Production and Characterization of Monoclonal Antibodies to ...

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Monoclonal antibodies (MAbs) against the nucleoprotein of Marburg virus (MARV-NP) with high specificity and activity would be useful for the diagnosis of ...

HYBRIDOMA Volume 27, Number 6, 2008 © Mary Ann Liebert, Inc. DOI: 10.1089/hyb.2008.0044

Production and Characterization of Monoclonal Antibodies to Nucleoprotein of Marburg Virus Zhang Jibin,1 Lu Xiumei,1 Wei Hongping,2 Cheng Longyan,2 He Jin,1 Zhang Zhiping,2 Zhang Xianen,2 and Yu Ziniu1*

Monoclonal antibodies (MAbs) against the nucleoprotein of Marburg virus (MARV-NP) with high specificity and activity would be useful for the diagnosis of MARV. In this report, a recombinant MARV-NP was successfully expressed by an Escherichia coli expression system. After immunization and cell fusion, three mouse hybridomas (1H4, 2G1, and 3B5) producing MAbs to MARV-NP were established. The MAbs obtained were fully characterized using indirect ELISA and Western blot analysis. The ELISA results showed that the MAbs’ titers were in the range of 1:6.400  103 - 1:1.280  104 in culture fluids, and 1:1.024  106  1:8.192  106 in ascitic fluids. The isotypes of the three MAbs were tested to be IgG1␬ and all the MAbs recognized the same antigenic epitope. Western blot analyses demonstrated that all the MAbs were directed against MARV-NP with the affinity constants (Kaff) of about 1.100  109 M1, 1.235  109 M1, and 1.408  109 M1 for the MAbs 1H4, 2G1, and 3B5, respectively.



ARBURG HEMORRHAGIC FEVER (MHF), which is caused by the Marburg virus (MARV), is an uncommon but severe disease occurring in humans and non-human primates with high mortality (30–90%).(1,2) Since MARV was initially recognized and isolated in 1967, there have been four occasions of sporadic cases recorded(3,4) and two severe large outbreaks (from 1998 to 2000 in the Congo; from 2004 to 2005 in Angola)(5,6) and over 400 deaths of 500 cases in all. With the increase of international trade and travel, MARV has attracted many researchers’ attention and public concerns because it is dangerous as an imported virus and bio-threat reagent.(7) Nowadays, the origin in nature of MARV still remains a mystery. However, neither a vaccine nor a curative treatment for MARV infection is yet available.(8) Therefore, specific and fast detection of MARV infection is of great importance during outbreaks of MHF even in countries without it. For developing immunodiagnostic kits, recombinant MARV proteins are commonly used instead of native viruses in recent years(9,10) because recombinant MARV proteins are not infectious and do not need biosafety level 4 (BSL-4) facilities for manipulations. MARV is a member of the family Filoviridae, which belongs to the order of Mononegavirales.

Seven structural proteins are encoded by a 19 kb non-segmented negative-strand RNA genome of MARV (EMBL nucleotide sequence database accession no. Z12132). These proteins are nucleoprotein (NP), L protein, two viral proteins (VP35 and VP30), a single surface protein (GP), and two putative matrix proteins (VP40 and VP24).(3) MARV-NP is the major nucleocapsid component of the ribonucleocapsid complex and consists of 695 amino acid residues.(11) Because of its abundance in MARV particles, MARV-NP is a good target for immunoassay of MARV. Some typical examples are enzyme-linked immunosorbent assay (ELISA) kits for detecting MARV-NP based on monoclonal antibodies (MAbs) against MARV-NPs.(12,13) In this study, we choose the whole MARV-NP as the target. The whole MARV-NP was successfully expressed in Escherichia coli, and three MAbs against the MARV-NP were obtained. Materials and Methods

Materials The plasmid pCRII-MARV-np, which contains the Marburg virus nucleoprotein gene, was donated by Manfred Weidmann of the Institute for Virology, University of Göttingen, Germany. A reference mouse monoclonal antiMARV-NP antibody was kindly provided by Thomas F.

1State Key Laboratory of Agricultural Microbiology, National Engineering Research Centre of Microbial Pesticides, Huazhong Agricultural University, Wuhan, Hubei, P.R. China. 2State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei, P.R. China.




