclonal antibodies (Mabs) against recombinant E2 protein (rE2) of the CHIK virus. The reactivity of Mabs for rE2 protein was demonstrated using ELISA. The specificity of the generated Mabs for rE2 was demonstrated by Western blot and indirect immunofluorescence. The application of this CHIK virus E2-specific monoclonal antibody in early clinical diagnosis was demonstrated by various analytical methods, such as immunoblotting, indirect immunofluorescence assay (IFA), and antigen-capture ELISA (AC-ELISA), for the detection as well as the identification of the novel ECSA genotypes of CHIK virus. These findings suggest that the high-affinity E2-specific monoclonal antibodies reported in this study will be useful for early clinical diagnosis and epidemiological studies of CHIK virus in developing countries.     

SARS冠状病毒N蛋白单克隆抗体的制备及鉴定

目的 制备SARS冠状病毒(SARS-CoV)N蛋白特异性单克隆抗体(McAb)，为SARS的快速诊断及致病机制的研究提供实验材料。方法 用纯化的重组SARS-CoVN蛋白免疫BALB/c小鼠，经细胞融合和亚克隆后获得分泌针对N蛋白的杂交瘤细胞株，用Western blot和间接免疫荧光法检测这些细胞株分泌的单克隆抗体特异性，并将N蛋白分3段表达初步定位单克隆抗体识别表位所在区域。结果 通过细胞融合和3轮克隆化，筛选出分泌抗N蛋白的6个杂交瘤细胞株。Western blot及免疫荧光显示，获得的McAb可与SARS-CoVN蛋白及SARS-CoV发生特异性反应，有4个细胞株分泌的抗体的识别位点位于N蛋白N端，2个位于C端。结论 获得了SARS-CoV特异性单克隆抗体并进行了初步定位，可用于SARS的早期诊断及致病机制研究。     

Development and application of monoclonal antibodies for detection and analysis of aquareoviruses

Monoclonal antibodies (mAbs) play an important role in detection of aquareoviruses. Three mAbs against grass carp reovirus (GCRV) were prepared. Isotyping revealed that all three mAbs were of subclass IgG2b. Western blot assay showed that all three mAbs reacted with GCRV 69 kDa protein (the putative VP5). In addition to the 69 kDa protein of GCRV, mAb 4B6 also recognize a 54 kDa protein. All three mAbs were used for detecting aquareovirus by Western blot assay and indirect immunofluorescence assay (IFA). All of them reacted with GCRV, and mAb 4A3 could also react with turbot Scophthalmus maximus reovirus (SMReV) and largemouth bass Microptererus salmonides reovirus (MsReV). Viral antigens were only observed in the cytoplasm of infected cells. Finally, syncytia formation was observed with light microscopy and fluorescence microscopy using fluorescein labelled 4A3 mAb at various times post-infection. Syncytia were observed at 36 hr post-infection (hpi) by light microscopy and at 12 hpi by fluorescence microscopy. The immunofluorescence based assay allowed earlier detection of virus than observation of virus-induced cytopathic effect (CPE) assay in inoculated cell cultures. The sensitivity and specificity of these mAbs may be useful for diagnosis and monitoring of aquareoviruses.     

A recombinant surface protein of Babesia bovis elicits bovine antibodies that react with live merozoites

Ten monoclonal antibodies (MoAbs) were generated against five surface-exposed proteins (16 kDa, 42 kDa, 44 kDa, 60 kDa, 225 kDa) on merozoites of Babesia bovis. A genomic library constructed in the lambda gt11 expression vector was screened with MoAbs in a plaque immunoassay for identification of clones expressing recombinant surface proteins. Two recombinant clones were identified (lambda Bo44-15 and lambda Bo44-16) that encoded a protein recognized by a MoAb specific for an epitope on the native 44-kDa surface protein. Southern blot analysis using radiolabeled Bo44-15 DNA (1.25 kb) against merozoite DNA and bovine leukocyte DNA confirmed the parasite-specificity of the cloned insert and revealed multiple bands of hybridization with merozoite DNA. Western blot analyses of lambda Bo44-15 lysogen preparations demonstrated that recombinant protein production in this clone was IPTG-induced and that the recombinant molecule was a beta-galactosidase fusion protein. Additionally, recombinant 44-kDa protein, purified by immunoaffinity chromatography, reacted with specific MoAb in Western blot assay indicating that the integrity of the epitope was retained during purification. Immune sera from calves immunized with purified recombinant Bo44-15 protein immunoprecipitated metabolically radiolabeled merozoite protein of 44 kDa indicating that antibody induced by recombinant Bo44-15 protein recognized native 44-kDa protein. Also, these sera reacted with the surface of live merozoites as evidenced by indirect immunofluorescence assay. Serum antibody titers determined by this assay had a wide range.     

