AU650864B2 - 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 capside protein VP1 and VP2 of parvovirus B19 are obtained which provide an economical, sensitive and specific test to detect antibodies against the human parvovirus B19. Short peptide sequences are identified which when used as an antigen serve to identify anti-B19 IgG-positive sera. The production of these peptides by gene-technological means is revealed. Other gene-technologically produced antigens which form stably in large quantities in E. Coli and can subsequently be separated from them act as additional antigens for IgG confirmation. Finally a set of antigens facilitates testing for the detection of IgM antibodies against the virus. In addition, the gene-technologically produced components of the surface proteins are substances which may be used for prophylactic immunisation.

Description

LEDERER, KELLER RIEDERER

MIKROGEN

molekularbiologische Entwicklungs-GmbH Westendstr. 125/G 8000 Munich 2 Immunologically active peptides or polypeptides of parvovirus B19 The human parvovirus B19 (for short hereinafter: B19) was discovered by chance in 1975 in plasma samples from blood donors (Cossart, Field, Cant, B., Widdows, Parvovirus-like particles in human sera.

Lancet I (1975) 72-73) by countercurrent electrophoresis.

In recent years it has been shown that B19 may cause an aplastic crisis in patients with chronic haemolytic crisis (sic), and is 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 CsC1 2 (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 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 parovirus (sic), 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 71kDa can be detected.

2 The DNA codes in the 5' region for the structural proteins of the capsid. The coding regions of the structural 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 are the target cells of this infection.

Inoculati of B19 cells in fetal erythropoietic cells and erythroblasts of a patient with chronic myeloid leukaemia has now succeeded.

B19 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 interuterinal (sic) death. Furthermore, B19 is associated with the occurrence of Schbnlein-Henoch purpura.

As a rule, B19 is transmitted by droplet infection but also by antigen-positive conserved blood and coagulation products.

L_ I_ I 3 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 blood from donors 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, detection of B19specific antibodies of the IgG and IgM class. This results in the following possible applications: 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 for 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 B19seronegative pregnant women.

It has emerged that utilisable immunologically active polypeptides cannot be prepared directly. Preparation 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 L, YLIYLI-~--~ IPI_ _II 4 impurities which interfere with the detection of antibodies directed -gainst parvovirus B19. 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 antibodies 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 active peptides according to the invention is that they can be prepared in good yield by the preparation process according 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 subsequent 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 Although these peptides are preferred, it is equally possible to employ other peptides with 8-50 amino acids, preferably to 32 amino acids, from the VP 1-VP 2 region. This region approximately corresponds to the polypeptide PAN1 which is depicted in Fig. 2-1.

In a preferred embodiment of the present invention, 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 peptides having the amino-acid sequences PAPEP 1 PAPEP 8 shown in Example 3.

In another preferred embodiment of the present invention, the amino-acid sequences which are depicted in l4 5 Figure 2 of the immunologically active peptides PAN-i, PAN-2, PAN-3, PAN-4, PCE, PANSE AND (sic) PAPST prepared by genetic 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 peptides 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, the 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 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 expression vectors is impossible because of various difficulties. For this reason, according to the invention, the viral protein segment is fused to a jrotein amenable to stable expression. This fusion protein can be employed directly after purification as antigen for IgG detection. However, the parvovirusspecific portion is preferably cleaved off by suitable methods, further purified and then employed for serological 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 principle be.used in all diagnostic test kits for detecting antibodies against parvoviL us B19. In a preferred embodiment of the test kits according to the invention, the solid phase of suitable microtitre plates or polystyrene 6beads 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 radioactivity then shows whether antibodies directed against parvovirus B19 are present in the serum sample.

The test kits according to the invention are normally supplied to laboratories of physicians, hospitals, 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 kits 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 polystyrene beads. After incubation of the test serum, and suitable washing and saturation steps, a specific enzymatically 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 for detecting IgM antibodies 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

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ij rr~rt-L-~M3ni, ri 7 radioactively labelled.

The principle of the so-called M-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 invention are then added. The extent 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 immunosorbent assay) test kits.

Also provided according to the invention are DNA sequences which 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 plasmid 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 Fig. 1.

Finally, vaccines against parvovirus B19 are also made available within the scope of the present invention.

This entails the immunologically activ peptides according to the invention being adiinistered, optionally several times, together with suitable adjuvants to the people to be protected. The production of antibodies 8 elicited by this can effect protection from infection with parvovirus B19.

EXAMPLE 1: Obtaining parvovirus B19 VP 1- and VP 2-encoding sequences from patient's serum Viral DNA was isolated from iml 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, Fritsch, E.F., Sambrook, J. (1982) Molecular cloning. Cold Spring Harbor, This DNA was taken up in 501p of TE buffer and then 1pl 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 oligodeoxynucleotides used as primers have at each of their ends sequences which are not homologous with the parvovirus 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 0-1 to 0-5 in Fig. 1 were used.

In each case five mixtures each containing 11 of isolated parvovirus DNA were amplified with the two pairs of primers in a volume of 100pl. The conditions for this were: 1.5 min denaturation at 94°C, 2 min attachment of the primers at 45 0 C, 4 min synthesis at 72°C; total of cycles; buffer, substrates and Taq polymerase were employed for this as stated by the manufacturer (Cetus/Perkin-Elmer, Oberlingen, FRG).

The amplified DNA fragments from the two different mixtures (for VP 1 and VP 2) were in each case 9 combined, precipitated by alcohol precipitation, washed with 70% alcohol, dried, dissolved in a volume of 2001 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 (id HindIII sites of the 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 pUC12PAN for the region encoding the VP 1 portion and pUC12VP2 for the VP 2-encoding region.

EXAMPLE 2 Preparation by genetic engineering of VP 1 portion and VP 2 from E.coli cells aj VPI portion: 1) PAN-1 The VP1-encoding region was isolated from the plasmid pUC12PAN 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 example pBD2) into the BamHI and HindIII restriction cleavage sites. E.coli cells with plasmids resulting therefrom express after induction with IPTG a p-gal::VP1 fusion protein (about 67kDA) in large quantity, which reacts very strongly with anti-parvovirus Bl9-positive sera in an immunoblot (Western blot).

Purification of this protein can be achieved very easily with conventional methods utilising the insolubility of tho protein. After lysis of the cells, the pellet fraction is washed with detergents such as Triton-Xl00 and octyl gluco-pyranoside, and the fusion protein is subsequently dissolved with 8M urea/l% mercaptoethanol and separated from cellular impurities by DEAE 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 fragments. After cleavage in 35% formic acid and 0.lmg/ml BrCN at room temperature for 4h, the sample was lyophilised, dissolved in 8M urea, 2mM DTE (sic) (dithiothreitol) and purified by DEAE chromatography in an NaCi gradient. The VP1 fragment resulting therefrom was called PAN-1 and ca; be used directly for serological determinations. The amino-acid sequence is indicated in Fig. 2-1.

Further constructs generated were plasmids which code for fusion proteins consisting of the glutathione Stransferase from Schistosoma japonicum (Smith, D.B. and Johnson, Single step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene, 67 (1988) 31-40) and the VP1 portion. However, it 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 pUC12PAN after BclI/PvuII digestion (618bp) and integrated into the BamHI and SmaI sites in pGEX1 (pGEXlPAN). The resulting 52kDA fusion protein was purified from the supernatant by means of glutathione-coupled agarose and employed as antigen for the serological tests in Example 4 (name:PCE). The amino-acid sequence of this antigen is shown in Fig. 2-2.

L 11 3) PAN-2: A 458bp fragment was isolated from pUC12PAN 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 oligodeoxynucleotides. 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 pUC12PAN with BclI/HincII 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.

51 PAN-4: The complete B19 DNA insert was obtained, from pUC12PAN 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 /f 4 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. 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 2, the B19 portion was cleaved off by digestion with trombin (sic) or factor X as stated by the manufacturer (Boehringer Mannheim). The fragments were then purified again by affinity chromatography. The Sglutathione (sic) transferase can in this case be selectively fished out, the parvovius protein portion is to be found in the flow-through and can be employed after a final DEAE 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 the fusion protein bound on. After an incubation time of about 1h, the VPI (sic) 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 vector, 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 a ino-acid sequence of the recombinant antigen is shown in Fig. 2-6.

