CA2075366C - Immunologically active peptides or polypeptides from the parvovirus b19 - Google Patents

Abstract

Immunologically active peptides or polypeptides with a partial amino-acid sequence of the capsid proteins VP1 and VP2 of parvovirus B19 which permit tests to be carried out at low cost, sensitively and specifically for the determination of antibodies against human parvovirus B19 are made available. Short peptide sequences which, employed as antigen, serve to identify anti-B19 IgG-positive sera are identified. Furthermore, the production of these peptides using genetic engineering measures is disclosed. Other antigens which are produced by genetic engineering and which can be stably produced in a high yield in E.coli and subsequently purified therefrom are used as additional antigens for IgG detection. Finally, a set of antigens permits tests to be carried out to determine IgM antibodies against the virus.
In addition, the components, produced by genetic engineering, of the surface proteins represent substances which can be used for prophylactic immunisation.

Description

The human parvovirus B19 (for short hereinafter:
B19) was discovered by chance in 1975 in plasma samples from blood donors (Cossart, Y.E., Field, A.M., Cant, B., Widdows, D.: Parvovirus-like particles in human sera.
Lancet I (1975) 72-73j by countercurrent electrophoresis.
In recent years it has been shown that B19 may cause an aplastic crisis in patients with chronic haemolytic anaemia, and i~~ the aetiological agent of erythema infectiosum (EI).
Under the electron microscope, B19 has a size of about 20 nm. The particles have an icosahedric symmetry.
Besides the virus particles there are also seen to be "empty" capsids which contain no DNA. The density in CsCl2 ( sic ) is 1. 36 - 1.40 g/ml. The virus genome consists of a single-stranded DNA of 5.4kb. The nucleotide sequence of the genome of a B19 parvovirus has been derived from a clone which contained virtually the complete viral genome (RØ Shade et al. Journal of Virology (1986) p. 921). In each case only one DNA
strand, either of the plus or the minus orientation, is packaged into each virus particle. B19 is an autonomous parvovirus, that is to say requires no helper virus for replication.
The capsid consists of two polypeptides with molecular weights of 83kDa (VP1) and 58kDa (VP2). In addition, three non-structural proteins of 52, 63 and 7lkDa can be detected.

The DNA codes in the 5' region for the structural proteins of the caps.id. The coding regions of the struc-tural proteins are identical apart from an additional N
terminus of VP1. This difference is caused by splicing processes at the mRNA level, in which in the case of VP2 the translational start for VP1 is taken out and thus translation can start only with the shorter VP2.
Investigations on various B19 isolates found world-wide have shown that these differ in part at the DNA level by the restriction enzyme pattern. These differences do not, however, correlate with the clinical spectrum of B19 infection.
It has not been possible to date to find a permanent cell line :in which B19 can be grown. There has been just as little success to date in establishing an experimental animal model for B19. B19 can, however, be grown in primary bone marrow cells in the presence of erythropoietin. It has thus been possible to clarify the mechanism of replication of the virus and show that cells of erythropoiesis a.re the target cells of this infection.
Inoculation of B19 cells in fetal erythropoietic cells and erythroblasts of a patient with chronic myeloid leukaemia has now su~~ceeded.
H19 causes erythema infectiosum (infectious erythema) which is an infectious disease which usually has a benign course and mostly occurs between the ages of childhood and early adulthood. B19 infection may in addition cause aplastic crises in patients with chronic haemolytic anaemia ( sickle cell anaemia etc . ) and chronic bone marrow aplasias in patients with inborn or acquired immunodefficiency states.
In pregnancy B19 infection may in about 10-15~
result in hydrops fetalis with resulting interuterine death. Furthermore, B19 is associated with the occurrer:~s of Schonlein-Henoch purpura.
As a rule, B19 is transmitted by droplet infec-tion but also by antigen-positive conserved blood and coagulation products.

_ Since no permanent cell line in which B19 can be obtained in large amounts is yet known, there is thus a lack of a source for obtaining antigen for diagnostic tests. To date one has made do with B19 virus discovered by chance in conserved blond from danors who are just in the viraemic stage of infection.
The object of the present invention is to provide immunologically active polypeptides which permit, with the test systems presented here, detention of B19-specific antibodies of the IgG and IgM class. This results in the following possible applicationso - Serodiagnosis of acute or previous B19 infections in dermatology, haematology, gynaecology, rheumatology and paediatrics.
- Determination of the B19 immune status in pregnant women - Investigation of conserved blood or donated plasma to exclude transmission of B19 antigen, since it is highly probable that transmission of B19 virus is no longer possible by anti-B19 IgG positive blood or plasma.
- Selection of anti--B19 positive plasma donors fox production of B19 hyperimmunoglobulin products.
There is a pressing need for the introduction of test reagents because of 'the broad clinical spectrum of the diseases caused by B19, and of the risk to B19 seronegative pregnant women.
It has emerged that utilisable immunologically active palypeptides cannot be prepared directly. Pre-paration of short peptides by genetic engineering is, just like that of large polypeptides, possible in a satisfactory yield only when suitable expression vectors are used. Although relatively short peptides can be easily prepared by synthesis, more accurate knowledge of the immunological activity is necessary.
The invention relates to immunologically active peptides which have a part of the amino-acid sequence of the capsid proteins VP 1 or VP 2 of parvovirus B19. These peptides are characterised in that they are free of r~ ,."~ 9a t b r.J~ ti '~ t impurities which interfere with the detection of anti-bodies directed against parvovirus 819, This property is of great importance since it is not possible to utilise those peptide preparations which contain, by reason of the preparation, components which .react with the anti-bodies to be detected. One example of an unwanted impurity of this type .is protein A, which is able to react specifically with the Fc portion of IgG antibodies.
A particular advantage of the immunologically actiue peptides according to the invention is that they can be prepared in good yield by the preparation process accord-ing to the invention. This is because, if the antigens required for a diagnostic test are not synthesised in an adequate amount in the preparation process, it is not possible to obtain the required yield after the subse-quent purification processes.
It has furthermore been possible within the scope of the present invention to determine short peptide segments from VP 1, more accurately from the region of VP 1 which does not coincide with VP 2, whose epitopes are suitable for reliable detection of antibodies against parvovirus B19 in the investigation fluids, especially sera. This region is called VP 1-VP 2 hereinafter. Fig. 3 shows by way of example the arrangement of some peptides (PAPEP 1-PAPEP 8) in the region (VP 1-VP 2). Although these peptides are preferred, it is equally possible to employ other peptides with 8-50 amino acids, preferably 10 to 32 amino acids, from the VP 1-VP 2 region, This region approximately corresponds to the polypeptide PAN:L
which is depicted in Fig. 2-1.
In a preferred embodiment of the present inven-tion, this small, immunodominant and B19-specific region is employed in the serological test. It is particularly preferable in this connection to employ a mixture of synthetic peptides, these pept ides having the amino-acid sequences PAPEP 1 - PAPEP 8 shown .i.n Exampl.e 3.
In another preferred embodiment of the present invention, the arnino-acid sequences which are depicted in Figure 2 of the imrnunologically active peptides PAN-1, PAN-2, PAN-3, PAN-4,, PCE, PANSE and PAPST prepared by ge::etic engineering are employed. It is as a rule sufficient in this case to use one peptide in the test.
It is possible, however, in special cases also to employ two or more of these peptides.
The peptide's according to the invention can be prepared either by synthesis or by genetic engineering.
The short peptides, which are explained in detail in Example 3, are preferably prepared by synthesis. The longer peptides are, however, preferably prepared by genetic engineering.
Firstly, they coding regions of the viral DNA were amplified from the serum of an infected patient by means of two polymerase chain reactions (PCR) and cloned in plasmids for further growth in Escherichia (E.) coli.
After further subcloning steps, various regions therefrom were then expressed by genetic engineering in E. coli, and the antigens resulting therefrom were investigated for their use for detecting antibodies against the virus.
Direct preparation of the peptides according to the invention in expres~;ion vectors is impossible because of various difficulties. For this reason, according to the invention, the viral protein segment is fused to a protein amenable t.o stable expression. This fusion protein can be employed directly after purification as antigen for IgG detection. However, the parvovirus specific portion is preferably cleaved off by suitable methods, further purified and then employed for sero logical tests.
The present invention furthermore relates to test kits for the determination of antibodies which are directed against parvovirus B19. The immunologically active peptides according to the invention can in prin-ciple be used in ai.l diagnostic test kits for detecting antibodies against parvovirus B19. In a preferred embodi-ment of the test kits according to the invention, the solid phase of suitable microtitre plates or polystyrene ~~

