Patent Application
20060127952
The present invention relates to a diagnostic method for Peste des Petits Ruminants (PPR), a viral disease in animals, and provides the a new diagnostic method for PPRV using recombinant Nucleocapsid (N) protein, expressed in insect cells, and the specific monoclonal antibody against N protein from PPRV(Peste des Petits Ruminants Virus).
More specifically, the invention relates to a PPR diagnostic method using a one-step Reaction Enzyme-Linked ImmunoSorbent Assay (ELISA) that detects peroxidase labeled monoclonal antibody competing with serum antibodies for 30 minutes to bind to pre-coated recombinant N protein of PPRV on the ELISA plates.
Peste des Petits Ruminants (PPR) is an infectious viral disease of ruminants including goats, sheep and cattle. Since PPR was first described in western Africa in 1942, it has become widely distributed in areas from south-Saharan desert regions in Africa and Middle east countries to southwest Asia, including Bangladesh.
Clinical signs include severe erosions in the mouth and lips, difficulty in breathing and diarrhea. Most deaths in affected animals result from severe diarrhea. PPR is one of the OIE list A diseases internationally and one of the obligatory notifiable animal diseases in Korea, because of its huge economic damage to the livestock industry of the countries in which it has occurred with very high morbidity and mortality in susceptible hosts. PPR is regarded as a foreign animal disease in Korea, since there has been no outbreak report so far.
Therefore, rapid detection by a rapid diagnostic test and destruction of PPR infected animals is very important to minimize the economic impact on the live stock industry.
PPR can be diagnosed by detection of viral antigen (antigen detection method) and by detection of antibodies from the infected animals (antibody detection method).
With regard to antigen detection methods; virus isolation and identification, reverse transcriptase-polymerase chain reaction (RT-PCR), and antigen detection ELISA have been applied, using tissue samples, pathological samples from mouth lesions and faeces from infected dead animals. However, these methods should be carried out in BL3 (biosafety level 3) laboratories.
As for antibody detection methods; virus neutralization test and ELISA have been applied for PPR diagnosis. The diagnostic method using ELISA can be performed general laboratories, whereas the virus neutralization test must be performed BL3 laboratories. For PPR diagnosis, inactivated viral particles, recombinant hemagglutinin(H) proteins or recombinant N proteins have been used for antigens in prior ELISA assays, and at least 3 hours has been required to perform the assays.
Therefore, the inventors recognized that the method using recombinant viral proteins would be the most effective method to perform in general laboratories for rapid diagnosis of PPR. The invention was accomplished by innovatively shortening the three or more steps of the former ELISA assays to a one-step ELISA that is able to detect PPR antibodies rapidly and precisely by using antigen coated ELISA plate and a peroxidase labeled monoclonal antibody.
In consequence, the purpose of this invention is to provide a new diagnostic method to detect PPRV antibodies by one-step reaction between antigen and test serum on antigen pre-coated plates.
To accomplish this purpose, the PPR antibody detection method of the invention has the characteristic that PPR antibodies in serum samples can be detected by one-step ELISA based on detection of bound peroxidase labeled monoclonal antibody following competition with serum antibodies on a plate pre-coated with recombinant N protein expressed in insect cells.
Lane M: Protein molecular size marker
Lane 1: Result of detection of recombinant N protein using an N protein specific monoclonal antibody
Lane 2 : Result of recombinant N protein using a PPRV specific anti-serum
PPR 2-65, 2-66, 2-69, 2-73 and 2-81: PPRV positive field sera
Pos Control: PPRV reference positive serum
33-4, 384, P-3H12, P-13A9, P-14C6, P-9H10 and P-11A6: monoclonal antibodies against N protein of PPRV
Recombinant baculovirus (abbreviated as Bacmid/PPRVN) of the invention has characteristics as follows;
The Bacmid/PPRVN contains the gene coding N protein of PPRV and produces antigens for one-step ELISA. Unlike recombinant antigens of the former ELISA assays, the recombinant proteins of Bacmid/PPRVN have 6× Histidine bound to the amino-terminal of the proteins to facilitate easy identification of protein expression and purification.
N protein gene of PPRV of the invention is originated from PPRV Nigeria75/1 strain isolated in Nigeria (NCBI Genbank L39878,
In addition, P-3H12, N protein-specific monoclonal antibody of the invention, has the characteristics that it is originated from B lymphocytes of Balb/c mice immunized with PPR Nigeria 75/1 strain, it binds to an immuno-dominant epitope on PPRV N protein which induces strong immunity, and it functions as the detector in one-step ELISA. Moreover, the monoclonal antibody has another characteristic in that it can be used as peroxidase conjugated antibody for one-step ELISA.