2 tet -i 13 2) PANSE: It emerged, surprisingly, that a truncation of the VP2-encoding sequence is associated with a considerable increase in the protein yield, that this truncated antigen can be stably expressed, is not degraded even during purification, and still has the same reactivity with anti-E19 positive sera too. This expression plasmid (pUC19PA.SE) was obtained by truncating the 5' region of VP2 by 355bp as far as an NsiI 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 prod an EcoRI site by intermediate cloning in order to be able to insert the required fragment into the PstI and EcoRI sites of pUC19.

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 amino-acid sequence of the antigen is indicated in Fig. 2-7.

3) PAPST: A fragment 716bp in size which encodes the Nterminal 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% of 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 DEAE chrcmatography. The aminoacid sequence is depicted in Fig. 2-8.

14 c) Complete VPl/VP2: The plasmid pUC12PAN was opened with PstI and HindIII, and the VP2 encoding region from pUC12VP2 was inserted after HindIII and partial PstI digestion as 1.7kb fragment (pUC12VP1/2).

Expression of VP1/2-containing antigens in E.coli: 1) PAV-1-B: pUC12VP1/2 was cut with EcoRI and BamHII and a DNA band 1466 bp in size was isolated and subsequently inserted into the EcoRI/BamHI sites of the vector pUC18stop. pUC18 stop resembles the abovementioned pUC vectors; however, it differs from the latter by containing between the PstI and HindIII site a synthetic oligodeoxynucleotide 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 ACG 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 CTGCAG, BgIII (sic) AGATCT and HindIII AACGTT (sic) are indicated by bold print) The vector (pUC-V1-B) obtained in this way encodes the VP-1 structural protein from the start up to amino acid 486 followed by some amino acids of the pUC polylinker and is terminated 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 kDa.

Its amino-acid sequence is depicted in Fig. 2-9, amino acids emphasised by bold print are not Bl9-specific and are encoded by pUC sequences. The reactivity with anit- B19-positive (sic) sera is very good and efficient purification can be achieved by removing soluble E.coli J5 proteins, dissolving in 8M urea and conventional ion 15 exchange chromatography (as described).

2) PAV-1-N: The vector pUCVP-1-B described above was digested with EcoRI and NsiI. The band 1137bp in size produced in this way was inserted into the vector pUC18stop into the EcoRI and PstI sites (see above). The resulting vector pUCVP-1-N encodes the structural protein from the start up to amino acid 377; the antigen (PAV-I-N) is produced after IPTG induction in the E.coli cells somewhat less well than the antigen PAV-I-B described above. It is kDa in size, and the reactivity with anti-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)l) and c)2) as GST fusion proteins The two vectors pUCVP-1-B 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 pUC18stop being transferred too. (The BglII site is indicated in the pUC18stop polylinker sequence indicated above, and the BclI site (TGATCA) immediately before the start of translation was introduced with the primers used for the DNA amplification see Fig. 1, The two fragments which encode the same 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 SmaI 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 SmaI (blunt end). This was effected by inserting the two DNA fragments into the EcoRI and BamHI (compatible with BglII) restriction sites of the vector pIC20H (The pIC plasmid and phage vectors with 16 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 HincII (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 HincII digestion.

The two pGEX vectors now express fusion proteins consisting of the glutathione 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-terminal 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 from the plasmid pUC12VP1/2 with the complete VP1 and VP2 encoding region and inserted into the eukaryotic expression vector pMDIII (Motz, M., Deby Jilg, Wolf, Expression of the Epsteinbarr (sic) virus major membrane proteins in Chinese hamster ovary cells. Gene, 44 (1986) 353-359. (Obtainable from ATCC)) after EcoRI and HindIII digestion. This plasmid was subsequently linearised again with a SalI, and a 2.4kb SalI fragment with a dihydrofolate reductase gene (DHFR) and regulatory sequences was also inserted.

The plasmid pMDIIIVP1/2 obtained in this way was transfected into DHFR-negative CHO cells. Colonies resulting after selection on alpha-ainus medium (gibco (sic)) were isolated-and amplified after washing out with increasing concentrations of methotrexate (MTX). Particles with VP1/VP2 can be purified from the culture supernatant from these cell cultures.

17 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 BamHI 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 parvovirus sequence.). After 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.

Expression of VP-2: Furthermore, expression of the smaller B19-VP-2 was obtained using recombinant baculoviruses. For this, the VP-2-encoding plasmid pUC12VP (see Example 1) was digested with EcoRI and HindIII, and the resulting 1.7kb fragment was inserted into an abovementioned vector which has, besides the HindIII site, also a BamHI site. The parvovirus portion was then isolated as BcII (sic) and BamHI fragment from this intermediat:e construct and inserted into the BamHI site of a baculovirus expression vector (various constructs can be used). (The BcII (sic) site is located 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 is also a site of this type still present .in the parvovirus sequence). After co-transfection of the resulting plasmid with wild-type baculovirus DNA into an insect cell culture line, cells which S* have no so-called inclusion bodies were isolated. VP2 can 18 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 particularly suitable for use in the p-capture test.

EXAMPLE 3 Synthetic peptides with immunodominant epitopes The reaction patterns of the bacterial expression products (especially pGEX::VP1 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 lie 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 Glu Asp Leu His Lys Pro Gly Gin Val Asn Tyr Val Gly Pro Gly Asn Glu Leu Gin Ala Gly Pro Pro Gin Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gin Leu L ._1 19 PAPEP-6: Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys d, Asn Ile Lys Asn Glu Thr Gly Phe PAPEP-7: Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Asn Ser 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 IgG or IgM antibodies in some cases different peptides or combinations thereof.

EXAMPLE 4 a) Determination of serum antibodies against parvovirus B19 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 concentration of 0.5 1 ig/ml in carbonate buffer, pH 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 Incubation with the sera for 2h took place in a /6 R2\ dilution of 1:100, and the subsequent washing procedures L riji_~ill _i*ji 20 and the detection of bound antibodies with a peroxidasecoupled anti-human IgG antibody took place by conventional test procedures.

Various serum panels were tested for anti-Bl9 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-Bl9 IgG positive even from the start of the clinical manifestation and remained positive over the observation period (19 weeks) both with the fusion protein from pGEX1PAN (PCE, see Example 2) and with a VP-1 region cleaved off by BrCN (PAN-1, see Example 2) and with 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-Bl9 IgG. The same sera were used for each antigen.

Result:

PCE:

Of the 21 pregnant women, 15 were anti-Bl9 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.

PAN-2: Of the 21 pregnant women, 14 were anti-Bl9 IgG negative and 7 were anti-Bl9 positive at the time of hospitalisation. On retesting serum samples taken from these women four weeks later, IgG was no longer detectable in one woman who was previously anti-Bl9 IgG positive.

L 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; IgMnegative, IgG-positive.

PAN-4 were tested by the procedure described above. The IgM antibodies were determined by the same test principle as for the IgG determination but PANSE and PAV-I-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 preadsorption 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-I-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 0.07 0.08 6 0.04 0.07 7 1.82 1.53 8 0.90 0.46 9 0.72 0.56 -i -22 1. 10 g 11 0.62 0.14 12 0.98 0.11 13 0.87 0.09 The results show a clear discrimination of the positive/negative sera both 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 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 particulate VP-2) and determining the binding (tt-capture assay).

These experiments demonstrate the high reliability of the test carried out using the immunolQgically 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 23 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 (p-capture assay).

EXAMPLE Use of Bl9-specific DNA primers for direct detection of pathogen Any B19 DNA present were obtained from the investigation samples (serum, biopsies) by proteinase K digestiop in the presence of 1% SDS (2h, 370C), phenol extraction and precipitation in 70% ethanol. This, and the DNA amplification wich then followed too, was carried out in analogy to the procedure described in Example 1. Primers 0-5 and 0-2 (see Fig. 1 for the sequence and position on the B19 genome) were used; in the case of Bl9-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 radioactively with 32P (sic) or digoxygenin (sic) by conventional methods (primer extension). The DNA fragment used for the hybridisation was obtained in the following way: a DNA fragment 260 bp long was isolated from the plasmid pUC12PAN after digestion with HincII and PstI and inserted into the HincII and PstI sites in pUC12. It is -w possible for the B19 fragment withot the sequences used for the amplification to be obtained from the resulting plasmid (pUC12PCRDIA) by EcoRI/PatI digestion and be employed after labelling as hybridisation probe.