beads is coated with the immunologically active peptides according to the invention. After incubation with the investigation fluid (serum sample) in a suitable dilution, and after customary washing steps, enzyme- or radioactively labelled anti-human IgG is added. The extent of substrate conversion or of the bound radio-activity then shows whether antibodies directed against parvovirus B19 are present in the serum sample.
The test kits according to the invention are IO normally supplied to laboratories of physicians, hospi tals, investigation facilities etc. They usually contain all the reagents required for carrying out the test.
Customary test reagents such as buffer solutions etc.
are, however, sometimes not included. As a rule, the test kits contain microtitre plates or polystyrene beads which are coated either with one or more peptides according to the invention or with anti-antibodies. The test kit:a may furthermore contain, depending on the test principle, one or more peptides according to the invention. Finally, the test kits also embrace an indicator component which makes it possible to quantify 'the test result.
In other preferred test kits, the antigens are bound to the solid phase of microtitre plates or poly-styrene beads. After incubation of the test serum, and suitable washing and saturation steps, a specific enzy-matically or radioactively labelled antibody against. the B19 antigens is added and its substrate conversion or the bound radioactivity is measured. Since this takes the form of an inhibition test, a small substrate conversion or low radioactivity indicates 'the presence of specific antibodies.
It is likewise possible to employ peptides according to the invention coupled to solid phases far detecting IgM ant ibadies against B19. In this detection method, firstly the IgG antibodies are eliminated by adding beads coated with protein A to the investigation fluids. Bound antibodies are then detected using an anti-human IgM antibody which is enzymatically or ~~~ ~t'~~~
_,_ radioacti.vely labelled.
The principle of the so-called ~-capture assay is used in another preferred test kit. First the IgM from the investigation fluid (serum) is bound by means of anti-human IgM antibodies bound to the solid phase. The immunologically active peptides according to the inven-tion are then added. The ewtent of 'the binding of the antigens and thus the amount of anti-B19 IgM present can be effected by either the antigens being radioactively labelled or labelled with other substances (digoxigenin, avidin) and thus being detectable, or by employing a second labelled antibody against the B19 antigens and measuring its binding.
Very particularly preferred within the scope of the present invention are ELISA (enzyme linked immuno sorbent assay) test kits.
Also provided according to the invention are DNA
sequences cahich can be used for direct detection of the virus in investigation samples (sera, biopsies, etc.).
Two DNA primers which attach themselves specifically to DNA regions in VP 1 are preferably used. It is then possible by means of a commercially available polymerase chain reaction kit to achieve amplification of the region lying between them. Amplified DNA which has then been immobilised in a suitable way is detected by a suitable DNA sequence. This DNA employed for the hybridisation is prepared with the aid of a plasmi.d which contains the DNA
region lying between 'the two primers.
It is self-evident that the primer sequences must not be present in the DNA employed for the hybridisation.
The sequence of the primers used, and the arrangement with respect to one another, is depicted in F'ig. 1.
Finally, vaccines against parvovirus B19 are also made available within the scope of the present invention.
This entails the immunologically active peptides accord ing to the invention being administered, optionally several times, together with suitable adjuvants to the people to be protected. The production of antibodies ~i~"~ ~ ~~~
_8_ elicited by this can effect protection from infection with parvovirus 819.
EXAMPLE 1:
Obtaining parvovirus B19 VP 1- and VP 2-encoding sequences from patient's serum Viral DNA was isolated from lml of serum from a patient with acute infection (erythema infectiosum) by proteinase K digestion in 1~ SDS, phenol extraction and subsequent alcohol precipitation (this and all the following steps for obtaining, processing and expressing DNA, as well as the preparation of recombinant proteins and fundamental steps for the purification thereof, are described in detail in: Maniatis, T., Fritsch, E.F., Sambrook, J. (1982) Molecular cloning. Cold Spring Harbor, N.Y.). This DNA was taken up in 501 of TE buffer and then 1~1 samples were employed for the amplification by means of the polymerase chain reaction and synthetic oligodeoxynucleotides. Two pairs of primers were used for the amplification of the coding regions of the surface proteins; one of these for obtaining the VP 1 portion, and the second pair for the complete VP p oligo-deoxynucleotides used as primers have at each of their 5' ends sequences which are not homologous with the parvo-virus sequence, code for restriction enzyme cleavage sites and are therefore suitable for cloning the DNA
fragments resulting from the PCR into suitable vectors.
The primers identified by O-1 to O-5 in Fig. 1 were used.
In each case five mixtures each containing 1~1 of isolated parvovirus DNA were amplified with the two pairs of primers in a volume of 100f~1. The conditions for this were: 1.5 min denaturation at 94°C, 2 min attachment of the primers at 45°C, 4 min synthesis at 72°C; total of 50 cycles; buffer, substrates and Taq polymerase were employed far this as stated by the manufacturer ;Cetus/Perkin-Elmer, LJberlingen, FRG) .
The amplified DNA fragments from the two dif-ferent mixtures (for VP 1 and VP 2) were in each; case _ g -combined, precipitated by alcohol precipitation, washed with 70% alcohol, dried, dissolved in a volume of 200.1 of TE buffer and digested with the restriction enzymes EcoRI and HindIII. Fractionation of the fragments by electrophoresis in a 1.2% agarose gel was then followed by isolation of the corresponding DNA bands ( 709bp for VP
1, 1704bp for VP 2) and insertion into the EcoRI and HindIII sites of th.e vector PUC12 (Pharmacia, Sweden).
After transformation of the plasmids into E.coli JM109 (Pharmacia, Sweden), bacterial clones with parvovirus DNA
inserts were characterised by restriction digestion. The corresponding zones were given the names pUCI2PAN for the region encoding the VP 1 portion and pUC12VP2 for the VP
2-encoding region.