The invention is explained more specifically as follows.
ELISA assay for PPR diagnosis in the invention has the following steps:
(1) the reaction step having a competitive reaction between the conjugated monoclonal antibody and test sera on recombinant N protein-precoated ELISA plates;
(2) the washing step to remove unbound antibodies on the antigen coated plates;
(3) the reading step to calculate PPR antibody level in test sera by measuring optical density(OD);
and all of the steps are schematized in
In addition, the ELISA assay of the invention includes two more steps; the preparation of ELISA plates pre-coated with recombinant N protein of PPR expressed in insect cells, and the preparation of monoclonal antibody conjugated to horseradish peroxidase. Procedures, reagents and reaction conditions related to the ELISA assay of the invention can be modified by broadly applied conventional methods in the industry.
The ELISA assay of the invention is excellent in its sensitivity and specificity because it uses the monoclonal antibody binding to the immuno-dominant epitope. It has another advantage in that it can detect rapidly any PPRV infection because amino acid sequences of the N protein are highly conserved among strains of PPRV.
The recombinant protein of the invention is expressed as fusion protein fused with 6× Histidines for easy identification of protein expression and purification. The recombinant N protein can be extracted from infected cells by conventional methods. In the invention, recombinant N proteins may be produced in insect cell expression systems after cloning of N protein gene and construction of the expression vector.
Production of recombinant N protein is not limited to the insect cell expression system, but also includes other recombinant protein expression systems, such as E. coli, adenovirus or yeast expression systems.
In addition, monoclonal antibody of the invention can be generally produced from hybridomas, in which mouse myelomas are fused with B lymphocytes from immunized mice with PPR viral antigen or recombinant N protein. Such hybridoma can be produced by conventional methods in the industry, such as the HAT selection method. It is possible to select some hybridomas with high affinity to N protein and reaction on immuno-dominant epitopes on N protein among various hybridomas producing monoclonal antibodies; for example, a hybridoma cell line deposited in Korean Cell Line Research, Seoul, Korea (deposit number KCLRF-BP-00104) is available.
The invention will be specified in detail in the following examples and tests. However, the invention is not restricted by such examples, and may also include any conventional modification, substitution, and insertion in the industry in the range of the invention.
PPR virus Nigeria75/1 strain, isolated from a goat in Nigeria in 1975 and suppled by CIRAD-EMVT, France, was inoculated into VERO (ATCC CCL81, USA) cells, then viral genome was extracted from the cell culture supernatant using RNeasy RNA extraction kit (Qiagen, USA) according to the manufacturer's instructions.
Complementary DNA of full-length N gene of PPR virus was synthesized by RT premix kit (bioneer, R.O.Korea) using genomic RNA (see example 1) and a primer 2 (reverse primer) below. The synthesized DNA was PCR-amplified by a PCR premix kit (Bioneer, Korea) using primers 1 and 2 below. The PCR amplication was carried out under conditions of pre-denaturation (10 min at 95° C.), a 30-cycle PCR reaction (denaturation for 60 sec at 94° C.; annealing for 60 sec at 58° C.; and extension for 60 sec at 72° C.), and final ligation (5 min at 72° C.) using a PCR machine (PE9600, Perkin Elmer). The primers were designed based on the published N gene sequence of PPR virus Nigeria 75/1 (Genbank accession number L39878) as follows:
Restriction enzyme sites (Nar1 site for primer 1 and Sac1 site for primer 2) were incorporated at the 5′ ends of each primer to facilitate cloning as indicated by the underlined nucleotides. Resulting DNA product amplified above was inserted into pGEM-Teasy vector (Promega,USA) to generate recombinant plasmid “pGEM/PPRVN”. Proper orientation of the insert was confirmed by the dideoxynucleotide chain termination sequencing method using the ABI Model 373A DNA sequencer (Applied Biosystem, USA) by comparison with the sequence of the N gene of PPR virus shown in
DNA fragment of 1,587 bps, extracted from the pGEm/PPRVN, was inserted into the Nar1 and Sac1 restriction enzyme sites of pFastBac HT vector (Invitrogen, USA), which were designed to express fusion protein with 6× His. Resulting recombinant plasmid with PPRV N gene was designated “pFastBac/PPRV-N”, as shown in
