Method for detecting acute Borna disease virus (BDV) infections, and diagnostic kit therefor, in particular in combination with methods for distinguishing acute from chronic and latent BDV infections, and diagnostic kits therefor

Abstract

One aspect of the present invention is directed to a method for detecting acute Borna Disease Virus (BDV) infections. According to the invention, the presence of heterodimers of p24 BDV phosphoprotein and p40 BDV nucleoprotein in a sample is determined by means of antibodies of both proteins using a sandwich ELISA. The invention also relates to a diagnostic kit for a sandwich ELISA for detecting mute BDV infections. Said kit uses a antibody of p24 BDV phosphoprotein and a second primary antibody of p40 BDV nucleoprotein, at least one reporter-molecule-labelled secondary antibody, means for immobilising a primary antibody on a surface, and instructions for carrying out the method according to the invention. The invention also relates to the combination of the method according to the invention and the new diagnostic kit with known methods for detecting circulating immune complexes (CIC) and antibodies. Acute BDV infections are thus distinguished from chronic and latent ones (humans and animals), allowing, for example, differential diagnosis and treatment monitoring for diseased individuals but also the identification of health risks depending on infection status in healthy carriers.

Claims

1. A method for detecting acute Borna disease virus (BDV) infections, comprising: the step of determining a presence of heterodimers composed of phosphorylated p24 BDV phosphoprotein and p40 BDV nucleoprotein in a sample using a sandwich enzyme-linked immunosorbent assay (ELISA), wherein a first primary antibody is present in an immobilized state and is incubated with the sample, wherein a second primary antibody is added in a non-immobilized state after incubation of the sample with the first antibody, wherein either the first primary antibody is directed against a phosphorylated p24 BDV phosphoprotein and the second primary antibody is directed against a p40 BDV nucleoprotein and/or wherein the first primary antibody and the second primary antibody are monoclonal antibodies.

2. The method as claimed in claim 1 wherein the immobilized first primary antibody is present in an immobilized state on a surface via a first secondary antibody or is directly immobilized on the surface.

3. The method as claimed in claim 1, wherein the non-immobilized primary second antibody is identified using a second secondary antibody comprising a reporter molecule.

4. The method as claimed in claim 1 wherein the first primary antibody and the second primary antibody originate from different species.

5. The method as claimed in claim 1 wherein the first primary antibody is present in an immobilized state directly on a surface or via a first secondary antibody on the surface, wherein the non-immobilized primary second antibody is identified using a second secondary antibody comprising a reporter molecule, wherein the first and second secondary antibodies are from different species or originate from the same species.

6. The method as claimed in claim 1 further comprising detecting circulating immunocomplexes (CIC) composed of BDV antigens and specific antibodies attached thereto.

7. The method as claimed in claim 1 further comprising detecting circulating free antibodies against p24 protein and/or the p40 protein.

8. A diagnostic kit for a sandwich ELISA for detecting acute BDV infections, comprising: a first primary antibody and a second primary antibody, a first of said first and second primary antibodies being directed against the phosphorylated p24 BDV phosphoprotein and a second of said first and second primary antibodies being directed against the p40 BDV nucleoprotein; at least one secondary antibody labeled with a reporter molecule; optionally means for immobilizing a primary antibody on a surface; and instructions for carrying out a method as claimed in claim 1.

9. The diagnostic kit for a sandwich ELISA for detecting acute BDV infections as claimed in claim 8 wherein the first primary antibody is present in an immobilized state directly on a surface or via a first secondary antibody on the surface, wherein the nonimmobilized primary second antibody is identified using a second secondary antibody comprising a reporter molecule, wherein the first and second secondary antibodies are from different species or originate from the same species.

10. The diagnostic kit as claimed in claim 8, further comprising means for detecting CIC composed of BDV antigens and specific antibodies attached thereto.

11. The diagnostic kit as claimed in claim 8, further comprising means for detecting circulating free antibodies against the p24 protein and/or p40 protein in the plasma/serum.

12. The method as claimed in claim 1 wherein p40 BDV nucleoprotein epitopes recognized by said second primary antibody do not overlap with a BDV phosphoprotein binding domain on the BDV nucleoprotein.

