Methods for detecting of human parvovirus

Abstract: A method for detection of human parvovirus B19, comprising the steps of: (1) bringing a sample into contact with fixed P-antigen positive red cells in a medium at pH 5.6 ± 0.6; and (2) determining whether or not hemagglutination occurs; and a reagent for detecting human parvovirus B19, wherein the reagent comprises fixed P-antigen positive red cells.

Description: The invention relates to a method for detecting human parvovirus B19. Human parvovirus B19 replicates in the nucleus of proliferating cells such as fetal liver cells and fetal cardiac myocytes. It is non-enveloped virus and induces remarkable viremia in humans. It causes aplastic crisis in patients of chronic hemolytic anemia, erythema infectiosum, hydrops fetalis, febrile diseases, arthropathy, transient bone marrow failure and so on. B19 virus resists various inactivation procedures such as heat and detergent treatment. B19 virus can be transmitted through blood, and it is important not to use infectious blood to prevent B19 virus infection by transfusion. There are two kinds of methods for detecting B19 virus infection, i.e., identification of the B19 virus itself and detection of IgM antibody specific to B19. The former methods include an indirect immunofluorescent (IF) method, a counter current immunoelectrophoresis (CIE) method, an enzyme-linked immunosorbent assay (ELISA) method, a radioimmunoassay (RIA) method, a dot blot hybridization method, and a polymerase chain reaction (PCR) assay. The latter includes the ELISA, RIA, and Western blotting methods. The conventional methods require a Long time and complicated manipulation and their sensitivity is not always sufficient. To date, B19 viremia could not be routinely checked on blood donations. Therefore, it is important to find a highly sensitive, quick and simple detection method for the B19 virus. Brown K. E. et al have reported hemagglutination by human parvovirus B19 (Journal of General Virology, Vol. 73, p2147 to 2149, 1992). They also demonstrated that hemagglutination occurred via P-antigen (red cell membrane antigen) as a receptor for the B19 virus. (Science, Vol. 262, p114 to 117, 1993). However, while Brown et al indicate that the B19 virus causes agglutination of human red cells, they do not suggest that this phenomenon can be used for detecting the B19 virus. The state of the art is also illustrated by documents WO-A-94/22014 and JP-A-64 000 467 as hereafter explained. WO-A-94/22014 describes a method of detecting the presence of the parvovirus B19 by contacting the sample with a purified receptor for a parvovirus, and detecting the presence of binding of parvovirus to the receptor, such presence of binding indicating the presence of parvovirus in the sample. JP-A-64 000 467 describes a method of measuring the hemagglutination inhibiting antibody comprising a reagent with glutaraldehyde fixed red blood cells which are likely to contain P-antigen. The present invention aims to provide a new method that is more sensitive and simple than previous methods. To resolve the drawbacks in the conventional detection methods, the present inventors have realized a highly sensitive and reproducible method of detecting B19 virus using both fixed red blood cells and a buffer of pH 5.0 to 6.2. Accordingly, the present invention provides a method for detection of human parvovirus B19 comprising the steps of bringing a sample into contact with fixed P-antigen positive red cells in a medium at pH 5.6 ± 0.6, and determining whether or not hemagglutination occurs, according to claim 1. Advantageously, the method of the invention is carried out by using an automated hemagglutination machine. Figure 1 shows the stability of the RHA reagent of the present invention. This invention relates to a new detection system for human parvovirus B19 and is characterized as follows. When fixed red cells having P-antigen and samples containing the virus are mixed together in a buffer of pH 5.0 to 6.2, hemagglutination occurs. The pH value positively influences the agglutination. According to this invention, B19 viruses in a sample combine with P-antigen on the surface of red cells. Consequently, red cells bind to each other via the virus and hemagglutination occurs. By observing whether or not hemagglutination occurs, the present invention can determine the presence or absence of B19 virus in a sample. This method is 100 to 1,000 times more sensitive than the ELISA method. The present invention uses 0-group blood cells, with the P blood-group antigen, that are fixed with glutaraldehyde. Approximately 30% of the Japanese population are 0-type as determined by ABO-blood typing. Almost all Japanese people are P-antigen positive, but very few Japanese are P-antigen negative (p-type). Therefore P-antigen positive blood must be confirmed by means of anti-P serum. The present invention uses red cells fixed with glutaraldehyde. As to specific antigens, fixed