WO1997013150A1 - Kit permettant de diagnostiquer la presence du virus respiratoire syncytial bovin - Google Patents

Kit permettant de diagnostiquer la presence du virus respiratoire syncytial bovin Download PDF

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Publication number
WO1997013150A1
WO1997013150A1 PCT/CA1996/000662 CA9600662W WO9713150A1 WO 1997013150 A1 WO1997013150 A1 WO 1997013150A1 CA 9600662 W CA9600662 W CA 9600662W WO 9713150 A1 WO9713150 A1 WO 9713150A1
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elisa
brsv
antibody
antigen
sample
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PCT/CA1996/000662
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English (en)
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WO1997013150B1 (fr
Inventor
Youssef Elazhary
Estela Cornaglia
Souhel Charara
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Universite De Montreal
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Priority to AU70817/96A priority Critical patent/AU7081796A/en
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Publication of WO1997013150B1 publication Critical patent/WO1997013150B1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses

Definitions

  • the invention relates to an ELISA test which per ⁇ mits a specific, sensitive and rapid diagnostic of the presence bovine respiratory syncytial virus.
  • the bovine respiratory syncytial virus was isolated in 1970. This virus belongs to the genes Pneumovirus and to the family Paramyxoviridae. It is encapsulated, pleomorphic virus which is composed of a single branch ribonucleic acid (ARN).
  • the BRSV is made of 10 proteins in which two are important for viral replication (Huang, Y. et al., 1985, Virus Res . , 2 : 157-173). These are glycoprotein F which is responsi ⁇ ble for fusion and protein G which is responsible for viral attachment (Walsh, E. and Hiruska, J., 1983, J. Virol . , 47:171-176).
  • Nucleocapside is made of ARN and three proteins: nucleoprotein (N), phosphoprotein (P) and polymerase (L).
  • N nucleoprotein
  • P phosphoprotein
  • L polymerase
  • protein N and pro- tein F have shown a high degree of homology with respect to their sequence of amino acids (Amann, V, et al., 1992, J. Gen . Virol . 73: 999-1003; Garcia-Barreno, B. et al., 1989; Lerch, R. et al., 1989, J. Virol . , 63: 833-840; Mulkey, K. and Anderson, G., 1991, J. Clin . Microbiol . , 29: 2038-2040).
  • BRSV Newcastle disease virus
  • the infection caused by BRSV has been described as the most important sickness causing an acute inflam ⁇ mation of the respiratory tracts in dairy cows and calves which are less than 12 months old (Elazhary et al., 1982, Cornell Vet . , 72: 325-333).
  • the first symp ⁇ toms of the sickness appear in the form of cough, pyr- exia and a nasal and ocular exsudat.
  • BRSV may cause anorexia, trachypnea and dyspnea which may lead to ter ⁇ minal pneumonia.
  • An infection by BRSV which is extremely contagious, spread horizontally by direct contact of rejects of respiratory tracts of infected animals. Morbidity is very high.
  • the infected animals suffer a lost of weight as well as a decrease of milk production and they are more susceptible to secondary infections (Elazhary, Y. et al. , 1982, Cornell Vet . , 72: 325-333).
  • the economical losses caused by morbid ⁇ ity and the death rate of this infection are therefore very high, in particular in intensive breeding. In Canada, these losses are estimated at more than 50 mil- lion dollars per year.
  • An efficient control of calves require the fast and reliable detection of a sickness in order to enable the isolation of affected animals and to decrease the risk of spreading of the disease to none infected flocks.
  • the tracking down of the infection in bovine may be carried out by viral isolation or by the detection of the virus in the nasal epithelial cells which are colored by the technique of immunofluorescence. These two techniques require the availability of a laboratory and qualified personnel. Moreover, because of the very fragile nature of the BRSV virus, the viral isolation is applicable only in the case where the inoculation may be carried out immediately after collecting the sample. It has been established that the termination of the presence of the antigen in the nasal epithelial cells is at least as sensitive as the isolation and consequently this technique has been accepted as cur ⁇ rent practice in laboratories. It would be highly desirable to be provided with a test for use in the field and in the laboratory and may rapidly determine the presence of the living or inactivated BRSV.
  • One aim of the present invention is to provide a test for use in the field and in the laboratory and may rapidly determine the presence of the living or inacti ⁇ vated BRSV.
  • Another aim of the present invention is to pro ⁇ vide an ELISA test which permits a specific, sensitive and rapid diagnostic of the presence BRSV.
  • the BRSV ELISA test essentially comprises the following: a) A polyclonal antibody which is specific to the BRSV is adsorbed on the surface of the wells of the
  • a blocking solution is used to obstruct the non-covered sites thus preventing adhesion of any other particle at the surface of the wells.
  • a blocking solution is used to obstruct the non-covered sites thus preventing adhesion of any other particle at the surface of the wells.
  • another washing is carried out.
  • a sample is incubated on the ELISA plate where only the antigen particles will bind to the antibody to which they are specific. The number of viral particles bound to the polyclonal antibody is dependent on the concentration of the virus in the sample.
  • Washing will permit the removal of the sample and of the particles which are not bound.
  • a second antibody which, this time is a mono ⁇ clonal antibody (MAb) is placed in the presence of the virus. Since this antibody is specific to the antigen, it will bind only to the virus which is held on the polyclonal antibody. Another washing will remove any excess of the MAb.
  • an anti-mouse antibody conjugated to peroxidase will be fixed on the bound MAb. A last washing will remove the excess of unbound anti-mouse antibody. Presence of peroxidase will thereafter enable the development of a blue coloration when the chromogenic substrates are added. The intensity of the coloring will be depend on the quantity of virus pre ⁇ sent in the sample. Presence of a coloring will be detected visually in the field and by spectrophotometer in the laboratory.
  • the BRSV ELISA test of the present invention relies on an indirect immuno-enzymatic technique for the detection of an antigen (structural nucleoprotein) which is common to all the strains of the syncytial respiratory virus in the bovine (BRSV). This test, directly carried out on a sample of nasal secretions, enables the determination of the virus BRSV.
  • an antigen structural nucleoprotein
  • the BRSV ELISA test of the present invention essentially includes 4 steps:
  • the controls and the samples to be tested are distributed in cupolas sensitized with anti-BRSV poly ⁇ clonal antibodies.
  • the viral protein if present in the sample, is fixed on the specific sites.