13. The method of claim 6 wherein the CIC BDV antigens include all or a portion of p24 protein and/or p40 protein.

14. A method for determining a prevalence of BDV infection differentiating acute, chronic, and latent states in an epidemiological study of a population's mental and/or physical health comprising performing the method of claim 1 on a plurality of different samples drawn from the population; detecting circulating immunocomplexes (CIC) composed of BDV antigens and specific antibodies attached thereto in the samples; and detecting circulating free antibodies against p24 protein and/or the p40 protein in the samples, wherein presence of the heterodimers is indicative of an acute BDV infection, wherein absence of the heterodimers and presence of the CIC is indicative of a chronic BDV infection, and wherein absence of the heterodimers and the CIC and presence of the circulating free antibodies is indicative of a latent BDV infection.

15. A method for diagnosing BDV infection differentiating acute, chronic, and latent states in a patient with a psychiatric and/or neurological disorder comprising performing the method of claim 1 on a sample taken from the patient; detecting circulating immunocomplexes (CIC) composed of BDV antigens and specific antibodies attached thereto in the sample; and detecting circulating free antibodies against p24 protein and/or the p40 protein in the sample, wherein presence of the heterodimers is indicative of an acute BDV infection, wherein absence of the heterodimers and presence of the CIC is indicative of a chronic BDV infection, and wherein absence of the heterodimers and the CIC and presence of the circulating free antibodies is indicative of a latent BDV infection.

16. A method for monitoring the course of a BDV infection in a patient comprising periodically differentiating acute, chronic, and latent states by performing the method of claim 1 on samples obtained from the patient at different time periods; detecting circulating immunocomplexes (CIC) composed of BDV antigens and specific antibodies attached thereto in the samples; and detecting circulating free antibodies against p24 protein and/or the p40 protein in the samples, wherein presence of the heterodimers is indicative of an acute BDV infection, wherein absence of the heterodimers and presence of the CIC is indicative of a chronic BDV infection, and wherein absence of the heterodimers and the CIC and presence of the circulating free antibodies is indicative of a latent BDV infection.

17. A method for differential diagnosis of acute BDV infections from chronic and latent infections comprising performing the method of claim 1 on a sample of a patient and comparing results obtained to standards for chronic and latent infections, wherein the standards are obtained by detecting circulating immunocomplexes (CIC) composed of BDV antigens and specific antibodies attached thereto in the sample; and detecting circulating free antibodies against p24 protein and/or the p40 protein in the sample wherein presence of the heterodimers is indicative of an acute BDV infection, wherein absence of the heterodimers and presence of the CIC is indicative of a chronic BDV infection, and wherein absence of the heterodimers and the CIC and presence of the circulating free antibodies is indicative of a latent BDV infection.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows the diagnosis schema for acute, chronic and latent BDV infections. In a first assay, what is assayed in accordance with the method according to the invention is the presence of the antigen. In the event of the presence thereof, there is an acute infection, whereas in the event of a negative detection, a further check is made to determine whether CIC are present or not. If CIC are present, a chronic infection is assumed, and in the case of CIC-negative samples, an additional check is made to determine whether antibodies are present. If no antibodies are present either, the existence of an infection is excluded, whereas in the case of the exclusive detection of antibodies (anti-p24 protein and/or anti-p40 protein antibodies), a latent infection is assumed.

(2) FIG. 2 shows a schema of the BDV antigen enzyme immunoassay (EIA) in accordance with the method according to the invention. In said assay, the monoclonal mouse antibody Kfu2 directed against phosphorylated p24 protein is immobilized on a microtiter plate via binding to a secondary antibody having anti-mouse IgG1 subclass specificity and then used for capture of the heterodimers from the plasma/serum sample. These bound heterodimers are then detected using the monoclonal mouse antibody W1 directed against p40 protein. To visualize the reaction, what follows is a secondary antibody having anti-mouse IgG2a subclass specificity, via the label of which with biotin reporter molecule and subsequent streptavidin-mediated binding of alkaline phosphatase, it is possible to effect the corresponding color reaction after addition of the substrate p-nitrophenyl phosphate.

(3) FIG. 3 shows a schema of the BDV immunocomplex EIA, as described in DE 19758017 C2 for example. There, the two monoclonal mouse antibodies W1 and Kfu2 are immobilized on a microtiter plate via binding to a secondary antibody having anti-mouse IgG specificity and then used for capture of the antigenantibody complexes from the plasma/serum sample. Said antigenantibody complexes (BDV CIC) are then detected using enzyme-labeled secondary antibodies directed against the IgG of the host species of the sample (alkaline phosphatase reporter molecule) and a corresponding color reaction (as in FIG. 2).