Preparation by genetic engineering of VP 1 portion and VP 2 from E.coli cells a1 VP1 portion:
1) PAN-1 The VP1-encoding region was isolated from the plasmi:d pUCI2PAN with BclI and HindIII (see Fig. 1, the HindIII site originates from the pUC vector) and inserted behind the 3' end of a truncated p-galactosidase gene of the vector (for exa~ruple pBD2) into the BamHI and HindLII
restriction cleavage sites. E.coli cells with plasmi.ds -ssulting therefrom express after induction with IPTG a ~;-gal::VP1 fusion protein (about 67kDA) in large quan-tity, which reacts very strongly with anti-parvovirus B19-positive sera in an immunoblot (Western blot.).
Purification of this: protein can be achieved very easily with conventional mE~thods utilising the insolubility of the Drotein. After lysis of the cells, the pellet frac-tion is washed with detergents such as TritonMX100 and octyl gluco-pyr=~zosi.de, and the fusion protein is subse-quently dissolved v~i.th 8M urea/1% mercaptoethanol and separated from cellular impurities by DEAF chromatography with an NaCl gradient.
The VP1 portion can be cleaved off the fusion protein by BrCN cleavage since the VP1 protein sequence starts with a methionine, and this amino acid no longer appears in the fragment itself; by contrast, methionine occurs relatively often in the bacterial fusion portion so that this portion is broken up into very small frag-ments. After cleavage in 35$ formic acid and O.lmg/ml BrCN at room temperature for 4h, the sample was lyophi-lised, dissolved in 8M urea, 2mM DTT (dithio-threitol) and purified by DEAF chromatography in an NaCl gradient. The VP1 fragment resulting therefrom was called PAN-1 and can be uses directly for serological determi-nations. The amino-acid sequence is indicated in Fig. 2-1.
Further con;~tructs generated were plasmids which code for fusion proteins consisting of the glutathione S-transferase from Sclz.istosoma japonicum (Smith, D.B. and Johnson, K.S.: Sing:Le step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene:, 67 (1988) 31-40) and the VP1 portion. However, i.t is also possible to use another fusion partner as long as it does not interfere with the diagnostic test.
2) PCE:
The B19 DNA fragment was isolated from pUCI2PAN
after BclI/PvuII digestion (618bp) and integrated into the BamHI and SmaI sites in pGEXl (pGEXIPAN) . The resul.t-ing 52kDA fusion protein was purified from the super-natant by means c~f glutathione-coupled agarose and employed as antigen f:or the serological tests in Example 4 (name:PCE). The amino-acid sequence of this antigen is shown in Fig. 2-2.

3) PAN-2:
A 458bp fragment was isolated from pUCI2PAN with BclI/HincII and, after intermediate clonings in other vectors, inserted into pGEX2 (pGEX2PAN). Insertion of the fragment in the same reading frame can also be achieved by using oligodeoxynucleot.ides. At the fusion site of glutathione S-transferase and the VP1 segment is the amino-acid sequence which is recognised by thrombin, so that the B19 portion can be cleaved off the fusion partner by this enzyme. It is also possible to use any other fusion partner as long as it has this protease recognition sequence. The amino-acid sequence of the antigen, as well as fused-on foreign amino acids ( in bold print) is indicated in Fig. 2-3.

4) PAN-3:
A 458bp fragment was isolated from pUCI2PAN ceith BcII/HincTI and, after intermediate clonings in other vectors, inserted into pGEX3 (pGEX3PAN). Insertion of the.
fragment in the same reading frame can also be achieved by using synthetic oligodeoxynucleotides. At the fusion site of glutathione S-transferase and the VP1 segment is the amino-acid sequence which is recognised by the protease factor Xa, so that the B19 portion can be cleaved off the fusion partner by this enzyme. It is also possible to use any other fusion partner as long as it has this protease recognition sequence. The amino-acid sequence of the antigen, as well as fused-on foreign amino acids (in bold print) is indicated in Fig. 2-4.

5) PAN-4:
The complete B19 DNA insert was obtained .from pUCI2PAN by BclI and PstI digestion and, after various intermediate cloning steps, inserted into the vector pGEX2. This resulted in the plasmid pGEX2PAN. Insertion of the fragment in the same reading frame can also be achieved by using synthetic oligodeoxynucleotides. At 'the fusion site of glutathione S-transferase and the VP1 segment is the amino-acid sequence for the protease thrombin, so that the B19 portion can be cleaved off the fusion partner by this enzyme. It is also possible to use another suitable fusion partner if it has this protease recognition sequence. The amino-acid sequence of the antigen, as well as fused-on foreign amino acids (in bold print) is indicated .in Fig. 2-5.
Purification of the antigens can be achieved by simple affinity chromatography with a glutathione-coupled gel matrix.
For further purification of the fusion proteins based on pGEX2 and _i, the B19 portion was cleaved off by digestion with thrombin or factor X as stated by the manufacturer (Boehringer Mannheim). The fragments were then purified again by affinity chromatography. The glutathione S-transferase can in this case be selec-tively fished out, the parvovirus protein portion is to be found in the flow-through and can be employed after a final DEAF chromatographic fractionation in serological tests.
However, as an alternative to this, the protease can also be added directly to the glutathione-coupled gel suspension with they fusion protein bound on. After an incubation time of about 1h, the VPl fragment which has been cleaved off can be washed out of the gel, while the glutathione S-transferase portion remains bound to the gel matrix.
b) VP-2 portion:
1) VP-2 Owing to the: choice of the PCR primers and of the vect..r, the coding region for VP2 is already in the correct reading frame in the plasmid pUC12VP2 and can be purified after induction with IPTG from the insoluble fraction of the bacterial lysate, in a similar way to that described for pBD2PAN. The amino-acid sequence of the recombinant antigen is shown in Fig. 2-6.