  • a MAb anti-BRSV antibody structural nucleoprotein
  • mice IgG of anti-IgG goat coupled to a peroxidase enzyme enable to reveal the binding of the MAb by forming the following complex: Polyclonal Antibody - Ag RSV (nucleoprotein) -
  • the enzyme bound to the complex is revealed by addition of a chromogenic sub ⁇ strate which it converts into a blue colored product.
  • a blue color indicates the presence of antigen in the sample.
  • the intensity of the blue color is directly in relation to the quantity of viral antigen present in the sample.
  • a visual reading of this coloration is carried out. In the laboratory, the reading is carried out by a spectrophotometer after having added a solution to stop the reaction of the substrate.
  • an ELISA test for qualitatively determining the presence of living or inactivated BRSV antigen in a bovine biological sample, wherein the sample has an unknown amount of BRSV antigen, which comprises the following: a) incubating a solid support having bound thereto a first anti-BRSV antibody with the biological sample for a time sufficient for an immune com ⁇ plex to form between the anti-BRSV antibody and any BRSV antigen present in the sample; b) incubating the incubated solid support of step a) with a second anti-BRSV antibody; and c) detecting the bound second antibody of step b) to determine the quantity of the BRSV antigen present in the sample.
  • the ELISA of the present invention may further comprise a step consisting of washing between steps a) and b). Also, the ELISA of the present invention may further comprise an additional step after the washing and before step b), wherein the step consists of react ⁇ ing the washed solid support of step a) with a blocking solution to obstruct sites the non-covered by the first antibody, thereby preventing adhesion of any other par ⁇ ticle on the solid support.
  • the preferred blocking solution which may be used in accordance with the ELISA of the present inven- tion, is selected from the group consisting of TRIS- casein-TWEENTM and PBS-TWEENTM-thimerosal.
  • the ELISA of the present invention may further comprise a step consisting of washing before step b), which is preferably effected between steps b) and c).
  • the preferred first antibody used in accordance with the ELISA of the present invention is a polyclonal antibody, while the preferred second antibody used is a monoclonal antibody.
  • the detecting step carried out in step c) in accordance with the ELISA of the present invention is effected by fixing an anti-mouse antibody conjugated to peroxidase on the bound antibody.
  • the ELISA may further comprise a a step consisting of washing after fixing the conjugated peroxidase.
  • the conjugated per- oxidase will develop a blue coloration upon addition of the chromogenic substrates.
  • the intensity of the blue color is read visually or by a spectrophotometer after addition of a stopping solution.
  • the preferred stop ⁇ ping solution is H2SO4.
  • the preferred bovine biological sample which may be used in accordance with the ELISA of the present invention, consists in nasal secretions.
  • Fig. 1 illustrates the protein specificity of the MAb RSQ89C
  • Fig. 2 illustrates the evaluation of the binding of IgG bovines specific to BRSV on the wall of the wells
  • Fig. 3 illustrates the determination of the im ⁇ munological specificity of the ELISA Test.
  • the first object of the present invention was the production of reagent required for the development of the ELISA test.
  • polyclonal and monoclonal antibodies (MAb) specific to BRSV have been produced and characterized.
  • the second object of the present invention was the standardization of the ELISA test. The optimum dilution of the polyclonal and MAb was established. The blocking solution, solutions for diluting each reagent and times of incubation have been determined.
  • the third objective was based on the evaluation of the ELISA test. The intra and inter-plate vari ⁇ ation, the sensitivity and immunological specificity as well as the relative sensitivity and specificity have been studied.
  • the fourth object of the present invention was to establish the conditions and times of preservation of the kit. For this purpose, different solutions and temperatures of preservation as well as the use or non- use of lyophiiization have been tested. I- PRODUCTION OF REAGENTS
  • the animals used for the production of polyclo- nal antibodies were hosting cows between 3 and 6 years old and 300 to 500 kg which show no clinical signs.
  • the antibodies in the sera from the cows were tested by immunofluorescence technique against the fol ⁇ lowing viruses as antigen: syncytial respiratory virus, bovine viral diarrhea virus, parainfluenza virus, and bovine adenovirus.
  • the cows which were found to be serologically negative to the above-men ⁇ tioned antibodies have been used.
  • the immunization of seronegative cows was carried out with the BRSV Ql strain at the concentration of IO 6 DICTso ml.
  • Each bovine was inoculated with 5 ml, at three different locations, by intramuscular injections. The injections were repeated twice at two weeks intervals.
  • the titer of the antibody against RSV was determined by the ELISA test described below in section 1-1.2.
  • BRSV BRSV
  • the viral suspension obtained as described in section 1-1.1, was clarified by centrifugation at 10 000 g, during 15 minutes. The supernatant was col ⁇ lected while taking note of the volume. Thereafter, four volumes (V) of viral supernatant were mixed with IV of polyethylene glycol (PEG) 50% W/V in the buffer TEN (TRIS HCl 0.05M, EDTA 0.001M, NaCl 0.15M, pH 7.5) and the mixture was incubated at 4°C, while stirring, during 2 hours and 30 minutes.
  • PEG polyethylene glycol
  • centrifugation was carried out at 10 OOOg, during 30 minutes at 4°C.
  • the supernatant was elimi ⁇ nated and the pellet was diluted in buffer TEN of pH 7.5 at 1/50 of the initial volume of the viral super ⁇ natant.
  • This new mixture was concentrated by ultracen ⁇ trifugation at 100 OOOg, on a sucrose cushion 30% and 60% W/W diluted in buffer TEN during 2h, in a centri ⁇ fuge Beckman L8-55RTM (Beckman Instrument, California, USA), by means of a SW28TM rotor.
  • the viral band was collected and dialyzed in a buffer TRIS lOmM at pH 7.2, at 4°C, while steering, during 12 to 18 hours.
  • the purified virus obtained is titrated, divided into small amounts and frozen at -70°C.
  • a viral solution of RSV was prepared containing 2 ⁇ g of viral protein per lOO ⁇ l, in a carbonated buffer (Chart No. 1) .
  • Nunc Polysorp F16TM plates having 96 wells were used (Gibco Laboratories, Ontario, Canada # 467679 Lot 131201). In each well lOO ⁇ l/wells of the viral dilu ⁇ tion was deposited. The plates were incubated, during 1 hour at 37°C. The plates were washed three times, then they were frozen at -70°C, wrapped in aluminum foil until ready for use. When needed, they are washed twice with the PBS and dried by inversion.