(4) FIG. 4 shows a schema of the BDV antibody EIA, as can be used according to the invention. Here, the monoclonal antibodies W1 and Kfu2 that were described are likewise immobilized on a microtiter plate via binding to a secondary antibody having anti-mouse IgG specificity and then used as capture antibodies for first binding native BDV antigens, which had been previously obtained in virus-free form from a standardized infected cell culture. These bound BDV antigens then make it possible to detect anti-BDV antibodies (anti-p24 or anti-p40 protein antibodies) from a plasma/serum sample. This is, after binding of anti-BDV antibodies present in the sample to the native antigen, then effected via enzyme-labeled secondary antibodies directed against the IgG of the host species of the sample (alkaline phosphatase reporter molecule) and a corresponding color reaction (as in FIG. 2).

(5) FIG. 5 shows the results of antigen assays for individual samples from test persons, wherein, firstly, BDV antigen is detected using the method according to the invention (New pAg) and, secondly, using the earlier antigen detection method described from DE 198 60 255 C2 (Old pAg).

(6) FIG. 6 shows the results of the combined use of the antigen assay according to the invention with the CIC assay and the antibody assay, the last two assays as described in DE 19758017 C2. The absorbance values are shown cumulatively.

(7) FIG. 7 shows the complete amino acid sequence of the BDV N protein (Seq. ID. No. 1). The five binding sites N1 to N5 (Seq. ID Nos. 3 to 7) for the described monoclonal antibody W1, identified by epitope mapping, form the conformational epitope on the native BDV N protein, wherein N1, N3 and N5 are interactive binding sites following crystal structure comparison (Bode, 2008). The binding sites are provided with a black frame. Provided with a dashed frame are the two domains which define the P protein binding sites on the N protein, amino acids 51-100 and 131-155 (Berg et al., 1998). These show no overlap with the W1 binding sites. As a result, N/P heterodimers can also be recognized.

(8) FIG. 8 shows the complete amino acid sequence of the BDV P protein (Seq. ID No. 2). The three binding sites P1 to P3 (Seq. ID. Nos. 8 to 10) for the described monoclonal antibody Kfu2, identified by epitope mapping, form the conformational epitope on the native P protein. The binding sites are provided with a black frame. P1 is the main binding site and includes the main phosphorylation site (S26/S28; Schwemmle et al., 1997) on the BDV P protein (provided with a dotted frame). Kfu2 recognizes only the active, phosphorylated BDV P protein (Bode, 2008).

(9) The invention will be further elucidated with the aid of examples without being restricted thereto.

EXAMPLES

(10) Isolated blood samples from individuals showing a BDV infection are provided, the status of the infection being acute, chronic or latent or no infection being present.

(11) TABLE-US-00001 Table of medical personnel from Chongqing study Antigen Code Medical heterodimers CIC Antibody Infection Sample personnel (MP) Sex Age Diluted 1:10 Diluted 1:20 Diluted 1:100 status E3875 No details female 50 0.066 0.055 none E4064 Administration male 51 0.073 0.027 none E3728 Ophthalmology female 29 0.059 0.033 none E3667 Gastroenterology male 28 0.074 0.067 none E3977 Infection department female 48 0.076 0.2835 chronic E4173 Breast surgery female 47 0.0815 0.2625 chronic E4272 Surgical preparation female 49 0.0605 0.6325 chronic E3731 Ophthalmology female 52 0.044 0.607 0.183 chronic E3693 Heart surgery female 59 0.0755 0.587 0.237 chronic E3838 No details female 26 0.142 0.8975 acute E3727 Ophthalmology female 55 0.029 0.155 chronic E4265 Experimental research female 41 0.2455 0.173 0.044 acute E3630 Retired female 35 0.214 0.1305 0.097 acute E4113 Rehabilitation male 81 0.166 0.306 acute E4278 Surgical preparation male 29 0.236 0.715 acute E4123 Rehabilitation male 30 0.2195 0.366 acute E3968 Positive control MP male 57 1.9835 0.3675 0.3025 acute Student Negative control 0.0495 0.068 none Assessment of the assay results (standard for all BDV EIA assays) Cut-off = 0.100. Negative <= 0.1; borderline > 0.1-0.12; (+) weakly positive > 0.12-0.15; 1+ positive > 0.15-0.3 2+ positive > 0.3-0.6; 3+ positive > 0.6-1.0; 4+ positive > 1.0.
The samples come from a large study which had been carried out in a university hospital in Chongqing, China, using the CIC assay and additionally the antibody assay and a real-time RT-PCR (Liu et al. 2015). The assays were carried out using the antibodies W1 and Kfu 2 that were used in DE 197 58 017 C2. In said assays, W1 is the primary antibody directed against the P40 BDV nucleoprotein and Kfu 2 is the primary antibody directed against phosphorylated P protein P24 phosphoprotein of BDV. The assay set-up for the CIC detection and for the antibody detection follow the respective schemas in FIGS. 3 and 4.
The assay set-up of the method according to the invention for antigen detection (heterodimers) follows the schema in FIG. 2.