Meyer of Max Planck Institute for Infection Biology (Germany). Recombinant nucleoprotein of Ebola virus (recombinant EBOV-NP) was supplied by State Key Laboratory of Virology, Wuhan Institute of Virology of Chinese Academy of Sciences (China). Anti-6  histine tag (His-tag) mouse monoclonal antibody, 50% polyethylene glycol-1450 (PEG1450), and Freund’s adjuvant were purchased from Sigma (St. Louis, MO). Ni-NTA Ni/NTA His-Bind sepharose resin was obtained from Novagen (Madison, WI). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (HL) was bought from Bio-Rad (Hercules, CA). Alkaline phosphatase (AP) labeled goat anti-mouse IgG (HL) and Isostrip mouse MAb isotyping kit were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). RPMI-1640 medium, HAT, and HT supplements were purchased from Gibco BRL (Grand Island, NY). 96-well plates were from Cellstar (Greiner, Frickenhausen, Germany). Nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyphosphate phosphate toluidine salt (BICP) were bought from Amersco (Dallas, TX). All other reagents used were of analyticalreagent grade.

Construction of gene MARV-np expression vector The full-length MARV-np gene was amplified by PCR from the source with primers MARV-np/F (5-GGAGGTACCCTTATGGATTTACAC-3) (the KpnI site is underscored) and MARV-np/R (5-TGTAAGCTTCGTCTTTCATCGCAA-3) (the HindIII site is underscored). The resultant DNA (2.0 kb fragment) was then sub-cloned into the vector pET-32a() to construct the recombinant vector pET-32a()-MARV-np with the sequences of His-tag and thioredoxin tag (Trx-tag) at the 5 terminus of MARV-np (Fig. 1). Subsequently, the inserted gene was sequenced and confirmed to be identical to the original sequence in order to exclude PCR errors. Finally, the recombinant vector pET-32a()-MARV-np was transferred into Escherichia coli (BL21 [DE3] stains).

Expression and purification of the recombinant MARV-NP The recombinant strains were grown overnight at 37°C in 5 mL LB medium containing 100 ␮g/mL ampicillin and 25␮g/mL kanamycin, then inoculated to 500 mL of the LB

FIG. 2. SDS-PAGE (A) and Western blot (B) analyses of the recombinant MARV-NP. M, protein marker (116 kDa); 1, pET-32a() total cell extract (uninduced); 2, pET-32a() total cell extract (induced with IPTG); 3, pET-32a()-MARVnp total cell extract (uninduced); 4, pET-32a()-MARV-np total cell extract (induced with IPTG); 5, the recombinant MARV-NP; 6, BL21 (DE3) cell lyaste containing pET-32a().

medium for large production and grown until an OD600 of 0.5. Recombinant MARV-NP was expressed in BL21 (DE3) by inducing with IPTG (1 mM in final concentration) and purified using Ni-NTA sepharose resin according to the instructions. The expressed MARV-NP was identified by SDSPAGE and further confirmed by Western blot analysis. In brief, the recombinant MARV-NP was separated by using a 10% SDS-PAGE with BL21 (DE3) cell lysate containing pET32a() as the negative control, and then transferred to a PVDF membrane at 15 volts for 20 min. The membrane was blocked with 5% non-fat milk overnight at 4°C, and then incubated with anti-His-tag mouse MAb (1:1000) for 2 h. The recombinant MARV-NP was detected with AP-labeled goat anti-mouse IgG (1:3000) for 1 h at room temperature. Following a final washing step, NBT/BICP substrate was used for color development.

Antigenic activity analysis of the recombinant MARV-NP

FIG. 1. Construction of recombinant expression vector pET-32a()-MARV-np.

The antigenic activity of the recombinant MARV-NP was preliminarily determined by an indirect ELISA. Briefly, the recombinant MARV-NP was diluted serially two-fold to different concentrations (80, 40, 20, 10, and 5 ␮g/mL) with carbonate buffer (pH 9.6) and coated on a 96-well plate at 4°C overnight. After washing the plates three times with phos-



