Monoclonal antibodies to Marburg virus proteins and their immunochemical characteristics]

Hybridomas producing monoclonal antibodies (MAb) to Marburg virus proteins are prepared. Positive hybridomas were selected by solid-phase enzyme immunoassay (EIA) by specificity of their immunoglobulin reaction with Marburg virus antigen. Virus proteins reacting with MAbs were identified by immunoblotting. Out of 20 examined hybridoma antibodies, 5 reacted with protein VP35, 5 with VP40, 3 with NP, 1 with protein complex VP35-VP40, MAb 7H10 detected 2 proteins (VP40 and NP), and 5 MAbs did not bind virus proteins in this assay. Marburg virus antigen adsorbed on the surface of plates were detected by indirect EIA with biotin-treated MAbs (PEIA-MAb) and indirect EIA (IEIA-MAb). The sensitivity of both methods differed with different hybridoma antibodies and was the maximum with MAb 5F1 specific to Marburg virus nucleoprotein: 5-10 and 1-2 ng/ml for the direct and indirect methods, respectively. Purified MAbs 7C4, 7D8, and 5F1 were used as antigen captures in EIA for detecting immunoglobulins to Marburg virus in a serum from convalescent after Marburg fever. The results recommend the above MAbs for use in test systems for the diagnosis of the disease and detecting virus antigen.     

Production and study of hybridomas, producing monoclonal antibodies to the structural glycoprotein of Marburg virus]

Hybridomas producing monoclonal antibodies (MAb) to Marburg virus glycoprotein were prepared by splicing of mouse myeloma cells (NSO and X.63-Ag6.653 strains) and splenocytes of immunized BALB/c mice. Cultural and MAb-producing properties of hybridomas were studied. The production of MAb during serial passages 15-30 was confirmed. MAb titers in culture fluids were 1:4096-1:8152, in immune ascitic fluids of BALB/c mice 1:409,600-1:638,400. The possibility of using MAbs as a component of ELISA test system was demonstrated. The sensitivity of ELISA-MAb method for Marburg virus was 1 x 10(3) PFU/mu.     

Detection of antibodies to individual proteins of rubella virus

Individual rubella virus structural polypeptides were electroeluted from SDS-polyacrylamide gels. The eluted polypeptides were used, without further purification, as antigens in ELISA assays for the detection of rubella-specific antibodies in patients' sera. This provided a more sensitive detection method than that involving classical serological assays such as HI or VN or that using immunoprecipitation. Antisera against individual viral polypeptides were raised in mice. No haemagglutination inhibition activity was observed in any of these sera and weak virus neutralizing activity was only detected with antiserum to the E1 protein. Antisera to either the E1 or E2(a,b) complex proteins cross-reacted with both the E1 and E2(a,b) complex proteins.     

Detection of antibodies specific to sodium dodecyl sulfate-treated proteins.

Heavy meromyosin (HMM) denatured by sodium dodecyl sulfate (SDS) was injected into guinea pigs, either in the presence of 1 mg SDS/mg protein or after chromatography on Sephadex G-10 to remove detergent excess. Antigen-antibody interactions were analyzed by the microcomplement fixation technique. When HMM was injected in the presence of excess of SDS, the microcomplement fixation curves exhibited two maxima; one was specific to the random coil configuration of heavy meromyosin or myosin, and the other was common to several SDS-protein complexes. The latter peak disappeared when the excess SDS was removed from the immunogen by chromatography. Results showed the presence of antibodies directed either against SDS or against the non-specific SDS protein link.     

Characterization and potential diagnostic application of monoclonal antibodies specific to rabies virus

Objective

Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. It is necessary to develop standard rabies virus diagnostic tools, especially for diagnosing the strains prevalent in China.

Methods

Monoclonal antibodies (MAbs) specific to rabies virus were produced and characterized by enzyme linked immunosorbent assay (ELISA), isotyping, affinity assay, immunofluorescence assay (IFA), and immunocytochemistry. The MAb, whose affinity was higher for antigen, was used to establish an antigen capture-ELISA (AC-ELISA) detection system and test the efficiency by using clinical samples.

Results

The heavy chain subclasses of two MAbs were all determined to be IgG2a. The 3C7 MAb showed stronger reactivity with rabies virus protein than the 2C5 MAb in an ELISA analysis, whereas the 3C7 MAb showed the highest affinity for antigen. IFA and immunocytochemistry results also indicated that the two MAbs could recognize rabies virus protein in its native form in cell samples. Data obtained using clinical samples showed that rabies virus could be detected by AC-ELISA detection system using the 3C7 MAb.