2) PANSE:
It emerged, surprisingly, that a truncation of the VP2-encoding sequence is associated with a consider-able increase in the protein yield, that this truncated antigen can be stably expressed, is not degraded euen during purification, and still has the same reactivity with anti-B19 positive sera too. This expression plasmid (pUCI9PANSE) was obtained by truncating the 5' region of VP2 by 355bp as far as an Nsil site. This fragment was inserted into pCUl9 (Pharmacia, Sweden) which has the same reading frame in the lacZ peptide as the B19 sequence. Since, because of the PCR primers, a HindIII
site is located at the 3' end, it was necessary also to produce an EcoRI site by intermediate cloning in order to be able to insert the required fragment into 'the Pstl and EcoRI sites of pUCl9.
The antigen with a size of about 38kDa ( PANSE ) can be separated from impurities very simply from the pellet fraction of the bacterial lysate after dissolving in 4M urea by DEAE chromatography. The ammo-acid sequence of the antigen is indicated in Fig. 2-7.
3) PAPST:
A fragment 7I6bp in size which encodes the N-terminal region of VP-2 was isolated from the plasmid pUC12VP2 by PstI digestion. After insertion of the fragment into the vector pUC9 (Pharmacia, Sweden] in the same orientation of the reading frames as the lacZ of the vector (characterised by restriction enzyme digestion), the B19 antigen with a size of about 33kDa is produced in very large quantity (about 10~ o.f the total E.coli protein). Purification can take place in a similar way as for pBDAN from the insoluble constituents by dissolving in 8M urea and subsequent DEAF chromatography. The amino-acid sequence is depicted in Fig. 2-8.

c) Complete VP1/VP2:
The plasmid. pUCI2PAN was opened with PstI and HindIII, and the VP~? encoding region from pUC12VP2 was inserted after HindIII and partial PstI digestion as l.7kb fragment (pUC:12VP1/2).
Expression of VP1/2-containing antigens in E.coli:
1) PAV-1-B:
pUC12VP1/2 'was cut with EcoRI and BamHI, and a DNA band 1466 by in size was isolated and subsequently inserted into the EcoRI/BamHI sites of the vector pUCl8stop. pUCl8 stop resembles the abovementioned pUC
vectors; however, it. differs from the latter by contain-ing between the Pstl and HindIII site a synthetic oligo-deoxynucleotide which codes for translation stop signals and for the stop of transcription. The polylinker region of the vector thus has the following sequence:
ATG ACC ATG ATT AGG ATT TCG AGC TCG GTA CCC GGG GAT CCT
CTA GAG TCG ACC TGC AGT AAT TAA TTA GAT CTC GAG CCC GCC
TAA TGA GCG GGC TTT TTT AAG CTT
(The restriction cleavage sites EcoRI - GAATTC, BamHI -GGATCC, PstI - CTGCA.G, BglII - AGATCT and HindIII -AAGCTT are indicated by bold print) The vector (pUC-V1-B) obtained in this way encodes the VP-1 stx-uctural protein from the start up to amino acid 486 followed by some amino acids of the pUC
polylinker and is germinated by the stop codon of the inserted oligodeoxynucleotide. The expressed antigen (PAV-1-B) is produced in very good yield after IPTG
induction in the E.coli cells and has a size of 60 kUa.
Its amino-acid sequence is depicted in Fig. 2-9, amino acids emphasised by bold print are not B19-specific and are encoded by pUC sequences. The reactivity with anti-B19-positive sera is very good and efficient purification can be achieved by removing soluble E.cali proteins, dissolving in 8M urea and conventional ion exchange chromatography (as described).
2) PAV-1-N:
The vector pUCVP-1-B described above was digested with EcoRI and Nsil. The band 1137bp in size produced in this way was inserted into the vector pUCl8stop into the EcoRI and Pstl sites (see above). The resulting vector pUCVP-1-N encodes the structural protein from the start up to amino acid 377; the antigen (PAV--1-N) is produced after IPTG induction in the E.coli cells somewhat less well than the antigen PAV-1-B described above> Tt is 45 kDa in size, and the reactivity with awti-B19 sera is good. The amino-acid sequence is indicated in Fig. 2-10, amino acids with bold print are encoded by the pUC vector and are not B19 specific. Purification of the antigen can be achieved as for PAV-1-B.
3) Expression of the antigens described under c)1) and c)2) as GST fusion proteins The two vectors pUCVP-1-H and pUCVP-1-N were digested with EcoRI/BglII and the resulting bands about 1480bp and 1150bp, respectively, in size were isolated, with the translation stop signals introduced together with the pUClBstop being transferred too. (The BglII site is indicated in the pUCl8stop polylinker sequence indi-cated above, and the Bcll site (TGATCA) immediately before the start of translation was introduced with the primers used for the DNA amplification - see Fig. 1, 0-1). The two fragments which encode the same 5' sequences but regions of different length of VP-1 were then inserted into the vector pGEX-1 described above.
Since pGEX-1 has only the Smal and EcoRI restriction cleavage sites available for insertion of the 3' end of a fragment, it was initially necessary also to produce a site compatible for SmaT (blunt end). This was effected by inserting the two DNA fragments into the EcoRI and BamHI (compatible with Bgl.II) restriction sites of the vector pTC20H (The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation, J.L. Marsh, M. Erfle and E.J. Wykes, Gene, 32. (1984) 481-485). The two fragments were then isolated in turn from the two resulting vectors with BclI and HincI:I (blunt end cleavage) and inserted into pGEX-1 in BamHI and SmaI. Since HincII also cuts in the B19 sequence, the two fragments were isolated by a partial HineII digestion.
The two pGEX vectors now express fusion proteins consisting of the g7.utathione S-transferase followed by the two VP-1 segments of different length. The fragment originating from pUCVP-1-B and now located in pGEXVP-1-B
yields a fusion protein of about 87kDa; the smaller fragment encoding only up to amino acid 377 a fusion protein 72kDa in size. The amino-acid sequences are indicated in Fig. 2--9 and 2-10. The only difference is that the five N-tex-minal amino acids are omitted and instead replaced by glutathione S-transferase.
4) Further expression of VP1/VP2:
A 2.4kb fragment was isolated after EcoRI and HindIII digestion fx:om the plasmid pUC12VP1/2 with the complete VPl and VP2 encoding region and inserted into the eukaryotic expression vector pMDIII (Motz, M., Deby G., Jilg, W., Wolf, H.: Expression of the Epstein-Barr virus major membrane proteins in Chinese hamster ovary cells. Gene, 44 (1986) 353-359. (Obtainab:Le from ATCC)) after EcoRI and HindIIT digestion. This plasmid was subsequently linearised again with a Sal:I, and a 2.4kb SalI fragment with a dihydrofolate reductase gene (DHFR) and regulatory sequences was also inserted.
The plasmid pMDIIIVPl/2 obtained in this way was trans-fected into DHFR-negative CHO cells. Colonies resulting after selection on a:Lpha-minus medium (GIBCO) were isolated and amplified after washing out with increasing concentrations of methotrexate (MTX). Particles with VPl/VP2 can be purified from the culture supernatant from these cell cultures.