(12) Material of Method According to the Invention:

(13) AffiniPure goat anti-mouse IgG1, Fc fragment-specific antibodies (concentration 1.3 mg of antibody per ml), minimal cross-reaction with human, bovine and leporine serum proteins (code number 115-005-205, Jackson ImmunoResearch).

(14) Biotin-SP-conjugated AffiniPure goat anti-mouse IgG2a, Fc fragment-specific antibodies, minimal cross-reaction with human, bovine and leporine serum proteins (code number 115-065-206, Jackson ImmunoResearch).

(15) Alkaline phosphatase-conjugated streptavidin (code number 016-050-084, Jackson ImmunoResearch).

(16) Alkaline phosphatase substrate kit (code number 172-1063, Biorad) or individual substances for substrate buffer and substrate (Bode et al., 2001) as follows:

(17) Substrate buffer, diethanolamine buffer pH 9.8 (diethanolamine 1.007 M+MgCl.sub.2.6H.sub.2O 0.5 mM+NaN.sub.3 0.003 M);

(18) Substrate, p-nitrophenyl phosphate hexahydrate (pNPP.6 H.sub.2O) (0.003 M); 5 mg tablets (code number N9389 Sigma Aldrich); always prepare fresh in the assay, 1 mg of pNPP per ml of substrate buffer.

(19) Conjugate buffer (alkaline phosphatase) TBS-Tween pH 8.0 (TRIS 0.02 M+NaCl 0.14 M+KCl 0.003 M+0.05% Tween 20).

(20) Wash buffer, NaCl-Tween (0.9% NaCl+0.05% Tween 20+NaN3 0.003 M).

(21) Sample dilution buffer (also for steps 2, 4 and 5 in FIG. 2),

(22) PBS-Tween pH 7.2 (KH.sub.2PO.sub.4 0.0015 M+Na.sub.2HPO.sub.4.12 H.sub.2O 0.008 M+NaCl 0.14 M+KCl 0.003 M+0.05% Tween 20+NaN.sub.3 0.003 M).

(23) Coating buffer, 0.01 M sodium phosphate/0.25 M NaCl pH 7.6 (NaH.sub.2PO.sub.4.2 H.sub.2O 0.02 M [stock A], Na.sub.2HPO.sub.4 0.02 M [stock B]; coating buffer contains 65 ml of stock A+435 ml of stock B+0.25 M NaCl in 1000 ml of double-distilled water).

(24) Stop solution, 3 M NaOH

(25) All chemical substances with degree of purity as specified by American Chemical Society (ACS grade), preferably by Sigma Aldrich.

(26) Nunc-Immuno 96-well microtiter plates for ELISA, MaxiSorp, F (flat bottom) (code number 442404, Nunc).

(27) BDV-Specific Key Reagents

(28) W1 (mouse subclass IgG2a) specifically directed against the nuclear BDV protein (N protein);

(29) Kfu2 (mouse subclass IgG1) specifically directed against the BDV phosphoprotein (P protein);

(30) W1 and Kfu2 are monoclonal mouse antibodies, first described by Ludwig et al., 1993.