  • Bovine serum was diluted 1/100 to 1/218700 (dilution factor of 3 and 8 dilutions per sample) in PBS.
  • lOO ⁇ l of each dilution were deposited in two wells and they were incubated, during 30 minutes at 37°C. Three washings then followed, with the washing solution (Chart No. 1) and drying was carried out by inversion.
  • lOO ⁇ l of anti-bovine rabbit antibodies IgG conjugated to peroxidase were added in each well, (Cappel-Oragon Tecknika, Ontario, Canada #3202-0082, lot 33703). After an incubation of 2 hours at 37°C, the product was washed three times.
  • lOO ⁇ l of sub- strate-chromogen were added in each well, reaction was allowed to take place during 10 minutes, and the reac ⁇ tion was stopped with 30 ⁇ l of H2SO4 (4N).
  • the plate was read by bichromatism, at a wave length of 450nm with a reference of 550nm, on a spec- trophotometer for ELISA (SLT Lab Instrument, Gr ⁇ dig, Australia). A sample is considered positive, if it gives an optical density of at least 0.1 and twice that of the negative control.
  • Chart No. 1 Record Solutions for the ELISA Test Carbonate Buffer
  • PBS Phosphate Buffer
  • Substrate B (chromogenic substrate)
  • TMB Tetramethyl benzidine
  • DMSO Dimethylsulphoxide
  • Casein 5 g mix during 15 min. adjust pH between 7.2 and 7.6 with NaOH 3N mix at 4°C until the next day allowing the solution be at room temperature ensure that the pH is between 7.2 and 7.6 otherwise adjusting it.
  • the serum was diluted 1/5 with PBS and it was filtered on MilliporeTM membrane having a porosity of
  • the module 1500 of protein G was used. Many cycles had to be carried out. Each cycle comprises the following steps: loading, washing, eluting and rebalancing.
  • the solutions used were: for loading, serum diluted 1/5 in PBS; for washing, PBS pH 7.4; for eluting, glycine-HCL 0.IM pH 2.5; for rebal- ancing, monobasic sodium phosphate 0.IM of pH 8.
  • the pH was adjusted at 7 with TRIS IM buffer.
  • the sample was deposited in a dialysis membrane (Tubulaires Spectra/Por MWCO 10,000, Fisher, Montreal, Canada) which is deposited in a 10L beaker (Fisher) containing PBS and a magnetic bar (Fisher); reaction was allowed to take place for at least 48 h, while stirring, at 4°C. PBS was changed 3 times at the minimum. Lyophilization
  • the purified and concentrated IgG were ⁇ lyophilized in a Lio-SanTM apparatus (Rolmex). The initial volume and the weight of the collected powder were noted in order to provide again in a water suspen ⁇ sion for use.
  • the polyclonal antibody thus treated is kept in sealed bags at -20°C.
  • the BCATM kit of Pierce (#23225) was used.
  • STD protein
  • distilled water were mixed in equal proportions.
  • Five plastic tubes were thereafter prepared as follows: a final volume of lOO ⁇ l/tube and a concentration of 5 to 50 ⁇ g/100 ⁇ l were needed.
  • a control was pre ⁇ pared with lOO ⁇ l of distilled water in a sixth tube.
  • Adequate dilutions of the samples were prepared, so that they can be found on the STD curve, always for a volume of lOO ⁇ l.
  • Reagent B was mixed with reagent A 1:50; these two reagents are supplied in the kit. The mixture is well mixed and 2ml are added to each of the previously prepared tubes. The tubes were incubated during 30 min. at 60°C, and they are read with a Spectronic 1001 plusTM spectrophotometer, at 562nm (Milton Roy Spec ⁇ tronic, New York, USA). Thereafter, the curve is made with the controls and the optical density of the sam- pies and their corresponding concentration of proteins are found. Titration of the polyclonal antibody
  • the polyclonal antibody was titrated with ELISA by using the method described above in section 1-1.2.
  • the titer represents the last dilution which gives an optical density at least twice higher than that of the negative control.
  • mice were inoculated by intraperitoneal injection with 250 ⁇ l of BRSV purified as described in section 1-1.2, mixed with 250 ⁇ l of incomplete Freund adjuvant, on days 0, 15 and 21. Three immunizations by utilizing only the purified virus were carried out on days -3, -2 and -1 before fusion. Preparation of spleen cells
  • the preparation of spleen cells is carried out after euthanasia of the mice with CO2, 24h after the last immunization.
  • the mice are disinfected with etha ⁇ nol alcohol 70% before removing the spleen.
  • the spleen is deposited in a Petri box containing 5ml of the medium Dulbecco's Modified Eagle Medium (DMEM rich in glucose, Gibco #430-2100EL) to which are added 3.7g/L of sodium bicarbonate, 4.8g/L of Hepes, 0.16g/L of sodium pyruvate, 2mM of L-glutamine and 0.2g/L of gen- tamicine.
  • DMEM rich in glucose Gibco #430-2100EL
  • the spleen is dissected in order to free the cells.
  • the DMEM containing the cells is col ⁇ lected and the pieces of tissue are allowed to be decanted for 2 min.
  • the supernatant is centrifuged at 250xg for 5 min. After two washings with DMEM, the cells are counted in DMEM.
  • NS-1 Ag4-1 (American Type Culture Collection, ATCC, 12301 Parklawn Drive, Rockville, MD 20852 USA, ATCC accession number TIB 18) myeloma cells are used as partner cells.
  • the NS-1 Ag4-1 cells contain 65 chromo ⁇ somes and synthesize the light kappa intracellular chain. The operation is carried out with cells in exponential growth phase and whose viability is at least 80%. They are counted after two washings in DMEM. Fusion
  • the volume may be completed to 50ml with DMEM containing 20 % of SFB. Centrifugation was carried out during 5 min., at 500xg (DamonTM/IEC HN-11) and the supernatant is vacu ⁇ umed. The residue is resuspended in 20ml of DMEM con ⁇ taining 20% SFB. The cells are capped and IO 5 cells/wells are deposited in plates containing 96 wells (CorningTM 25860). Thereafter, IO 4 spleen cells from a normal mouse are added, in each well.