Mapping of epitopes of VP2 protein of chicken anemia virus using monoclonal antibodies

To map the epitopes of VP2 protein of chicken anemia virus (CAV), VP2 was expressed as a fusion protein in Escherichia coli BL21 (DE3). The Western blot demonstrated that recombinant VP2 protein could be recognized by sera of chickens infected with CAV. Female BALB/c mice were immunized with purified recombinant VP2 produced in E. coli BL21 (DE3) and seven VP2-specific monoclonal antibodies (MAbs) were developed. The results of Western blot showed that all the seven MAbs recognized the recombinant VP2 protein expressed in the baculovirus and reacted with MDCC-MSB1 cells infected with CAV by indirect immunofluorescence assay. The VP2 protein was dissected into 21 overlapping fragments, expressed as fusion peptides in E. coli and used for epitope mapping by pepscan analysis. ELISA and Western blot assays indicated that most of MAbs reacted with the 12th and 13th fragments (amino acids 111-136) and one of them reacted with the 3rd fragment (amino acids 21-36). The linear immunodominant epitope of VP2 was located mainly in amino acid residues 111-126 and 121-136.     

Purification and amino-terminal sequence of the dengue virus-induced cytotoxic factor.

The present study was undertaken to purify the dengue type 2 virus (DV)-induced cytotoxic factor (CF) and analyse its amino-terminal sequence. Spleens collected from DV-infected moribund mice were made into a single cell suspension and cultured for 24 h. The culture supernatant was purified using fast protein liquid chromatography (FPLC) and polyacrylamide gel electrophoresis (PAGE). CF could be purified in a single step by FPLC and native PAGE. The isoelectric point of CF was pH 6.5. Purified CF had a molecular weight of 45 kDa on native-PAGE while on SDS-PAGE it dissociated into two bands of 20 and 25 kDa. Anti-CF-antisera reacted specifically with CF bands separated on native and SDS-PAGE in a Western blot assay. A sequence of 19 amino-acids of the N-terminus of CF was analysed which on comparison with that of other known cytotoxic proteins indicated that CF differs from them. Thus, CF appears to be a unique cytotoxic protein induced by a virus.     

Molecular cloning, expression of testicular transcript abundant in germ cells and immunobiological effects of the recombinant protein

PROBLEM: It has been well documented that antisperm antibodies can be causative factors for infertility. In this report we have identified a protein on human sperm referred as human sperm-associated protein (HSAP) using serum of an immunoinfertile woman; it is thus a sperm-specific protein--a candidate molecule for control of fertility. METHOD OF STUDY: An immunoinfertile woman serum showing head-head sperm agglutination and acrosomal localization, reacted with human sperm protein of apparent molecular weight of 48 kDa on Western blot. Anti-48 kDa antiserum was raised in rabbit by eluting 48 kDa protein and was used to screen the human testis cDNA expression library. A putative positive hsap cDNA clone was obtained, sequenced and subjected to tissue specificities studies by Northern blotting. The cell type-specific expression was done using in situ RNA hybridization studies. To obtain recombinant HSAP (r-HSAP), hsap cDNA was cloned in pET 22b(+) expression vector. r-HSAP was expressed as polyhistidine fusion protein in Escherichia coli and purified. Rabbits were immunized with the purified r-HSAP, which led to generation of antibodies. In order to evaluate in vitro immunocontraceptive potential, the anti-r-HSAP antibodies were characterized by agglutination assay, zona-free hamster egg penetration assay, indirect immunofluorescence (IIF) assay, and by flow cytometry analysis. RESULTS: We have cloned a human testis gene encoding a protein (HSAP) of 328 amino acids. Antibodies against the purified recombinant protein specifically recognized approximately 40 kDa r-HSAP, and a cognate 48 kDa protein band in human sperm extract in Western blot procedure. The anti-r-HSAP antibodies localized acrosomal compartment, inhibited sperm binding/attachment in zona-free hamster penetration assay and revealed surface binding with human live sperm by flow cytometry. The cDNA sequence has been submitted to EMBL and has been given the accession number Y16676. CONCLUSION: This study has put in evidence that novel sperm-specific r-HSAP has role in sperm function and may have application in the development of a contraceptive vaccine. The availability of the recombinant protein will facilitate studies on the assessment of its potential as a contraceptive immunogen.     