24 DESCRIPTION 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 B19 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 Nterminal region (shaded bar), identical to VP2 (black bar). Underneath this are indicated the regions of oligodeoxynucleotides 0-1 to 0-4 which were used as primers for the amplification (PCR) of the B19 sequences located between them (0-1 and 0-2 for the VPl region, and 0-3 and 0-4 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 oligodeoxynucleotide sequences are identified by bold print, non-homologous regions, that is to say sequences which do not hybridism 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 0-1, for EcokI, BclI and BspHII (TC-ATGA) in the case of 0-3, and for HindIII (AAGCT-T) in the case of 0-4. The amplified VP2 encoding fragment (0-3 and 0-4) 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,

CTGCAG).

25 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- Bl9-authentic foreign amino acids are also contained at the N-termini and at the C-termini (apart from PANSE AND (sic) VP-2) and are emphasised by buld 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. 2-4: PAN-3 Fig. 2-5: PAN-4 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 26 lip '2 0- 1 0-41 0-2 0 -1 GTG AAT TCT GAT CAT ATG AGT AAA AAA AGT GGC AAA TOG 3 GCT TTG TAG ATT ATG AGT AAA AAA AGT GGC AAA TGG TGG 3 1 3 TTT CCA AAC ATC TAA TAC TCA TTT TTT TCA CCG TTT ACC ACC CTG CAG AAG CCA GCA CTG GTG CAG GAG GGG GGG GCA 3 3 'TAGC~C TTC GGT CGT GAC CAC GTC CTC CCC CCC CGT 5 1 3C TTC GGT CGT GAC CAC GTC CTC CCC 0- 3: .1AGG AAT TCT CTG ATC ATG ACT TCA GTT AAT TCT GCA GAA GCC 3 1 GXA AAA TAC OCA AGC ATG ACT TCA GTT XAT TCT G3CA GAA GCC 3 1 3 1CTT TTT ATG GG? T CC TAC TGA AGT CA.A TTA AGA CGT CTT 0-4: TA.A ACA CTC CCC ACC GTG CCC TC-A GCC AGG ATG CGT A" 3AAC ATT TGT GAG GGG TCC CAC GGG AGT CGG TCC TAC GCA T51 '1GAG GGG TGG CAC GGG AGT COG TCC T GCTA CALA GCT GGG CCC CCG CAA AG 31 'GAG CTA CAA GCT GGG CCC CCG CAA ACT GCT GTT GAC ACT GCT 3 31CAC GAT G TT CGA CCC CGG COC OTT TCA CGA CAA CTC TCA CGA- FIG. 1 -27 Fig. 2 Fig. 2-1 PAJ. I: Met Set Lys Lys Ser Gly Lys Trp Trp Glu Set Asp Asp Lys Pbe Ala Lys Al.i VaJ Tyr GIn Gin Pbe Val Ulu Pbc Tyr Glu Lys Val Thx Gly The Asp Lcu Giu Leu Ilie Gi Ilie Leu Lys Asp His Tyr As Ile Set Leu ksp isn Pro Leu Giu sn Pro Set Set Leu Pbe Asp Leu Vai A1a Atg Be Lys Asn iA Lcu Lys A-s Set Pro Asp Lzu TyT Set His His ?he Gin Set His Gly GIn I-eu &er Asp His Pro His Ala Leu Set Set See Set Set His Ala Glu Pro Arg Gly Giu Asa Ala Val LUu Set Ser Glu Aisp I-eu His Lys Pro Gly Gin Val Set Val Gin Lcu Pro Gly The Asa Tyr Val Gly Pro Gly Asa Giu Lzu Gin Ala Gly Pro Pro Gin Set Ala Val Asp Set Ala Ala Mg Ile His Asp Pbe AMg Tyr Set Gin Lcu Ala Lys L4u Gly Ile Asa Pro Tyr Thi His Trp Thr Val Ala Asp Giu Giu Ltu -eu Lys A- Ilie Lys Asa Giu Ther Gly Phe Gin Ala Gin Val Val Lys Asp Tyr Phe 11e I-eu Lys Giy Ala Ala Ala~ Pro V31 Ala His hbe Gin Gly Set LUu Pro Glu Val Pro Ala Tyr sa Ala Set Giu Ly's Tyrf Pro Set Fig. 2-2

PCE:

