Institut für Medizinische Mikrobiologie und Hygiene, Universität Regensburg, Franz-Josef-Strauß-Allee 11, 93053 Regensburg, Germany1
Institut für Physikalische Biochemie, Universität Potsdam, Im Biotechnologiepark, 14943 Luckenwalde, Germany2
Author for correspondence: Susanne Modrow. Fax +49 941 9446402. e-mail simone.dorsch{at}klinik.uni-regensburg.de
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Soon after infection, antibodies appear directed against the structural proteins VP1 and VP2, which both contain neutralizing epitopes (Sato et al., 1991a , b
; Yoshimoto et al., 1991
). These two proteins are encoded in the same reading frame located in the 5'-half of the viral single-stranded DNA genome (Cotmore et al., 1986
). VP1 and VP2 are identical except for an additional 227 amino acids at the amino terminus of the VP1 protein, the so-called VP1-unique region (Ozawa & Young, 1987
; Cotmore et al., 1986
). A study using empty capsids produced by recombinant baculovirus indicated that only VP1/VP2 capsids are able to create an efficient immune response, due to the neutralizing epitopes present in both the VP1-unique region and the VP2 proteins (Brown et al., 1991
; Bansal et al., 1993
; Rosenfeld et al., 1994
).
After the onset of neutralizing antibody production, the virus is cleared rapidly from the circulation in the peripheral blood. Viraemia may be controlled by the application of immunoglobulin preparations containing neutralizing antibodies against parvovirus B19 (van Elsacker-Niele & Kroes, 1999 ). We have described previously the generation of human MAbs directed against the VP1-unique region that have the capacity to neutralize parvovirus B19 in vitro (Gigler et al., 1999
). In order to investigate whether these antibodies have broad reactivity with various naturally occurring B19 isolates, and may therefore be of therapeutic use, we analysed the influence of amino acid variations in the VP1-unique region on antibody binding and affinity. Virus isolates from patients with various B19-correlated disease manifestations were sequenced. Amino acid variations that would potentially influence the epitope, either directly or indirectly by tertiary effects, were introduced in a vector system expressing a VP1-unique regionintein fusion protein in E. coli. The affinity of interaction with MAbs was then determined with a quartz crystal microbalance (QCM) biosensor.
![]() |
Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antisera and human MAbs.
For generation of polyclonal antisera directed against the VP1-unique region, rabbits were immunized with His-tagged protein that had been expressed in E. coli and purified by Ni2+-affinity chromatography and preparative gel electrophoresis. All sera reacted specifically in ELISA and Western blot assays (data not shown). Cell lines secreting human MAbs against the VP1-unique region and VP2 virus-like particles were established as described previously (Gigler et al., 1999 ) and the antibodies were purified from supernatants by protein G-affinity chromatography.
Amplification of the part of the genome encoding the VP1-unique region by nested PCR.
Routine tests for the presence of parvovirus B19 DNA were done by nested PCR for amplification of the VP1/VP2-encoding region with primers that have been published previously (Hemauer et al., 1996 ) (first round: forward nt 29012918, reverse nt 35113895; second round: forward nt 29562972, reverse nt 34313448).
An additional PCR was established to amplify in total the genomic region encoding the VP1-unique region, using the following primer pairs. First round: forward nt 22712288, reverse nt 33193337; second round: forward nt 23272347, reverse nt 32633281. Primer sequences were designed from the published B19 genome sequence (Shade et al., 1986 ) and synthesized by Metabion (Munich, Germany).
Serum samples were diluted 1:1 with water and incubated at 95 °C for 10 min. Aggregated proteins were removed by centrifugation. Two µl aliquots of the supernatants were used in the first cycling reaction. In the second round, 2 µl of the first amplification reaction was applied. The PCR parameters were: 1 min at 95 °C (denaturation), 45 s at 94 °C (hybridization) and 90 s at 72 °C (polymerization). Forty PCR cycles were performed. Identical conditions were used for the first and second rounds of amplification. Ten µl aliquots of each reaction were analysed on a 2% agarose gel and stained with ethidium bromide. Bands with sizes of approximately 500 bp for the VP1/VP2-encoding region and 950 bp for the VP1-unique region indicated positive results.