Recombinant baculovirus Bacmid/PPRVN was generated using a commercialized Baculovirus expression system (Invitrogen, USA). For this purpose, recombinant baculovirus DNA, Bacmid/PPRVN DNA was constructed by transforming expression vector pFastBac/PPRVN (see Example 3) into E. coli DH10Bac cells. The mixture of Bacmid/PPRVN DNA (5 μg in 100 μl serum-free Grace's medium) and Cellfectin (Invitrogen) (6 μl in 100 μl serum-free Grace's medium) was incubated at room temperature for 30 min. During the incubation, Spodoptera frugiperda(S-f21) cell monolayer (GibcoBRL, 2×106 cells in well of 35 nm cell culture petridish) was washed using serum-free Grace's medium. The mixture was then co-transfected into the S-f21 cells for 5 hrs at 27° C. After incubation, the mixture solution was replaced with 2 ml volume of fresh Grace's medium containing 10% fetal bovine serum. The cells were incubated for 4 to 6 days at 27° C. until cytopathic effects were observed. Cell culture supernatant containing recombinant baculoviruses was harvested to purify recombinant baculoviruses by using plaque assay. Briefly, a 100 μl volume of recombinant baculoviruses harvested was inoculated onto a monolayer of S-f21 cells (2×106 in well of 35 mm petridish) for 1 hr at 27° C. The cell monolayer was overlaid using Grace's medium containing 1.5% low-melting agarose for 3 to 4 days of incubation at 27° C. to pick up plaque containing recombinant virus. The plaques were infected into fresh S-f21 cells for 3 to 4 days. Cells showing cytopathic effects were examined in indirect immunofluorescence assay and Western blot using PPRV-specific monoclonal antibody to select recombinant baculovirus Bacmid/PPRVN expressing PPR N protein.
Recombinant N protein of PPRV (rPPRV-N) was prepared in insect cells infected with a recombinant baculovirus (Bacmid/PPRV-N) expressing PPRV N protein. Briefly, the S-f21 cells in a 175 cm2 tissue culture flask were infected with the recombinant baculovirus (Bacmid/PPRV-N) at a multiplicity of infection of 0.1. After 90 min incubation at 27° C., the infected cells were removed from the flask and then added into 200-ml S-f21 cell culture (2×106/ml) of a spinner culture vessel. The infected cells in the spinner culture vessel were incubated for 4 or 5 days at 27° C., then the cells were harvested from the culture vessel. After centrifugation at 500×g for 20 min at 4° C., the cells were collected and resuspended in 1/20 volume of a lysis buffer (0.01 M phosphate buffered saline containing 1% Triton X-100) containing protease inhibitor cocktail (Roche Molecular Biochemicals, Germany). After 5 min incubation at room temperature, the cell lysates were sonicated to extract the recombinant N protein. Finally, the lysate was clarified by centrifugation at 500×g for 20 min at 4° C. and the supernatant was used as an ELISA antigen.
Semi-purified PPRV (Nigeria75/1, CIRAD-EMVT, France) was used to produce monoclonal antibody. Briefly, PPRV-infected Vero cells were harvested and frozen-thawed three times. The resulting lysates were clarified by centrifugation at 5000×g for 30 min. Polyethylene glycol 800 (23g/l) and sodium chloride (70 g/l) were added to the supernatant containing PPRV and left overnight at 4° C.
The aggregated PPRV was precipitated by centrifugation at 5000×g for 30 min and the pellets were resuspended in 10 ml of 0.01M PBS. The concentrated PPRV were semi-purified by using 25% (w/v) Sucrose-gradient ultracentrifugation at 100,000×g for 90 min. The PPRV was quantified using GenQuanil(Pharmacia Biotech, USA) to adjust to 0.1 mg/ml.
BALB/c mice were immunized with purified viral antigen (50 μg per dose in Freund's incomplete adjuvant) via foot-pad route. Ten to fifteen days after immunization, the lymphocytes derived from popliteal lymph nodes of immunized mice were harvested and fused with the SP2/0-Ag14 myeloma cells using polyethylene glycol 1500 (Boehringer Mannheim, Germany) by the conventional method. Briefly, popliteal lymphocytes were washed with serum-free medium (SFM) and mixed with SP2/0-Ag14 mouse myeloma cells at a ratio of 5:1 to 10:1. One ml of PEG1500 solution was added to the mixture of lymphocytes and myeloma cells. PEG-mediated fusion cells were diluted in HAT(Hypoxanthine Aminopterin Thymidine) medium containing 10% fetal bovine serum and distributed into 96 wells (100 μl per well) of tissue culture microplates, which had previously been cultured with mouse peritoneal macrophage cells. The plates were incubated in a CO2 incubator at 37° C. Hybridoma cells secreting N protein of PPRV monoclonal antibody were screened by indirect ELISA using recombinant N protein (see Example 5). The positive hybridoma cells were subjected to cloning by the limiting dilution method, and finally inoculated intraperitoneally into BALB/c mice, which were primed by Freund's incomplete adjuvant. Ascitic fluid was collected 1 to 2 weeks later.