  • DMEM-HAT IX DMEM to which there are added 10% SFB, 13.61mg/L of hyposanthine, 3,78mg/L of thymidine and 0.76mg/L aminopterine.
  • Cells of clones secreting the desired MAb are incubated at least during three days with more than 80% confluence to permit the production of antibodies.
  • the product is subject to centrifugation for 5 min., at 500xg (DamonTM/IEC HN-SII).
  • the supernatant obtained is used for all the subsequent steps.
  • lOO ⁇ l of the sample are deposited in each of the wells and are incubated during 30 min. at 37°C. This is followed by three washings, with PBS-TweenTM and drying is carried out by inversion. There is then added in each of the wells lOO ⁇ l of anti-mouse goat antibody IgG (H+L), conjugated to peroxidase (Bio-Rad) diluted 1/4000. After a 30 min. incubation at 37°C, the prod ⁇ uct is washed three times with PBS-TweenTM and twice with the PBS. lOO ⁇ l of chromogenic substrate mixture (50:50) are added in each well, the reaction is allowed to be carried out, for 10 min. at room temperature, and the reaction is stopped with 30 ⁇ l of H 2 SO4 (4N).
  • Reading of the plate is made by bichromatism, at a wave length of 450nm with a reference of 550nm, on a spectrophotometer for ELISA (SLT Lab Instrument, Gr ⁇ dig, Australia). A sample was considered positive, if it gives a optical density which is higher than that of the control. 2.2. Characterization of the MAb Type of immunoglobulin
  • the type of immunoglobulin of the MAb is deter ⁇ mined by means of the INNO-LIA Mouse MAB IsotypingTM kit (Innogenetics, Belgium). Technique of immunoblot
  • BRSV antigen 0.4ml of purified BRSV antigen and 0.4ml of the migration buffer (TRIS HCl 0.5 M pH 6.8, glycerol 10%, SDS 2%, bromophenol blue 0.2%) are mixed together and the mixture is boiled for 5 min.
  • the migration is car- ried out on a polyacrylamide mini-gel, with a 4% con ⁇ centration gel and a 10% migration gel.
  • a print of the gel is carried out on nitrocellulose paper using the buffer Tris 50mM pH 8.3, glycine 15mM and 20% methanol.
  • These nitrocellulose papers were incubated with a solution of PBS, 0.3% of fish gelatin (PBS-G) during 1 hour while steering, after which MAb diluted in PBS-G containing 0.05% TweenTM 20 are added and the mixture is incubated during 1.5 hours while steering. After wash- ing with PBS, incubation is carried out in the presence of anti-i munoglobulin mouse antibodies conjugated to peroxidase during 1 hour while steering.
  • each MAb 50 ⁇ l of each MAb are mixed with lOO ⁇ l of BRSV containing 200 DICT50 and the mixture is incubated dur- ing 1 h at 37°C. Plates containing 96 wells are used, which plates have flat bottoms (CorningTM 25860, Fisher), with confluent MDBK cells (see section 1-1.1). lOO ⁇ l/well of the antibody-virus are deposited in four wells. Incubation follows, during 1 h, at 37°C, in the presence of 2% of CO2. Then, lOO ⁇ l of a maintenance medium is added (Chart No. 2) in each well and incuba ⁇ tion is allowed to proceed, during three days.
  • a maintenance medium is added (Chart No. 2) in each well and incuba ⁇ tion is allowed to proceed, during three days.
  • Verification of the cytopathogenic effect is carried out every day during 3 days.
  • the presence of the cytopathic effect indicates that the MAb is not a neutralizing agent.
  • MEM Minimum Essential Medium
  • SB bovine fetal serum
  • gentamicine 5mg/ml of Schering Canada
  • Pointe Claire, Quebec 1% anti-PPLO 100X (pleuropneumonia-like organisms) of GIBCO Laboratories, New York, USA.
  • MAb which are specific to BRSV have been developed and tested by ELISA using two RSV bovine strains (Ql and VR794ATCC) and a strain of human origin as antigens.
  • Two MAb, RSQ89C and RSQ90 reacted with all the strains used. Considering that the MAb RSQ89C has given the higher optical density, it was selected as second antibody to develop the double sandwich ELISA.
  • the MAb RSQ89C presents a type of immunoglobulin
  • Fig. 1 shows that the MAb RSQ89C reacts with nucleoprotein.
  • the cellular division is carried out every third to fourth day.
  • the culture flask is rinsed (Corning, Pointe Claire, Canada) twice with PBS in order to remove all trace of serum which would risk to inactivate trypsin-EDTA.
  • the cellular monolayer is rinsed twice with trypsin-EDTA (Chart No. 2) having a final dilution of 0.25%. Tryp- sin-EDTA is allowed to react, during 3 to 10 minutes, at 37°C.
  • the flask is knocked to remove the cells and then a medium MEM is added (Chart No. 2).
  • the cells thus removed are passed through a needle 23G1TM (Becton Dickinson, Franklin Lakes NJ, #305145). Thereafter, they are distributed at the rate of 60,000 cells per cm 2 on a flat bottom plate having 96 wells (CorningTM 25860, Fisher), or at the rate of 50,000 cells per cm 2 in a 150cm 2 flask (CorningTM 25120, Fisher). The flask is filled to 40 ml with MEM (Chart No. 2). The cells are incubated at 37°C.
  • the BRSV virus used is the Quebec strain Ql iso- lated and characterized by Dr. Y. Elazhary (1980, Can . J. Comp. Med. , 44:299-303).
  • the strain has been iden ⁇ tified as virus BRSV by the neutralization test.
  • the virus which is inoculated on the cells is at passage number 20. 4.2 Inoculation of cells with the virus
  • MDBK cells which are confluent at 70-90% and are 24 to 48 hours old are used. Their culture medium is removed by washing with PBS. An adequate dilution of virus is thereafter added, which is made in the mainte- nance medium (Chart No. 2) to have a multiplicity of infection of 0.005 to 0.1 (10ml for a 150cm 2 ).
  • reaction is allowed to proceed during lh, at 37°C, while stirring.
  • the volume is then completed, depending on the flask used, with a maintenance medium. Incubation is carried out at 37°C.
  • the inoculated cells are observed daily to detect the cytopathic effect. When the latter reaches 50% of the cells, the virus is collected. It is col ⁇ lected by means of a policeman and it is frozen at -70°C.