Gluthion-S-Transferase -His Met Set Lys Ls S, Gly Lys Trp Trp Giu Set A-sp Asp Lys Phe.Ala Lys Ala Vad Ty;. Gin Gin Phe Val Giu hec Tyr Giu Lys Vial Thr Gly The Asp I-eu Giu I-u Ilie Gin Ile I-eu Lys Asp His Tyr Asa Ilie Set I-eu Asp Asa ?to Leu Giu Am Pro Set Set I-eu Phe Asp U-u Val Ala Arg Ile Lys Asn sn U-u Lys isn Set Pro Asp Leu Tyr Set His His Phe Gin Set His Gly Gin U-u Set Asp His Pro His Ala U-u Set Set Set Set Set His Ala Glu Pro Arg Gly Giu Asa Ala Val I-eu Set Set Giu Asp I-eu His Lys Pro Gly Gin Val Set Val Gin I-eu Pro Gly The sa Tyr Val Gly Pro Gly Asa Giu I-eu Gin Ala Gly Pro Pro Gin Set Ala Val Asp Set Ala Ala Arg Ile His Asp Phe AMg Tyr Set Gin I-eu Ala Lys I-eu Gly Ile sa Pro Tyr Ther His Trp Th. Val Ala Asp Glu Giu Laeu Leu Lys sn Ile Lys Asa Glu The Gly Pbe Gin Ala Gin Val Val Lysisp TyT Phe Thr I-eu Lys Gly Ala Gly Giu be Ile Val The 4 sp Fig. 2-3 Gly Ser "Ar Ag Pro Asp His Met Set Lys Lys Set Gly Lys Trp Trp Giu Set Asp Asp Lys Pbe Ala LYS Ala Val Tyr Gin Gin Phe Val Giu Phe Tyr Giu Lys Val Ther Gly The sp I-eu Giu Ieu Ile Gin fle I-eu Ly's Asp His Tyr Asa Ilie Set I-eu Asp Asa Pro U-u Giu Asa Pro Set Set Phe Asp I-eu Val Ala Arg lRe Lys isn isa U-u Lys Aso Set Pro Asp I-eu Tyr Set His His Phe Gin Set His Gly Gin Lnu Set isp His Pro His Ala I-eu Set Scr Set Set Set His Ala Giu Pro Arg Gly Giu Asa Ala Val 1-eu Set Set Giu isp L-eu H is Lys Pr o Gly Gin Val Set Val Gin I-eu Pro Gly The Asa Tyr Val Giy Pro Gly Asa Giu Ltu Gin Ala Gly Pro Pro Gin Set Ala Val Gly Asp Pro Arq Giu Pit Ile Val Thr Asp -28- Fig. 2-4 Gly lie Leu Scr At r Pro Asp His Met Set Ly's Lys 3cr Gly Lys Trp Trp Giu Scr Asp Asp LYS Pbe AJa Lys Ala Val Tyr Gin GIn Pbe VaI Giu Phe Tyr Giu Lys Val Thi Gly Thr Asp Leu Glu Leu lie Gin Ile Lzu Lys Asp His Tyr Asa Ile Scr Ltu Asp Asa Pro Lcu Giu Aso Pro Scr Ser Leu Phe Asp Leu Val Ala Aig Ile Lr.. Aso Asti Leu Lys Asa Ser Pro Asp Ltu Tyr Set His His Phe Gin 3cr His Gly GIn Lcu Scr Asp His Pro His Ala Ut Set Set Scr Ser 3Ser His Ala Giu Pro Arg Giy Giu Asm Ala Val Lzu Scr Scr Giu Asp Lcu His LS Pro Gly Gin Val Ser Val Gin Lzu Pro Gly Thi Aso Tyr Vad Gly Fro Gly Asa Giu Leu Gin Ala Gly Pro Pro Gin Ser Ala Val Gly Asp Pro Leu Giu Asp Pro Argj Val Pro S3cr Ser Asn Ser Eig. PAY04 Gly Ser .Aqg Arg Pro Asp His Met Set Lys Lys Scr Gly LYS Trp Trp Glu Ser Asp Asp L's Pbe Ala Lys,-Ala Val~i TyT Gin Gin Pbe Val Glu Pbe Tyr Giu Lys Val Thr Gly Thur Asp Leu Glu Lzu Ile Gin lie Ltu Lys Asp His Tvr Asa lie Ser Lcu Asp Asa Pro Lcu Giu Asa Pro 3cr Scr Leu Pbe Asp Lcu Val Ala Akrg lie Lys Aso Aso Lcu Lys Asa Ser Pro Asp Lcu Tyr 3cr His His Phe Gin Scr His Gly Gin Leu Scr Asp His Pro His Ala Lcu 3cr Set Ser Ser 3cr His Ala Glu Pro Arg City Giu Asa Ala Val Leu 3cr Ser Giu Asp Leu His Lys Pro Giy Gin Val 3cr Vad Gin L--u Pro Gly Thr Asn Tyr Val Gly Pro Gly Asa Giu Ltu Gin Ala Gly Pro Pro Gin Ser Ala Val Asp Ser Ala Ala Arg fle His Asp Phe Axg Ty'r Scr Gin Leu Ala Lys Leu Giy fle Asn Pro TyT TVh His Trp, Thr Val Ala Asp Giu Giu Leu Leu Lys Asa Ile Lys Asz Giu ThV Gly Pbe Gin Ala Gin Val Val Lys Asp Tyr Pbe Tar Lu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gin Gly 3cr Lcu Pro Giu Val Pro Ala Tyr Asa Ala Ser Giu Ly.s Tyr Pro 3cr Met Thr 3cr Val Asa Ser Ala Gly Arg Ar lie Pro Gly Asn Ser 3cr Pig. 2-6 VP2: Met Thr Met lie 7%r so Ser Leu lie Met Tbr 3cr Val Asa 3cr Ala Glu Ala 3cr Tbx Gly Ala Gly Gly Gly Gly 3cr Asa 3cr Val Lys Set MuA Trp 3cr Glu Gly Ala Thr Pbe Ser Ala Asa Scr Val Th~r Cys Thr Pbc 3cr Arg Gin Phe Lcu Ile Pro Tyr Asp Pro Giu His His Tyr Lys Val ?he 3cr Pro Ala Ala 3cr 3cr Cys His Asa Ala Scr Gly Lys Glu Ala Lys Val Cys Thr lie Scr Nro Ile Met Gly Tyr 3cr Thr Pro Trp Arg Tyr Lcu Asp Phe Aso Ala Lcu Asa Ltu Phe Pbe 3cr Pro iLcu Giu Phe Gin His Leu le Glu Asa Tyr Gly Set Ile Ala Pro Asp Ala Leu Thar Val TVa lie 3cr Giu lie Ala Val Lys Asp Val TVr Asp Lys Thz Gly Giy Gly Val Gin Val Thr ,Asp 3cr Thar Thr Gly Arg Leu Cys Met Leu Val Asp His Glu Tyr Lys Tyr Pro Tyr Val Lcu Gly Gin Gly Gin Asp Tlu Lcu Ala Pro Giu LUu Pro rie Trp Val Tyr Pbe Pro Pro Gin Tyr Ala Tyr L~cu Thr Val Gly Asp Val Asn Thi Gin Gly lie 3cr City Asp Set Lys Lys Leu Ala 3cr Glu Glu Scr Ala Phe Tyr Vad Lcu Giu His 3cr Scr Phe Gin Lzu Lcu Gly Tht Gly Gly Tbx Ala 3cr Met Set Tyr Lys Phe Pro Pro Val Pro Pro Giu Aso Le u Giu Gly Cys Set Gin His Pbe Tyr Giu Met Tyr As. 7ro Lcu Tyr Gly 3cr Arg Leu Gly Val Pro Asp Thr Leu Gly Gly Asp Pro Lys Pbe Arg 3cr Lcu Thr His Glu.-Asp His Ala lie Gin Pro Gin Asa Phe Met Pro Gly Pro Lz u V al Aso 3cr Val 3cr Thr Lys Glu Gly Asp 3cr 3cr Asa Thr Gly Ala Gly Ly's Ala Lcu Thar Gly Leu 3cr TVx Gly TV r Gin Asa Th-r Arg Ilie Ser Lcu Arg Pro Gly Pro Val 3cr Gin Pro Tyr His His Trp Asp Tlr As p I TyrValyTrl Il AsnAla e StHisGy GIn TV T yGy As Aa Gu sp Lys GluTyr Gin Gtn Gly Val Gly Arg Phe Pro A~i Glu Lys '-lu Gin Leu Lys Gin Lzu Gin Gly Leu Asn Met His Thr Tyr Pbe Pro Aso Lys Gly ThV Gin Gin Tyr Th; Asp Gin lie Giu Arg Pro Lzu Met Val Gly 3cr Val Trp As~n Akrg Ar; Ala Lcu His Tyr Glu 3cr Gin Lcu Trp 3cer Lys lie Pro Asa Lceu Asp Asp 3cr Phe Lys TVw Gin ?be Alta Ala Lzeu Gly Gly Tr-p Gly Leu His Gin Pro Pro Pro Gin flc Phe Lcu Lys Gin Tyt Ala Val Gly lie Met TbiVIITVMetThrPh Ly Lu Gly Pro .krg L-,S Ala TVm Gly. Arg TrpAsn Pro Gin Pro Gly Va TVT Pro f Pro His Ala,.Ala Gly His L--u Pro Tar Val Lcu Th A-sp Pro TbV Ala Thr.Asp.Ala Lys Gin HiLs His krg Hi-s -iv Tvr Glu L's Pro Glu Glu Wz T,-p Thi kl.a Lys Scr,"r Vii His Pro Lcu I N'HANG I

ANNEXANNEXE

-29- Fig. 2-7 P.AN S E.

Met Thr Met lie TVr Pro Ser Leu His .Ala Cys Met Lzu Val Asp His Giu Tyr Lys Tyr Pro Tyr Va] Lcu Gly Gin Gly Gin Asp ThV Leu Ala Pro Giu Le-u Pro Ile Trp Val Tyr Pbe Pro Pro Gin Tyr Ala Tyr Leu Thr Val Gly Asp Val Xsn ThV Gin Gly Ilie Set Gly Asp Set Lys Lys Leu Ala S-er Glu Glu Set Ala Phe Tyr Va] Lcu Giu His Ser Set Phe Gin Lzu Lcu Gly TVx Giy Gly TVr Ala Ser Met Ser Tvr LYS Pbe Pro Pru Val Pro Pro Glu Asn Lecu Giu Gly Cys Set Gin His Pbe Tyr Giu Met Tyr Asn Pro Leu Tyr G1y Set Arg Lzu Gly Va] Pro ,As p TVx Lcu Gly Gly Asp Pro Lys Pbe Arg Set Ltu Thmr His Giu Asp His Ala tie Gin Pro Gin Asa Pbe Met Pro Gly Pro Lzu Val Asa Set Va] Ser TVr Lys Giu Gly Asp Set Set Asn TVr C~y Ala Gly L 'ys Ala Le u TVr Gly Lteu Ser Ther Gly Thr Ser Gin Asa Thr Arg lie Ser Leu Arg Pro Gly Pro Val Set Gin, Pro Tyr His His Trp Asp TVr Asp Lys Tyr Val Thr Gly Ile Asa Ala Ile Set His Gly Gin Thr Tbr Tyr Gly Asn Ala Giu Asp Ly's Giu Tyr Gin Cin Gly Val Giy Arg Phe Pro Aso Giu Lys Giu Gin Lzu Lys Gin Lcu Gin Giy Leu Aso Met His TVx Tyr Phe Pro Asa Lys Giy TVr Gin Gin Tyr TVr Asp Gin lie Giu Arg Pro Lou Mfet Val Gly Set Val Trp Asa Arg ,Vg Ala Leu His Tyr Glu Set Gin Lcu Trp, Set Lys lie Pro Asn Lzu Asp Asp Set Pbe Lys TVr Gin Phe Ala Ala Lau Gly Gly Trp Gly Leu His Gin Pro Pro Pro Gin Ile Pbe Lzu Lys Gin Tyr Ala Val Gly Ilie Met Thr Val Ther Met Thr Phe Lys Lceu Gly Pro Arg Lys Ala Thr Gly Aeg Trp Asn Pro Gin Pro Gly Va] Tyr Pro Pro His Ala Ala Gly His Leu Pro Tyr V31 Leu Tyi A-sp Pro TVr Ala Tbr A-sp Ala Lys Gin His His Aeg His Gly Tyr Giu Lys Pro Giu Glu Lau Trp TVx Ala Lys See Artg Val His Pro Leu Fig. 2-8