DNA sequence analysis.
Nested PCR fragments of the VP1-unique region were purified via QIAquick spin columns (Qiagen). Nucleotide sequences were obtained with a 373A Sequencer (Applied Biosystems) by the cycle sequencing method using primers derived from nt 24472466 (forward) and 31053124 (reverse). The nucleotide sequence data from this study have been deposited in the GenBank database under accession numbers AF293862AF293881.
Construction of plasmids, expression and purification of the VP1-unique region.
The amplified DNA fragments VP1N, for the chosen standard pJB (Shade et al., 1986 ), VP1N-1, encoding an exchange of 28E to D, and VPN-2, with exchanges 17K to R, 18A to D, 39Q to H and 43D to H, were cloned into the T7 expression vector pET21a_int, kindly provided by Uli Schmidt (Institut für Biotechnologie, University of Halle-Wittenberg, Germany). The correct integration of the fragments into the vector was confirmed by sequence analysis. The constructs were introduced into the E. coli strain BL21 (DE3). Bacteria were incubated in LB medium containing 100 µg/ml ampicillin at 37 °C. Expression of the recombinant protein was induced by addition of 1 mM IPTG for at least 3 h of culture. The bacteria were harvested by centrifugation and resuspended in 30 ml of 20 mM HEPESNaOH, 1 mM EDTA, 100 mM NaCl, pH 8·5. Protein expression was checked by SDSPAGE followed by silver staining or by Western blot analysis using the polyclonal rabbit serum directed against the VP1-unique region. Bacteria were lysed by the use of a French press and the debris was pelleted at 10000 g. The supernatant was loaded on a chitin column (NEB) using an FPLC system (Pharmacia Biosystems). The column was washed with 2 vols of 20 mM HEPESNaOH, 1 mM EDTA, 100 mM NaCl, pH 8·5, 8 vols of 20 mM HEPESNaOH, 1 mM EDTA, 2 mM NaCl, pH 8·5 and 2 vols of 20 mM HEPESNaOH, 1 mM EDTA, 100 mM NaCl, pH 8·5. The protein was eluted with 3 vols of 50 mM DTT in a buffer containing 20 mM HEPESNaOH, 1 mM EDTA, 100 mM NaCl, pH 8·5. Fractions were tested for recombinant proteins by SDSPAGE and silver staining. Positive fractions were pooled and concentrated by using a Centriplus concentrator (3 kDa exclusion limit; Amicon). The protein concentration was determined after dialysis against PBS (0·9 mM KH2PO4, 8·0 mM Na2HPO4, 2·7 mM KCl, 137 mM NaCl) using a Bradford assay (Bio-Rad Laboratories).
Non-infectious VP2 capsids and VP1/VP2 capsids were kindly provided by Bärbel Kaufmann (University of Potsdam, Germany). Expression was performed in a bac-to-bac baculovirus system (Gibco) with a pFast Bac DUAL vector. The capsids were purified by caesium chloride gradient centrifugation.
Western blot assays.
For Western blot analysis, purified proteins were separated by SDSPAGE and transferred to a nitrocellulose membrane as described previously (Towbin et al., 1979 ). Protein-free regions were blocked with 5 % low-fat dried milk in Tris-buffered saline (pH 7·5) and incubated overnight at room temperature with MAbs or rabbit antiserum diluted 1:500 in PBS. Secondary antibodies diluted 1:2000 in PBS were added after washing. For the detection of rabbit IgG, alkaline phosphate-conjugated swine antibodies against rabbit immunoglobulins were used (Bio-Rad). For the detection of human IgG, an alkaline phosphate-conjugated goat anti-human antibody was used (Sigma-Aldrich).
Determination of antibody affinity.