  • Verification of the virus produced Titration of virus on cells MDBK There are used plates of 96 wells, prepared with the MDBK cells. Dilution of the virus are made with factors of 10, in the maintenance medium, and there are deposited lOO ⁇ l/well of each dilution, in four wells. Incubation is carried out during lh, at 37°C, in the presence of 2% of CO2• Then, lOO ⁇ l of maintenance medium is added in each well and incubation is allowed to proceed during three days.
  • the verification of the cytopathic effect is carried out every day and the viral titter is calcu- lated according to the Karber formula.
  • AL3092VRS 200 10 4 DICTso/ml OE1292VRS 100 IO 5 • 5 DICT5 ⁇ /ml AL0294VRS 100 IO 5 • 5 DICT5p/ml
  • the objective was to determine the dilution of the polyclonal antibody to be used for the sensibiliza- tion of the plates.
  • Concave bottom plate with 96 wells, made of polystyrene MaxisorpTM U16 (Nunc) were used.
  • the lyophilized polyclonal antibody lot AL0692PRS-1 was resuspended at the rate of 0.0147mg of powder/ml of distilled water.
  • the thus resuspended polyclonal anti ⁇ body contains 4.28mg of protein/ml. It was diluted in PBS at the following concentrations: 8.5, 4.2, 2.1, 1.0, 0.5 and 0.2 ⁇ g of protein/lOO ⁇ l. These dilutions were used to coat the wells at the rate of lOO ⁇ l/well.
  • the anti-mouse antibody conjugated to peroxi ⁇ dase (Jackson ImmunoResearch Laboratories, West Grove, USA) diluted 1/2000 in PBS was added at the rate of lOO ⁇ l/well and incubated 30 min. at room temperature. After 3 washings, lOO ⁇ l of chromogenic substrate were added in each well. After 10 min., the reaction was stopped with 30 ⁇ l of H2SO4 4N. The plate is read by bichromatism, at a wave length of 450nm with a refer ⁇ ence of 550nm, on a spectrophotometer for ELISA (SLT Lab Instrument, Gr ⁇ dig, Australia).
  • the objective was to determine the working dilu ⁇ tion of the MAb RSQ89C Lot #SE2192M89C. This lot of MAb was used during the entire project.
  • An ELISA test as described above in section II-l was carried out. The polyclonal antibody was used at the rate of l ⁇ g/well and the BRSV diluted 1/10. The dilutions per factor of 2, 1/4 to 1/64 of MAb were tested. The working dilution was defined as the last dilution giving the highest optical density.
  • the best ratio between the optical density of the positive control and that of the negative control was detected at a dilution 1/400 of the polyclonal an ⁇ tibody and at a dilution 1/3000 of the conjugate.
  • the working dilution was established at 1/3000.
  • the lot of conjugate No. 115-035-062 was titrated and used during the entire project. Its work ⁇ ing dilution was established in function of the optical densities obtained with the first lot of the conjugate used (#115-030-061).
  • the objective was to compare two ELISA methods: with and without a pre-incubation of the antigen and the MAb.
  • the optical densities obtained by using the positive control Lot #AL3092VRS diluted 1/10 by the two methods were compared.
  • the ELISA test with a pre-incu ⁇ bation of the antigen and the MAb was carried as described in section II-l. During the second test, the antigen and the MAb were added in to subsequent steps and each were incubated during 15 min. at room tempera ⁇ ture.
  • the method without pre-incubation of the antigen and the MAb was selected.
  • the plates were sensitized with the polyclonal antibody at the rate of l ⁇ g/well during 1 hour at 37 C C. After blocking with the TRIS-C-T during 30 min. at 37°C, the positive control diluted 10 "1 and IO "2 was added. The MAb was diluted 1/8 and the conjugate 1/3000.
  • the time of incuba ⁇ tion of the antigen was incubated during 5, 15, 30 and 45 min.
  • the MAb was incubated during 15 min. and the conjugate during 30 min.
  • the incubations were carried out at room temperature. The strongest reaction was detected when the antigen was incubated during 45 min.
  • the antigen was incubated during 45 min., the MAb during 5, 15, 30 and 45 min. and the con ⁇ jugate during 30 min. The incubations were carried out at room temperature. The strongest reaction was detected when the MAb was incubated during 5 min.
  • the antigen was incubated during 45 min., the MAb during 5 min. and the conjugate during
  • the developing solution (chromogenic substrate) is added at the rate of lOO ⁇ l/well.
  • the objective was to prepare a panel of refer ⁇ ence antigens made of two strongly positive samples, two weakly positive and two negative, in order to use it for the validation of the reagents and the kits.
  • the antigen panel is made of 6 samples:
  • HighRl RSV of lot OE1292VRS non diluted HighR2: RSV of lot AL0294VRS non diluted LowRl: RSV of lot OE1292VRS diluted 1/20 LowR2: RSV of lot AL0294VRS diluted 1/60 NegRl: Negative control lot # MI2194CMDBK
  • NegR2 Negative control lot # MI2594CMDBK Each antigen was kept in a 20 aliquots of 1 ml and frozen at -70°C.
  • Each antigen was tested in 32 wells on 5 plates (at the rate of 8 or 4 wells per plate) independently test by two operators.
  • the average optical density (O.D.) as well as the standard deviation, the percent ⁇ age of the standard deviation and the lower and upper limits of acceptance were calculated for each antigen.
  • Table 9 shows that the percentage of standard deviation of the positive antigens (strong and weak) in all case is lower than 10. Consequently, the noted variation is acceptable since a percentage of standard deviation of 15% is recommended as limit of acceptance (Gall, D. et al. , 1992, Guidelines for the development , optimization and standardization of the targeting ELISA procedure, ADRI, Nepean). This value may not be con ⁇ sidered in the case of negative antigens, since very small variations may represent a very high deviation percentage.
  • the lower and upper O.D. limits indicate the limits between which the optical density values must be found each time the panel of antigen will be used so that the test be validated.
  • Table 10 shows the lower and upper O.D. limits between which the optical density values should be found each time C+ and C- will be used. 2. Variation intra-plate
  • the objective was to verify if the localization of the sample on the plate had an influence on the optical densities obtained.
  • Tables 12 to 16 show that the intra-plate vari ⁇ ation is acceptable since the standard deviation per ⁇ centage, for all the positive antigens of each plate, was in all cases lower than 15%. Therefore, the results obtained with the samples are independent of the localization on the plate.