Furthermore, the 2.4kb fragment from pUC12VP1/2 was inserted after EcoRI/HindIII digestion into a vector which has, besides the HindIII site, also a HamHI site.
The parvovirus portion from this intermediate construct was then isolated as. BclI and BamHI fragment and inserted into the BamHI site: of a baculovirus expression vector (various constructs can be used). (The BclI site is located immediately in front of the translational start of VP1, it is encoded by the PCR primers, see Fig. 1; the BamHI digestion must. be carried out partially since there is also a site of this type still present in the parvo-virus sequence.). Ai_ter co-transfection of the resulting plasmid with wild-type baculovirus DNA into an insect cell culture line, cells which have no so-called inclusion bodies were isolated. The VP1 which is produced intracellularly can be purified in large quantity from those cells in which the baculovirus polyhedrin gene is replaced by the VP1/2 gene.
5 ) Expression of VP~-2 Furthermore, expression of the smaller B19-VP-2 was obtained using recombinant baculoviruses. For this, the VP-2-encoding plasmid pUCI2VP (see Example 1) was digested with EcoRI and HindIII, and the resulting l.7kb fragment was inserted into an abovementioned vector which has, besides the H:indIII site, also a BamHI site. The parvovirus portion was then isolated as BclI and BamFiI fragment from this intermediate construct and inserted into the BamHI site of a baculovirus expression vector (various constructs can be used). (The BclI
site is ~:~cated immediately in front of the translational start of BP2, it is encoded by the PCR primers, see Fig. 1, 0-3; the BamHI digestion must be carried out partially since there i~ also a site of this type still present in the pa:rvovirus sequence). After co-trans-fection of the resulting plasmid with wild-type baculo-virus DNA into an insect cell culture line, cells which have no so-called .inclusion bodies were isolated. VP2 can ~'~~r~
be purified in large quantity as particles from those cells in which the baculovirus polyhedrin gene is replaced by the VP2 gene . These particles are particu-larly suitable for use in the ~-capture 'test.

Synthetic peptides with imrnunodominant epitopes The reaction patterns of the bacaerial expression products (especially pGEX::VPl fusion constructs) with parvovirus-positive sera in a Western blot lead to the surprising conclusion that a short fragment from the VP1 portion suffices to identify all IgG-positive parvo sera.
The fragment can be covered with the following peptides which can be produced by synthesis:
PAPEP-1:
Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr PAPEP-2:
Leu Lys Asp His Tyr Asn Tle Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser PAPEP-3:
Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala PAPEP-4:
Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser G1u Asp Leu His Lys Pro Gly Gln Val PAPEP-5:
Asn Tyr Val G1y Pro Gly Asn Glu Leu Gln Ala Gly Pro Fro G1n Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg '.Cyr Ser Gln Leu _ 19 _ PAPEP-6:
Pro Tyr Thr Isis Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr G1y Phe PAPEP-7:
Asn Ala Ser Glu Lys Tyr Pro Sex° Met Thr Ser Val Asn Sex Ala Glu Ala Ser PAPEP-8:
Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys His Ile Lys These antigens can be prepared in large quantities by synthesis without problems, purified and then employed in the ELISA in concentrations between 100-200 ng per mixture.
Even when good results can be achieved with just one of the peptides, conjoint use of two or three peptides is preferred.
It is possible to use for detecting TgG or IgM
antibodies in some cases different peptides or com-binations thereof.

a) Determination of serum antibodies against parvovirus The antigens purified and described in Example 2 were used to test a relatively large quantity of sera for their reactivity with these antigens. For this, the various recombinant proteins were added in a cancentration of 0.5 - 1 ug/rnl in carbonate buffer, pFI
9.5, to the wells of commercially available ELISA plates for 16h for binding on. After unbound material has been washed out it is possible then to store these plates in the dry state at 4°C.
Tncubation with the sera for 2h took plane in a dilution of 1:100, and the subsequent washing procedures ~~~ ~~,~.~~' and the detection of bound antibodies with a peroxidase-coupled anti-human IgG antibody took place by conven-tional test procedures.
Various serum panels were tested for anti-B19 IgG:
1. Sera from a patient with acute B19 infection were investigated consecutively from the appearance of erythema infectiosum up to 19 weeks after the illness.
Result :
All the sera were recognised as anti-B19 IgG
positive even from the start of the clinical manifes-tation and remained positive over the observation period (19 weeks) both with the fusion protein from pGEXIPAN
(PCE, see Example 2) and with a VP-1 region cleaved off by BrCN (PAN-1, see Example 2) and daith a VP1 portion cleaved off by thrombin (PAN-4) too as antigens.
2. Serum pairs from pregnant women (n=21) from whom a serum sample was taken on hospitalisation and four weeks later were tested for anti-B19 TgG. The same sera were used for each antigen.
Result:
PCE:
Of the 21 pregnant women, 15 were anti-B19 negative and 6 were anti-B19 IgG positive at the time of hospitalisation. ~'he serological result on the second serum sample four weeks later produced an identical result.
PAN-2:
Of the 21 pregnant women, 14 were anti-B19 2gG
negative and 7 were anti-B19 positive at the time of hospitalisation. On retesting serum samples taken from these women four weeks later, IgG was no longer detect-able in one woman who was previously anti-B19 TgG
positive.

_ 21 _ PAN-4:
Of the 21 pregnant women, 15 were anti-B19 negative and 6 were anti-B19 IgG positive at the time of hospitalisation. The serological result on the second serum sample four weeks later produced an identical result.
b) Testing of a definitely B19 IgG/M~-positive/negative serum collection (n=13) The sera used were obtained from clinically defined cases and had previously been checked in an IgG/M
test which uses purified virus as antigen. Sera 1-6:
anti-B19 negative, 7-9: IgM/IgG-positive, 10-13~ IgM
negative, IgG-positive.
PAN-4 were tested by the procedure desa~:ibed above. The IgM antibodies were determined by the same test principle as for the IgG determination but PANSY and PAV-1-B were bound as antigens to the plates in a 1:1 mixture with a 10-fold higher concentration, furthermore the serum IgG antibodies were eliminated by pre adsorption with protein A-coupled beads.
The following values for the absorption were obtained:
IgG determination with PAN-4 ( about 20ng per test well), IgM with a 1:1 mixture of PANSE and PAV-1-B (about 150ng per test well total protein) Serum IgG IgM
1 0.09 0.07 2 0.05 0.06 3 0.10 0.08 4 0.07 0.06 5 0.07 0.08 6 0.04 0.07 7 1.82 1.53 8 0.90 0.46 9 0.72 0.56 ~'"~~~~f~