FIG. 3. Antigenic activity analysis of the recombinant MARV-NP determined by indirect ELISA (A) and Western blot analyses (B). (A) The recombinant MARV-NP (䊏) and BL21 (DE3) cell lysate containing pET-32a()(ⵧ) reacted with the reference anti-MARV-NP mouse MAb by indirect ELISA. The mean plus 3 SD was set at 0.2 by calculating OD450 from the negative control. Error bars represent the SD from independent experiments in duplicate. (B) M, protein marker (116 kDa, pre-stained); 1, the recombinant MARV-NP (108 kDa); 2, BL21 (DE3) cell lysate containing pET-32a(). phate-buffered saline (PBS), 200 ␮L of PBS containing 5% non-fat milk (PBSM) was added to the well for blocking for 1 h at 37°C to avoid non-specific binding. The PBSM was then removed, and the reference anti-MARV-NP mouse monoclonal antibody was added and incubated for 1 h at 37C. After washing again, HRP-conjugated goat anti-mouse IgG was added. The plate was washed again and incubated with 100 ␮L of 3,3,5,5-tetramethylbenzidine (TMB) substrate at room temperature for 15 min. After stopping the reaction with 2 M H2SO4, the absorbance was measured at 450 nm. Recombinant EBOV-NP with the same dilutions was used as the negative control. The mean and standard deviation (SD) of the adjusted ODs were calculated from the negative control. The cut-off value was set by calculating the mean plus 3 SD. Moreover, the antigenic activity of the recombinant MARV-NP against the anti-MARV-NP mouse monoclonal antibody (1:1000) was further testified by Western blot analysis.

Development of MAbs against the recombinant MARV-NP protein Six-week-old female BALB/c mice were immunized subcutaneously and intraperitoneally with a mixture containing 50 ␮g purified recombinant MARV-NP protein in 100 ␮L PBS and an equal volume of Freund’s complete adjuvant. A booster injectiosn with the same amount of antigen in Freund’s incomplete adjuvant was administered at 2-week intervals. The antibody titer was assayed with the recombinant

MARV-NP at a concentration of 3 ␮g/mL by indirect ELISA. One mouse was splenectomized at day 3 after the last boosting. Then the conventional hybridoma technique was performed for developing specific MAbs with some modifications. In brief, the immunized splenocytes were mixed with the SP2/0-Ag14 (SP2/0) myeloma cells at a ratio of 5:1, and fusion was carried out with 50% PEG-1450. The hybridoma cells were resuspended in RPMI 1640 medium supplemented with 20% fetal calf serum, HAT, and antibiotics (100 U/mL penicillin, 100 ␮g/mL streptomycin). Fusion products were seeded in 96-well plates containing the feeder cells and cultured in a CO2 incubator. The cultured supernatants from each well were screened by detecting their reactivity to MARV-NP by indirect ELISA. To ensure monoclonality, single cells producing the desired antibody were re-cloned successively four to six times by limiting dilution. Clones positive for producing anti-MARV-NP antibody were expanded stepwise and then injected into the intraperitoneal cavity of BALB/c mice primed with liquid paraffin for antibody production.

MAb characterization Titer and isotype. Titers of the culture supernatant and ascitic fluids of hybridoma cells were detected by indirect ELISA after serial dilutions. The isotypes of the immunoglobulins were analyzed using the Isostrip mouse MAb isotyping kit, according to the manufacturer’s instructions. The harvested ascitic fluids were purified by octanoic


JIBIN ET AL. EBOV-NP and BL21 (DE3) cell lysate were used as the negative controls. The affinity constants (Kaff). Non-competitive enzyme immunoassay(14) was used to measure the affinity constants of MAbs against MARV-NP, which were serially diluted to concentrations of 6, 3, 1, and 0.5 ␮g/mL. The sigmoid curve was plotted to represent the relationship of OD450 value versus MAb dilution. Kaff could be calculated according to equation 1: Kaff  (n  1) / {2 (n [Ab] t  [Ab] t)}


where n  [Ag]t / [Ag⬘]t, [Ag]t and [Ag⬘]t are the total antigen concentrations in the wells while [Ab⬘]t and [Ab]t are the measurable total antibody concentrations in the wells at OD50 ( 50% of OD-100, the upper plateau ) and OD-50 for plates coated with [Ag]t and [Ag⬘]t. Primary analysis of antigenic epitopes. A modified ELISA double antibody binding system as described by Friguet et al.(15) was used to calculate the additivity index (AI) to analyze whether the corresponding MAbs recognize different epitopes on MARV-NP. The purified MAbs were serially diluted to concentrations of 5, 0.1, and 0.05 ␮g/mL and then the two tested MAbs were added either separately or simultaneously into a 96-well plate pre-coated with the recombinant MARV-NP. The amounts of the bound antibodies were quantitatively measured by use of goat anti-mouse IgG-HRP. Additivity of the bound enzymatic activity was measured and the AI was calculated according to equation 2: AI  [2A1 ⫹ 2 / (A1  A2)  1]  100%


Where A1, A2, and A1 ⫹ 2 represent the OD450 values of MAb1, MAb2, and the mixture of the two MAbs, respectively. If the AI is above 50%, it shows that the two MAbs recognize different antigenic epitope; if below 25%, it demonstrates that the two MAbs recognize the same antigenic epitope.