(31) The inventors have hybridoma supernatants of these two antibodies, which were already produced and which are suitable for assay kit production on an industrial scale. The inventors also have hybridoma cells which allow unlimited production of further batches of the monoclonal antibodies W1 and Kfu2.
Further characterization of W1 and Kfu2 (Bode, 2008)

(32) TABLE-US-00002 Short description W1 Species Mouse Subclone TL9 IgG subclass IgG 2a Specificity N protein p40 of BDV (reference strain V) Dissociation constant (K.sub.D) 2.31 × 10.sup.−9 +− 0.26 × 10.sup.−9M (native N protein) Epitope type Conformational epitope (discontinuous) Hybridoma supernatant 1:500 usage dilution in BDV ELISAs (=ready-to-use) Recognition of recombinant (r) Yes (assay with earlier antigen ELISA) N protein Purified rN detection limit 1.5-3 nanograms per mL Kfu2 Species Mouse Subclone 57-4-33 IgG subclass IgG 1 Specificity P protein p24 of BDV (reference strain V) Dissociation constant (K.sub.D) 3.33 × 10.sup.−9 +− 0.34 × 10.sup.−9M (native P protein) Epitope type Conformational epitope (discontinuous) Hybridoma supernatant 1:500 usage dilution in BDV ELISAs (=ready-to-use) Recognition of recombinant (r) No (assay with earlier antigen ELISA) P protein, nonphosphorylated Recognition of rP, Yes (assay with earlier antigen ELISA) phosphorylated in vitro
First Description of the Epitope Sequences of W1 and Kfu2

(33) TABLE-US-00003 Epitope on N protein p40 of BDV SEQ ID No. W1 Epitope type Conformational epitope (discontinuous) Number of binding sites Five on 5mer peptides (N1-N5) in each case N1 3 Amino acid sequence 32_KFLQY_36 N2 4 Amino acid sequence 116_AKFYG_120 N3 5 Amino acid sequence 168_TMMAA_172 N4 6 Amino acid sequence 294_LAPRS_298 N5 7 Amino acid sequence 307_FYWSK_311
See also FIG. 7

(34) TABLE-US-00004 Epitope on P protein p24 of BDV SEQ ID No. Kfu2 Epitope type Conformational epitope (discontinuous) Number of binding sites Three (15mer, 12mer, 5mer peptides), P1-P3 P1, main domain, 8 Amino acid sequence phosphorylation site serine 22_RRERSGSPRPRKVPR-36 26/28 P2 9 Amino acid sequence 46_LLKDLRKNPSMI_57 P3 10 Amino acid sequence 142_DRSMK_146
See also FIG. 8
Protocol of Method According to the Invention:

(35) 100 μl each of a 1:1000 dilution of the goat anti-mouse IgG1 secondary antibody diluted with coating buffer are pipetted into the MaxiSorp F microtiter plates and incubated at 37° for one hour. Thereafter, three wash cycles are carried out with the wash buffer (120 ml total). Afterwards, the monoclonal antibody Kfu2 in a dilution of 1:500 (hybridoma supernatant) in PBS-Tween is incubated, at 100 μl per well, at 37° C. for one hour, followed by three wash cycles with the wash buffer. The samples (diluted 1:10 with PBS-Tween) are pipetted, at 100 μl each per well, into the microtiter plate and incubated at 37° C. for one hour, followed by three wash cycles with wash buffer. Thereafter, the monoclonal antibody W1 in a dilution of 1:500 as hybridoma supernatant diluted in PBS-Tween is added at 100 μl per well, incubated at 37° C. for one hour, followed by three wash cycles. Thereafter, the goat anti-mouse IgG2a secondary antibody labeled with biotin in a dilution of 1:1000 in PBS-Tween is added in an amount of 100 μl per well, incubated at 37° C. for one hour and subsequently washed with three wash cycles.

(36) Then, the alkaline phosphatase-labeled streptavidin, diluted 1:1000 in conjugate buffer TBS-Tween pH 8.0, is added in an amount of 100 μl per well and incubated at 37° C. for 30 min. This is followed by again three wash cycles with wash buffer.

(37) Thereafter, the detection reaction takes place with p-nitrophenyl phosphate (1 mg/ml) p-NPP in substrate buffer pH 9.8. 100 μl each of the corresponding p-NPP solution are added, followed by an incubation at room temperature for 2.5 minutes. Note: continuous visual monitoring is necessary, the negative control must remain colorless, whereas the positive control changes color. The color reaction is stopped by addition of the stop solution (3 M NaOH) at 50 μl each per well. The absorbance is immediately determined at 405 nm using a suitable multichannel photometer. “Blank” measurement against unconverted substrate.