1.10 0.08 11 0.62 0.14 12 0.98 0.11 13 0.87 0.09 5 The results show a clear discrimination of the positive/negative sera bath for the IgG test and for the IgM test.
The IgM-positive sera used were obtained from clinically defined cases and had previously been checked 10 in an IgM test which uses purified virus as antigen. A
test mixture with recombinant antigens from the VP1 and VP2 regions also recognised all IgM-positive sera. It emerged that the "PAPST, VP2 but especially PANSE" VP2 portions reacted better in this case than in the IgG
test. Both regions will therefore be represented in a commercial test kit for IgM.
A further improvement in the sensitivity can be achieved by selectively binding the serum IgM antibodies to the solid phase by means of monoclonal antibodies, adding recombinant antigen (baculovirus-expressed par-ticulate VP-2) and determining the binding (~c-capture assay).
These experiments demonstrate the high reliability of the test carried out using the immunologi cally active polypeptides according to the invention.
The VP2 region contained in the antigens called "PANSE, PAPST and VP-2" results in no additional increase in the sensitivity for the determination of antibodies from patients with long-passed infection. On the other hand, a good reaction with these antigens is to be found in the case of sera within infection only in the recent past. This antigen is therefore suitable for providing information about the 'timing of the infection.
In a test kit it is possible to admix one or a mixture of these antigens either with the VP1 portions produced by genetic engineering or with the synthetic peptide, or else to use these in separate mixtures where the discrimination of the reactivity with these two regions provides additional information about the timing of the infection.
A further improvement in the sensitivity can be achieved by selective binding of the serum IgM antibodies to the solid phase by means of monoclonal antibodies, adding recombinant antigen and determining the binding (~a-capture assay).

Use of B19-specific DNA primers for direct detection of pathogen Any H19 DN.A present were obtained from the investigation samples (serum, biopsies) by proteinase K
digestion in the presence of 1~ SDS (2h, 37°C), phenol extraction and precipitation in 70~ ethanol. This, and the DNA amplification which then followed too, was carried out in ana:Logy to the procedure described in Example 1. Primers 0-5 and O-2 (see Fig. 1 for the sequence and position on the B19 genome) were used; in the case of B19-positive samples, the amplified fragment has a size of 319bp. Demonstration of the B19-specificity of the DNA fragment was carried out after fractionation of the PCR mixtures by a 1.5$ agarose gel, transfer of the DNA to a nitrocellulose membrane (Southern blot) and hybridisation with a piece of DNA which was located between them and which had been labelled either radio-actively with P-3c. or with digoxigenin by conven-tional methods (primer extension). The DNA fragment used for w:ze hybridisation was obtained in the following way:
a DNA fragment 260 by long was isolated from the plasmid pUCI2PAN after digestion with HincII and PstI and in serted into the HincII and Pstl sites in pUCl2. It is now possible for the B:19~ fragment without the sequences u~~ed for the amplification to be obtained from the resulting plasmid (pUCI2PCRDIA) by EcoRI/PstI digestion and be employed after labe~_bing as hybridisation probe.

- 23a -In summary, this invention relates to an immunologically active peptide or polypeptide which has part of the amino-acid sequence of the capsid proteins VP1 or VP2 of parvovirus B19, characterised in that it is free of impurities which may interfere with the detection of parvovirus Bl9 specific antibodies, and the polypeptide is a partial sequence of 8 to 50 amino-acid residues, in particular 10 to 32 amino-acid residues, of the peptide PAN-1, as depicted in Fig. 2-1, or has one or more amino acid sequences selected from a group consisting of:
Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys His Ile Lys;
Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr;
Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser;
Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala;
Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val;
Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu;
Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Tly Phe;
Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Glu Ala Ser;

- 23b -His Met Ser Lys Lys Ser Gly Lys Trp Trp G1u Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Gly Glu Phe Ile Val Thr Asp;
Gly Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Gly Asp Pro Arg Glu Phe Ile Val Thr Asp;
Gly Ile Leu Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Gly Asp Pro Leu Glu Asp Pro Arg Val Pro Ser Ser Asn Ser;

- 23c -Gly Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Gly Arg Arg Ile Pro Gly Asn Ser Ser;
Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser;
or partial sequences thereof - 23d -or selected from a group consisting of:
Met Thr Ile Thr Asn Ser Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala G1n Val Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Glu Ala Ser Thr Gly Ala Gly Gly Gly Gly Ser Asn Ser Val Lys Ser Met Trp Ser Glu Gly Ala Thr Phe Ser Ala Asn Ser Val Thr Cys Thr Phe Ser Arg Gln Phe Leu Ile Pro Tyr Asp Pro Glu His His Tyr Lys Val Phe Ser Pro Ala Ala Ser Ser Cys His Asn Ala Ser Gly Lys Glu Ala Lys Val Cys Thr Ile Ser Pro Ile Met Gly Tyr Ser Thr_ Pro Trp Arg Tyr Leu Asp Phe Asn Ala Leu Asn Leu Phe Phe Ser Pro Leu Glu Phe Gln His Leu Ile Glu Asn Tyr Gly Ser Ile Ala Pro Asp Ala Leu Thr Val Thr Ile Ser Glu Ile Ala Val Lys Asp Val Thr Asp Lys Thr Gly Gly Gly Val Gln Val Thr Asp Ser Thr Thr Gly Arg Leu Cys Met Leu Val Asp His Glu Tyr Lys Tyr Pro Tyr Val Leu Gly Gln Gly Gln Asp Thr Leu Ala Pro Glu Leu Pro Ile Trp Val Tyr Phe Pro Pro Gln Tyr Ala Tyr Leu Thr Val Gly Asp Val Asn Thr Gln Gly Ile Ser Gly Asp Ser Lys Lys Leu Ala Ser Glu Glu Ser Ala Phe Tyr Val Leu Glu His Ser Ser Phe Gln Leu Leu Gly Thr Gly Gly Thr Ala Ser Met Ser Tyr Lys Phe Pro Pro Val Pro Pro G1u Asn Leu Glu Gly Cys Ser Gln His Phe Tyr Glu Met Tyr Asn Pro Leu Tyr Gly Ser Ser Arg Val Asp Leu Gln;

- 23e -Met Thr Ile Thr Asn Ser Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Glu Ala Ser Thr Gly Ala Gly Gly Gly Gly Ser Asn Ser Val Lys Ser Met Trp Ser Glu Gly Ala Thr Phe Ser Ala Asn Ser Val Thr Cys Thr Phe Ser Arg Gln Phe Leu Ile Pro Tyr Asp Pro Glu His His Tyr Lys Val Phe Ser Pro Ala Ala Ser Ser Cys His Asn Ala Ser Gly Lys Glu Ala Lys Val Cys Thr Ile Ser Pro Ile Met Gly Tyr Ser Thr Pro Trp Arg Tyr Leu Asp Phe Asn Ala Leu Asn Leu Phe Phe Ser Pro Leu Glu Phe Gln His Leu Ile Glu Asn Tyr Gly Ser Ile Ala Pro Asp Ala Leu Thr Val Thr Ile Ser Glu Ile Ala Val Lys Asp Val Thr Asp Lys Thr Gly Gly Gly Val Gln Val Thr Asp Ser Thr Thr Gly Arg Leu Cys Ser Asn This invention further relates to a test kit for the detection of antibodies against human parvovirus B19, characterised in that it has at least one immunologically active peptide or polypeptide according which is able to react with the antibodies present in the investigation fluids, and in that it has at least one indicator component which makes it possible to detect complexes of immunologically active peptide and antibody.