Determination of the affinities of antibodies reacting with purified proteins representing the various VP1-unique regions, non-infectious VP1/VP2 and VP2 particles and infectious parvovirus B19 was performed on an AFFco 2000. This immunosensor, supplied by the Fraunhofer Institute of Microelectronic Circuits and Systems, Munich, Germany (Kößlinger et al., 1992 ), is based on a quartz crystal microbalance with flow-injection analysis system and peristaltic pump and 20 MHz quartz crystals with gold electrodes. For activation, the gold electrodes were incubated with dithiobissuccinimidyl propionate at a concentration of 0·4% in DMSO at room temperature for 20 min and washed with PBS, pH 7·4. Subsequently, 10 µl protein A (1 mg/ml) or 10 µl antigen (0·5 mg/ml) was added to the quartz crystals and incubated at 4 °C for 12 h. This immobilization method produces a stable protein layer (Uttenthaler et al., 1998
). After rinsing with PBS and insertion of the quartz crystal into the clip holder, the protein variants were pipetted into the flow system via a 100 µl syringe at concentrations of 1 µM and the kinetics were visualized directly. BSA (0·025% in PBS) was used as a blocking reagent to prevent unspecific binding. The curves were fitted by using the program Origin version 5.0 (Microcal Software Inc.).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The nucleotide sequences encoding the VP1-unique region and the adjoining region encoding the amino terminus of the VP2 protein (nt 24443194; Shade et al., 1986 ) were determined from all virus isolates. The corresponding amino acid sequences and the standard isolate sequences AU and pJB were compared with those of a consensus sequence derived from all sequences (Table 1
). The variability ranged from 0% (isolates B, E, 2_980414 and 7_980326) and 0·4% (one amino acid exchange; isolates A, C, D, I, 2_980728, 6_960625, 8_980219, AU and pJB) to 0·8% (two amino acid exchanges; isolates F and H) and 2% (five amino acid exchanges; isolates G and J). Sequences obtained from a child with chronic B19 infection displayed a distinctly higher degree of variability (3·2% or eight amino acid exchanges; isolate 5940) (Hasle et al., 1994
). Comparison of the virus sequences isolated from maternal sera with those obtained by analysing the corresponding amniotic fluids or ascites did not reveal any variation in protein sequence except for isolate I, with one mutation in isolates I-FO (foetal serum) and I-AS (ascites). As the mutation observed in isolate I was detected in foetal serum and ascites samples, but not in the isolate from amniotic fluid, it probably occurred after infection of the foetus. Amino acid exchanges that could be associated with the transmission of the virus to the amniotic fluid or the development of hydrops foetalis were not observed.
|
Expression of recombinant proteins in E. coli
In order to analyse the influence of naturally occurring amino acid variations on the protein conformation, the amino-terminal 227 residues representing the VP1-unique region of the prototype B19 isolate pJB (VP1N-pJB) were produced in E. coli using the impact T7 system (NEB), which allows protein production and purification under native conditions. The sequences encoding the VP1-unique region were amplified by PCR, fused to a heterologous construct consisting of the region encoding a modified vacuolar membrane ATPase subunit I (VMA-I)intein (Saccharomyces cerevisiae) and a chitin-binding domain (CBD; Bacillus circulans) and introduced into the vector pET21a_int. After induction with 1 mM IPTG, the fusion protein consisting of the VP1-unique region, intein and CBD, with a molecular mass of approximately 85 kDa, was produced and positive clones were identified by Western blot analysis using a polyclonal rabbit serum and human MAbs directed against the VP1-unique region (Fig. 1). Purification of the protein was performed by chitin-affinity chromatography. Addition of DTT as reducing agent resulted in the cleavage of the amino-terminal part, representing the VP1-unique region, from the intein domain. The VP1-unique region could be eluted with the inteinCBP part still bound to the chitin-modified Sepharose and cleared of residual DTT by dialysis.