Conclusion

It was potentially useful for the further development of highly sensitive, easily handled, and relatively rapid detection kits/tools for rabies surveillance in those areas where rabies is endemic, especially in China.     

Characterization of monoclonal antibodies to Marburg virus (strain Musoke) glycoprotein and identification of two protective epitopes

Monoclonal antibodies (MAbs) reactive with Marburg virus (strain Musoke) were evaluated for both biological activity and specificity. Several of the Marburg virus- (MBGV) specific MAbs reduced the size and/or number of MBGV plaques in vitro. The ability of the MAbs to affect plaque formation in vitro was demonstrated to be specific for the glycoprotein (GP) of the strain of MBGV used for vaccination. Using deletion analysis and peptide mapping, the binding epitopes of several of these neutralizing MAbs were identified. Not unexpectedly, the epitopes were shown to lie in the most hypervariable and highly glycosylated region of MBGV GP. An analysis of the in vivo activity of several MAbs revealed that some antibodies provided substantial but incomplete protection of naive guinea pigs by passive transfer. These data suggest that neutralizing epitopes exist within MBGV GP but that induction of antibodies to these neutralizing epitopes may not be sufficient for protection from lethal infection.     

Detection of specific human immunodeficiency virus IgM antibodies

This study was done to demonstrate whether the use of the antigen-sandwich human immunodeficiency virus (HIV) antibody-screening assays (3rd generation assays), which detect all classes of anti-HIV immunoglobulins, leads to an earlier detection of HIV IgM compared to the 2nd generation HIV antibody-screening assays. We tested sequential bleeds of three donors obtained from commercially available seroconversion panels. Anti-HIV testing was done before and after high-performance liquid chromatography separation of IgG and IgM fractions. The positive result of the first bleedings from all three panels was linked to the IgM fraction, while at that time the IgG fraction was still negative. For subsequent samples drawn 5–9 days later, a positive signal was obtained with the IgG fraction in addition to a stronger positive signal obtained with the IgM fraction. We conclude that an assay capable of simultaneously detecting different immunoglobulin classes, including IgM, will help to narrow the window period for serological detection of seroconversion to HIV by detecting anti-HIV IgM-containing samples earlier than conventional assays using only anti-human IgG enzyme conjugates (indirect anti-HIV-screening assay, 2nd generation assays).     

Production and characterization of monoclonal antibodies specific to sweet clover necrotic mosaic virus

Monoclonal antibodies were produced in vitro by fusing mouse myeloma cells P3-X63-AgU1 with spleen cells derived from BALB/c mice immunized with purified sweet clover necrotic mosaic virus (SCNMV). Twenty-one out of 47 clones which secreted monoclonal antibodies of high titres against SCNMV were injected intraperitoneally into mice previously primed with Pristane. The ascites fluid harvested 10 to 14 days later showed a strong and specific anti-SCNMV activity in reverse passive haemagglutination inhibition, passive haemagglutination and indirect enzyme linked immunosorbent assay. The monoclonal antibodies of the 21 clones did not react with red clover necrotic mosaic virus (Swedish isolate). The class and subclass of immunoglobulins of the monoclonal antibodies secreted by established cultures were determined to be IgG2a for 15 clones and IgG3 for 6 clones.     

The induction and characterization of monoclonal antibodies specific to GP of Ebola virus

The Ebola virus is highly infectious and characterized by hemorrhagic fever, headache, and so on with a high mortality rate. Currently, there are neither therapeutic drugs or vaccines against the Ebola virus nor fast diagnostic methods for the detection of Ebola virus infection. This study reported the induction and isolation of two monoclonal antibodies that specifically recognized the glycoprotein (GP) and secreted glycoprotein (sGP) of the Ebola virus. Plasmids encoding either GP or sGP were constructed and immunized BALB/c mice, accordingly purified sGP was boosted. The antisera were analyzed for binding activity against sGP protein in enzyme-linked immunosorbent assay (ELISA) and neutralization activity in a pseudotyped virus neutralization assay. A number of reactive clones were isolated and two monoclonal antibodies T231 and T242 were identified to react with both GP and sGP. Western blot and ELISA assays showed that the monoclonal antibodies could react with GP and sGP, respectively. Moreover, they could recognize Ebola pseudovirus by cellular immunochemistry assay. We labeled the monoclonal antibody T231 with biotin and analyzed the competitiveness of the two antibodies by the ELISA test. The results showed that the binding epitopes of the two monoclonal antibodies to sGP were partially overlapped. In summary, two GP-specific mAbs were identified, which will be used to detect the Ebola virus or investigate GP.     