- 23f -This invention further relates to a process for the purification of immunologically active peptides or polypep.tides characterised in that it comprises the dissolving of unpurified peptide or polypeptide in 8M urea, and fractionation by a DEAF SephacelTM column with an NaCl gladient.
This invention further relates to the use of at least one DNA sequence selected from 0-1:
GTG AAT TCT GAT CAT ATG AGT AAA AAA AGT GGC AAA TGG
0-2:
C TTC GGT CGT GAC CAC GTC CTC CCC
0-3:
G AGG AAT TCT CTG ATC ATG ACT TCA GTT AAT TCT GCA GAA GCC
0-4:
GAG GGG TGG CAC GGG ACT CGG TCC TTC GAA GAG
0-5:
G CTA CAA GCT GGG CCC CCG CAA AG
for the direct detection of parvovirus B19 by means of DNA
amplification, especially by means of polymerase chain reaction.
This invention further relates to the use of immunologically active peptides as vaccines against infections with parvovirus B19.

- 24 -°
DESCRTPTION OF THE FIGURES
Fig. 1: Diagrammatic representation of the VP1/2 encoding region of parvovirus B19 with the primer sequences used for the amplification.
The structure of the single-stranded 819 genome with the inverse regions at the ends (double strand) and with coding regions is depicted diagrammatically in the upper part. The coding region for the non-structural proteins (NS) which are synthesised as polypeptide and then processed is in the left region. The right region codes for the surface proteins of the viral capsid (VP1/2), with VP1 being, apart from an additional N-terminal region (shaded bar), identical to VP2 (black bar). Underneath this are indicated the regions of oligodeoxynucleotides 0-1 to O-4 which were used as primers for the amplification (PCR) of the H19 sequences located between them (0-1 and O-2 for the VP1 region, and O-3 and O-4 for VP-2).
The DNA sequences of the corresponding B19 regions as well as of the oligodeoxynucleotides are indicated in the lower part of the figure. The oligo deoxynucleotide sequences are identified by bold print, non-homologous regions, that is to say sequences which do not hybridise with B19, are contrasted by a line spacing.
These non-hybridising sequences represent restriction enzymes sites for EcoRI (GAATTC) and BclI (TGATCA) in the case of O-l, for EcoRI, BclI and BspHII (TC-ATGA) in the case of 0-3, and for HindITI (AAGCT-T) in 'the case of 0-4. The amplified VP2 encoding fragment (O-3 and O-4) was digested with EcoRI and HindIII before insertion in pUC vectors, the VP1 encoding fragment with EcoRI and PstI, the PstI cleavage site being located in the B19 DNA
(from position no. 4 in the indicated sequence for 0-2, C'PGCAG ) .

Fig. 2: Amino-acid sequences of the antigens described in Example 2 and produced by recombination in E.coli cells.
Owing to cloning steps, in each case some non B19-authentic foreign amino acids are also contained at the N-termini and at. the C-termini (apart from PANSE and VP-2) and are emphasised by bold print.
The amino-acid sequences of the antigens described in Example 2 are described:
Fig. 2-1: PAN-1.
Fig. 2-2: PCE
Fig. 2-3: PAN-2:
Fig. c-4: PAN-3~
Fig. 2-5: PAN-9 Fig. 2-6: VP2 Fig. 2-7: PANSE~
Fig. 2-8: PAPST
Fig. 2-9: PAV-1B
Fig. 2-10: PAV-1N
Fig. 3: Diagrammatic' representation of the arrangement of some peptides with respect to one another

Claims (20)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Immunologically active peptide or polypeptide which has a part of the amino-acid sequence of the capsid proteins VP1 or VP2 of parvovirus B19, characterised in that it is free of impurities which may interfere with the detection of parvovirus B19 specific antibodies, and the polypeptide is a partial sequence of 8 to 50 amino-acid residues of the peptide PAN-1, as depicted in Fig.
2-1, or has one or more amino-acid sequences selected from a group consisting of:

Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys His Ile Lys;

Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr;

Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser;

Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala;

Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val;

Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu;

Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Tly Phe;

Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Glu Ala Ser;

His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val G1y Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Gly Glu Phe Ile Val Thr Asp;
Gly Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Gly Asp Pro Arg Glu Phe Ile Val Thr Asp;

Gly Ile Leu Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Sex Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Gly Asp Pro Leu Glu Asp Pro Arg Val Pro Ser Ser Asn Ser;

Gly Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys G1y Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Gly Arg Arg Ile Pro Gly Asn Ser Ser;

and partial sequences thereof.

2. Immunologically active peptide or polypeptide which has a part of the amino-acid sequence of the capsid proteins VP1 or VP2 of parvovirus B19, characterised in that it is free of impurities which may interfere with the detection of parvovirus B19 specific antibodies, and the peptide is PAN-1, as depicted in Fig. 2-1.

3. Immunologically active peptide or polypeptide which has a part of the amino-acid sequence of the capsid proteins VP1 or VP2 of parvovirus B19, characterised in that it is free of impurities which may interfere with the detection, of parvovirus B19 specific antibodies, and the polypeptide is a partial sequence of 14 to 32 amino-acid residues of the peptide PAN-1, as depicted in Fig.
2-1, or has one or more amino-acid sequences selected from a group consisting of:

Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys His Ile Lys;

Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr;

Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser;

Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala;

Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val;

Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu;

Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Tly Phe;

Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Glu Ala Ser;

His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Sex Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Gly Glu Phe Ile Val Thr Asp;

Gly Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Gly Asp Pro Arg Glu Phe Ile Val Thr Asp;

Gly Ile Leu Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Gly Asp Pro Leu Glu Asp Pro Arg Val Pro Ser Ser Asn Ser;

Gly Ser Arg Arg Pro Asp His Met Ser Lys Lys Sex G1y Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Va1 Lys Asp Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Gly Arg Arg Ile Pro Gly Asn Ser Ser;
and partial sequences thereof.