(38) CIC Determination as Per the Methods Described in DE 19758017 C2

(39) CIC determination in the samples was carried out as per the methods described in DE 19758017 C2 (see above schema in FIG. 3). To optimize the diagnostic assay for CIC determination in practice, the incubation times and temperatures were simplified to respectively 1 h at 37° C. for all steps, except for the visualization steps, i.e., the substrate incubation (C3), stop-solution addition (C4) and measurement (C5). With regard to the monitoring of the development of the color reaction, the procedure was as above for the invention described here. The initial dilution of the samples to be assayed of 1:20 in single determination with validation on positive and negative control offers the same reliability as the inclusion of further dilutions.

(40) In the description of the materials, the code numbers are added below: AffiniPure goat anti-mouse IgG, Fc fragment-specific antibodies (code No. 115-005-071; Jackson ImmunoResearch).

(41) Alkaline phosphatase-conjugated AffiniPure goat anti-human IgG, Fc fragment-specific antibodies, minimal cross-reaction with bovine, equine and murine serum proteins (code No. 109-055-098; Jackson ImmunoResearch) for human samples.

(42) Alkaline phosphatase-conjugated AffiniPure goat anti-horse IgG, Fc fragment-specific antibodies (code No. 108-055-008; Jackson ImmunoResearch) for horse samples. Key reagents W1 and Kfu2, substrate kit or substrate buffer and substrate tablets, buffer and Nunc MaxiSorp F ELISA plates as described above for the new invention.
Antibody Determination in Plasma/Serum as Per the Methods Described in DE 19758017 C2

(43) The determination of the antibodies against the p24 protein and/or p40 protein in plasma/serum samples was in principle carried out as per the method described in DE 19758017 C2 (see above scheme in FIG. 4). Analogously to the CIC assay, the incubation times and temperatures were simplified to respectively 1 h at 37° C. for all steps, except for the visualization steps, to optimize the diagnostic kit for the antibody determination in practice. Furthermore, instead of the described initial dilution of the samples of 1:50, the dilution was adjusted to 1:100. It became apparent that the initial dilution of the samples to be assayed for antibodies of 1:100 in single determination with validation on positive and negative control offers the same reliability as the inclusion of further dilutions.

(44) To carry out the BDV antibody EIA described in DE 19758017 C2, detection antigen from BDV-infected cells is required. The usage dilution of the detection antigens from infected cell cultures is, depending on the batch, between 1:100 and 1:300.

(45) A human oligodendroglial cell line (OLIGO/TL) persistently infected with BDV strain V (Briese et al., 1994) that allows the unlimited production of detection antigen was deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ) in Braunschweig on 12 Dec. 1997 under No. DSM ACC2334.

(46) For the description of the remaining materials, see the CIC assay.

(47) Results:

(48) FIG. 5 shows a comparison of the method according to the invention with the prior antigen assay. Since the prior antigen assay works with a sample dilution of 1:2, the results thereof are included both for this dilution and for the dilution of 1:10 applicable to the assay according to the invention.

(49) FIG. 5 shows that sample E4265 was positive only with the assay according to the invention, whereas it was not recognized with the prior antigen assay, neither at 1:10 nor at 1:2 sample dilution. Sample E3630 was weakly positive with the assay according to the invention. The positive signal was confirmed in a further analysis (table and FIG. 6). The prior antigen assay likewise showed a weakly positive to borderline positive signal, though only at the 1:2 dilution. The next dilution step 1:4 was already distinctly negative with absorbance value 0.034 (not depicted), and this led to a negative assessment. Because of the high sample volume for a dilution series from 1:2, i.e., 100 μl, a repetition was no longer possible.

(50) Samples E3727 and E3731 were negative in both antigen assays, even at the dilution of 1:2 in the case of the prior antigen assay. Both samples originate from the physician/nurse team in the eye care department of the hospital. However, the combination with the CIC assay shows that both were chronically infected with BDV (see the table). Team member E3731 had high CIC values and additionally antibodies, indicating an acute bout of virus that had recently taken place.

(51) In the case of samples E4265 and E3630, who were positive only with the new assay at 1:10 sample dilution, it should be emphasized that the prior assay, even at its sample dilution of 1:2 that is five times lower as standard, was not capable or not unambiguously capable of detecting BDV antigens. The assay according to the invention thus had at least a five-times higher sensitivity than the prior assay and was able to demonstrate that the medical coworkers (E4265 female, research; E3630 female, retired) went through an acute BDV infection at the sampling time point. If only the prior antigen assay had been available, the diagnosis “chronic infection” would have had to been made on the basis of the simultaneous detection of CIC, and the acute exposure would have been overlooked (see the table).