|
|
|
Testing the variant VP1-unique regions VP1N-1 and VP1N-2 revealed only slight differences in frequency shifts compared with the standard VP1N-pJB (-180 Hz for VP1N-1, -110 Hz for VP1N-2 in comparison with VP1N-pJB, -150 Hz). The deduced kon rates (9587 and 7875 M-1 s-1) and koff rates (0·00041 and 0·0004 s-1) are in the same ranges as those observed for VP1N-pJB. The corresponding dissociation constants result in similar values: 4·1x10-8 M for VP1N-1 and 5·2x10-8 M for VP1N-2. As control antigen, virus-like VP2 capsids lacking the VP1-unique region were used, which were produced via recombinant baculovirus and purified by caesium chloride gradient centrifugation. No specific binding to the VP1-specific antibodies immobilized on the crystal surface was observed. Binding of the VP2 capsids could, however, be shown by coating the crystals with VP2-specific human MAbs (data not shown). Application of polyclonal VP1-specific rabbit serum samples resulted in a KD of 1·7x10-7 M, indicating a clearly lower affinity in comparison with the human MAbs. In addition, concentrations of 10 µM (rather than 1 µM) were necessary to obtain a signal, with a distinctly smaller frequency shift of about -10 Hz.
In further experiments, the human MAbs were tested for their capacity to interact with virus-like capsids consisting of VP1 and VP2 proteins produced via recombinant baculovirus and with native B19 virions isolated from the amniotic fluids of B19-infected pregnant women. The corresponding dissociation constants were 7·3x10-8 and 7·5x10-8 M, respectively, indicating a slightly lower, but similar affinity to that obtained for the MAbs interacting with the purified versions of the VP1-unique region (KD=5·4x10-8 M). As a control, we used samples of amniotic fluids that were determined as PCR-negative and did not contain parvovirus particles. Interaction with human MAbs bound to the crystal surface could not be observed (data not shown).
Combining these data, neither naturally occurring amino acid variations nor the structural environment of the VP1-unique region as part of the VP1 protein in virus-like particles or B19 virions influenced antibody binding or affinity, suggesting that the epitope is surface exposed and highly stable.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently, we published the establishment of two human MAbs directed against the VP1-unique region of parvovirus B19 (Gigler et al., 1999 ). These antibodies are identical except for one amino acid in the variable region of the light
chain. Both antibodies are highly neutralizing, as shown in assays with human bone marrow cells. In order to analyse the specificity and affinity of the antibodies further, the VP1-unique region of the standard isolate pJB was expressed as an intein-fusion protein and purified via affinity chromatography. By using a QCM biosensor, antibodyantigen interactions can be visualized at very low concentrations. Additionally, this system allows the detection of minor differences, since the dissociation constants can be calculated directly from the kon and koff rates. Both MAbs interacted with the standard isolate VP1N-pJB with a similar affinity, indicated by a KD of 5·4x10-8 M, showing that the amino acid alteration in the
chain did not influence the interaction with the epitope. This value is in the range of high-affinity antibody interactions as described for other neutralizing immunoglobulins (Stryer, 1991
).
In order to analyse differences in binding of the MAbs to naturally occurring variants of the VP1-unique region, we sequenced a series of different B19 isolates from acutely infected pregnant women, arthritis patients and chronically infected patients and compared the amino acid sequences. Sample 5940 was derived from a chronically infected child with an underlying hyper-IgM immunodeficiency (Hasle et al., 1994 ). As shown in previous publications, the variability of the VP1-unique region ranged from 0 to 8·2% at the amino acid level (Hemauer et al., 1996
). With respect to the reading frame of the structural protein, the variability ranged from 0 to 1·7% at the amino acid level (Erdman et al., 1996
). The highest variabilities were found in isolates derived from patients with persistent infections and patients with transient aplastic crisis. This is in accordance with the results shown by Hokynar et al. (2000)
. They described variability at the amino acid level of the VP1-unique region ranging from 1·2 (three amino acids) to 2·8% (seven amino acids). In our study, the variability of the VP1-specific region was found to range between 0 and 3·2% (eight amino acids). No relationship between specific amino acid exchanges and particular disease manifestations, e.g. hydrops foetalis, or transmission of the virus to the foetus could be observed. By comparing isolates from maternal sera with those from the foetus or the amniotic fluid, it could be shown that transmission of the virus was not associated with mutations or variations.