4. ~Immunologically active peptide or polypeptide which has a part of the amino-acid sequence of the capsid proteins VP1 or VP2 of parvovirus B19, characterised in that it is free of impurities which may interfere with the detection of parvovirus B19 specific antibodies, and the polypeptide has one or more amino-acid sequences selected from a group consisting of:

Met Thr Ile Thr Asn Ser Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Sex Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Glu Ala Ser Thr Gly Ala Gly Gly Gly Gly Ser Asn Ser Val Lys Ser Met Trp Ser Glu Gly Ala Thr Phe Ser Ala Asn Ser Val Thr Cys Thr Phe Ser Arg Gln Phe Leu Ile Pro Tyr Asp Pro Glu His His Tyr Lys Val Phe Ser Pro Ala Ala Ser Ser Cys His Asn Ala Ser Gly Lys Glu Ala Lys Val Cys Thr Ile Ser Pro Ile Met Gly Tyr Ser Thr Pro Trp Arg Tyr Leu Asp Phe Asn Ala Leu Asn Leu Phe Phe Ser Pro Leu Glu Phe Gln His Leu Ile Glu Asn Tyr Gly Ser Ile Ala Pro Asp Ala Leu Thr Val Thr Ile Ser Glu Ile Ala Val Lys Asp Val Thr Asp Lys Thr Gly Gly Gly Val Gln Val Thr Asp Ser Thr Thr Gly Arg Leu Cys Met Leu Val Asp His Glu Tyr Lys Tyr Pro Tyr Val Leu Gly Gln Gly Gln Asp Thr Leu Ala Pro Glu Leu Pro Ile Trp Val Tyr Phe Pro Pro Gln Tyr Ala Tyr Leu Thr Val Gly Asp Val Asn Thr Gln Gly Ile Ser Gly Asp Ser Lys Lys Leu Ala Ser Glu Glu Ser Ala Phe Tyr Val Leu Glu His Ser Ser Phe Gln Leu Leu G1y Thr Gly Gly Thr Ala Ser Met Ser Tyr Lys Phe Pro Pro Val Pro Pro Glu Asn Leu Glu Gly Cys Ser Gln His Phe Tyr Glu Met Tyr Asn Pro Leu Tyr Gly Ser Ser Arg Val Asp Leu Gln; and Met Thr Ile Thr Asn Ser Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr Gln Gln Phe Val Glu Phe Tyr Glu Lys Val Thr Gly Thr Asp Leu Glu Leu Ile Gln Ile Leu Lys Asp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Ile Lys Asn Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gln Ser His Gly Gln Leu Ser Asp His Pro His Ala Leu Ser Ser Ser Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gln Val Ser Val Gln Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gln Ala Gly Pro pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gln Leu Ala Lys Leu Gly Ile Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys Asn Ile Lys Asn Glu Thr Gly Phe Gln Ala Gln Val Val Lys Asp Tyr Phe Thr Leu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gln Gly Ser Leu Pro Glu Val Pro Ala Tyr Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser Ala Glu Ala Ser Thr Gly Ala Gly Gly Gly Gly Ser Asn Ser Val Lys Ser Met Trp Ser Glu Gly Ala Thr Phe Ser Ala Asn Ser Val Thr Cys Thr Phe Ser Arg Gln Phe Leu Ile Pro Tyr Asp Pro Glu His His Tyr Lys Val Phe Ser Pro Ala Ala Ser Ser Cys His Asn Ala Ser Gly Lys Glu Ala Lys Val Cys Thr Ile Ser Pro Ile Met Gly Tyr Ser Thr Pro Trp Arg Tyr Leu Asp Phe Asn Ala Leu Asn Leu Phe Phe Ser Pro Leu Glu Phe Gln His Leu Ile Glu Asn Tyr Gly Ser Ile Ala Pro Asp Ala Leu Thr Val Thr Ile Ser Glu Ile Ala Val Lys Asp Val Thr Asp Lys Thr Gly Gly Gly Val Gln Val Thr Asp Ser Thr Thr Gly Arg Leu Cys Ser Asn.

5. Immunologically active peptide or polypeptide according to Claim 1, 2, 3 or 4, characterised in that it is in the form of a fusion protein, where this fusion protein has at least a part of .beta.-galactosidase or of glutathione S-transferase.

6. Test kit for the detection of antibodies against human parvovirus B19, characterised in that it has at least one immunologically active peptide or polypeptide according to Claim 1, 2, 3 or 4, which is able to react with the antibodies present in the investigation fluids, and in that it has at least one indicator component which makes it possible to detect complexes of immunologically active peptide and antibody.

7. Test kit according to Claim 6, characterised in that the indicator component is an antibody which is directed against the antibody to be detected and has a label.

8. Test kit according to Claim 7, characterised in that the label consists of a radioactive isotope.

9. Test kit according to Claim 7, characterised in that the label consists of an enzyme which is able to catalyse a colour reaction.

10. Test kit according to Claim 7, characterised in that the immunologically active peptide or polypeptide is biotinylated, and the indicator component is avidin or streptavidin with enzyme covalently bonded thereto.

11. Test kit according to Claim 10, characterised in that peroxidase is covalently bonded to said indicator component.

12. Test kit according to any of Claims 7 to 11, characterised in that it is an ELISA kit.

13. Test kit according to Claim 12, characterised in that at least one immunologically active peptide or polypeptide according to Claim 1, 2, 3 or 4 is coupled to microtitre plates, and in that the indicator component consists of anti-human IgG and/or IgM antibodies to which an enzyme catalysing a colour reaction is coupled.

14. Test kit according to Claim 12, characterised in that monoclonal antibodies against human IgM
antibodies are coupled to microtitre plates, and in that the indicator component is a biotinylated immunologically active peptide or polypeptide according to Claim 1, 2, 3 or 4 which cooperates with avidin or streptavidin with enzyme covalently bonded thereto.

15. Process for the purification of immunologically active peptides or polypeptides according to Claim 1, 2, 3 or 4 characterised in that it comprises the dissolving of unpurified peptide or polypeptide in 8M urea, and fractionation by a DEAE Sephacel .TM. column with an NaCl gladient.

16. Process according to Claim 15, additionally comprising an affinity chromatography.

17. Process according to Claim 16, characterised in that the affinity chromatography is carried out with a glutathione-coupled gel matrix.

18. Use of at least one DNA sequence selected from 0-1:
GTG AAT TCT GAT CAT ATG AGT AAA AAA AGT GGC AAA TGG
0-2:
C TTC GGT CGT GAC CAC GTC CTC CCC
0-3:
G AGG AAT TCT CTG ATC ATG ACT TCA GTT AAT TCT GCA GAA GCC

0-4:
GAG GGG TGG CAC GGG ACT CGG TCC TTC GAA GAG and 0-5:
G CTA CAA GCT GGG CCC CCG CAA AG
for the direct detection of parvovirus B19 by means of DNA amplification.

19. Use of at least one DNA sequence as claimed in claim 18, for the direct detection of parvovirus B19 by means of polymerase chain reaction.

20. Use of immunologically active peptides according to Claim 1, 2, 3 or 4 as vaccine against infections with parvovirus B19.