PAPST:

Met Thr Met lie nre Pro Ser Leu Ala Giu Ala Set TVr Gly Ala Gly Giy Gly Gly Set Asa Set Val Lys Set Met Trp Set Giu Gly Ala The Pbc Set Ala Asa Set Val TV Cys TVr Phe Set Arg Gin Phe Leu lie Pro Ty s r l i i y y a b e r l l e e i s e l y l l Lys Val Cys TV Ile Set Pro Ilie 'Met Giy Tyr See ThV Pro Trp Arg Tyr Leu Asp Phe Asn Ala Lzu Xsn Le*u Phe phe set Pro Lceu Glu Phe Gin His Lcu Ilie Giu Aso Tyr Giy Set lie Ala Pro Asp Ala Lzu TV Val Thr- Ile See Glu lie Ala Val Lys Asp Va] The Asp Lys TVx Giy Gly Giy Val Gin Val TVr Asp Set TV~ TVr Giy .Aeg Leu C:ys M4et Leu Va] Asp His Glu Tyr Lys Tyr Pro Tyr Val Leu Gly Gin Gly Gin Asp TVr Leu Ala Pro Glu Lau Pro Ilie Trp Val Tyr Phe Pro Pro Gin Tyr Ala Tyr Lcu, TVr Val Gly Asp Val Aso TVm Gin Giy tie Setr Gly Asp Set Lys Lys Lcu Ala See Giu Giu See Ala Phe Tyr Val Leu Glu His Set Set Pbe Gin Lcu L4eU Gly TVr Gly Gly Thr Ala Set Met Ser Tyr Lys Pbe Pro Pro Val Pro Pro Glu Aso Lzu Glu Gly Cys kn~r S-er Thir Asp Pro.Ar Glu mbe Thr Giy,Arg kr te Thr Thir Ser Fig. 2-9 PA. V1-B3 Met Thr Ile Thar Aso Sir Asp His Met Set Lys Lys Set Gly Lyvs Trp, Trp Glu Set Asp Asp L ys Pbe Ala Lys Ala Val Tyr Gin Gin Phe Val Giu Phe Tyr Giu Lys Val Th~r G1y Thr Asp 1-eu Glu 1-eu le Gin Ile 1-eu Lys Asp His Tyr Asn le Set 1-eu Asp Aso Pro L-eu Giu Asa Pre Set Ser Laeu Phe Asp Laeu Val Ala Arg Ile Lys Asn Asn L-eu Lys Asa Ser Pro Asp 1-eu Tyr Ser His His Phe Gin Set His Gly Gin L-eu Set Asp His Pro His Ala 1-eu Set Set Set Ser Set His Ala Gin Pro Arg Gly Glu Asn Ala Val Lau Set Set Glu Asp 1-eu, His Lys Pro Gly Gin Val Set Val Gin 1-eu Pro Gly Thir Asa Tyr Val Gly Pro Gly Asa Glu Leu Gin Ala Gly Pro Pro Gin Ser Ala Val Asp Set Ala Ala Arg lie His Asp Phe Arg Tyr Set GIn L-eu Ala Lys 1-eu Gly lie Asn Pro Tyr Thr His Trp Tbr Val Ala Asp Glu Glu L-eu La.u Lys Asai le Lys Asia Giu Thr Gly Phe Gin Ala Gin Val Val Lys Asp Tyr Phe Thr 1-eu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gin Gly Set 1-eu Pro Giu Val Pro Ala Tyr Asoa Ala Set Glu Lys Tyr Pro Ser Me. Thr Set Val Asn Ser Ala Glu Ala Set Thr Gly Ala Gly Gly Gly Gly Set Asia Set Val Lys Set Met Trp Set Giu Gly Ala Thr Phe Set Ala Asn Ser Val Thr Cys Thr Phe Ser Arg Gin Phe Laeu lie Pro Tyr Asp Pro Giu His His Tyr Lys Val Phe Set Pro Ala Ala Set Set Cys His Asn Ala Set Gly Lys Giu Ala Lys Val Cys Tr le Ser Pro Ile Met Gly Tyr Set Thx Pro Trp Arg Tyr 1-eu Asp Phe Asa Ada Leu Aso L-eu Phe ?he Ser Pro 1-eu ONu Phe Gin His I=e lie Giu Asn Tyr Gly Ser Ile Ala Pro Asp.Alaa1-eu Tlir Val Thr lie Set Giu le Ala Va!l Lys Asp Val Thr Asp L ys Thr Gly Gly Gly Val Gin Val Thr Asp Set Thr Thr Gly Arg 1-eu Cvs Met 1-eu Val Asp His Glu Tyr Lys Tyr Pro Tyr Val 1-eu Gly G~n Gly Gin Asp Tbx 1-u Ala Pro Glu 1-eu Pro Ile Trp Val Tyr Phe Pro Pro Gin Tyr Ala Tyr 1-eu Thr Val Gly Ap Val Asoa Thr Gin Gly Ile Set Gly. Asp Set 1-ys Lys 1-eu Ala Ser Giu Giu Set Ala Phe Tyr Val L-cu Glu His Ser Set Phe Gin 1-eu L-eu Giy Thr Gly Gly Thr Ala Ser Met Set Tyr Lys Phe Pro Pro Val Pro Pro Giu Asn 1-eu Glu Gly Cys Set Gin His Phe Tyr Giu Met Tyr Asn Pro 1-eu Tyr Gly Ser Ser Arg Val Asp Leu Gin Fig. 2-10 PA V.1-,N Miet Thr fle Thir Asn 5cr Asp His Met Set Lys Lys Set Gly LY5 Trp Trp Giu Set Asp Asp Lys Fhe Ala Lys Ala Vad Tyr Gin Gin Phe Val Giu Phe Tyr Giu Lys Val Thr Gly Thr Asp 1-u Giu 1-eu le Gin Ile 1-eu Lys Asp His Tyr Am Ile Ser 1-eu Asp Asia Pro Leu Giu Asn Pro Ser Set 1-eu Phe Asp 1-au Val Ala Arg lie Lys .,sn Asn 1-eu Lys -ksn Set Pro Asp Leu Tyr Set His His ?he Gin Set His Gly Gin Leu Ser Asp His Pro His Ala 1-cu Ser Set Set Set Set His Ala Giu Pro Axg Gly Glu Asri Ala Val 1-au Set Set (iu. Asp 1-eu His Lys Pro Gly Gin Val Set Val Gin 1-eu Pro Gly Tbr Asia Tyr Val Giv Pro Gly Asia Giu Leu Gin A\la Gly Pro Pro Gin Set Ala Val Asp Set Ala Ala Arg Ile His Asp Phe Arg Tyr Set Gin 1-cu Ala L ys 1-cu Gly Ile Asn Pro Tyr Thr His Trp TIu Veil Ala Asp Giu Glu 1-eu 1-eu Lys Aso Ile Lys Aso Giu Thr Gly Phe Gin Ala Gin Val Val Lys Asp Tyr Phe Tbhr 1-eu Lys Gly Ala Ala Ala Pro Val Ala His Phe Gin Gly 5cr 1-cu Pro Giu Val Pro Ala Tyr Asn Ala Set Giu 1-vs Tyr Pro Set Met Thr Set Val Asn Set Ala Glu Ala Set Thx Gly Ala Gly Gly Gly Gly Ser Asia Set Val Lys Set Met Trp Set Giu Gly Ala Thr Phe Set Ala Asia Ser Val Thr Cys, Thr Phe Ser Arg Gin Phe 1-au Ile Pro Tyr Asp Pro Giu His His Tyr Lys Val Phe Ser Pro Ala Ala Set Set Cys His Asia Ala Set Gxy Lys Giu.Ala Lys Val Cys Thr Ile Set Pro Ile Met Gly Tyr Set Thr Pro Trp Arg Tyr 1-cu Asp Phe Aksn Ala 1-cu Asn 1-eu Phe Phe Set Pro 1-eu Glu Phe Gin His 1-eu Ile Giu Aso Tyr Gly Set le Ala Pro Asp Ala 1-u Thr Val Thr lie Set Giu Ile Ala Val Lys Asp Vai Thr Asp Lys Thy Gly Gly Gly Val Gin Val Thr Asp Set Thr Thr Gly Arg Lau Cys Ser Asoa 31 Vp- t VP-2 PAN I PCE 1, PAN 2 PAN 3I PAN 4 VP -2