  • the objective was to determine the variation of optical densities obtained with the same sample tested on different plates.
  • Table 17 shows that the variation inter-plate is acceptable since the percentage of standard deviation for all the positive antigens was in all cases lower than 15%. Therefore, the results obtained with the samples are reproducible on different plates.
  • the objective was to test the immunological sen ⁇ sitivity of the ELISA test developed.
  • an ELISA test was provided by using BRSV as antigen at different dilutions (10 _1 , 10 ⁇ 2 and IO" 3 ).
  • the BRSV used a titer of 10 DICT 50 / m ⁇ .
  • the objective was to determine the immunological specificity of ELISA.
  • the ELISA was made by utilizing the following viral strains as antigens: bovine adenovirus; equine viral arteritis; bovine herpes virus type 4; bovine viral diarrhea virus; bovine coronavirus; feline leuke- mia virus; virus of infectious bovine rhinotracheitis; equine influenza virus; bovine parainfluenza virus; canine parvovirus; bovine rotavirus; purified bovine syncytial respiratory virus Ql; bovine syncytial respi ⁇ ratory virus VR794 ATCC; human syncytial respiratory virus; bovine syncytial respiratory virus Ql.
  • the first experiment consists in the detection of bovine RSV antigen in the nasal secretions of calf infected experimentally, while the second experiment consists in the detection of bovine RSV antigen in the nasal secretions of calf of different herds.
  • the MDBK cells were distributed in plates of 24 wells as described in point 1-3.1. After 3 hours, while the cells are 50% confluent, the growth medium was removed and the cells were washed with MEM contain ⁇ ing 1% antibiotic. Each sample of nasal secretions is inoculated on two wells, 200 ⁇ l per well. Previously, the nasal secretions have been filtered on 0.45 ⁇ m. For each plate, a maintenance medium (Chart No. 2) is used as negative control and BRSV virus diluted 1/10 in this medium is used as positive control. The plates are incubated during 1 hour at 37°C with 2% C ⁇ 2- Then, 1 ml of the maintenance medium is added in each well. The plates were examined daily during four days and if necessary a second passage is carried out.
  • a maintenance medium (Chart No. 2) is used as negative control and BRSV virus diluted 1/10 in this medium is used as positive control.
  • the plates are incubated during 1 hour at 37°C with 2% C ⁇ 2- Then, 1 ml of
  • the plates are fixed with for ⁇ maldehyde.
  • 1 ml of formaldehyde diluted 1:7 in distilled water is put in.
  • the cells were washed 3 times with PBS-TweenTM.
  • a solution of 5% skim milk in PBS is deposited on the cells, 250 ⁇ l per well and incubated 30 minutes at 37°C.
  • the cells are washed 3 times with PBS-TweenTM and the MAb 89c is introduced, 250 ⁇ l per well, and incubated 60 minutes at 37°C.
  • the cells are again washed 3 times and at 250 ⁇ l of anti-mouse conjugated peroxidase, produced on goat, diluted 1/200 in a solution of PBS-0.1% TweenTM-1% gelatin are placed in each well.
  • the plates are incu ⁇ bated 90 minutes at 37°C.
  • the cells are washed 3 times and 250 ⁇ l per wells of substrate 3-Amino-9-Ethylcarbo- zole (AEC) is applied. After a 10 min. incubation fol ⁇ lowed by 3 last washings with PBS-TweenTM, the plates are dried and a reading is carried out on an inversion microscope with a small objective 10X.
  • the presence of a brownish color in the cytoplasm indicates the pres ⁇ ence of virus.
  • na nasal secretions not available
  • Isolated viral isolation *: optical density
  • the relative sensitivity (Se) and the relative specificity (Sp) have been calculated and the correlation between the two tests, viral isola ⁇ tion and ELISA was measured by the kappa factor (K).
  • the limit threshold of 0.100 is the threshold which enables to have a maximum and sensitivity and specificity (100%).
  • this threshold of 0.100 it will be noted that we have 100% sensitivity and specificity for the entire range of ratio between 1 and 1.5.
  • 6 kits have been prepared and the test was made by 6 operators having little or no experience with ELISA (results; Table 22). It appears that the ratio 1.5 really establishes the limit since none of the operators has exceeded this value with the negative controls. However, with operator E, a ratio of 1.494 is obtained. This value is very close to 1.5, and it is therefor concluded that the ratios between 1.5 and 1.7 should be considered as doubtful.
  • the interpretation of the results could be summarized as follows: if the optical density of a sample is lower than 0.100, this sample is negative. - if the optical density of a sample is higher or equal to 0.100 and the ratio (SAM od /NC ocj ) is smaller than 1.5, than the sample is negative. if the optical density of a sample is higher or equal to 0.100 and the ratio (SAM od /NC oc ;) is between 1.5 and 1.7 then the sample is doubtful and the test should be taken over again, if the optical density of a sample is higher or equal to 0.100 and the ratio (SAM od /NC oc j) is higher or equal to 1.7 then the sample is posi- tive.
  • the rela ⁇ tive sensitivity of the test is 92%, the specificity is 100% and the kappa factor is 0.957.
  • Ratio 1 Optical density sample 1/ optical density of negative control
  • Ratio 2 Optical density sample 21 optical density of negative control
  • the visual reading and the reading with spectro ⁇ photometer was realized on 653 different wells (Table 23). Visual reading was carried out at least by two persons. In all cases the reading obtained by differ ⁇ ent persons was the same.
  • Table 23 shows that the visual reading 100% cor ⁇ relation with the spectrophotometer when the optical density is higher than 0.2 or less than 0.1.
  • the specificity and sensitivity of the visual reading towards spectro ⁇ photometer reading are 100 and 67% respectively. It can be concluded that the specificity of the visual reading is very good in all cases.
  • its sensitivity is smaller than that of the spectro- photometer reading, when the optical densities of posi ⁇ tive samples are between 0.1 and 0.2.
  • the polyclonal antibodies lot #AL0692PRS-2 was purified according to the method described in section 1-1.3. Its working dilution was established at 1/400 according to the methodology described in section II-l. The ELISA test was carried out by utilizing the poly ⁇ clonal antibody purified diluted 1/400 in order to sensitize the plates. The panel of antigen (section III-l) as well as the C+ and the C-, have been used as antigens. A sensitized plate with the polyclonal antibody lot #Av0692PRS-l was used as a reference plate.