(52) Results of the combined use of the antigen assay according to the invention with the CIC assay and the antibody (Ab) assay as per DE 19758017 C2 are shown by FIG. 6. The values from Table 1 are thus clearly depicted by cumulative columns.

(53) FIG. 6 illustrates that the assay according to the invention was able to identify six acute infections in the example panel (E4123, E 4278, E4113, E3630, E 4265 and E3838). Negative results in the assay according to the invention were, in combination with the CIC assay, able to identify six chronic infections (E3727, E3693, E3731, E4272, E4173, E3977). Negative CIC and antibody assays were, in four cases from the panel, able to exclude a BDV infection (E3667, E3728, E4064, E3875).

(54) The sample panel of medical personnel for which there was no mental disorders according to a structured interview, neither earlier nor currently, was chosen in order to show that the assay according to the invention, alone and in combination with CIC assay and antibody assay, is, independently of clinical symptoms, capable of differentiating between acute, chronic and latent infections and of excluding a BDV infection with analysis of a single blood sample. Chronic stress is common in everyday life in the medical sector, and so an increased activation risk does not appear unusual. In addition, there is an increased infection risk in the daily care of sick people.

(55) The volume of 100 μl for the prior antigen assay, with initial 1:2 dilution and subsequent dilution series, which volume is distinctly higher in comparison with the antigen assay according to the invention, was only available for selective parallel tests, as shown in FIG. 5, for the study samples already analyzed multiple times elsewhere.

(56) The advantages of the new method according to the invention for antigen detection are also multifarious and practical without direct comparison.

(57) Firstly, what is possible is a broad applicability for a very wide variety of different sample materials, i.e., for any type of body fluid or tissue fluid. BDV activity is measured independently of the host species. Furthermore, the determination of the phosphorylated heterodimers composed of p24 and p40 allows the verification of acute BDV infections, since the primary antibody Kfu2 only binds the active phosphorylated form of the BDV P protein. For the monoclonal antibody Kfu2 used here, this is globally a unique feature.

(58) In contrast to the assay described in DE 19758017 C2, what is presently determined is the heterodimer. In contrast, the prior method is based on two capture antibodies, i.e., immobilized primary antibodies against both p24 and p40, being used in order to also determine monomeric forms thereof. Accordingly, however, it was not possible to differentiate the present phosphorylated heterodimers therewith. In one embodiment, the method according to the invention uses, in addition, secondary antibodies which allow a high level of reproducibility. In the embodiment with the binding pair biotinstreptavidin coupled to an enzyme such as alkaline phosphatase, a signal enhancement and thus improved detectability is achieved compared to the prior visualization of the signal solely via enzyme-labeled anti-antibodies. Furthermore, significantly less patient sample (10 μl for one-time determination, 20 μl for dilution series from 1:10) is required, and the sensitivity of the method is higher according to the five-times higher initial dilution of the sample of 1:10. This became especially apparent by it being possible to identify an acute BDV infection in more individuals than with the prior antigen assay.

(59) The present invention provides a completely new assay method for acute BDV infections that has a higher sensitivity than the prior antigen assay and is independent of limited resources, as represented by rabbit antibodies for example. By combination with CIC assay and antibody assay, the new method is capable of reliably distinguishing between acute, chronic and latent infections (humans and animals) and of thereby raising BDV diagnostics to a hitherto unreached level of informative value with a high standard of quality.

(60) The method according to the invention is therefore suitable especially for diagnosis, but also for course monitoring of a BDV infection, especially also for determination of the therapy plan. Furthermore, the method according to the invention can be part of a treatment method.

(61) The previously unpublished amino acid sequences of the binding sites which form the respective conformational epitopes are named for the first time here. They are essential molecular constituents of the properties of the monoclonal antibodies W1 and Kfu2.

(62) The present invention provides a completely new assay method for acute BDV infections that is independent of limited resources and can be used in humans and animals in an overarching manner as regards species. By combination of the new diagnostic kit with the use of known methods or kits for detecting circulating immunocomplexes (CIC) and antibodies in the plasma or serum, the new method is capable of reliably distinguishing between acute, chronic and latent infections (humans and animals) and of reaching a hitherto unreached level of informative value in BDV diagnostics.

CITED LITERATURE (IN ALPHABETICAL ORDER)

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