PANSE

PAPST

Section %P I -VP2) PAPEPI PAPEP2 P A PEP 1 PAPEP4 PAPEP5 PAPEP6 PAPEP 8 Fig. 3

Claims (16)

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, which is free of impurities which may interfere with the detection of parvovirus B19 specific antibodies, and wherein 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 the amino acid sequence Asn Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Leu Lys His Ile Lys and/or Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Ala Val Tyr and/or Leu Lys ,.sp His Tyr Asn Ile Ser Leu Asp Asn Pro Leu Glu Asn Pro Ser Ser and/ ;r Ile Lys Asu Asn Leu Lys Asn Ser Pro Asp Leu Tyr Ser His His Phe Gin Ser His Gly Gin Leu Ser Asp His Pro His Ala and/or Ser Ser His Ala Glu Pro Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Asp Leu His Lys Pro Gly Gin Val and/or AsH, Tyr Val Gly Pro Gly Asn Glu Leu Gin Ala Gly Pro Pro Gln Ser Ala Val Asp Ser Ala Ala Arg Ile His Asp Phe Arg Tyr Ser Gin Leu and/or J o nn o n n u n oa~i~os I ct o r aoa t n u r, it .lo rc lir o c oo a o aoco o s o0 II ~O(ID O (lb0~ Pro Tyr Thr His Trp Thr Val Ala Asp Glu Glu Leu Lys Asn Glu Thr Gly Phe and/or Asn Ala Ser Glu Lys Tyr Pro Ser Met Thr Ser Val Ala Ser and/or the amino-acid sequence Leu Lys Asn Ile Asn Ser Ala Glu I 0' .4: 33 His Met Ser Lys Lys Ser Gly Lys Trp Trp Glu Ser Asp Asp Lys Phe Ala Lys Gly Thr Ser Leu Ala Arg His Phe Ser Ser Ser Glu Thr Asn Ser Ala Leu Ala Glu Glu Val Val Thr Asp Ala V Asp L Asp A Ile L Gin S Ser S Asp L Tyr V Val A., Lys L Leu L Lys Ac and/or 'a1 eu sn ys er er eu 3a pU au uP Tyr Glu Pro Asn H.s His His Gly Ser Gly Lys Tyr Gin Leu Leu Asn Gly Ala Lys Pro Ala Ile Asn Phe Gin Ile Glu Leu Gln Glu Pro Gly Ala Asn Ile Thr Phe Gin Asn Lys Leu Pro Gly Asn Arg Pro Lys Leu Val Ile Pro Asn Ser Arg Gin Glu Ile Tyr Asn Lys Glu Leu Ser Ser Asp Gly Va1 Leu His Thr Glu Gly Phe Lys Ser Pro His Glu Ser Gin Asp His Thr Ala Tyr Asp Leu Asp Pro Asn Val Ala Phe Trp Gly Gly Glu His Phe Leu His Ala Gin Gly Arg Thr Phe Glu Lys Tyr Asp Tyr Ala Va1 Leu Pro Tyr Val Gin Phe Val Asn Leu Ser Leu Leu Pro Pro Ser Ala Ala Ile Thr Ile Val His Ser Ser Gly Gin Gin Asp Gin Val a so oo 0 o 8 n 0 On i;, O nOoBOU i)LI 0 0 10 ii Oo D 0 010 (IOLU L C LLOI- 0 0 O D 0 OU oO d L)r ;i i r r) O i) 9 lid 00 I (IQ CI Jil O :i(ls 4&iy Glu Phe Lys Ser Pro His Glu Ser Ser Tyr Asp Leu Asp Pro Asn Arg Asp Glu His Phe Leu Hl,,7 Ala Arg Asp Lys Tyr Asp Tyr Ala Val Pro Lys Val Asn Leu Ser Leu Leu Asp Phe Thr Ile Val His Ser Ser His Ala Gly Ser Ala His Ser Ser Met Lys Thr Leu Arg Phe Ser Glu Ser Ala Asp Asp Ile Gin Ser Asp Lys Va1 Leu Asn Lys Ser Ser Leu Lys Tyr Glu Pro Asn His His His Ser Gin Leu Leu Asn Gly Ala Lys Gly Gin Ile Glu Leu Gin Glu Pro Lys Phe Gln Asn Lys Leu Pro Gly Trp Val Ile Pro Asn Ser Arg Gin Trp Glu Leu Ser Ser Asp Gly Val Ser Val Gin Leu Pro Gly Thr Asn Tyr Val Gly Pro Gly Asn Glu Leu Gin Ala Giy Pro Pro Gin Ser Ala Val Giy Asp Pro Arg Giu Phe Ile Val Thr Asp and/or Gly Ile Leu Ser Arg Arg Pro Asp His Met Trp Trp Giu Ser Asp Asp Lys Phe Ala Lys Val Glu Phe Tyr Giu Lys Val Thr Gly Thr Ile Leu Lys Asp His Tyr Ash Ile Ser Leu Pro Ser Ser Leu Phe Asp Leu Val Ala Arg Asn Ser Pro Asp Leu Tyr Ser His His Phe Ser Asp His Pro His Ala Leu Ser Ser Ser Arg Gly Glu Asn Ala Val Leu Ser Ser Glu Gin Val Ser Val Gin Leu Pro Gly Thr Asn Giu Leu Gin Ala Gly Pro Pro Gin Ser Ala Asp Pro Arg Val Pro Ser Ser Asn Ser and/or Ser Ala Asp Asp Ile Gin Ser Asp Tyr Val Lys Val Leu Asn Lys Ser Ser Leu Val Gly Lys Tyr Glu Pro Asn His His His Gly Asp Ser Gin Leu Leu Asn Gly Ala Lys Pro Pro Gly Lys Gin Phe Ile Gin Giu Asn Leu Lys Gin Leu Glu Pro Pro Gly Gly Asn Leu Glu f0i- 34 Gly Ser Arg Arg Pro Asp His Met Ser Lys Lys Ser Gly Lys Trp Ti-p Git Phe Lys Ser Pro His Glu Ser Gin Aksp His Thr Ala Ala Ser Tyr Asp Leu Asp Pro Asn Val Ala Phe Trp Gly Ala Tyr Asp Glu His Phe Leu His Ala Gi7n Gly Arcy Thr Phe Ala Asn Asp Lys Tyr Asp Tyr Ala Val1 Leu Pro Tyr Val1 Gin Pro Ala Lys Val Asn Leu Ser Leu Leu Pro Pro Ser Ala Ala Val1 Ser Phe Thr Ile Val His Ser Ser Gly Gin Gin Asp Gin Ala Giu Ala Lys Gly Thr Ser Leu Ala Arg His Phe Ser Ser Ser Glu Thr Asn Ser Ala Leu Ala Giu Giu Val Val His Phe Lys Tyr Ala Asp Asp Ile Gin Ser Asp Tyr Val Lys Leu Lys Gin Val Leu Asn Lys Ser Ser Leu Val1 Asp Leu Leu Asp Gly Tyr Giu Pro Asn His His His Gly Ser Gly Lys Tyr Ser Gin Leu Leu Asn Gly Ala Lys Pro Ala Ile Asn Phe Leu Gin Ile Glu Leu Gin Giu Pro Gly Ala Asn Ile Thi- Pro Ser Phe Gin Asn Lys Leu Pro Gly Asn Arg Pro Lys Leu Giu Val Val1 Ile Pro Asn Ser Arg Gin Giu Ile Tyr Asn Lys Val Asn Giu Leu Ser Ser Asp Gly Val Leu His Thi- Giu Gly Pro Ser Pro Ser Met Thi- Ala Gly Arg Arg Ile Pro Gly Asn Ser Ser and/or Met Thi- Met Ile Thi- Pro Ser Leu His Ala Cys Met Giu Pro Thi- Leu ~Gln Pro Met Gin Lys ~Leu Pro Giy Asp) Gin] Lys Gly Ser Leu Gin Pro I Pro f Thi- P Tyr Giu Val Ala Leu Val Tyr Gly Pro Giu Ser V'al Ile Lys Leu Ser .qs yr ~rg is *Lys Leu Gly Ser Leu Pro Asn Asp Gin Gly Thr Ser Asn Giu Lys Thi- Val Ile Gly Ala Lys Ala Ala TY Prc ASE Giu Gly Pro Pro Pro Asn Asp Gly Gin Ala Tyr Gin Gin Ti-p Pro Trp Val Ala Ala Lys rPro Tyr Vai Leu Gly Ile Ti-p Val Tyr Phie Val Asn Thr Gin Gly Giu Ser Ala Phe Tyr Thr Gly Gly Thr Ala Giu Asn Leu Glu Gly Leu Tyr Gly Ser Arg Eqs Phe Arg Ser Leu Phe Met Pro Gly Pro Ser Ser Asn Thi- Gly Thr Ser Gin Asn Thr Pro Tyt~ His His Trp Ile Ser His Gly Gin Gin Gin Giy Val Gly Leu Gin Gly Leu Asn Gin Tyr Thr Asp Gin Asn Arg Arg Ala Leu Asn Leu Asp Asp Ser Gly Leu His Gin Pro Gly Ile Met Thi- Val IJ Thi- Gly Arg Ti-p Asn I Gly His Leu Pro Tyr Gin His His Arg His G Gli Prc I lE Val Ser Cys Leu Thr Leu Ala NArg Asp Tri Arg (e t Ile Us ?he ro Lhr ro al1 ;iy iGly SPro Sar Leu Met Ser Gly His Val Gly Ile Thi- Thr Phe His Giu Tyr LysI ProC Met I Gin I Leu I1 Tyr C Gir Gin Gly Glu Ser Gin Val Giu Asn Lys Ser ksp ryr Pro rhr krg ;1u ~hr ;ln hr 'ro ~yr ;iu -Leu Asp Tyr *Asp His Tyr His Pro Asp Ser Ala Leu Lys Gly Asn Tyr ProI Ser GinI Ile PheI Gly Asp I Lys E Val Thr Ala Ser Ser Lys Phe Asp His Val Leu krg Tyr