Characterization and application of monoclonal antibodies specific to West Nile virus envelope protein

Recent epidemics of West Nile virus (WNV) around the world have been associated with significant rates of mortality and morbidity in humans. To develop standard WNV diagnostic tools that can differentiate WNV from Japanese encephalitis virus (JEV), four monoclonal antibodies (MAbs) specific to WNV envelope (E) protein were produced and characterized by isotyping, reactivity with denatured and native antigens, affinity assay, immunofluorescence assay (IFA), and epitope competition, as well as cross-reactivity with JEV. Two of the MAbs (6A11 and 4B3) showed stronger reactivity with E protein than the others (2F5 and 6H7) in Western blot analysis. 4B3 could bind with denatured antigen, as well as native antigens in indirect ELISA, flow cytometry analysis, and IFA; whereas 2F5 showed highest affinity with native antigen. 4B3 and 2F5 were therefore used to establish an antigen capture-ELISA (AC-ELISA) detection system. The sensitivity of this AC-ELISA was 3.95 TCID(50)/0.1 ml for WNV-infected cell culture supernatant. Notably, these MAbs showed no cross-reactivity with JEV, which suggests that they are useful for further development of highly sensitive, easy handling, and less time-consuming detection kits/tools in WNV surveillance in areas where JEV is epidemic.     

Detection of IgG antibodies to measles virus using monoclonal antibodies for antigen-capture in enzyme immunoassay

An enzyme immunoassay (EIA) for the detection of IgG antibodies to measles viral hemagglutinin (H) has been developed. Monoclonal antibodies were employed as antigen-capture reagents on a polystyrene ball solid phase. The antigen-capture EIA was significantly more sensitive than hemagglutination inhibition (HAI) and somewhat more sensitive than indirect immunofluorescence and indirect EIA for the detection of antibodies to measles virus. The importance of selecting a monoclonal antibody with a high binding affinity, and controlling for non-specific adherence of antigen or antibodies to the solid phase were demonstrated. This method offers not only greater sensitivity than HAI, but also the practicality of reagents capable of standardization and long term storage.     

Detection of antibodies to caprine arthritis-encephalitis virus using recombinant gag proteins

Summary The coding sequences of the core proteins p17 and p28 of caprine arthritis-encephalitis virus (CAEV) were amplified using the polymerase chain reaction and cloned into the plasmid expression vector p-GEX-2T. Both p17 and p28 were expressed as fusion proteins with glutathione S-transferase. The recombinant proteins were affinity purified from induced bacterial lysates using glutathione-agarose beads. The purified proteins were used in an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against CAEV in goat sera and milk samples. Three different ELISA tests were developed based on p17, p28 or the combination of these two recombinant proteins (p17+p28). A comparison was made to an ELISA based on purified whole virus particles and to agar immunodiffusion test (AGID). Sera with conflicting results in the different ELISA tests were examined by Western blotting. There was a high correlation between the ELISA tests based on p17+p28 recombinant proteins and whole virus ELISA, with an estimated value of 0.92. Only 72–75% of the sera that tested positive in these two ELISA tests were positive in AGID. Antibodies to CAEV were detected in significantly more animals when serum samples were tested compared to milk samples. Based on the time and materials required to prepare the reagents, the recombinant based ELISA test was less expensive than the whole virus ELISA.     

Psoriasis specific chromosomal proteins, antibodies against them and disease activity

It has been demonstrated recently that the lymphoid cells of patients with psoriasis have antibodies directed against the psoriasis specific non-histone proteins. A conceptual hypothesis for the role of these antibodies in the pathogenesis of psoriasis presented in this communication, is as follows. Perhaps the psoriasis specific non-histone proteins following phosphorylation bind to histone which keeps psoriasis gene(s) repressed. This may result in the displacement of histone from the DNA. Antibodies against psoriasis specific non-histone proteins may facilitate the displacement of histone. DNA set free, possibly containing psoriasis gene(s), can then be transcribed into RNA causing a shift of the resting pool of keratinocytes in the symptom-free skin of psoriasis patients to the proliferating pool of keratinocytes in the psoriasis lesions.     