FIG. 4. Detection of MAb titers in culture supernatants (A) and ascitic fluids (B) of hybridoma cells 1H4 (ⵧ), 2G1 (䊏), and 3B5 (䉱) against MARV-NP by indirect ELISA. The recombinant MARV-NP was used as antigen, and the recombinant EBOV-NP and BL21 (DE3) cell lysate containing pET32a() were used as negative controls. The mean plus 3 SD was set at 0.20 and 0.18 by calculating OD450 from the negative controls (the recombinant EBOV-NP and BL21 (DE3) cell lysate containing pET-32a(), respectively). Error bars represent the SD from independent experiments in duplicate.

acid/ammonium sulfate precipitation assay, and the concentrations of the obtained MAbs were measured by ultraviolet and visible spectrophotometry. The specificity. The specificity of the MAbs were evaluated with the respective hybridoma cell supernatants by the Western blot method described above. The recombinant

FIG. 5. Western blot analyses of specificity of MAbs 1H4 (A), 2G1 (B), 3B5 (C) against MARV-NP. M, protein marker (116 kDa, pre-stained); 1, the recombinant MARV-NP (108 kDa); 2, BL21(DE3) cell lysate containing pET-32a(); 3, the recombinant EBOV-NP (116 kDa).







MABS 1H4, 2G1,





[Ag] (ng/mL)

OD-50a (OD450  SD)

[Ab] at OD-50 (ng/mL)

Kaff (M1)

Average Kaff (M1)

6000 3000 1000 500 6000 3000 1000 500 6000 3000 1000 500

1.037  0.022 1.023  0.023 0.987  0.030 0.708  0.009 1.050  0.011 0.988  0.022 0.919  0.015 0.741  0.018 1.049  0.018 1.014  0.021 0.996  0.013 0.845  0.016

62.654 64.818 66.018 68.557 55.267 57.291 60.109 60.773 51.105 51.967 52.707 53.295

1.120  109 1.126  109 1.055  109

1.100  109

1.264  109 1.219  109 1.221  109

1.235  109

1.420  109 1.413  109 1.392  109

1.408  109

aOD-50 represents the half maximum optical density obtained for a given concentration of the recombinant MARV-NP ([Ag]) and the corresponding MAb ([Ab]). The affinity constant (Kaff) for each selected concentration of Ag and Ab was determined using the formula described in the Materials and Methods section. OD450 and SD represent the mean of OD450 value and standard deviations obtained from independent experiments in duplicate.


Antigenic activity analysis of the recombinant MARV-NP

Expression and purification of recombinant MARV-NP

Antigenic activity of the recombinant MARV-NP was determined by indirect ELISA and Western blot, as shown in Figure 3. The ELISA results demonstrated that the recombinant MARV-NP could react with the reference MAb (Fig. 3A). Moreover, the Western blot analysis (Fig. 3B) indicated that the recombinant MARV-NP appeared as a distinct reaction band. In conclusion, the ELISA and the Western blot analyses revealed that the recombinant MARV-NP protein expressed in E. coli remained antigenically active. Therefore, the recombinant MARV-NP was a promising antigen candidate for MARV-NP antibody detection system.

After induction, the crude proteins of E. coli were separated by SDS-PAGE to examine if the recombinant MARVNP was expressed. According to the sequence of MARV-NP, the molecular weight of MARV-NP should be 78 kDa. However, MARV-NP migrates on an SDS gel with an apparent molecular weight of 92–94 kDa because MARV-NP is strongly negatively charged.(16) After adding the masses of His-tag and Trx-tag, which are at a molecular mass of 3 and 12 kDa, respectively, the recombinant MARV-NP expressed should show a mass about 107–109 kDa. As shown in Figure 2A, a band with the molecular mass of approximately 108 kDa was shown as expected. To further confirm if the protein band is the recombinant MARV-NP, a Western blot immunoassay was conducted using a MAb specific for Histag (Fig. 2B). The results showed that a single reaction band against the recombinant MARV-NP in lane 1 (Fig. 2B) was detected while no corresponding band in the BL21 (DE3) cell lysate containing pET-32a() (Fig. 2B, lane 2) was observed. The above results showed that the recombinant MARV-NP protein was expressed successfully in E. coli. Subsequently, the induced protein was purified by Ni-NTA sepharose resin for immunization. TABLE 2.