ksn Giu Phe j6 u 31n ?he ?he dys ra 1 'ro ~ro *Asj LeL Tyl Lys Ser Phe Tyr Thi- Ala Ser Thi- Pro Val1 Ala Lys Pro Met Leu Ala Leu Leu Tyr Thr G iu ?His i Ala Leu ILys Phe Pro Giu Leu Ile Thi- Gly G ly Thr Giu Glu Asn Val Ti-p Ala Ly s Gly Pro Ala Glu '1' Leu Ti-p Thr Ala Lys Ser Arg Val His Pro Leu and/or m" I- 35 Met Thr Met Ile Thr Pro Ser Leu Ala Ala Giu Ala Ser Thr Gly Ala Gly Gly Thr Phe lie Pro Ser Ser Ser Pro Ala l-:u Asn I *Ile Ala Thr Asp Lys Tyr Leu Pro Gly Asp Ser Glu Gly Ser Tyr cys Ile Asn Gly Val Ser Pro Ile Val Glu Gly Ala Asp His Met Leu Ser Lys Thr Tyr Trp Asn Ser Ser Asn Asn Ser Pro Glu Asn Ala Gly Tyr Phe Phe le Ala Asp Val Thr Gly Val Leu Val Tyr Thr Gin Ala Phe Ser Val Val Thr His His Ser Gly Ser Thr Ser Pro Pro Asp Thr Asp Arg Leu Giy Gin Phe Pro Giy Ile Tyr Val Lys Cys Tyr Lys Pro Leu Ala Lys Cys Gly Pro Ser Leu Ser Thr Lys Glu Trp lu Leu Thr Met Gln Gin Gly Glu Met Phe Val Ala Arg Phe Thr Giy Leu Asp Tyr Asp His Tyr Thr Trp Ser Glu Gly Ala Ser Arg Gin Phe Leu Phe Ser Pro Ala Ala Lys Val Cys Thr Ile Tyr Leu Asp Phe Asn Gin His Leu Ile Glu Val Thr Ile Ser Glu Gly Gly Val Gin Val Val Asp His Giu Tyr Thr Leu Ala Pro Glu Ala Tyr Leu Thr Val Ser Lys Lys Leu Ala Ser Ser Phe Gin Leu Lys Phe Pro Pro Val Asp Pro Arg GIU Phe Leu Gly Thr Gly Gly Thr Ala Ser Met Ser Pro Pro Glu Asn Leu Glu Gly Cys Arg Ser Thr Gly Arg Arg Phe Thr Thr Ser and/or iiooon~ nii a u i c oo o L)13 a O D010 0oOo iDvi 13 O a (i a 011~ d a a a DCi D a DO Met Lys Thr Leu Arg Phe Ser Glu Asn Ala Ala Glu Val Phe Ser Ala Asp Asp Ile Gin Ser Asp Tyr Val Lys Leu Lys Gin Lys Val Leu Asn Lys Ser Ser Leu Val Asp Leu Leu Asp Gly Lys Tyr Glu Pro Asn His His Hig Gly Ser Gly Ly- Syr Ser Ser Gin Leu Leu Asn Gly Ala Lys Pro :1 le Asn Phe Gly Gin Ile Glu Leu Gin Glu Pro Gly Ala Asn Ile Thr Lys Trp Phe Val Gin Ile Asn Pro Lys Asn Leu Ser Pro Arg Gly Gin Asn Glu Arg Ile Pro Tyr Lys- Asn Leu Lys Trp Glu Glu Phe Leu Lys Ser Ser Ser Pro Asp His Gly Glu Val Ser Leu Gin His Asp Thr His Glu Thr Gly Ala Ser Asp Tyr Glu Asp His Leu Phe Asp Leu Pro His Asn Ala Vi1 Gin Ala Gly Phe Arg Trp Thr Gly Phe Ala Ala Asp Lys Tyr Asp Tyr Ala Val Leu Pro Tyr Val Gin Pro Lys Val Asn Leu Ser Leu Leu Pro Pro Ser Ala Ala Val Phe Thr Ile Val His Ser Ser Gly Gin Gln Asp Gin Ala Ala Gly Ser Ala His Ser Ser Thr Ser Leu Glu Va1 His Leu Pro Giu Val Pro Ala Tyr Asn Ala Ser Giu Lys Tyr Pro Ser and/or L-_ 36 Met Thr Ile Thr Asn Ser Asp His Met Ser Lys Lys Ser Gly Lys Trp Trp GiU Leu Ser Ser Asp Giy Val Leu His Thr Glu Gly Pro Ser Cys Gl1u Phe Lys Ser Pro His Glu Ser Gin Amsp His Thr Ala Ala Ala Ser Thr Liys Giu Trp Giu Leu Thr Met Gin Gin Gly Glu Ser Gin Ser Tyr Asp Leu Asp Pro Asr Val Ala Phe Trp Gly Ala Tyr Giu Met Phe Val Ala Arg Phe Thr Gly Leu As3p Tyr Asp His Tyr His *Asp *Glu His Phe Leu His Ala Gin Gly Arg Thr Phe Ala Asn Ala Trp Ser Phe Lys Tyr Gin Val Gly Val Thr Ala Ser Ser Lys Phe Asp ILys Tyr Asp Tyr Ala Val Leu Pro Tyr Val Glin Pro Ala Ser Ser Arg Ser Val Lau His Thr Gly Asp Lau Tyr Ly s Ser Phe Tyr Lys Val Asn Leu Ser Leu Lau Pro Pro Ser Ala Ala Val1 Ser Thr 31u 31n Pro Cys Asp Leu Ile Val His Ala Leu Lys Phe Pro Glu Phe Thr Ile Val His Ser Ser GIl. Gin Gin Asp Gin Ala Giu Gly Gly Phe Ala Thr Phe Ile Ser Gin Glu Pro Thr Leu Gin Prc Met Ala Gly Ser Ala His Ser Ser Thr Ser Leu Giu Val His Las Ala ELeu Lys Thr Leu Arg Phe Ser Giu Asn Ala Ala Giu Val Phe Tyr Gly Thr Ile Ala Asp Asp Ile Gin Ser Asp Tyr Val1 Lys Leu Lys Gin Pro Gly Phe Pro Val Tyr Leu Glu Asn Pro Lys Asn Ser His Ser His Leu His Vai Gly Asp Ser Leu Giy Leu Lys Asp Tyr Gly Ser Ser Met Gly Gly Ser Ala Tyr Asp Cys His 11 e Met Asn Leu Gly Ser Val Lys Ser Thr Pro Tyr Ile Trp Val Asn Giu Ser Thr Gly Giu Asn Leu Tyr Gin Lau Leu Asn Giy Ala Lys Pro Ala Ile Asn Phe Leu Thr Ser Asn Pro Asn Gly Phe Ile Asp Thr Val Val Thr Ala Giy Leu Gly G-in Ile Glu Leu Gin Glu Pro Gly Ala Asn Ile Thr Pro Ser Asn Ser Glu Ala Tyr Phe Ala Val Gly Leu Tyr Gin Phe Thr Giu Ser Phe Gin Asn Lys Leu Pro Gly Asn Arg Pro Lys Le'i Giu Val Ser Val His Ser Ser Ser Pro Thr Arg Gly Phe Gly Tyr Ala Giy Ser Val1 Ile Pro Asn Ser Arg Gin Giu Ile Tyr Asn Lys Val Asn Val1 Thr His Gly Thr Pro Asp Asp Leu Gin Pro Ile Val1 Ser Cys Arg Tyr Lys Pro Lau Ala Lys Cys Gly Pro Ser Lou Met Ser Ala Ser Ser Ile Ser Pro Asn Ala Leu Giu Asn Tyr Glu Ile Ala Val Thr Asp Tyr Lys Tyr Giu Leu Pro Val Giy Asp Ala Ser Giu Leu Leu Gly Val Pro Pro Tyr Asn Pro Val Asp Lau Gin and/or K 37 mfet Thr Ile Thr Asn Ser Asp His Met Ser Lys Lys Ser Gly Lys -Trp Tr-p Giu Leu Ser Ser Asp Gly Val Leu His~ Thi- Glu Gly Pro Ser Lys Cys Tyr Lys GiU Phe Lys Ser His GlU Ser Gin Asp His Ala Ala Ala Ser Thr Lys Glu ISer Tyr Asp Leu Asp Pro Asn Val1 Ala. Phe Trp Gly Ala Tyr Glu Met Phe Val Ala Asp Giu Phe Leu His Ala Gin Gly Arg Thr Phe Ala Asn Ala Trp Ser Phe Lys Asp L-S Tyr Aspj Tyr Ala Val Leu Pro Val1 Gin Pro Ala Ser Ser Arg Ser Val Lys Val1 Asn Leu Ser Leu Leu Pro Pro Ser Ala Ala Val Ser Thr Glu G in Pro y~s Phe Ala *Thr Gly Sle Ser Val Ala His His Ser Ser Ser Ser Giy Thr Gin Ser Gin Leu Asp Glu Gin Val Ala His Giu Lys Gly Ala Gly Ala Phe Leu Ala Ala Thr Ile Lys T.hr Leu Arg Phe Ser Giu Asn Ala Ala Giu Val1 Phe Tyr Gly Thr Ile Ser Ser Ala Asp Asp Ile Gin Ser Asp Tyr Val Lys Leu Lys Gin Pro Giy Phe Pro Ser Pro Val Leu Asn Lys Ser Ser Leu Val1 Asp Leu Leu Asp Gly Ser Gly Ser Tyr Cys Ile Tyr Giu Pro Asn His His His, cGly Ser Gly Lys Tyr Ser Met Gly Ala Asp H~is ,le t Gin Leu Leu Asn Gly Ala Lys Pro Ala Ile Asn Phe Leu Thr Ser Asn, Pro Asn Gly Phe Gin Ile Giu Leu Gin Pro Gly Ala Asn Ile Thr Pro Ser Asn Ser Giu Ala Tyr Phe Phe Gin Asn Lys Leu Pro G ly Asn Arg Pro Lys Leu Giu Val Ser Vai His Ser Ser Ser Val Ile Pro Asn Ser Arg Gin Giu Ile Tyr Asn Lys Val Asn Val Thr His Gly Thr Pro Pro Trp Arg Tyr Leu Asp Phe Asn Ala Leu Asn Leu Leu. Glu Phe Gin His Lea Ile Giu Asn Tyr Gly Ser Ala Leu Thr Val Thr Ile Ser Glu Ile Ala Val Lys Lys Thr Gly Gly Gly Val Gin Val Thr Asp Ser Thr 9ys Ser Asn or partial sequences thereof. Ile Ala Pro Asp Val Thr Thr Gly Arg Asp Asp Leu