  • SUBSTITU ⁇ Table 25 shows that the values of optical den ⁇ sity obtained by utilizing the three lots of MAb are found between the upper and lower limits established for each of the lots of antigens in section III-l. Therefore, the production of MAb was realized and validated three times. It can be concluded that the method of production of the MAb is reproducible.
  • the choice of the conditions of preservation was based on the following conditions: a minimum time of preservation of 22 weeks (based on the comparison of the values of optical density between the element to be tested and the reaction control) , the conditions of non-lyophilization were pre ⁇ ferred to the conditions of lyophiiization; and - facility of utilization in the field.
  • the second objective was to validate the kit made of the elements selected in points 1 to 7 (point 8, below).
  • SUBSTITUTE SHEE The third objective was to establish the time of preservation of each element of the kit at 22 and 37°C (point 9, below).
  • the fourth objective was to establish the time of preservation of the elements of the kit after the kit has been opened (point 10, below). 1.
  • the objective was to test the stability of the plates previously sensitized and blocked.
  • the plates were sensitized with the polyclonal antibody diluted 1/400 in PBS (l ⁇ g/well) during 1 h at 37°C. After 3 washings, the plates were blocked with TRIS-C-T during 30 min. at 37°C. The plates were closed with a sheet of acetate, sealed in an aluminum wrapper and preserved at 4°C.
  • the plates were tested at weeks 10 and 22 after their preparation. A sensitized plate and blocked the day of the test was used as control plate.
  • Table 25 shows that the plates previously sensi ⁇ tized and blocked preserved at 4°C give values of opti- cal density equivalent to those of the control plates. Therefore, the plates preserved at 4°C appear stable at least during 22 weeks. 2. Positive Control (virus BRSV)
  • the objective was to test the stability of posi ⁇ tive control kept at 4°C.
  • the BRSV Lot #OE1292VRS was diluted at the rate of 2:1, 1:1 and 1:5 in SPGA (0.218M sucrose, 0.0038M KHPO4, 0.0072M K 2 P0 4 , 0.0049M sodium glutamate, pH between 6.2 and 7). These viral preparations, lyophilized and non-lyophilized were kept at 4°C. Moreover, BRSV was diluted at the rate of 1:1,
  • Table 27 shows that the positive controls lyophilized and kept at 4°C give values of optical den- sity which are equivalent to those of the controls freshly prepared. Therefore, the positive controls which are lyophilized and kept at 4°C appear stable at least during 22 weeks.
  • Table 28 shows that the positive controls, non- lyophilized and diluted in the SPGA solution are not stable at 4°C. However, when the positive control is diluted in the SS solution, it appears stable at least during 10 weeks. A follow up of these positive con ⁇ trols during 22 weeks offers the future possibility of utilizing a non-lyophilized positive control.
  • the objective was to verify the stability of the negative control kept at 4°C.
  • the cells MDBK Lot #AL1192CMDBK were diluted at the rate of 2:1, 1:1 and 1:5 in SPGA. These prepara- tions were kept at 4°C, lyophilized and non- lyophilized.
  • Tables 29 and 30 show that the negative controls kept under all the conditions tested gave the values of optical density which are equivalent to those of the controls freshly prepared. In order to keep the same conditions as in the positive control, the lyophilized pure cells were selected as negative control of the kit.
  • MAb Monoclonal Antibody
  • the MAb Lot SE2192M89C was diluted 1/4 and 1/8 in the SPGA solution and in TRIS 50mM pH 8.6. These preparations were kept at 4°C lyophilized and non- lyophilized.
  • Table 31 shows that the lyophilized and non- lyophilized MAb gives values of optical density which are equivalent to those of the freshly prepared MAb. Therefore, the MAb appears stable at least during 22 weeks.
  • All the conditions tested may be selected to preserve the MAb of the kit.
  • MAb non-lyophilized and diluted 1/4 in TRIS was selected .
  • the conjugate Lot #115-035-062 was diluted at the rate 1/100 and 1/500 in TRIS-C-T in the presence of 1% casein or 3% albumin or 1% gelatin or 10% bovine fetal serum (SFB) or 1% calf serum.
  • the conjugate was also diluted 1/100 in the SS solution.
  • the conjugate was verified at weeks 10 and 22.
  • the conjugate unfrozen the day of the test was used as control of the reaction.
  • the conjugates 1/100 were diluted at 1/3000 before their use.
  • Table 32 shows that the conjugate diluted in TRIS-CT contains either 1% casein or 3% albumin or 1% gelatin, and when lyophilized gives values of optical density which are equivalent to those the freshly pre ⁇ pared conjugate. Therefore, the conjugate under the above-described conditions, appear stable at least dur ⁇ ing 22 weeks.
  • Table 33 shows that the conjugate, non- lyophilized and kept at 4°C, gives values of optical density which are equivalent to those of the controlled conjugate only when it is diluted solution SS. There ⁇ fore, the conjugate, non-lyophilized and diluted 1/100 in the SS solution and kept at 4°C, has been chosen as the conjugate of the kit.
  • Table 34 shows that the solutions kept at 4 °C give values of optical density which are equivalent to those of freshly prepared solutions . Therefore, the solutions appear stable at least during 12 months at
  • the objective was to select the method of pres ⁇ ervation of each component of the kit.
  • kits All these elements were combined to constitute kits.
  • the kit thus assembled was validated at 5 months and 7.5 months after its preparation (see point 8, below) .
  • the objective was to validate kits composed of elements preserved during 5 and 7.5 months at 4°C.
  • the ELISA test was carried out by utilizing as antigens the panel of antigens (section III-l) as well as C+, C-, a positive sample and a negative sample.
  • the values of the optical density obtained with the panel of antigen was compared to those fixed as low limit and upper limit in section III-l.
  • the values of optical density obtained with C+, C-, the positive sam ⁇ ple and negative sample were compared to those obtained by utilizing a freshly prepared kit.
  • Table 35 shows that the values of optical den ⁇ sity by utilizing the kits kept during 5 and 7.5 months at 4°C are found between the superior and lower limits established for each of antigens composing the panel of antigens in section III-l. Moreover, the values of optical density obtained are equivalent to those obtained with the control kit, given a difference of optical density ⁇ 0.1. Therefore, it can be concluded that 5 and 7.5 month old kits are validated.