Modification of vaccinia virus penetration proteins analyzed by monoclonal antibodies

Modifications induced in structural vaccinia virus proteins that elicit the high infectious state by virus activating treatments involving trypsin and phosphatidylserine were analyzed using antivaccinia monoclonal antibodies (MABs). MABs reactive against each of the five outer layer proteins (VP54K, 34K, 32K, 29K, and 17K-25K) neutralized infectivity. VP54K possesses at least two neutralizing epitopes. Treatment with trypsin or with isolated plasma membrane cleaved VP54K into TVP41K carrying epitope A and removed a fragment containing epitope B from the virus. MABs against either of the epitopes could neutralize the virus. The exposure of epitope A concomitantly activated virus infectivity, and it was an essential step of penetration. MABs against VP17K-25K reacted more efficiently with trypsin-treated virus than with untreated virus, but the size of VP17K-25K was not affected by trypsin; this finding indicated that trypsin treatment rendered the VP17K-25K epitopes more accessible to antibody and hence to neutralization. MABs against VP32K and VP29K neutralized infectivity to the same extent irrespective of the state of activation. Virus treated with phosphatidylserine (PS) was neutralized more efficiently by MAB against VP34K than untreated virus, but the amount of antibody that reacted with the virus was the same before and after treatment with PS. Phosphatidylserine did not modify epitope structure itself, but it activated the function of VP34K. It was concluded that blocking of the functions attributed to any of the five proteins resulted in neutralization of virus infectivity, and treatment with trypsin and phosphatidylserine activates infectivity of vaccinia virus by modifying three of them (VP54K, VP34K, VP17K-25K) with characteristic behavior for each protein.     

Recombinant human antibodies specific for hepatitis C virus proteins

Summary.  Human antibodies to hepatitis C virus core, NS4A and NS3 were cloned in a prokaryotic vector and expressed as soluble Fab fragments and as phage-displayed Fabs. The recombinant Fabs were shown to be a suitable tool for immunohistochemistry, since they recognize the cognate antigen expressed in mammalian cells. The nucleotide sequence of the cDNA for the variable domains of these antibodies was determined and the V-gene usage was derived. On the basis of the deduced amino acid sequence, a structural model of the V domains of the Fabs was constructed.     

Detection of monoclonal antibodies specific for carbohydrate epitopes using periodate oxidation

A method is described for determining whether particular monoclonal antibodies are specific for carbohydrate or non-carbohydrate antigenic determinants. In a model system consisting of the Lewis a human blood group determinant attached to either protein or lipid, mild periodate oxidation destroyed the carbohydrate determinant without altering protein or lipid epitopes. The technique was readily applied to antigens bound to plastic wells for ELISA, to nitrocellulose sheets for Western blots, and to thin layer chromatography (TLC) plates for TLC immunostaining. Mild periodate oxidation can prove useful during the early stages of hybridoma screening in order to select for or against anti-carbohydrate antibodies.     

Production of monoclonal antibodies specific to theophylline-treated lymphocytes

The T11 molecule is reported to play a key role in T lymphocytes activation. Moreover, theophylline is known to modify the functional properties of T lymphocytes probably inducing early changes in T11 molecule during T cell activation. Aim of our work was to clarify the effect on T lymphocyte surface after in vitro treatment with theophylline. In this paper, we describe the production and the functional properties of several monoclonal antibodies obtained by immunizing Balb/c mice with theophylline treated cells. Some of the monoclonal antibodies reacted only with the theophylline-treated lymphocytes and showed a promitogenic activity, enhancing the expression of T activated cell antigen. These monoclonal antibodies seem to demonstrate the existence of a membrane molecule which appears on lymphocytes surface after a trigger signal occurring in the early stages of T cell activation likely related to the T11 dependent alternative pathway.     

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.     

Construction and characterization of monoclonal antibodies specific to Epstein-Barr virus latent membrane protein 1

Epstein-Barr virus (EBV) has been implicated in the development of many human neoplasias including B lymphomas and nasopharyngeal carcinoma (NPC). The EBV latent membrane protein 1 (LMP-1) has been found to participate in diverse cellular signaling pathways and is essential for virus-induced B-cell immortalization. In order to determine quantitatively the amount of LMP-1 in cells, five monoclonal antibodies (Mabs) specific to LMP-1 were generated. The epitopes recognized by these Mabs were found to cluster within the repeat region between the CTAR1 and CTAR2 domains, corresponding to amino acid positions 254-319 of LMP-1. These Mabs were capable of recognizing LMP-1 proteins of both lymphoid and epithelial origin as revealed by immunoblot, enzyme-linked immunosorbent assay (ELISA) and immunocytofluorescence analysis. A sandwich ELISA for the quantification of LMP-1 has been established using these Mabs. Taken together, our results indicate that the Mabs generated in this study are suitable for the detection of LMP-1 in biomedical research.     

The immunogenic properties of Marburg virus proteins]

Immunogenic and protective properties of VP40 and NP proteins of Marburg virus were studied. VP40 protein was shown to have insignificant immunogenicity and NP protein to be capable of protecting the animals from lethal infection by stimulation of cell-mediated immunity. No significant increase in the specific antibody level was found.