red cells can remain in the same condition as untreated red cells for a long time. When using untreated cells, hemolysis occurs gradually within a few weeks, whereas fixed red cells keep the same condition for several years. Therefore using fixed cells, a stable result can be obtained. The fixation with glutaraldehyde should be done in 1% glutaraldehyde at 15 to 35°C for 10 to 30 hours. In the present invention, the fixed cells, as described above, and the samples are mixed together in a solution, containing a citrate-phosphate buffer or an acetate buffer, of pH 5.0 to 6.2. This buffer should preferably include a polymer, a protein, a sugar, sodium chloride, and a preservative. The present methods uses polyvinylpyrrolidone and arabic gum as polymers. Polyvinylpyrrolidone enhances hemagglutination and arabic gum reduces non-specific reactions. Both of the polymers should be used at a concentration of 0.05 to 1.0%. Seronegative human AB serum should be added to the buffer at a concentration of 0.1 to 2.0%, because serum protein reduces non-specific reactions. The addition of 0.5 to 3.0% sucrose in a buffer is preferable for long-term storage of fixed red cells. The concentration of sodium chloride influences the detection sensitivity and the frequency of non-specific agglutination. The optimal concentration of sodium chloride is approximately 0.45 to 2.0%. 0.1 to 1.0% sodium azide may be added, as a . preservative, to the buffer. In this detection system, a blood sample is mixed with fixed red blood cells in a buffer as previously described. This assay can be done on a microplate at room temperature and a result can be obtained after 30 to 120 minutes. If a sample contains human parvovirus B19, hemagglutination occurs. EXAMPLES Next, the present invention is described in detail by means of Examples. Example 1 Preparation of red cells Red cells as a reagent for detecting B19 virus were prepared as follows. Human O-blood cells with the P-antigen were washed more than three times with 1/15M phosphate buffered saline (PBS), pH 7.2. A 10% suspension of the red cells in PBS was fixed with glutaraldehyde at a final concentration of 1%. The fixed cells were washed twice with physiologic saline and once with PBS. The fixed red cells were suspended in PBS at a concentration of 25% and stored at 4°C. Before the assay, the concentration of the fixed cells was adjusted in the suspension buffer to 1%. The suspension buffer is described as follows. The suspension buffer consists of 0.1M citrate-phosphate buffer, at pH 5.6, containing 0.1% polyvinylpyrrolidone (PVP-K30), 0.1% arabic gum, 1% sodium chloride, 1% sucrose, and 0.1% sodium azide. A sample dilution buffer was prepared by adding seronegative human AB serum, at a final concentration of 0.6%, to the suspension buffer. Example 2 We compared this invention, a receptor-mediated hemagglutination (RHA) test, with a PCR test, an ELISA test, and a CIE test on twenty-one B19 virus positive samples. (1) The method of the present invention Twenty-five µl of sample diluent buffer was added to a P6 microplate (OLYMPUS) from the first to the eighteenth well. Then we made two-fold serial dilutions of 25 µl of serum up to the eighteenth well. Twenty--five µl of 1% fixed red cell solution was added to each well and mixed together using a microplate mixer for 15 to 30 seconds. After 1 hour at room temperature, the results were obtained. (2) ELISA The diluted samples were added to the microwells coated with the IgM antibody specific to the B19 virus. After incubation and a washing procedure, a monoclonal-antibody specific to B19 was added to each well. After a washing step, peroxidase-conjugated antimouse IgG was added. Excess conjugate was removed by washing and the bound peroxidase was developed by adding the substrate. The intensity of the color is positively correlated with the concentration of B19 virus. (3) Measurement of copies of B19 (PCR) At first, the detection limit in PCR was determined with plasmid containing B19 genome. A ten-fold dilution series of plasmid was amplified, and standard curve was obtained. Virus DNAs from ten-fold serial dilution of B19 viremic samples were obtained by a phenol-chloroform extraction method and used for PCR assay. The number of copies of the virus in sample serums were determined from the standard curve. (4) CIE At first, three-lined holes on agarose plates were made. Human parvovirus B19 antiserum was added to the plus terminal side, blood samples to central part, human parvovirus B19 positive serum to minus terminal side, and then the plates were electrophoresed in 30 mM barbiturate buffer (pH 8.6). The result are indicated in table 1. Detection of 21 B19 positive samples in RHA, PCR, ELISA and CIE Sample-No. Lot-No. RHA titer copies of the virus (/ml) ELISA CIE antigen antigen antibody 194 086-0975 4096 4 × 10 10 1.581 + - 195 471-6610 2048 4 × 10 10 0.907 + - 196 090-0314 8192 4 × 10 11 NT + - 197 849-4973 8192 4 × 10 11 1.542 + - 257 067-5238 8192 4 × 10 10 2.243 + - 258 812-3182 4096 4 × 10 10 1.990 + - 259 318-5775 8192 4 × 10 10 1.486 + - 260 475-3114 2048 4 × 10 10 1.593 + - 261 491-7509 1024 4 × 10 10 0.488 + - 262 647-0846 8192 4 × 10 10 1.665 + - 263 034-8953 8192 4 × 10 10 1.028 + - 264 455-2829 8192 4 × 10 10 2.068 + - 265 098-8479 8192 4 × 10 11 1.921 + - 266 085-9943 8192 4 × 10 10 1.523 + - 267 021-2032 4096 4 × 10 10 0.975 + - 268 491-7184 8192 4 × 10 10 2.335 + - 269 480-5296 4096 4 × 10 10 2.385 + - 270 489-8200 8192 4 × 10 10 2.256 + - 271 831-3167 2048 4 × 10 10 0.825 + - 272 400-4427 4096 4 × 10 10 1.407 + - 273 092-4293 512 4 × 10 10 0.623 + - negative negative-1 <2 <4 × 10 3 0.076 - - control negative-2 <2 <4 × 10 3 0.083 - - NT: not tested As shown above, positive samples for ELISA and CIE were also positive for the RHA test. The detection limit of RHA is 10 5 -10 6 virus per ml. On the other hand, ELISA, CIE, and PCR can only detect more than 6.3 × 10 7 virus per ml, 3.6 × 10 9 virus per ml, and 4 × 10 3 virus per ml, respectively. These results suggest that RHA test is more sensitive than ELISA and CIE. Example 3 We examined 27,265 samples including B19 positive serum by both methods, RHA and CIE. RHA test was applied to PK 7200 (OLYMPUS, automated hemagglutination machine). Seventeen µl of sample serum were mixed with 250 µl of sample diluent buffer. Ten µl of the diluted sample was added, with 40 µl of 0.625% fixed red cells, to a P6 microplate. [Dilution rate; 1/39.25] The result of these samples was determined automatically after one hour. This result indicates table 2. Comparison of RHA with CIE RHA (+) (-) CIE (+) 21 0 (-) 19 27,225 Total sample number: 27,265 Twenty-one positive samples in CIE and RHA were also positive in PCR assay. Out of 19 samples, CIE(-) and RHA(+), 6 samples were positive in PCR. This observation suggests that RHA is more sensitive than CIE. RHA rarely gives a false-positive reaction (13/27,265: 0.048%). Example 4 From September to November in 1995, 211,701 donor samples were examined by RHA using PK7200. Positive samples in PK 7200 were subjected to titration of RHA, RHA inhibition test shown below and PCR assay. (1) RHA inhibition test for confirmation To confirm the positive reactions, inhibition of RHA was done by specific antibodies against B19 virus. Optimal concentration of B19 antibodies was determined by box titration. In RHA inhibition test, two lines of microwells per sample in a P6 microplate were used. On one line, sample serum was serially diluted with sample diluent buffer. On the other line, sample was also serially diluted with buffer containing B19 antibodies. One hour later, the fixed cells were added to each well. The RHA inhibition titer was determined after 1 hour. The result is-shown in table 3. (A) Screening of blood donors Screening of B19 virus by RHA n % Screening by RHA (using PK7200) 211,701 100.00 Positive samples 680 0.32 RHA titer 2 6 ≤ 249 0.12 Inhibition titer 2 1 ≤ 29 0.01 PCR (+) 13 0.01 (B) Comparison of RHA titer with PCR RHA titer 2" 12 11 10 9 8 7 6 total No. 4 2 4 5 30 65 139 249 PCR(+) 4 2 2 2 1 0 2 13 % positive 100.0 100.0 50.0 40.0 3.3 0.0 1.4 As shown in table 3(A), among 211,701 samples, 680 positive samples, including suspicious hemagglutination, were selected for a second confirmation test. 249 samples were more than 2 6 of titer on RHA. Twenty-nine out of 249 samples were more than 2 1 of titer on inhibition test. The non-specific rate of RHA by PK 7200 was 0.32%. Table 3(B) indicates that all samples with more than 2" of RHA titer were also positive in PCR assay. Considering the results of inhibition test and PCR, it is suggested that inhibition test as a second screening is desirable to confirm the results of the RHA test. Example 5 Stability of RHA reagents We checked the titers of B19 viremic samples and negative samples by RHA at several time points. The test started 4 months after preparation of the RHA reagent. The result is shown in figure 1. As shown in Figure 1, the reagents for RHA were stable without the significant changes of titer for at least two months, from day 120 to day 191, after preparation. The test may be a feasible method for detecting human parvovirus B19 because it is simple, sensitive, and applicable to automated hemagglutination machine that enable routine large-scale screening for the virus.

Claims: A method for detection of human parvovirus B19, comprising the steps of : (1) bringing a sample into contact with fixed P-antigen positive red cells in a medium at pH 5.6 ± 0.6; and (2) determining whether or not hemagglutination occurs. A method according to claim 1, wherein the method is carried out by using an automated hemagglutination machine.

Inventors: MAEDA YOSHIAKI, SATO HIROYUKI, TAKAKURA FUMIHIRO, MAEDA, YOSHIAKI, SATO, HIROYUKI, TAKAKURA, FUMIHIRO
Applicants: JAPAN RED CROSS, THE JAPANESE RED CROSS SOCIETY
Application year: 1996
Main classification: G01N33
IPC codes: C12Q1, G01N33