As the next step, we analysed whether the observed amino acid alterations were associated with changes in the structure of the VP1-unique region. As the epitope recognized by the VP1-specific MAb has been mapped to residues 3042 (M. Kohlmann, S. Dorsch, S. Modrow and M. Przybylski, unpublished results), we selected two representative protein variants with mutations in and around this area: one was derived from an isolate associated with hydrops foetalis (patient G, VP1N-1; exchange of 28E to D) and the second isolate was from a chronically infected child (Hasle et al., 1994 ) (patient 5940, VP1N-2; exchanges of 17K to R, 18A to D, 28E to N, 39Q to H and 43D to H). By comparing the sequences of the epitope region of the VP1-unique region with those published previously, this region was found to be highly conserved. Therefore, the selected naturally existing variants are good representatives to test the affinities of the human MAbs and to correlate these with potential changes in the conformation of the VP1-unique region. On testing the variant proteins with the microbalance biosensor, no differences were observed in the calculated dissociation constants (Fig. 4
). Even though the amino acid variations were in the neighbourhood of or within the determined epitope, the affinities of the human MAbs were identical. Obviously, the variations do not affect the structural conformation of this region, indicating that the epitope must be easy accessible and lying on the surface of the protein and not packed in a tight globular structure. This leads to the suggestion that the amino-terminal part from amino acid 1 to 80 could be folded in a loop-like structure. Variations of individual amino acid residues apparently have no effect on antibody binding; the conformation of the epitope is still presented in the correct form. Furthermore, the affinity of the human MAbs did not change when VP1/VP2 capsids and native virions from amniotic fluid were used as antigen. This confirms the hypothesis that the epitope is not only presented on the surface of the unique region but also on the capsid surface. The broad reactivity of the VP1-specific MAbs renders them optimal reagents to be applied in immunotherapeutic approaches for the treatment of severe or persistent B19 infections. Our data, however, also indicate that the VP1-unique region is an important component for future vaccines.
|
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, M. J., Higgins, P. G., Davis, L. R., Willman, J. S., Jones, S. E., Kidd, I. M., Pattison, J. R. & Tyrrell, D. A.(1985). Experimental parvoviral infection in humans. Journal of Infectious Diseases 152, 257-265.[Medline]
Bansal, G. P., Hatfield, J. A., Dunn, F. E., Kramer, A. A., Brady, F., Riggin, C. H., Collett, M. S., Yoshimoto, K., Kajigaya, S. & Young, N. S.(1993). Candidate recombinant vaccine for human B19 parvovirus. Journal of Infectious Diseases 167, 1034-1044.[Medline]
Brown, T., Anand, A., Ritchie, L. D., Clewley, J. P. & Reid, T. M. (1984). Intrauterine parvovirus infection associated with hydrops fetalis. Lancet ii, 10331034.
Brown, C. S., Van Lent, J. W., Vlak, J. M. & Spaan, W. J.(1991). Assembly of empty capsids by using baculovirus recombinants expressing human parvovirus B19 structural proteins. Journal of Virology 65, 2702-2706.[Medline]
Cossart, Y. E., Field, A. M., Cant, B. & Widdows, D. (1975). Parvovirus-like particles in human sera. Lancet i, 7273.