The reactivity of monoclonal antibodies against orf virus with other parapoxviruses and the identification of a 39 kDa immunodominant protein

Summary.  A panel of 27 mouse monoclonal antibodies (Mabs) was raised against orf virus. Sixteen of these Mabs reacted with a protein with a molecular mass of 65 kDa, 8 reacted with a protein with a molecular mass of 39 kDa and three remain uncharacterised. Reactivity of the Mabs with a library of recombinant vaccinia viruses expressing various regions of the NZ-2 orf virus genome identified the approximate positions of the genes encoding these 2 immunodominant orf virus proteins. The gene encoding the 39 kDa protein was identified and sequenced. The protein was detected in an envelope fraction of orf virus and was shown to be homologous to the envelope protein encoded by the H3L gene of vaccinia virus. The 65 kDa protein has not been fully characterised, but the gene encoding it has been localised to a 10 kbp region of the orf virus genome. The Mabs were used to discriminate 4 parapoxviruses derived from sheep, 2 from cattle and 1 each from a seal and squirrel. Eighteen Mabs reacted with all 4 sheep viruses, 19 Mabs reacted with both cattle viruses, 6 recognised seal parapoxvirus and 2 recognised the squirrel parapoxvirus. Only one of the 27 Mabs reacted with all 8 parapoxviruses suggesting it recognises a conserved epitope within the genus. Received March 11, 1998 Accepted July 24, 1998     

Antigen-antibody reaction in solution in capture competition immunoassay for human immunodeficiency virus antibodies.

In the capture competition immunoassay, undiluted serum was reacted in solution with purified human immunodeficiency virus (HIV) antigen in wells of microtest plates coated with anti-HIV immunoglobulin G antibodies (HIV capture antibodies). HIV antibodies present in the serum being tested combined with the HIV antigen and thus blocked (completely or partially) the fixation of the antigen to the capture layer. Unblocked antigenic activity was measured in subsequent steps by the use of biotinylated anti-HIV immunoglobulin G and peroxidase-conjugated avidin. The assay was evaluated in comparison with indirect enzyme-linked immunosorbent assay and Western (immuno-) blot (WB). A total of 180 serum samples which reacted repeatedly as positive in indirect enzyme-linked immunosorbent assay but negative in WB were found to be negative by the capture competition assay. Of 54 serum samples showing dubious reactions (single p24 bands in WB), 53 were clearly separated into positive or negative reactions, whereas 1 serum sample gave a borderline reaction. It was concluded that a characteristic feature of this kind of inhibition assay is a very low frequency of equivocal results.     

Purification of a baculovirus-expressed hepatitis E virus structural protein and utility in an enzyme-linked immunosorbent assay.

We report on the purification of the full-length structural protein encoded by open reading frame 2 (ORF-2) of hepatitis E virus. The ORF-2 protein, expressed in Sf9 cells by using a recombinant baculovirus vector system, was successfully purified to homogeneity. Gel electrophoresis of the purified ORF-2 protein showed a single polypeptide of 75 kDa by Coomassie blue staining and by Western blot (immunoblot) analysis. We demonstrated that the partially purified ORF-2 protein could be used successfully in a sensitive and specific enzyme-linked immunosorbent assay for the detection of antibodies to hepatitis E virus.     

Characterisation of an avian influenza virus nucleoprotein expressed inE. coli and in insect cells

The nucleoprotein (NP) gene from influenza virus A/Shearwater/Australia/72 has been expressed intracellularly in both E. coli and insect cells. E. coli-derived NP was identified by Western blot analysis as a 56 kDa protein which co-migrates with virion-derived NP. This protein was purified by immunoaffinity chromatography and a nitrocellulose binding assay showed that NP formed complexes with positive- and negative-sense influenza neuraminidase RNA transcribed in vitro. ELISA and Western blot analysis revealed that recombinant NP of 56 kDa was produced in high yields in insect cells using a baculovirus vector. Immunofluorescence microscopy revealed that NP was localised to the nucleus of infected insect cells.     

Anti-Golgi Antibody in Rheumatoid Arthritis Patients Recognizes a Novel Antigen of 79 kDa (Doublet) by Western Blot

We have detected cytoplasmic anti-Golgi antibody (AGA) during a routine immunofluorescence test for detecting autoantibodies. Two sera from patients with rheumatoid arthritis (RA) reacted to the Golgi complex by an indirect immunofluorescence technique on HEp-2 cells. Localization of AGA in the Golgi complex was confirmed by double-staining with antibodies to beta-COP. The effect of monensin on the integrity and morphology of the Golgi complex was also studied. To confirm the presence of AGA further, we performed immuno-electron microscopy. Both sera reacted with cytoplasmic antigen located in the Golgi complex of various animal tissues. Furthermore, by using the Western blot technique, both sera reacted to a relative molecular weight (MW) of 79 kDa (doublet) Golgi antigen purified from rat liver. To our knowledge, this study may be the first to identify the relative MW of Golgi antigen by the Western blot method. Identification of this antibody could provide better understanding of protein synthesis and secretion. The presence of AGA in RA patients further substantiates the diversified nature of autoantibody production seen in this disease.