MABS 1H4, 2G1,


Preparation of MAbs against MARV-NP BALB/c mice were immunized with the recombinant MARV-NP. Following three booster immunizations, the titers of the antibody against the antigen in mice sera could reach up to 1:6.400  104. Three days after the last booster, the mouse with the highest antibody titer was splenectomized and the spleen cells were fused with SP2/0 myeloma cells. After continuously screening and cloning, three stable monoclonal hybridoma cell lines of 1H4, 2G1, and 3B5, which could constitutively produce specific MAbs to MARV-NP, were successfully established. 3B5



OD450 value of different MAb concentrations (O 苶D 苶苶4苶5苶0  SD) MAbs 1H4 2G1 3B5 1H4  2G1 1H4  3B5 2G1  3B5

5 ␮g/mL

0.1 ␮g/mL

0.05 ␮g/mL

2.177  0.015 2.164  0.022 2.188  0.019 1.784  0.029 1.923  0.026 2.157  0.016

2.058  0.028 2.014  0.026 2.113  0.012 1.916  0.015 2.005  0.006 2.263  0.011

2.086  0.021 2.119  0.029 2.117  0.014 1.912  0.027 1.945  0.024 2.106  0.023

The standard deviations from independent experiments in duplicate were in the range of 0.002 and 0.031. 苶 OD 苶4苶5苶0苶 and SD represent the mean of OD450 value and standard deviations obtained from independent experiments in duplicate.


Characterizations of the MAbs produced The ELISA results showed that the titers of the MAbs 1H4, 2G1, and 3B5 were 1:6.400  103, 1: 6.400  103, and 1:1.280  104 in culture supernatants; and 1:1.024  106, 1:4.096  106, 1:8.192  106 in ascitic fluids, respectively (Fig. 4). The MAb 3B5 has the highest MAb titers in culture supernatants as well as in ascitic fluids. All the MAbs selected belonged to IgG1 subclasses and all had kappa light chains evaluated by the Isostrip kit. After purification, the concentrations of the MAbs 1H4, 2G1, and 3B5 were 0.713, 1.166, and 3.702 mg/mL, respectively. The specificity of the MAbs obtained were further investigated by Western blot analyses using the recombinant MARV-NP, the recombinant EBOV-NP, and the BL21 (DE3) cell lysate. The Western blot analyses revealed that the three MAbs were directed against the recombinant MARV-NP and had no reactivity with the EBOV-NP, His-tag, or Trx-tag (Fig. 5A, 5B, 5C), respectively. The affinity constants (Kaff) of MAbs to MARV-NP were measured by the non-competitive enzyme immunoassay described using serial dilutions of both coated antigen and MAbs (Table 1). The results showed that the mean Kaff of MAbs 1H4, 2G1, and 3B5 with MARVNP were 1.100  109 M1, 1.235  109 M1, and 1.408  109 M1, respectively. The additivity assay (Table 2) showed that all the AIs of the mixed two MAbs were below 10%, which indicated that the three MAbs recognized the same antigenic eiptope. Discussion The choice of an appropriate screening assay is one of the most important parts of hybridoma production. The practical constraints on screening of assays are reliability, speed, cost, and labor. Generally, the screening assay is selected according to the characteristic of the antigen and the laboratory conditions, as well as the use of the monoclonal antibody in the future. However, regardless of the method used, it should be fast, reliable, and simple; otherwise it would miss the best opportunity for harvesting desired hybridoma cells. Because the recombinant MARV-NP contained His-tag and Trx-tag, and some host proteins may contaminate the MARV-NP solution, it is vital to develop an appropriate screening method to avoid false-positive results. EBOV and MARV belong to the same family but are serologically distinct; moreover, the recombinant EBOV-NP contained Histag and Trx-tag also. Therefore, choosing the recombinant EBOV-NP as the negative control in our experiments could effectively eliminate the false-positive MAbs to His-tag and Trx-tag. Meanwhile, BL21 (DE3) cell lysate containing pET32a() was used as another negative control to further exclude the interference from the host proteins. The results proved that the method was efficacious, and the Western blot analyses showed that the three MAbs obtained had no reactivity with His-tag, Trx-tag, and host proteins. In this study, the whole recombinant protein MARV-NP was expressed successfully in E. coli with a His-tag and a Trx-tag at the N-termimus of MARV-NP, and the recombinant MARV-NP obtained remained antigenically active, which means that it is feasible to use the recombinant MARV-NP as a specific diagnostic antigen for seroepidemi-