Cotmore, S. F., McKie, V. C., Anderson, L. J., Astell, C. R. & Tattersall, P.(1986). Identification of the major structural and nonstructural proteins encoded by human parvovirus B19 and mapping of their genes by procaryotic expression of isolated genomic fragments. Journal of Virology 60, 548-557.[Medline]
Erdman, D. D., Durigon, E. L., Wang, Q.-Y. & Anderson, L. J.(1996). Genetic diversity of human parvovirus B19: sequence analysis of the VP1/VP2 gene from multiple isolates. Journal of General Virology 77, 2767-2774.[Abstract]
Foto, F., Saag, K. G., Scharosch, L. L., Howard, E. J. & Naides, S. J.(1993). Parvovirus B19-specific DNA in bone marrow from B19 arthropathy patients: evidence for B19 virus persistence. Journal of Infectious Diseases 167, 744-748.[Medline]
Gigler, A., Dorsch, S., Hemauer, A., Williams, C., Kim, S., Young, N. S., Zolla-Pazner, S., Wolf, H., Gorny, M. K. & Modrow, S.(1999). Generation of neutralizing human monoclonal antibodies against parvovirus B19 proteins. Journal of Virology 73, 1974-1979.
Hasle, H., Kerndrup, G., Jacobsen, B. B., Heegaard, E. D., Hornsleth, A. & Lillevang, S. T.(1994). Chronic parvovirus infection mimicking myelodysplastic syndrome in a child with subclinical immunodeficiency. American Journal of Pediatric Hematology and Oncology 16, 329-333.[Medline]
Hemauer, A., von Poblotzki, A., Gigler, A., Cassinotti, P., Siegl, G., Wolf, H. & Modrow, S.(1996). Sequence variability among different parvovirus B19 isolates. Journal of General Virology 77, 1781-1785.[Abstract]
Hokynar, K., Brunstein, J., Söderlund-Venermo, M., Kiviluoto, O., Partio, E. K., Konttinen, Y. & Hedman, K.(2000). Integrity and full coding sequence of B19 virus DNA persisting in human synovial tissue. Journal of General Virology 81, 1017-1025.
Johansen, J. N., Christensen, L. S., Zakrzewska, K., Carlsen, K., Hornsleth, A. & Azzi, A.(1998). Typing of European strains of parvovirus B19 by restriction endonuclease analyses and sequencing: identification of evolutionary lineages and evidence of recombination of markers from different lineages. Virus Research 53, 215-223.[Medline]
Kößlinger, C., Drost, S., Aberl, F., Wolf, H., Koch, S. & Woias, P.(1992). A quartz crystal biosensor for measurement in liquids. Biosensors and Bioelectronics 7, 397-404.[Medline]
Kurtzman, G. J., Ozawa, K., Cohen, B., Hanson, G., Oseas, R. & Young, N. S.(1987). Chronic bone marrow failure due to persistent B19 parvovirus infection. New England Journal of Medicine 317, 287-294.[Medline]
Kurtzman, G. J., Cohen, B. J., Field, A. M., Oseas, R., Blaese, R. M. & Young, N. S.(1989a). Immune response to B19 parvovirus and an antibody defect in persistent viral infection. Journal of Clinical Investigation 84, 1114-1123.[Medline]
Kurtzman, G., Frickhofen, N., Kimball, J., Jenkins, D. W., Nienhuis, A. W. & Young, N. S.(1989b). Pure red-cell aplasia of 10 years duration due to persistent parvovirus B19 infection and its cure with immunoglobulin therapy. New England Journal of Medicine 321, 519-523.[Medline]
Mori, J., Beattie, P., Melton, D. W., Cohen, B. J. & Clewley, J. P.(1987). Structure and mapping of the DNA of human parvovirus B19. Journal of General Virology 68, 2797-2806.[Abstract]
Naides, S. J., Scharosch, L. L., Foto, F. & Howard, E. J.(1990). Rheumatologic manifestations of human parvovirus B19 infection in adults. Initial two-year clinical experience. Arthritis and Rheumatism 33, 1297-1309.[Medline]
Ozawa, K. & Young, N.(1987). Characterization of capsid and noncapsid proteins of B19 parvovirus propagated in human erythroid bone marrow cell cultures. Journal of Virology 61, 2627-2630.[Medline]
Pont, J., Puchhammer-Stockl, E., Chott, A., Popow-Kraupp, T., Kienzer, H., Postner, G. & Honetz, N.(1992). Recurrent granulocytic aplasia as clinical presentation of a persistent parvovirus B19 infection. British Journal of Haematology 80, 160-165.[Medline]
Reid, D. M., Reid, T. M., Brown, T., Rennie, J. A. & Eastmond, C. J. (1985). Human parvovirus-associated arthritis: a clinical and laboratory description. Lancet i, 422425.