2. immunologically active peptide or polypeptide according to claim 1, wherein- the polypeptide is a partial sequence of 10 to 32 amino acid residues of the peptt de PAN-i as depi(,'-ed in Fig. 2-1 or any one of the amino acid sequences listed in claim 1.

3. Immunologically active peptide or polypeptide according to claim, 1 or claim 2, which is in the form of a fu~sion protein, where this fusion protein has at least a part of P-galactopidase or of giutatbhione S-transferase.

4. Test kit for the detection of antibodies against human parvovirus B19, which comprises at least one immunologically active peptide or polypeptide according to any one of claims 1 to 3, which is able to react with the /0) 00 00 0 0 38 antibodies present in the sample to be tested, and in that it has at least one indicator component which makes it possible to detect complexes of immunologically active peptide and antibody.

Test kit according to claim 4, in which the indicator component is an antibody which is directed against the antibody to be detected a:,d is labelled with a detectable marker.

6. Test kit according to claim 5, in which the marker is a radioactive isotope.

7. Test kit according to claim 5, in which the marker is an enzyme which is able to catalyse a colour reaction.

8. Test kit acco-rding to Claim 5, in which the immunologically active peptide or polypeptide is biotinylated, and the indicator component is avidin or streptavidin with an enzyme, especially peroxidase, covalently bonded thereto.

9. Test kit according to any of Claims 5 to 8, which it is an ELISA kit.

Test kit according to claim 9, in which at least one immunologically active peptide or polypeptide according to claim 1 is coupled to microtitre plates, and in that the indicator component consists of anti-human IgG and/or IgM antibodiec to which an enzyme catalysing a colour reaction is coupled,

11. Test kit according to Claim 9, in which monoclonal antibodies against human IgM antibodies are coupled to microtitre plates, and in that the indicator component is a biotinylated imnunologically active peptide or polypeptide according to claim 1, which cooperates with avidin or streptavidin with an enzyme covalently bonded thereto.

12. Process for the purification of immunologically active peptides or polypeptides according to claim 1 or claim 2, comprising the steps of removal of insoluble constituents, ,s^2"kv T r l <f ^..00 L_ 1; -39 (ii) a fractionation by a DEAE-sephacell column, and (iii) another purification by means of an anion exchanger column in HPLC in 8M urea.

13. Process according to claim 12, which additionally comprises an affinity chromatography.

14. Process according to claim 13, in which the affinity chromatography is carried out with a glutathione- coupled gel matrix.

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 o o 0-3: o 2 G AGG AAT TCT CTG ATC ATG ACT TCA GTT AAT TCT GCA GAA GCC S.0-4: GAG GGG TGG CAC GGG AGT CGG TCC TTC GAA GAG G CTA CAA GCT GGG CCC CCG CAA AG for the direct detection of pathogen by means of DNA amplification, especially by means of polymerase chain reaction.

16. Use of an immunologically active peptides s according to any one of claims 1 to 3 as a vaccine against infections with parvovirus B19. DATED THIS 2ND DAY OF MARCH 1994 MIKROGEN MOLEKULARBIOLOGISCHE ENTWICKLUNGS-GMBH By Its Patent Attorneys GRIFFITH HACK CO Fellows Institute of Patent Attorneys of Australia Immunologically active peptides or polypeptides with a partial amino-acid sequence of the capsid proteins VP1 and VP2 of parvovirus B19 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-Bl9 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. T r I 1