  • the objective was to test at different tempera ⁇ tures the stability of the selected kit.
  • Kits have been prepared and kept at 37°C, 22°C and 4°C. Each element of the kit was tested on days 1.5, 3, 9, 14, 20 and 32 after their preparation. The tests were realized by utilizing all the freshly pre ⁇ pared elements except for the element to be tested. The values of optical density obtained with the ele ⁇ ments at 37°C and at 22°C were compared to the values obtained with the elements at 4°C. The values have been considered as equivalent if the difference was ⁇ 0.1.
  • Table 36 shows that the plates are stable during 1.5 days at 37°C and during at least 20 days at 22°C.
  • Table 37 shows that the positive control has a decrease of 58% of its intensity of reaction after 1.5 days at 37°C and that it appears to be stable during 3 days at 22°C. However, even if after 32 days at 22°C the optical density represents 31% of the optical den ⁇ sity of the control, it may be used as positive control of the kit since it gives a positive reaction upon vis ⁇ ual reading. The negative control appears stable at least during 32 days at 22°C.
  • Table 38 shows that MAb is stable 14 days at °C and at least during 32 days at 22°C.
  • Table 40 shows that the chromogenic substrate are stable during 9 days at 37°C and during at least 32 days at 22°C
  • Table 41 shows that the washing solution is sta ⁇ ble during at lea ⁇ t 20 days at 37 and 22°C. 10. Stability of the elements making up the kit after its opening.
  • the objective was to test the stability of each element of the kit after its opening, i.e. after a first utilization.
  • a kit was opened at kept at 4°C. Each element of the kit was tested on days 1.5, 3, 9, 14, 20 and 32 after opening of the kit. The tests were carried out by utilizing all the freshly prepared elements except for the element to be tested. The values of optical density obtained were compared with the values obtained on days 0. The values were considered as equivalent if the different was ⁇ 0.1.
  • Table 42 shows that the values of optical den ⁇ sity between day 0 and day 32 are equivalent since the difference found was _0.1. Therefore, it can be con-
  • SUBSTITUTE SHEET eluded that the elements of the kit are stable at least during 32 days after the opening of the kit.
  • MAb which are specific to BRSV was carried out and MAb RSQ89C was selected as a second antibody for double sandwich ELISA.

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Abstract

La présente invention concerne un test ELISA pour déterminer quantitativement l'antigène de VRSB, vivant ou inactivé, dans un échantillon d'origine bovine contenant une quantité inconnue d'antigène de VRSB, consistant à: a) incuber un support solide portant un premier anticorps anti-VRSB immobilisé, avec un échantillon biologique pendant un temps suffisant pour former un complexe immun entre l'anticorps anti-VRSB et tout antigène de VRSB présent dans l'échantillon; b) incuber le support solide après l'étape a) avec un second anticorps anti-VRSB; et c) détecter le second anticorps lié à l'étape b), pour déterminer la quantité d'antigène de VRSB présente dans l'échantillon.
PCT/CA1996/000662 1995-10-03 1996-10-02 Kit permettant de diagnostiquer la presence du virus respiratoire syncytial bovin WO1997013150A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033417A1 (fr) * 2000-10-17 2002-04-25 Besst-Test Aps Dosage servant a la detection directe d'une cellule biologique associee au virus rs, dans un echantillon de fluide organique
WO2002033418A1 (fr) * 2000-10-17 2002-04-25 Besst-Test Aps Analyse permettant de detecter directement une cellule biologique dans un echantillon de fluide corporel
CN107858357A (zh) * 2017-11-22 2018-03-30 北京农学院 牛呼吸道合胞体g蛋白特异性核酸适配体及其筛选方法

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EP0242082A2 (fr) * 1986-04-16 1987-10-21 City Of London Polytechnic Méthode de détection et de numération de bactéries réduisant le sulfate
JPS6316266A (ja) * 1986-07-09 1988-01-23 Toyobo Co Ltd 自己抗体測定用試薬組成物
RU1814075C (ru) * 1991-09-03 1993-05-07 Научно-Исследовательский Институт Охраны Материнства И Детства Мз Таджсср Способ определени трипсина в крови

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Publication number Priority date Publication date Assignee Title
EP0242082A2 (fr) * 1986-04-16 1987-10-21 City Of London Polytechnic Méthode de détection et de numération de bactéries réduisant le sulfate
JPS6316266A (ja) * 1986-07-09 1988-01-23 Toyobo Co Ltd 自己抗体測定用試薬組成物
RU1814075C (ru) * 1991-09-03 1993-05-07 Научно-Исследовательский Институт Охраны Материнства И Детства Мз Таджсср Способ определени трипсина в крови

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Title
A. CLAYTON ET AL.: "The selection and performance of monoclonal and polyclonal anti-respiratory syncytial virus (RS) antibodies in capture ELISAs for antigen detection.", JOURNAL OF VIROLOGICAL METHODS, vol. 17, no. 3,4, September 1987 (1987-09-01), pages 247 - 261, XP000617816 *
DATABASE WPI Section Ch Week 9429, Derwent World Patents Index; Class B04, AN 94-240423, XP002024901 *
DATABASE WPI Week 8809, Derwent World Patents Index; AN 88-060559, XP002024900 *
R. M. HENDRY ET AL.: "Monoclonal capture antibody ELISA for Respiratory Syncytial Virus: detection of individual viral antigens and determination of monoclonal antibody specificities.", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 77, 1985, pages 247 - 258, XP002024899 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002033417A1 (fr) * 2000-10-17 2002-04-25 Besst-Test Aps Dosage servant a la detection directe d'une cellule biologique associee au virus rs, dans un echantillon de fluide organique
WO2002033418A1 (fr) * 2000-10-17 2002-04-25 Besst-Test Aps Analyse permettant de detecter directement une cellule biologique dans un echantillon de fluide corporel
US6790611B2 (en) 2000-10-17 2004-09-14 Besst-Test Aps Assay for directly detecting a RS virus related biological cell in a body fluid sample
CN107858357A (zh) * 2017-11-22 2018-03-30 北京农学院 牛呼吸道合胞体g蛋白特异性核酸适配体及其筛选方法
CN107858357B (zh) * 2017-11-22 2020-12-22 北京农学院 牛呼吸道合胞体g蛋白特异性核酸适配体

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