Rosenfeld, S. J., Yoshimoto, K., Kajigaya, S., Anderson, S., Young, N. S., Field, A., Warrener, P., Bansal, G. & Collett, M. S.(1992). Unique region of the minor capsid protein of human parvovirus B19 is exposed on the virion surface. Journal of Clinical Investigation 89, 2023-2029.[Medline]
Rosenfeld, S. J., Young, N. S., Alling, D., Ayub, J. & Saxinger, C.(1994). Subunit interaction in B19 parvovirus empty capsids. Archives of Virology 136, 9-18.[Medline]
Sato, H., Hirata, J., Kuroda, N., Shiraki, H., Maeda, Y. & Okochi, K.(1991a). Identification and mapping of neutralizing epitopes of human parvovirus B19 by using human antibodies. Journal of Virology 65, 5485-5490.[Medline]
Sato, H., Hirata, J., Furukawa, M., Kuroda, N., Shiraki, H., Maeda, Y. & Okochi, K.(1991b). Identification of the region including the epitope for a monoclonal antibody which can neutralize human parvovirus B19. Journal of Virology 65, 1667-1672.[Medline]
Sauerbrey, G. Z.(1959). The use of oscillators for weighting thin layers and for microweighting. Zeitschrift für Physik 155, 209-212.
Serjeant, G. R., Serjeant, B. E., Thomas, P. W., Anderson, M. J., Patou, G. & Pattison, J. R.(1993). Human parvovirus infection in homozygous sickle cell disease. Lancet 341, 1237-1240.[Medline]
Shade, R. O., Blundell, M. C., Cotmore, S. F., Tattersall, P. & Astell, C. R.(1986). Nucleotide sequence and genome organization of human parvovirus B19 isolated from the serum of a child during aplastic crisis. Journal of Virology 58, 921-936.[Medline]
Stryer, L. (1991). Molecular immunology. In Biochemistry, 3rd edn, pp. 926947. San Francisco: W. H. Freeman.
Towbin, H., Staehelin, T. & Gordon, J.(1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences, USA 76, 4350-4354.[Abstract]
Ueno, Y., Umadome, H., Shimodera, M., Kishimoto, I., Ikegaya, K. & Yamauchi, T.(1993). Human parvovirus B19 and arthritis. Lancet 341, 1280.[Medline]
Uttenthaler, E., Kosslinger, C. & Drost, S.(1998). Characterization of immobilization methods for African swine fever virus protein and antibodies with a piezoelectric immunosensor. Biosensors and Bioelectronics 13, 1279-1286.[Medline]
van Elsacker-Niele, A. M. W. & Kroes, A. C. M.(1999). Human parvovirus B19: relevance in internal medicine. Netherlands Journal of Medicine 54, 221-230.[Medline]
Willwand, K. & Hirt, B.(1993). The major capsid protein VP2 of minute virus of mice (MVM) can form particles which bind to the 3'-terminal hairpin of MVM replicative-form DNA and package single-stranded viral progeny DNA. Journal of Virology 67, 5660-5663.[Abstract]
Yoshimoto, K., Rosenfeld, S., Frickhofen, N., Kennedy, D., Hills, R., Kajigaya, S. & Young, N. S.(1991). A second neutralizing epitope of B19 parvovirus implicates the spike region in the immune response. Journal of Virology 65, 7056-7060.[Medline]
Received 18 August 2000;
accepted 5 October 2000.