Characteristics of monoclonal antibody selected are shown in Table 1.
A competitive ELISA was carried out to select monoclonal antibody for one-step ELISA using recombinant N protein antigen(see Example 5) and monoclonal antibodies (see Example 6). Maxisorp™ ELISA plates (Nunc, Denmark) were coated with 50 μl of purified PPRV antigen (1:1500 dilution in 0.01M PBS) for 1 hour at 37° C. After washing three times with wash buffer (0.002 M PBS containing 0.05% Tween 20), 25 μl of optimally diluted monoclonal antibody and each test serum at a 1:400 dilution in blocking buffer (PBS containing 0.5% bovine serum and 0.05% Tween 20) were added to duplicate wells. Reference positive and negative serum controls were included in each plate. After a 1-hour incubation at 37° C. with shaking, the plates were washed three times and incubated at 37° C. for 1 hour with peroxidase-labeled anti-mouse immunoglobulins (Kirkegaard-Perry Laboratories, Inc., 50 μl per well) diluted 1:2000 in blocking buffer. Following another washing step, the plates were incubated for 10 min with the substrate O-phenylenediamine (Sigma, USA) in 0.05M citrate phosphate buffer (pH 5.0). Colormetric reaction was stopped by adding 50 μl volume of 1.25 M sulfuric acid to all wells. Optical density (OD) readings were measured at the 492 nm wavelength. The reaction was considered as competition positive when the OD of the monoclonal antibody in the presence of test serum showed a 50% or greater reduction of that of the monoclonal antibody alone.
Recombinant N proteins prepared as the above Example 5 were added 50 ul/well diluted 1500-fold in 0.01M PBS onto ELISA plates (Maxisorp, Nunc) and incubated at 37° C. for 1 hour. After incubation the plates were washed three times with 0.01M PBS to remove the unreacted remnants. Protein stabilization solution (Sigma,USA) was added to block the unreacted sites on the plates and incubated at room temperature for 1 hour, and the plates were dried and stored at 4° C. until use. Optimal concentration of recombinant N protein was determined by indirect ELISA as shown in
Monoclonal antibody (P-3H12) was purified according to the manufacturer's protocol. The purified monoclonal antibody was conjugated to peroxidase using peroxidase labeling kit (Roche,USA). The conjugated monoclonal antibody was mixed with the stablilization solution supplied within the kit, frozen under liquid nitrogen, and stored at −70° C.
One-step ELISA for diagnosis of PPR uses recombinant N protein-coated plates and peroxidase-conjugated monoclonal antibody, and the process is as follows. The N protein-coated plates were incubated with peroxidase-conjugated monoclonal antibody diluted 400-fold in blocking solution and an equal volume of sera diluted 10-fold in blocking solution at 37° C. for 30 min. After incubation, the plates were washed three times with PBST and developed with OPD (O-PhenyleneDiamine) solution at room temperature for 10 min. The reaction was stopped by adding 50 ul of 1.25M sulfuric acid and optical density was measured at 490 nm.The serum is determined positive if the percent inhibition (PI) is above 50% as compared to the control (containing peroxidase-conjugated monoclonal antibody only).
The ELISA was performed with sera collected from domestic cattle and goats (n=510) that proved negative for PPR.
The one-step ELISA was conducted for sera derived from goat infected with four lineages of PPRVs (3 goats per each lineage of PPRV). The results were compared to the standard ELISA recommended by FAO and OIE.
As shown in the above, the one-step ELISA curtailed the process dramatically as compared to the standard ELISA without losing sensitivity and specificity. The use of monoclonal antibody reactive to the immunodominant epitope of PPRV ensures the efficient detection of antibodies to PPRV. In addition, the short turnaround time and simple process of the one-step ELISA enables sera surveillance for PPR on a large scale. As PPR causes severe damage to the livestock industry in many African and Asian countries, if H or F structural protein were to be used as a vaccine, the one-step ELISA should be useful in differentiating infected from vaccinated animals.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2004-0089816 | Nov 2004 | KR | national |