JIBIN ET AL. ological studies of MARV. Moreover, three hybridoma cell lines secreting MAbs against the recombinant MARV-NP were generated and identified with high affinity, stability, and specificity. These MAbs might be applied to develop diagnostic systems for MARV infection. However, the availability of the three MAbs against authentic MARV virus should be further validated. Acknowledgment We thank Manfred Weidmann of the Institute for Virology, University of Göttingen, Germany, for providing pCRIIMARV-np plasmid containing the NP gene of Marburg virus, and Thomas F. Meyer of Max Planck Institute for Infection Biology, Germany, for supplying a reference mouse antiMARV-NP MAb. This work was supported partially by a grant from the Chinese Academy of Sciences. References 1. Geisbert TW, and Jahrling PB: Exotic emerging viral diseases: progress and challenges. Nat Med 2004;10:110–121. 2. Sanchez A, Khan AS, Zaki SR, Nabel GJ, Ksiazek TG, and Peters CJ: Filoviridae: Marburg and Ebola viruses. In: Fields’ Virology, 4th ed. Knipe DM and Howley PM (Eds.). Lippincott, Williams & Wilkins, Philadelphia, 2001, pp. 1279– 1304. 3. Brigitte B, and Reinhard K: Characteristics of Filoviridae: Marburg and Ebola viruses. Naturwissenschaften 1999;86: 8–17. 4. WHO: Marburg haemorrhagic fever in Uganda. Epidemic and Pandemic Alert and Response (EPR), 2007. 5. WHO: Marburg fever, Democratic Republic of the Congo. Wkly Epidemiol Rec 1999; 74:145. 6. WHO: Outbreak of Marburg virus hemorrhagic fever-Angola, October 1, 2004–March 29, 2005. Morbidity Mortality Wkly Rep 2005:54. 7. Bray M: Defense against filoviruses used as biological weapons. Antiviral Res 2003;7: 53–60. 8. Bausch DG, and Geisbert TW: Development of vaccines for Marburg hemorrhagic fever. Expert Rev Vaccines 2007; 6(1):57–74. 9. Kachko AV, Cheusova TB, Sorokin AV, Kazachinskaia EI, Cheshenko IO, Belanov EF, Bukreev AA, Ivanova AV, Razumov IA, Riabchikova EI, and Netesov SV: Comparative study of the morphology and antigenic properties of recombinant analogs of a Marburg virus. Mol Biol (Mosk) 2001;35(3):492–499. 10. Saijo M, Niikura M, Ikegami T, Kurane I, Kurata T, and Morikawa S: Laboratory diagnostic systems for Ebola and Marburg hemorrhagic fevers developed with recombinant proteins. Clin Vaccine Immunol 2006;13:444–451. 11. Sanchez A, Kiley MP, Klenk HD, Klenk HD, and Feldmann H: Sequence analysis of the Marburg virus nucleoprotein gene: Comparision to Ebola virus and other non-segmented negative-strand RNA viruses. J Gen Virol 1992;73:347–357. 12. Saijo M, Georges-Courbot MC, Fukushi S, Mizutani T, Philippe M, Georges AJ, Kurane I, and Morikawa S: Marburg virus nucleoprotein-capture enzyme-linked immunosorbent assay using monoclonal antibodies to recombinant nucleoprotein: detection of authentic Marburg virus. Jpn J Infect Dis 2006;59(5):323–325. 13. Saijo M, Niikura M, Maeda A, Sata T, Kurane I, and Morikawa S: Characterization of monoclonal antibodies to

MAbs TO NUCLEOPROTEIN OF MARBURG VIRUS Marburg virus nucleoprotein (NP) that can be used for NPcapture enzyme-linked immunosorbent assay. J Med Virol 2005;76(1):111–118. 14. Beatty JD, Beatty BG, and Vlahos WG: Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Meth 1983;100(1–2):173–179. 15. Friguet B, Djavadi-Ohaniance L, Pages J, Bussard A, and Goldberg M: A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site and application to hybridomes specific for the ␤2-subunit of Escherichia coli tryptophan synthase. J Immunol Meth 1983;60(3):351–358. 16. Becker S, Huppertz S, Klenk HD, and Feldmann H: The nucleoprotein of Marburg virus is phosphorylated. J Gen Virol 1994;75(4):809–818.

429 Address reprint requests to: Yu Ziniu State Key Laboratory of Agricultural Microbiology National Engineering Research Centre of Microbial Pesticides Huazhong Agricultural University Wuhan, Hubei 430070 P.R. China E-mail: [email protected] Received: May 25, 2008 Accepted: July 10, 2008

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